Caspr2 Antibodies in Patients with Thymomas

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provided by Elsevier - Publisher Connector MALIGNANCIES OF THE THYMUS

Angela Vincent, FRCPath,* and Sarosh R. Irani, MA*

neuromuscular junction. Neuromyotonia (NMT) is due to Abstract: Myasthenia gravis is the best known autoimmune disease motor nerve hyperexcitability that leads to muscle fascicula- associated with thymomas, but other conditions can be found in tions and cramps. A proportion of patients have antibodies patients with thymic tumors, including some that affect the central that appear to be directed against brain tissue-derived volt- nervous system (CNS). We have become particularly interested in age-gated potassium channels (VGKCs) that control the ax- patients who have acquired neuromyotonia, the rare Morvan disease, onal membrane potential.4,5 VGKC antibody titers are rela- or limbic encephalitis. Neuromyotonia mainly involves the periph- tively low in NMT. eral nerves, Morvan disease affects both the peripheral nervous Morvan disease is a rare condition first described in system and CNS, and limbic encephalitis is specific to the CNS. 1876 but until recently hardly mentioned outside the French Many of these patients have voltage-gated potassium channel auto- literature.6 The patients exhibit NMT plus autonomic distur- antibodies. All three conditions can be associated with thymomas bance (such as excessive sweating, constipation, and cardiac and may respond to surgical removal of the underlying tumor arrhythmias) and central nervous system (CNS) involvement together with immunotherapies and symptomatic treatments. Herein, that includes insomnia and confusion. Many reported cases we review the results of our recent studies that show that voltage- are associated with thymomas,7,8 although other tumors have gated potassium channel autoantibodies are not principally directed been identified.9 Morvan disease can also be nonparaneoplas- against the potassium channels themselves but in some patients are tic10 and occasionally self-limiting.11 Antibodies to VGKCs directed against a protein that is complexed with potassium channels have been found at moderately high titers in a few reported in both the peripheral nervous system and CNS, contactin-2 associ- cases of MoS.9 ated protein (Caspr2). These antibodies are common in the subgroup Limbic encephalitis (LE) was first described in the of patients with thymic malignancies. 1960s12 in association with small cell lung cancer. LE patients develop a subacute onset of amnesia, which is often Key Words: Thymoma, VGKC antibody, VGKC complex, Neuro- profound, and is usually accompanied by seizures and psy- myotonia, Caspr2. chiatric disturbances. Patients traditionally have magnetic (J Thorac Oncol. 2010;5: S277–S280) resonance imaging evidence of inflammation within the me- dial temporal lobes. Although originally considered a paraneoplastic condition,13 there are increasing numbers of pa- he presence of autoantibodies to proteins that are shared tients with nonparaneoplastic LE, and many of these have Tbetween tumors and the nervous system is well known. VGKC antibodies at high titers.14–16 A relatively specific Such paraneoplastic or “onconeural” antibodies are usually feature in these cases is the presence of a serum hyponatre- directed at intracellular proteins and are unlikely, therefore, to mia. The VGKC antibody titers fall when the patients are be pathogenic, but the detection of the antibody helps identify treated in parallel with clinical improvements. the presence of a tumor. In thymomas, such antibodies NMT, MoS, and LE have all been described in patients 17,18 include those against titin in myasthenia gravis (MG)1 and with MG and thymoma, and in patients with thymoma 18 against a variety of neuronal antigens such as Hu and CV2/ without MG, usually with VGKC antibodies. These condi- CRMP5 in other cases.2 tions can also occur with other tumors (principally small cell By contrast, autoantibodies that are causative are usu- lung cancer) and other antibodies, but in this study, we will ally directed toward membrane receptors or ion channels. only discuss the patients with VGKC antibodies and review MG is caused in most patients by antibodies to acetylcholine recent data on the specificity of antibodies to VGKCs. receptors (AChRs3) that lead to loss of AChRs from the MATERIALS AND METHODS *Department of Clinical Neurology, John Radcliffe Hospital, Oxford, United The data and methods are based on the results of studies Kingdom. Disclosure: Angela Vincent, FRCPath, and her department receive royalties over a number of years, which are described in detail in Ref. and payments for antibody assays. 19. Patient serum samples were mainly referred for testing by Address for correspondence: Angela Vincent, FRCPath, Department of our clinical neuroimmunology service, and the clinical fea- Clinical Neurology, Level 6 West Wing, John Radcliffe Hospital, Oxford tures were obtained subsequently by questionnaires and dis- OX3 9DU, UK. E-mail: [email protected] cussion with the referring neurologists. VGKC antibodies Copyright © 2010 by the International Association for the Study of Lung 125 Cancer were measured by radioimmunoprecipitation of I-␣-dend- ISSN: 1556-0864/10/0510-0277 rotoxin-labeled VGKCs extracted from rabbit brain tissue.4,5

Journal of Thoracic Oncology • Volume 5, Number 10, Supplement 4, October 2010 S277 Vincent and Irani Journal of Thoracic Oncology • Volume 5, Number 10, Supplement 4, October 2010

The brain tissue extracts were made in 2% digitonin, a Of the 82 patients to be described, 61 had LE, 5 had detergent which is mild and does not dissociate protein MoS, and 16 had NMT. Six patients had thymomas including complexes. Some experiments were performed with addi- one patient with MoS and five with NMT; one patient with tional sodium dodecyl sulfate (SDS), a harsher detergent. We NMT had a metastatic endometrial carcinoma. The thymoma also transfected human embryonic kidney cells (HEK293) classifications were obtained from the patients’ clinical records cells with cDNAs for VGKC subunits (Kv1.1, 1.2, 1.6) or and are given in Table 1. Eight of the remaining patients had past contactin-2 associated protein (Caspr2) or Caspr2-EGFP. The histories of other tumors that appeared to be inactive at the time latter construct was made by fusing the cDNA for EGFP onto of presentation of their VGKC-Ab–related diseases (five LE, the C-terminal end of the Caspr2 (see Ref. 19). one MoS, and two NMT, see Ref. 19). The results of their VGKC antibodies are shown in RESULTS Figure 1A; as expected from observations on smaller cohorts The patients to be discussed here were chosen on the or individual cases, those with NMT had relatively low basis of their high VGKC-Abs (values Ͼ400 pM; normal antibodies compared with those with LE, whereas the few values Ͻ100 pM), because patients with high titers most MoS cases had intermediate titers. Correspondingly, the an- often have CNS disease. We will compare those patients with tibody titers in the patients with thymomas were lower than in thymomas or other tumors with those who did not have those without (Figure 1B). detectable tumors. It should be made clear, therefore, that A series of experiments were undertaken to define the many patients with thymomas and antibody-mediated dis- specificities of the antibodies for the VGKCs. The VGKCs 125 ␣ eases, e.g., MG, do not have VGKC antibodies and would not that bind I- -dendrotoxin and are used for measuring have been studied nor did we include patients with lower antibodies are tetramers composed of differing proportions of VGKC-Ab titers. Kv1.1, 1.2, 1.4, and 1.6. However, when we expressed these Kv1 subunits individually or collectively in human embryonic kidney (HEK293) cells, we could not detect Kv1- specific binding by immunofluorescence, a technique that we TABLE 1. Thymoma Types in Patients with Thymoma and now use for detecting antibodies to many other membrane VGKC Antibodies antigens such as the AChR (termed a cell-based assay; as in Description of Thymoma Refs. 19–22). Moreover, there was no effect of the antibodies Sex Age from Histology Reports from patients with high titers of VGKC antibodies on the M 71 Type B3, Stage 2 voltage-dependent currents in these cells (See Ref. 19). M 60 Type B2, Stage 2 We hypothesized that the antibodies might be binding, F 53 Thymic carcinoma, Type B2, Stage 2 instead, to other proteins that were part of the brain mem- M 37 Type AB brane protein complexes that contain VGKCs. To investigate M 49 Malignant metastatic thymoma this, we first added increasing amounts of harsh detergent, M 44 Malignant metastatic thymoma SDS, to the VGKC assays. Although the binding of commer- F 74 Malignant metastatic thymoma, Type B2 with areas of B3 cial antibodies to Kv1.1 or 1.2 was not substantially affected by 0.025% SDS, binding of the patients’ antibodies was MG, myasthenia gravis. reduced to approximately 20% (Figure 1C). This suggested

FIGURE 1. A, Voltage-gated potassium channel antibody (VGKC-Ab) titers in healthy controls (HCs) and in neuromyotonia (NMT), Morvan disease (MoS), and limbic encephalitis (LE) patients. B, VGKC-Ab titers in patients with and without thymomas. C, The binding sites for patients’ antibodies on 125I ␣-dendrotoxin-labeled digitonin- solubilized VGKC antibody complexes are more sensitive to a low concentration of sodium dodecyl sulfate (SDS) than binding sites for Kv1.1 antibodies. D, Healthy control sera (HC) do not show binding to the surface of human embryonic kidney cells expressing Caspr2. The Caspr2 was tagged with enhanced green fluorescent protein (Caspr2-EGFP) so that the transfected cells could be identified. Some VGKC-Ab positive sera (1:100 dilution), including six of the seven sera from patients with thymomas, bound to Caspr2-transfected cells.

S278 Copyright © 2010 by the International Association for the Study of Lung Cancer Journal of Thoracic Oncology • Volume 5, Number 10, Supplement 4, October 2010Caspr2 Antibodies in Patients with Thymomas that the SDS had dissociated the target of the patients’ TABLE 3. Clinical Features of Patients with Thymomas and antibodies from the Kv1 channels. Work by others had shown Caspr2 Antibodies that proteins such as Caspr2, Lgi1, contactin-2, and PSD95/93 were part of the Kv1 VGKC complexes.23 We Neuromyotonia (muscle twitching and cramps) found that commercial antibodies to Caspr2 were able to Sweating precipitate 125I-␣-dendrotoxin-labeled VGKCs from digito- Autonomic disturbance nin extracts of rabbit brain. This showed that Caspr2 must be Pain (often burning) part of the protein complex that contains VGKCs and sug- Confusion or amnesia (MoS) gested that some patients might have antibodies to Caspr2 Hallucinations (MoS) rather than to the VGKCs themselves. Insomnia (MoS) Only one patient in this cohort had thymoma and MoS, but other patients with MoS Antibodies to Caspr2 have been identified with thymomas and Caspr2 antibodies in subsequent studies (Irani To determine whether the patients had antibodies to et al., unpublished data). Caspr2, we expressed the protein in HEK293 cells and looked MoS, Morvan disease. for binding of the patients’ serum IgG to the unpermeabilized cells. Healthy/disease control sera did not show binding (Figure 1D, upper panels). Caspr2 was expressed on the cell few case reports that exist, it is clear that thymomas are surface as demonstrated by binding of a commercial antibody relatively frequent in these patients, but there have been no (not shown). Antibodies to Caspr2 were found in 19 of the 82 comprehensive reviews as yet. patients (23%; e.g., Figure 1D, lower panels). Caspr2 is The most frequent antibody associated with thymomas strongly expressed at the juxtaparanodes of myelinated axons and CNS disturbance are VGKC antibodies. In studies de- in the CNS and in the peripheral nerves (reviewed in Ref. 24). signed to determine the antigenic specificity of these antibod- Antibodies to Caspr2 in Thymoma ies, we discovered that they are not directed against the VGKCs themselves but to other proteins that are part of the Of the 6 patients with thymomas, 5 had Caspr2-Abs, complexes that anchor VGKCs to the juxtaparanodes of compared with 13 of the 75 patients without thymomas the axonal membrane or to synapses and are also likely to (Fisher’s exact test p ϭ 0.0005). The one patient with an modulate their function. In particular, we found that active endometrial carcinoma also had Caspr2-Abs. The di- Caspr2, a protein known to be complexed with VGKCs agnosis and clinical features of the patients with Caspr2 and essential for their localization at the juxtaparanodes,25 antibodies are shown in Table 2, which is divided between is a target for antibodies in around 20% of the patients with those with and without tumors in Table 3. “VGKC” antibodies. The other major target for the “VGKC” antibodies is leucine-rich glioma inactivated 1 DISCUSSION (Lgi1), and our data show a very low rate of tumors in Thymomas have been associated with MG for many association with Lgi1 antibodies.19 years, but more recently it has been clear that other In this review, we summarize the clinical findings in conditions can be found in these patients, including CNS patients with high VGKC antibodies. Nineteen had Caspr2 diseases that are likely due to specific antibodies. The most antibodies, and this included six of the seven with thymomas. striking clinical condition is Morvan disease, which is a These patients mainly had NMT with muscle fasciculations; rare but highly interesting disorder in which patients suffer twitching and cramps; sweating; some autonomic distur- from a combination of peripheral nervous system (NMT), bance; and often pain in the skin, muscle, or joints. Only one autonomic, sleep, and higher mental dysfunction. From the patient in the thymoma group had Morvan disease, but two patients without detected thymomas had this syndrome, which includes the features of NMT with additional insom- TABLE 2. Demographics and Clinical Diagnoses in Patients nia, confusion, and memory loss. with Caspr2 Antibodies We did not find Caspr2 antibodies at detectable Caspr2-Abs levels in patients with typical MG and thymoma without VGKC antibodies.19 It is difficult to predict from this Tumors No Tumors study, therefore, how common Caspr2 antibodies will be in Number 7 12 thymoma patients in general. MG is a much more frequent Thymoma 6a 0 thymoma-associated disease (at least 10 times higher in- Age range 44–71 59–77 cidence, and around 10% of patients have thymomas) than M:F 4:3 12:0 VGKC-Ab–associated CNS disorders. The incidence of MG 3 0 the syndromes associated with thymomas and these Caspr2 NMT 6 3 antibodies is probably low by comparison. Although MoS 1 2 Caspr2 antibodies are an infrequent accompaniment to LE 0 7 thymoma, however, if a patient with thymoma has CNS or a The seventh tumor patient had an active endometrial carcinoma. peripheral nerve involvement, and if they have Caspr2 Caspr2, contactin-2 associated protein; MG, myasthenia gravis; NMT, neuromyo- antibodies, one should assume that the thymoma may be tonia; MoS, Morvan disease; LE, limbic encephalitis. particularly active.

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