Clinical and aetiopathogenetic considerations
The latter hypothesis would be well in line with the fact that ITPR1 is not exclusively expressed in the brain but is present also in the anterior and posterior (including the substantia gelatinosa) horns of the spinal cord [
2,
3], in the sensory DRG, in the trigeminal ganglia, in the sympathetic ganglia [
2], and in the peripheral nerves. Accordingly, signs and symptoms of peripheral nervous system (PNS) involvement (reduced sensory conduction velocity of sural nerve and motor conduction velocities of the median and tibial nerves; reduced muscle stretch reflexes and vibration sense) have been described in a subset of patients with mutations in the
ITRP1 gene [
23,
24], and dysregulation of ITPR1 has been found to cause allodynia and hyperalgesia in an animal model [
5].
The presence of lung cancer suggests a paraneoplastic aetiology in patient 1. Lung cancer is the most common type of tumour in patients with paraneoplastic neurological disorders (PND). However, no antibodies typically found in patients with lung cancer and PND [
25], including anti-Hu, anti-CV2/CMRP5, anti-amphiphysin, anti-VGCC, anti-SOX1, anti-GABABR and anti-AMPAR, were present in our patients. ITPR1 has been shown to be expressed in a broad variety of tumours (partly ectopically), particularly in Hodgkin disease (HD) and malignant lymphoid and myeloid cell lines, but also lung cancer, melanoma, prostate cancer and many other tumour entities [
18]. In patient 2, Bence Jones proteinuria in fact indicated the presence of a non-Hodgkin (B cell) lymphoma and thus a possible paraneoplastic aetiology in this patient as well. By contrast, no evidence of cancer was present in patient 3 at last follow-up.
Patient 1 presented with ascending motor and sensory symptoms suggestive of GBS. Subacute sensory neuronopathy (Denny-Brown syndrome) and subacute motor neuropathy both represent “classical paraneoplastic syndromes” according to an international consensus definition [
25]; GBS, acute sensorimotor neuropathy and autonomic neuropathy, on the other hand, are well-recognized “non-classical”, non-obligatory paraneoplastic syndromes [
25]. While GBS is considered to be of non-paraneoplastic origin in the vast majority of cases, numerous reports of GBS in oncological patients exist, and a population-based study found an increased frequency for tumours in patients with GBS (odds ratio ~2.4) [
26]. Associated tumours reported in GBS were mainly lung cancer, Hodgkin lymphoma, non-Hodgkin lymphomas and leukaemia, though several other types of cancer were occasionally described (see Additional file
1: Table S1 for a comprehensive list and references).
A paraneoplastic aetiology of ITPR1-IgG-associated autoimmunity in patient 1 is further suggested by the finding of ITRP1 expression in the tumour tissue in structures also stained by the patient’s IgG. According to proteomic data, ITPR1 is not constitutively expressed in normal lung tissue or only at low level [
18]. Of note, ITPR1 has been detected at protein level only in a subset of adenocarcinomas of the lung [
18,
19], leaving the possibility that anti-ITPR1 seropositivity in patients may denote a subtype of adenocarcinoma in patients with lung cancer and PND.
Whether anti-ITPR1 autoimmunity has a protective role in patients with PND, as suggested for other antibody-related syndromes such as anti-Hu, is currently unknown but warrants further investigation. Of potential interest in this context, ITPR1 has been reported to protect renal cancer cells from NK-mediated lysis [
27]. In vivo, ITPR1 targeting significantly enhanced NK-mediated tumour regression [
27].
Some paraneoplastic disorders, including anti-Hu, anti-CV2/CRMP5 and anti-amphiphysin syndrome, have been shown to be capable of causing both central and peripheral neurological symptoms, in line with the presence of the respective autoantigens in both central and peripheral nervous tissue. Similarly, ITPR1 is expressed both in the CNS and in the PNS. Why one and the same paraneoplastic neurological disorder affects the CNS in some patients but the PNS in others (and both systems in yet others) is not well understood. However, differences in blood-brain/CSF barrier function among patients or variations hereof over time in the same patient, the presence or absence of antigen-specific intrathecal B and T cell clones, differences in antigen/epitope availability among tissues and, importantly, differences in antibody epitope/isoform specificity could play a role. To date, eight isoforms of ITPR1 produced by alternative splicing have been described [
28]. Future studies need to address (a) possible cross-reactivity of anti-ITPR1-IgG with ITPR2 and ITPR3 as well as among the various ITPR1 isoforms and (b) differences in epitope/isoform specificity between patients with ITPR1-IgG-associated ACA and ITPR1-IgG-positive patients with peripheral nervous system involvement.
In the adult brain, ITPR1 is not only expressed in PCs of the cerebellum but also in hippocampal neurons (particularly in CA1 pyramidal cells) [
2‐
4], in the putamen and the caudate nucleus, and in the cerebral cortex (most pronounced in pyramidal layer V and non-pyramidal layer II) [
2,
3]. Given this wide expression profile, it is likely that the spectrum of ITPR1 autoimmunity will broaden further as more patients are identified in the future. In this context, it is worth noting that patient 2 developed severe depression in the course of disease. Signs of possible limbic involvement (confusion, severe memory loss, suspected temporal lobe epilepsy) were also present in two recent ITPR1-IgG-positive patients identified by the authors, one of whom also had cerebellar ataxia (unpublished data).
In the light of ITPR1 being expressed in the autonomic nervous system, in particular in the sympathetic ganglia [
2], it is of special note that patient 1 exhibited reduced pathological heart frequency variability, a typical sign of autonomic neuropathy of the heart, and later developed repeated stroke episodes attributed to intermittent atrial fibrillation. Interestingly, ITPR1 is also present in human and murine cardiac myocytes [
18,
29,
30], where it is involved in crucial regulation of intracellular calcium pathways: The cardiac ITPR receptors have recently been shown to modulate the electromechanical properties of the human myocardium and its propensity to develop arrhythmias [
29]. Further studies on anti-ITPR1 autoimmunity should therefore pay attention also to signs and symptoms of autonomic heart dysregulation and autoimmune myocarditis.
It is of further interest in this context that patient 1 reported continuous bowel dysfunction with diarrhoea starting several months before onset of the polyradiculoneuropathy. Whether these symptoms were caused by infection and whether an infectious agent was involved in the pathogenesis of the patient’s neurological disorder (e.g. molecular mimicry) remains unknown. No evidence was found for an infection with
Campylobacter jejuni, which is present in around 30 % of patients with classical GBS. Alternatively, the patient’s bowel dysfunction could have been a first sign of autonomic neuropathy. The latter hypothesis is supported by the fact that he simultaneously developed bladder dysfunction. ITPR1 is expressed in the sympathetic ganglia as well as in the smooth muscle cells of the bowel and the bladder. Accordingly, we found binding of IgG from this and other ITPR1-IgG-positive patients to smooth muscle cells in the enteric wall in a pattern identical to that seen with a commercial antibody to ITPR1 [
1]. Moreover, paraneoplastic enteropathy is a well-known complication of cancer, and diarrhoea has recently been recognized as a typical prodromal symptom in DPPX (dipeptidyl-aminopeptidase-like protein 6) syndrome, another novel antibody-related autoimmune disease of the CNS [
31‐
34]. Studies evaluating the frequency of ITPR1-Ab in patients with paraneoplastic and other types of suspected autonomic enteropathy seem therefore warranted.
Finally, the facts that ITPR1 is expressed in the sensory DRG, the trigeminal ganglia and the substantia gelatinosa [
3], in which C fibre axons synapse with neurons of the pain-transmitting lateral spinothalamic tract and damage to which can cause pain and hyperalgesia, and that ITPR1 dysregulation has been implicated in hyperalgesia and allodynia in animal studies [
5], along with the presence of a severe pain syndrome in our patient, provide a preliminary rationale for seeking a potential role of ITPR1-related autoimmunity also in patients with pain syndromes of unknown aetiology.
Immunological considerations
Passive transfer experiments using IgG from anti-ITPR1-positive patients have not yet been performed. Therefore, no direct evidence for a pathogenic impact of the antibody is currently available. ITPR1 is thought to be primarily an intracellular antigen located in membranes encompassing the endoplasmatic and, in muscle cells, sarcoplasmatic reticulum. Many researchers believe that intracellular antigens are not accessible to antibodies in vivo. In fact, most neurological autoantibodies of proven pathogenic impact, such as antibodies to AQP4 in neuromyelitis optica [
35‐
38], acetylcholine receptor in myasthenia gravis, VGCC in Lambert-Eaton syndrome [
39] and mGluR1 in paraneoplastic cerebellar degeneration [
40], target cell-surface-expressed proteins. Moreover, passive transfer of antibodies to intracellular antigens such as anti-Yo [
41‐
43] has not produced clinical disease in animal studies. Instead, T cell-mediated immune mechanisms directed against the target antigen of the accompanying antibody have been proposed to play a role in those disorders [
44‐
47]. It is therefore possible that the antibody has diagnostic but no pathogenic impact, similar to the situation in many paraneoplastic neurological disorders.
However, surface localization of ITPR1 has been reported to occur under certain circumstances [
48‐
52], warranting further investigation. Moreover, studies demonstrating cell damage following uptake of paraneoplastic antibodies by neurons have somewhat challenged the above-mentioned paradigm (see [
20‐
22] for a summary). Indirect evidence suggesting a potential pathogenic role of anti-ITPR1 includes the fact that the antibodies mainly belonged to the complement-activating IgG1 subclass, the very high serum titres in two of our patients (titres were lower in patient 2, in whom ITPR1 was tested late in the disease course), the decline in titres after tumour removal, which was accompanied by clinical stabilization and improvement, and the good concordance of the antigen’s tissue expression profile with the clinical symptoms present in our patient, in particular in patient 1. The absence of intrathecal ITPR1-IgG synthesis does not
per se argue against a pathogenic role of the antibody: First, the antibody can still reach the CSF via passive diffusion and/or a leaky blood-brain/CSF barrier (ITPR1 was in fact present in the CSF and Q
Alb markedly elevated in patient 1). Second, the blood-CSF barrier is thought to be particularly leaky around the nerve roots. Third, there is no need for intrathecal synthesis when it comes to peripheral nerve damage. Finally, AIs are regularly negative also in AQP4-IgG-positive neuromyelitis optica spectrum disorders (NMOSD) [
53] and in MOG-IgG-positive encephalomyelitis [
54‐
58], two diseases in which a direct pathogenic impact of the antibody has been proven or is highly likely.
Of note, anti-Sj/ITPR1-IgG was of the strongly complement-activating IgG1 subclass in the severely affected patient 1 (as well as in the severely disabled anti-Sj/ITPR1-IgG index patient [
1]), but exclusively of the very weakly complement-activating IgG2 subclass in patient 2, who had relatively mild disease with no major progression of his polyneuropathy after 1.5 years. Future studies investigating the clinical associations of ITPR1-related autoimmunity should therefore include IgG subclass analyses.
To investigate the role of cell-mediated immunity in this disease, we studied the proliferative response of the PBMCs from patient 1 and from a control donor to stimulation with purified ITPR1 (boosted with radiated antigen-presenting cells) and a control protein in vitro, and assessed the PBMC surface phenotypes before and after stimulation. Phenotypic analysis of the PBMCs revealed a marked difference in proportions of B cells, CD4 T cells and CD8 memory T cells between patient and control donor after exposure to ITPR1. Moreover, compared with the healthy donor the patient’s PBMCs proliferated strongly as measured by 3H-thymidine uptake after ITPR1 stimulation. Taken together, these findings indicate that the patient’s PBMCs contained ITPR1-specific B cells and autoreactive CD4 and CD8 T cells, which could be directly involved in injury of the nervous system as well as in antitumour immunity.
Serological and therapeutic considerations
Anti-Sj/ITPR1-IgG titres were extraordinarily high both in the IHC assay (up to 1:15,000) and in the CBA (up to >1:1000) during acute disease in patient 1; after removal of the tumour (and thus of the ectopically presented antigen), CBA titres declined to 1:320 and later 1:100. Similarly, relatively high titres of anti-Sj/ITPR1-IgG were noted in patient 2 (IHC 1:5,000) and in all of the previously reported ITPR1-IgG-positive cases (IHC 1:5,000, 1:3,200, 1:3,200, 1:1,000) [
1]. In direct comparison, IHC titres were higher on average than those in the majority of patients with AQP4-IgG-positive NMOSD or MOG-IgG-positive encephalomyelitis tested by the authors in recent years using the same methodology [
59,
60].
Patients 1 and 2 yielded a positive result in all three assays employed, largely ruling out false-positive results due to insufficient assay specificity. While mouse ITPR1 was used in the CBA, purified rat was employed in the dot-blot assay, and primate, rat and mouse ITPR1 was the antigenic substrate in the IHC assay. Similarly, all previously reported patients reacted with all three antigens. This suggests that the epitope targeted by ITPR1-IgG in these patients was located in a region not relevantly affected by differences in amino acid sequence and/or confirmation between those species. Patient 3 was positive in the CBA and IHC assay but negative in the dot-blot assay; while the reason remains unknown, it is likely that this was due to sensitivity issues related to the dot-blot assay, given that the sample repeatedly yielded low titres in the CBA and in the IHC assay.
Interestingly, serum anti-Sj/ITPR1-IgG was still detected at a titre of >1:1000 both in the IHC assay and in the CBA after seven PEX treatments in patient 1, and CSF ITRP1 was positive 18 days after the first PEX treatment in that patient. This is in line with the authors’ experience from other autoantibody-related neurological syndromes, in which the pathogenic antibodies occasionally persist after five to seven PEX treatments, indicating that the number of PEX treatments usually administered in neurological indications may be too low to achieve complete autoantibody removal in some cases and may need to be adjusted according to serological findings. As a caveat, however, it should be kept in mind that evidence for a direct pathogenic role of the antibody is lacking so far and that the immune response in patients with paraneoplastic neurological syndromes may contribute to controlling tumour growth and spread. Moreover, T cells may be involved in the pathogenesis, which would make it necessary to employ treatment modalities targeting not only the B cell and antibody arm of the immune reaction but also the T cell arm.