BRIEF RESEARCH REPORT published: 12 April 2019 doi: 10.3389/fimmu.2019.00769 Caveats and Pitfalls of SOX1 Autoantibody Testing With a Commercial Line Blot Assay in Paraneoplastic Neurological Investigations Raquel Ruiz-García 1 , Eugenia Martínez-Hernández 2,3 , Milagros García-Ormaechea 1 , Marta Español-Rego 1 , Lidia Sabater 2 , Luis Querol 4,5 , Isabel Illa 4,5,6 , Josep Dalmau 2,7,8 and Francesc Graus 2* Edited by: 1 Roland S. Liblau, Immunology Department, Centre Diagnóstic Biomédic, Hospital Clínic, Barcelona, Spain, 2 Neuroimmunology Program, Institut National de la Santé et de la Institut d’Investigacions Biomédiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 3 Service of Neurology, Hospital Clinic, Recherche Médicale (INSERM), Barcelona, Spain, 4 Neuromuscular Disorders Unit, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 5 Centro de France Investigaciones Biomédicas en Red en Enfermedades Raras (CIBERER), Valencia, Spain, 6 Universitat Autónoma de Barcelona, Barcelona, Spain, 7 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain, 8 Department of Reviewed by: Neurology, University of Pennsylvania, Philadelphia, PA, United States Patrick Joseph Waters, University of Oxford, United Kingdom Bonaventura Casanova, SOX1 autoantibodies are considered markers of small cell lung cancer (SCLC) and Hospital Universitari i Politècnic La Fe, Spain paraneoplastic neurological syndromes (PNS) and are usually determined by commercial *Correspondence: line blot in many clinical services. Recent studies suggested that SOX1 autoantibodies Francesc Graus also occur in patients with neuropathies unrelated to SCLC, questioning the value of francesc.graus@idibaps.org SOX1 autoantibodies as paraneoplastic biomarkers. Here, we compared the specificity Specialty section: and sensitivity of a commercial line blot (Euroimmun, Lübeck, Germany) with those of an This article was submitted to in house cell-based assay (CBA) with HEK293 cells transfected with SOX1. Overall, 210 Multiple Sclerosis and patients were included in the study, 139 patients with polyneuropathies without SCLC, Neuroimmunology, a section of the journal and 71 with disorders associated with SOX1 autoantibodies detected with the in-house Frontiers in Immunology CBA. Forty one of these 71 cases had been referred to our laboratory for onconeuronal Received: 29 January 2019 antibody assessment and 30/71 were patients with known PNS and SCLC. None of Accepted: 25 March 2019 Published: 12 April 2019 the patients with polyneuropathies had SOX1 autoantibodies by either line blot or CBA Citation: (specificity of the immunoblot: 100%; 95%C.I.: 97.8–100). Among the 71 patients with Ruiz-García R, Martínez-Hernández E, CBA SOX1 autoantibodies, only 53 were positive by line blot (sensitivity: 74.6%; 95%C.I.: García-Ormaechea M, 62.9–84.2). Lung cancer was detected in 37/41 (90%; 34 with SCLC) patients referred Español-Rego M, Sabater L, Querol L, Illa I, Dalmau J and Graus F (2019) for onconeuronal antibody assessment and 34 of them also had a PNS. Our study Caveats and Pitfalls of SOX1 confirms the association of SOX1 autoantibodies with SCLC and PNS. The line blot test Autoantibody Testing With a Commercial Line Blot Assay in misses 25% of the cases; therefore, to minimize the frequency of false negative results Paraneoplastic Neurological we recommend the use of a confirmatory test, such as CBA, in patients suspected to Investigations. have a SCLC-related PNS. Front. Immunol. 10:769. doi: 10.3389/fimmu.2019.00769 Keywords: SOX1, autoantibodies, small-cell lung cancer, paraneoplastic neurological syndromes, line blot Frontiers in Immunology | www.frontiersin.org 1 April 2019 | Volume 10 | Article 769 Ruiz-García et al. SOX1 Autoantibody Testing in Paraneoplastic Syndromes INTRODUCTION onconeural antibodies and whose serum showed the presence of SOX1 autoantibodies by CBA. The clinical information SOX1 autoantibodies are serological markers of small cell lung of these 41 patients was used to assess the association of cancer (SCLC).They occur in up to 15% of patients with SCLC SOX1 autoantibodies with PNS and SCLC. Serum samples independently of the presence of a paraneoplastic neurological were deposited in the Biobank of the Institut d’Investigacions syndrome (PNS) (1, 2). The frequency of SOX1 autoantibodies Biomèdiques August Pi i Sunyer, and Hospital of Sant Pau increases to 60% in patients with SCLC and paraneoplastic (Barcelona, Spain). Written informed consent for the storage and cerebellar degeneration (PCD) or Lambert-Eaton myasthenic use of the samples for research was obtained from patients or syndrome (LEMS) (3, 4). SOX1 was first identified as the antigen representative family members. The study was approved by the recognized by anti-glial nuclear antibodies (AGNA) which were ethics committee of the Hospital Clínic. characterized by immunohistochemistry showing a characteristic pattern of reactivity with the nuclei of Bergmann glia cells (5). Diagnostic Criteria for SOX1 belongs to the group B of the Sry-like high mobility SOX1 Autoantibodies group box family of proteins, which are highly expressed in the In initial studies, SOX1 autoantibodies were detected by an developing nervous system and the Bergmann glia of the adult avidin-biotin immunoperoxidase technique on nitrocellulose cerebellum, and in SCLCs (6–8). filters with purified phage plaques expressing SOX1 (3). Samples Unlike other onconeural antibodies, screening by of 30 patients from the initial study that had been positive with immunohistochemistry is not recommended for the detection this technique were examined here with a CBA of HEK293 of SOX1 autoantibodies because the sensitivity is low compared cells transfected with GFP tagged SOX1 (see below). All 30 with that of immunoblot of recombinant SOX1 protein (3). samples were also found positive with the CBA assay, which we Currently, there are several commercial antibody tests based considered the “gold standard” technique for the diagnosis of on line blots of recombinant SOX1 but comparison with other SOX1 autoantibodies in the present study. techniques is lacking and their sensitivity and specificity is unknown. Recent studies using immunoblot or ELISA suggested Cell Based Assay that SOX1 autoantibodies can occur in serum of patients with HEK293 cells transfected with a plasmid containing the human idiopathic polyneuropathies questioning the value of SOX1 SOX1 sequence tagged with the green fluorescent protein autoantibodies as biomarkers of PNS associated with SCLC (GFP) sequence (Origene, RG218236) were fixed with 4% (9, 10). paraformaldehyde for 10 min, permeabilized with 0.3% Triton In this study we assessed the specificity and sensitivity of a X-100 for 5 min, and subsequently incubated with patients’ sera, commercial line blot test for SOX1 autoantibodies in a large diluted 1/40, in PBS-1% bovine serum albumin (BSA) overnight series of patients with several types of neuropathies or PNS. In at 4◦ C, and a fluorescent secondary antibody diluted 1/2000 addition, we examined the clinical features of the patients who (goat anti-human Alexa Fluor 594 [Jackson ImmunoResearch, were referred at our laboratory for the diagnosis of onconeural PA, USA]) for 1 h at room temperature. Slides were then mounted antibodies and had SOX1 autoantibodies. with Prolong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA) and the reactivity visualized with an Axioscope METHODS Zeiss microscope. Patients Line Blot Sera from 210 patients were investigated for the presence of SOX1 Serum samples were tested by the commercial immunoblot autoantibodies by a commercial line blot of recombinant SOX1 kit EUROLINE Paraneoplastic Neurological Syndromes 12 Ag and a cell based assay (CBA) of HEK293 cells transfected with (DL 1111-1601-7 G; Euroimmun, Lübeck, Germany) following SOX1, and the findings were used to calculate the sensitivity and the manufacturers’ instructions at serum dilution 1/100. Test specificity of the commercial line blot test. The cohort included strips were scanned and evaluated for band intensity using three subgroups of patients: (1) 139 patients with different the EUROLineScan software (Euroimmun Lübeck, Germany). types of neuropathies [41 had idiopathic sensory neuropathy, Following the manufacturer’s recommendations, band intensity 49 chronic inflammatory demyelinating polyneuropathy (CIDP), values >10 were considered as SOX1 autoantibody-positive. 18 paraneoplastic neuropathy associated with tumors other Results ≤5 were considered negative and weak bands that showed than lung, and 31 anti-MAG positive monoclonal gammopathy; values between 6 and 10 (borderline range) were also evaluated this subgroup was included because SOX2 autoantibodies have as negative. been described in patients with paraproteinemias (11)]; (2) 30 patients with paraneoplastic LEMS, PCD, or other PNS and RESULTS SCLC with SOX1 autoantibodies that had been initially tested by an avidin-biotin immunoperoxidase technique on nitrocellulose All 139 sera from patients with polyneuropathies were SOX1 filters containing purified phage plaques expressing SOX1 (these autoantibody-negative by the commercial line blot and SOX1 patients were included in the initial study that reported the CBA. The serum of one patient with sensory neuropathy and characterization and clinical value of SOX1 autoantibodies) (3); parotid cancer was positive by immunoblot with a score of 8 and (3) 41 patients referred to our laboratory for analysis of (borderline range) provided by the automated band intensity Frontiers in Immunology | www.frontiersin.org 2 April 2019 | Volume 10 | Article 769 Ruiz-García et al. SOX1 Autoantibody Testing in Paraneoplastic Syndromes FIGURE 1 | (A) HEK293 cells transfected with GFP-SOX1 (green) incubated with (S1) a negative control and two sera positive for SOX1 antibodies (S2 and S3 red). (B) The three sera were incubated with strips of the commercial immunoblot (EUROLINE Paraneoplastic Neurological Syndromes 12 antigens). Note that one of the positive sera in the CBA assay is negative by the commercial immunoblot (strip 3). assessment of EUROLineScan software. However, the same (90%) patients, 34 [83%] of them SCLC. Only 2 (5%) patients serum was negative in CBA of SOX1. In the remaining 71 patients had tumors other than lung cancer (breast, prostate), and no with CBA SOX1 autoantibodies (30 with PNS and SCLC from cancer was detected in the other two patients (5%). A PNS was the initial study and 41 referred to our laboratory for antibody confirmed in 34 of 41 (83%) patients. Among the seven patients testing) the immunohistochemistry on rat cerebellar sections without PNS, five had cancer but the cause of the neurological showed the pattern compatible with AGNA antibodies in 50 of symptoms was metastasis, Wernicke encephalopathy, or non- 60 (83%) sera, and the other 11 cases were not assessable due specific complains, and the other two patients did not have to co-existence of other immunoreactivities. The line blot was cancer and the cause of neurological symptoms (cerebellar ataxia positive for SOX1 antibodies in 53 of 71 patients (74.6%) and and fasciculations) was unclear. negative in 18 (24.4%). Ten of the 18 samples showed weak bands of reactivity (borderline range 6–10) and the other eight DISCUSSION were negative (≤5) (Figure 1). No clinical differences were noted between these patients and the 18 (25.4%) cases that were SOX1 The findings of this study confirm the robust association between autoantibody negative by line blot (Table 1). the occurrence of SOX1 autoantibodies and the presence of lung The specificity of the line blot for diagnosis of SOX1 cancer and show the limitations of the immunohistochemical and autoantibodies (proportion of samples without SOX1 line blot assays in the detection of these antibodies. Currently, autoantibodies that were also negative by the line blot) was the preferred screening test for onconeural antibodies in many 100% (95%C.I.: 97.8–100) and the sensitivity (proportion of diagnostic laboratories is the use of commercial line blots which samples with SOX1 autoantibodies that were also positive in can identify multiple onconeural antibodies in the same strip. the line blot) 74.6% (95%C.I.: 62.9–84.2). If we exclude the The main advantage in using these kits is the simultaneous 30 patients with SOX1 autoantibodies that were selected from assessment of multiple onconeural antibodies in a single assay. our database of PNS, the clinical data of the remaining 41 On the other hand, when these commercial line blots are used patients whose samples were sent for onconeuronal antibody as the only antibody screening test, there is an increased risk, testing confirmed the specificity of SOX1 autoantibodies for which varies for each antibody, of reporting false positive results, PNS and lung cancer. Lung cancer was diagnosed in 37 of 41 downplaying the clinical significance of the autoantibodies. For Frontiers in Immunology | www.frontiersin.org 3 April 2019 | Volume 10 | Article 769 Ruiz-García et al. SOX1 Autoantibody Testing in Paraneoplastic Syndromes TABLE 1 | Demographic, clinical, and immunological data of 71 patients with (9, 10). A study that investigated the frequency of SOX1 antibodies according to the commercial immunoblot results. SOX1autoantibodies in 92 patients with paraneoplastic and Immunoblot Immunoblot P (t-student, non-paraneoplastic neuropathies, found nine positive patients positive negative Chi2 ) and only 5 (55.5%) of them had cancer (SCLC: four patients; N = 53 (75%) N = 18 (25%) bronchial carcinoid: 1) (9). SOX1 autoantibodies were detected by immunoblot of HEK293 cells transfected with mouse SOX1 Median age 63 (22–87) 63 (52–74) 0.93 clone and the findings were not confirmed with additional (range) techniques (9). Although the homology between human and Male/Female (%) 75/25 83/17 0.16 mouse SOX1 proteins is high we cannot rule out that the Cancer 52 (98) 17 (94) 0.42 discordant results compared with our current findings are due to SCLC 49 (94) 15 (88) 0.41 the use of clones from different species. For example, only 60% of Lung or NSCLC 2 (4) 1 (6) 0.72 patients with antibodies against human myelin oligodendrocyte Other 1 (2) 1 (6) 0.40 glycoprotein (MOG) react with mice MOG (14). In another No cancer 1 (2) 1 (6) 0.42 study, SOX1 autoantibodies were screened by ELISA in a series Paraneoplastic 49 (92) 15 (83) 0.15 of 1,493 patients with paraneoplastic and non-paraneoplastic syndrome disorders. Fifteen patients (1.0%) showed anti-SOX1 reactivity PCD 9 (18) 6 (40) 0.08 and nine of them had neuropathies. Only 6/15 patients had LEMS 12 (24) 2 (13) 0.36 cancer, none of them SCLC. However, the presence of SOX1 LE 13 (27) 2 (13) 0.29 antibodies was not confirmed with other techniques, a usual Other 15 (31) 5 (34) 0.84 requirement in many ELISA assays, and the concentrations of Non- 4 (8) 3 (17) 0.15 SOX1 antibodies were significantly lower compared with those paraneoplastic of controls that had PNS and SCLC, raising doubt on the clinical Other antibodies 35 (66) 9 (50) 0.55 significance of the findings (10). AGNA 38/45 (84) 12/15 (80) 0.69 immunoreactivity We confirmed in the 41 samples with SOX1 autoantibodies that were sent to our laboratory for onconeuronal antibody assessment the robust association with SCLC. example, line blot is more sensitive than immunohistochemistry The frequency of patients without cancer was only 4.9%, in detecting low titer Hu autoantibodies (12). However, these and SCLC was detected in 83% of the patients, figures low Hu antibody titers indicate the presence of a SCLC but similar to those seen with anti-Hu autoantibodies (15). In do not necessarily confirm that the associated neurological contrast, and compared with other antibodies associated symptoms are paraneoplastic (2, 12). A second limitation of with SCLC and PNS (Hu or CRMP5), the frequency commercial line blots is that in some patients they fail to of patients with the final diagnosis of PNS was lower detect the presence of onconeural antibodies, suggesting they (87%) emphasizing the need to rule out alternative are negative. This is clinically important because the disorder diagnoses for the neurological symptoms that may lead may no longer be considered paraneoplastic and therefore, the to antibody testing even when the presence of cancer search for a tumor felt to be unnecessary. Some autoantibodies is confirmed. seem to be more undetected than others; for example, we In summary, findings from this study reveal a diagnostic previously reported that CV2 (CRMP5) autoantibodies were limitation of a commercial line blot for the detection of SOX1 missed in 7.5% (4/53) of samples that were otherwise anti-CV2- autoantibodies. A logical approach to circumvent this problem positive by immunohistochemistry or CBA (13). The reason would be the use of a CBA as the primary assay. However, no for the negative immunoblot reactivity was unclear but the commercial CBA is available, so in order to minimize the risk possibility of very low CV2 antibody titers and the reaction of false negative results by line blot, we suggest using in-house with conformational epitopes were reasonably excluded. In this CBA testing (available in centers of reference) in all patients study, we found the same problem with SOX1 autoantibodies whose samples show weak or absent reactivity with line blot but that were undetected in 25.4% of the samples using the whose clinical syndrome strongly suggest a PNS associated with commercial line blot. This figure could be reduced to 11.3% SCLC (3, 4). if we consider SOX1 autoantibody positive those cases that showed a weak, but visible, immunoblot band (score 6–10 by the EUROLineScan software). However, if this approach is taken ETHICS STATEMENT there is a risk of false positive results as demonstrated by the weak positive sample in the group of neuropathies that was Written informed consent for the storage and use of the samples negative by CBA. A potential solution to increase sensitivity, for research was obtained from patients or representative family keeping specificity, would be to test by CBA all samples that members in agreement with the principles of the Declaration show a weak band in the line blot and that are currently of Helsinki. The study was approved by the ethics committee considered negative. of the Hospital Clínic. The experimental procedure that We were unable to confirm previous findings of SOX1 include animals complies with European and local regulations antibodies in patients with non-paraneoplastic polyneuropathies (2010/63/UE and RD53/2013). Frontiers in Immunology | www.frontiersin.org 4 April 2019 | Volume 10 | Article 769 Ruiz-García et al. SOX1 Autoantibody Testing in Paraneoplastic Syndromes AUTHOR CONTRIBUTIONS JR17/00012 to EM-H by Instituto de Salud Carlos III, Spain. LQ is supported by Fondo de Investigaciones Sanitarias (FIS), FG designed the study and wrote the manuscript in collaboration Instituto de Salud Carlos III, Spain and FEDER under grant with JD, RR-G, and EM-H. RR-G, EM-H, MG-O, ME-R, and LS FIS16/00627, and personal grant SLT006/17/00131 of the Pla performed laboratory work and/or data analysis. LQ and II send estratègic de recerca i innovació en salut (PERIS), Departament patient samples. All authors discussed the results and commented de Salut, Generalitat de Catalunya. on the manuscript. FUNDING ACKNOWLEDGMENTS This study was supported in part by Fondo de Investigaciones We thank Mrs. Eva Caballero for her excellent technical Sanitarias (FIS), FEDER, Spain (FIS 15/00377, FG; FIS 14/00203, assistance in the CBA, immunohistochemistry, and JD), NIH RO1NS077851 (JD), Fundació Cellex (JD) and immunoblot assays. REFERENCES 11. Spisek R, Kukreja A, Chen LC, Matthews P, Mazumder A, Vesole D, et al. 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The use, distribution or reproduction in other forums is permitted, provided the doi: 10.1016/j.jneuroim.2010.07.005 original author(s) and the copyright owner(s) are credited and that the original 10. Berger B, Dersch R, Ruthardt E, Rasiah C, Rauer S, Stich O. Prevalence of publication in this journal is cited, in accordance with accepted academic practice. anti-SOX1 reactivity in various neurological disorders. J Neurol Sci. (2016) No use, distribution or reproduction is permitted which does not comply with these 369:342–6. doi: 10.1016/j.jns.2016.09.002 terms. Frontiers in Immunology | www.frontiersin.org 5 April 2019 | Volume 10 | Article 769