Immunopathological characterization of ovarian teratomas associated with anti-N-methyl-D-aspartate receptor encephalitis

Encephalitis (E) with anti-NMDA receptor (NMDAR) antibodies (NMDAR-E) is a recently described severe autoimmune neurological disorder, defined by a clinical presentation of encephalitis and presence of IgG targeting the GluN1 subunit of the NMDAR in patients’ cerebrospinal fluid (CSF) [7]. An underlying neoplasm is found in 25 to 40% of patients, primarily in young females, and this associated tumor is an ovarian teratoma in 90% of the cases [2, 8, 29]. This strong association suggests a role of the tumor in the immunopathogenesis of the autoimmune disease. Histopathological studies reporting presence of neuroglial tissue expressing NMDAR in ovarian teratoma associated with NMDAR-E [7, 14, 27, 30] raised the hypothesis that immunization against the NMDAR might be triggered by NMDAR expression by teratoma neuroglial elements. However, later studies including sporadic teratomas without associated NMDAR-E showed that presence of an ovarian teratoma with neuroglial tissue expressing NMDAR is not sufficient to induce anti-NMDAR auto-immune response [14, 20, 27], suggesting tumor specificities in NMDAR-E patients. Moreover tumor-like features of the teratomatous neuroglial component have been reported in the recent literature [9, 14]. Furthermore, a marked intratumoral lymphoid infiltrate colocalizing with mature neuroglial elements is reported in a few patients with NMDAR-E but is yet to be fully characterized [6, 9, 14, 19, 27, 30]. In the present study, we compared ovarian teratomas associated with NMDAR-E to those in patients with sporadic teratomas in order to describe histological features characterizing these tumors.

This histopathological study emphasizes the specificities characterizing ovarian teratomas associated with NMDAR-E. Notably, all but one NMDAR-E associated ovarian teratomas contained a nervous tissue component, while only just over a third of control ovarian teratomas did so. Furthermore, expression of the NMDAR GluN1 subunit by the teratomatous nervous tissue was significantly more often glial in NMDAR-E teratomas than in control teratomas. Another striking particularity was that among these 27 mature teratomas, 3 contained neuroglial tissue exhibiting histopathological features of CNS neuroglial tumor, while it was exceptionally reported in the literature on sporadic ovarian teratomas ([1, 3, 10, 15, 24, 25, 31‐34] the histological classification of these 20 cases is summarized in Additional file 1: Table S2 in Online Resource). We also confirmed the particular immune environment of the nervous tissue of these teratomas that exhibited massive inflammatory infiltrates.

Although the frequency of immature teratomas among NMDAR-E associated cases herein is lower than that reported in the previously published case series [6, 9, 30], 14% of all reported cases (including those herein) are immature which is much greater than sporadic ovarian teratomas; only 3% of the latter are diagnosed as immature, mostly on the presence of immature neural tissue. [4, 22]. This difference could be related to ovarian teratomas of NMDAR-E patients more frequently containing neural tissues that have de facto the potential to be immature; neural tissue was found in all but one ovarian teratoma from patients with NMDAR-E but only just over a third of controls herein, and 30% of mature ovarian teratomas described in the literature [4, 21]. This suggests that the presence of nervous tissue may be essential to trigger the breaking of immune tolerance against NMDAR associated with encephalitis. Interestingly, the expression of this receptor in the nervous component of NMDAR-E teratoma has been detected in some cases by immunofluorescence using patient serum IgG [30] or immunohistochemistry against the GluN1 subunit [14, 27] and the GluN2B subunit of NMDAR [27]. Furthermore, using a commercial antibody we confirmed herein the positivity of teratoma nervous tissues for the GluN1 subunit of NMDAR in more than 80% of NMDAR-E teratomas of this large cohort. We also, however, report GluN1 expression in the same proportion of control teratomas with a neural differentiation, confirming that the mere ectopic expression of GluN1 is not per se sufficient to trigger the cross-immune reaction leading to neurological symptoms. This is supported by the report of GluN1 and GluN2B expression in epithelial ovarian carcinoma without occurrence of paraneoplastic NMDAR-E [23]. A possible explanation could be related to the NMDAR subunit composition profile or the cellular phenotype of cells expressing NMDAR. Herein, GluN1 was expressed both by neuronal and glial cells composing the nervous component of ovarian teratomas associated or not with NMDAR-E; neuronal expression is expected but glial expression is noteworthy, even if NMDAR expression by human astrocytes has been described [5, 17]. This glial GluN1 expression was more frequent in NMDAR-E associated teratoma than in sporadic cases, which might suggest that type of neural cells expressing the antigen NMDAR could be involved in the outbreak of NMDAR-E.

The present study establishes that other neuroglial features also distinguish NMDAR-E teratomas from sporadic ovarian teratomas. For instance, in three patients the mature nervous component presented tumor-like areas characterized by histological features of CNS tumors [18]. Such foci of nervous tissue forming a histological pattern reminiscent of glioblastoma can be observed in ovarian teratoma [28]. However, less than 25 cases of mature ovarian teratoma with nervous tissue exhibiting histological features of glioma (mostly glioblastoma and oligodendroglioma) have been described since 1960 [1, 3, 10, 15, 24, 25, 31‐34], making these extremely rare events. The presence of a singular neuronal component in NMDAR-E teratomas has been previously suggested by the report of “abnormal neuronal elements” in 3 cases of mature ovarian teratomas [9]. Consistently, Iemura et al. have recently reported an “abnormal monotonous appearance” of the mature neuroglial tissue of 4 NMDAR-E teratomas with densely aggregated small neurons [14]. Herein, we precisely characterized these previously reported histological phenotypes that were consistent with neuroglial tumors; we observed obvious neuroglial changes that were not limited to the presence of “dysplastic or monotonous neurons”, but which also included gliomatous features, characterized by increased glial cell cellularity and glial marker expression, and clearly distinct from reactive abnormalities often observed in teratomas [9]. In one case, we further investigated the molecular phenotype of this glioma-like component. No gliomatous mutations were detected in this ganglioglioma-like proliferation, suggesting that adult CNS gliomagenesis pathways might not be involved in the glioma-like phenotype of the teratomatous tissue.

Besides these tumoral specificities, NMDAR-E-associated ovarian teratoma were also characterized by a massive and systematic infiltration of immune cells in close contact with nervous tissue component. Dense inflammatory infiltrates around neural tissue have been previously shown in NMDAR-E teratomas [6, 9, 14, 19, 27]. These lymphoid aggregates are composed of segregated B- and T-cells and sometimes organized in reactive tertiary lymphoid structures [6, 19]. We further revealed that immune infiltrates in NMDAR-E associated teratoma frequently contained mature dendritic cells in the vicinity of nervous tissue. These features are characteristics of tertiary lymphoid organs, that have already been reported in other tumors [13] and are believed to be generated as a result of local antigen presentation in a context of chronic inflammation and to perpetuate adaptive immune responses providing local source for antibody production [12]. Herein, we observed immunoglobulin deposits and immunoglobulin secreting cells in direct contact with the nervous tissue of NMDAR-teratomas, while previous studies had already detected plasma cells in NMDAR-teratomas [19, 32]. The presence of the B-cell response effectors in the tumor associated with B-mediated immunity supported the idea that in young females with NMDAR-E and ovarian teratoma, the tumor triggers the anti-NMDAR immune reaction. In particular, the detection of intra-tumoral IgA is consistent with the existing correlation between the presence of an associated teratoma and anti-NMDAR IgA in the CSF of patients with NMDAR-E [11]. All these data strongly suggest a link between the tumor immune environment and the CNS humoral auto-immunity directed against the onconeural antigen NMDAR. Recently, Makuch et al. demonstrated this link by showing that NMDAR ovarian teratoma tissue contained B cells which can produced IgG directed against GluN1 in culture [19]. We previously reported that genetic alteration of onconeural antigens, including gains and mutations, can play a major role in the induction of the immune response associated with paraneoplastic neurological syndromes [26]. Neoantigen production due to such genetic alterations in NMDAR coding genes might be also a potential mechanism leading to this singular anti-tumor immune response characterized by the intratumoral presence of B- and T-cell response effectors of the auto-immune disease.

Further molecular characterization of the expressed NMDAR antigens and gene expression profiling of tumor infiltrating immune cells in NMDAR-E teratoma are needed. This will require microdissection to improve the sampling of nervous tissue and infiltrates, and the prospective collection of fresh resected tumors to obtain a better quality of extracted DNA/RNA and to allow flow cytometry analysis of infiltrating immune cell population, as previously done in the cystic aspirate from one NMDAR-E teratoma by Makuch et al. [19].

In conclusion, the present study finds that the particular immune environment of the neuroglial tissue in NMDAR-E teratomas is associated with teratoma specificities. These histological features are unlikely to be sufficient per se to increase the immunogenicity of this ectopic nervous tissue; further investigation of teratoma genetic alterations is required to identify the molecular triggers of the immune tolerance breakdown leading to the auto-immune disease.