Metabotropic glutamate receptor 1 is associated with unfavorable prognosis in ER-negative and triple-negative breast cancer.

New therapies are an urgent medical need in all breast cancer subgroups. Metabotropic glutamate receptor 1 (mGluR1) is suggested as a potential new molecular target. We examined the prevalence mGluR1 expression in different clinically relevant breast cancer subgroups and determined its association with prognosis. In this retrospective cohort, 394 consecutive primary breast cancer tissues were incorporated into a tissue microarray and immunohistochemically stained for mGluR1. The prevalence of mGluR1 protein expression in different breast cancer subgroups was evaluated and correlated with metastasis-free survival (MFS) and overall survival (OS). In total, 56% (n = 219) breast cancer tissues had mGluR1 expression. In estrogen receptor (ER)-negative tumors, 31% (n = 18/58) had mGluR1 expression that was significantly associated with MFS (HR 5.00, 95% CI 1.03-24.35, p = 0.046) in multivariate analysis, independently from other prognostic factors. Of the 44 triple-negative breast cancer (TNBC), 25% (n = 11) expressed mGluR1. mGluR1 expression in TNBC was significantly associated with shorter MFS (HR 8.60, 95% CI 1.06-20.39, p = 0.044) and with poor OS (HR 16.07, 95% CI 1.16-223.10, p = 0.039). In conclusion, mGluR1 is frequently expressed in breast cancer. In ER-negative breast cancer and in TNBC mGluR1 protein expression is an unfavorable prognostic marker. This study provides rationale to explore mGluR1 as a novel target for breast cancer treatment, especially for the more aggressive TNBC.

Neurologic syndromes related to anti-GAD65: Clinical and serologic response to treatment.

OBJECTIVE: Antibodies against glutamic acid decarboxylase 65 (anti-GAD65) are associated with a number of neurologic syndromes. However, their pathogenic role is controversial. Our objective was to describe clinical and paraclinical characteristics of anti-GAD65 patients and analyze their response to immunotherapy. METHODS: Retrospectively, we studied patients (n = 56) with positive anti-GAD65 and any neurologic symptom. We tested serum and CSF with ELISA, immunohistochemistry, and cell-based assay. Accordingly, we set a cutoff value of 10,000 IU/mL in serum by ELISA to group patients into high-concentration (n = 36) and low-concentration (n = 20) groups. We compared clinical and immunologic features and analyzed response to immunotherapy. RESULTS: Classical anti-GAD65-associated syndromes were seen in 34/36 patients with high concentration (94%): stiff-person syndrome (7), cerebellar ataxia (3), chronic epilepsy (9), limbic encephalitis (9), or an overlap of 2 or more of the former (6). Patients with low concentrations had a broad, heterogeneous symptom spectrum. Immunotherapy was effective in 19/27 treated patients (70%), although none of them completely recovered. Antibody concentration reduction occurred in 15/17 patients with available pre- and post-treatment samples (median reduction 69%; range 27%-99%), of which 14 improved clinically. The 2 patients with unchanged concentrations showed no clinical improvement. No differences in treatment responses were observed between specific syndromes. CONCLUSION: Most patients with high anti-GAD65 concentrations (>10,000 IU/mL) showed some improvement after immunotherapy, unfortunately without complete recovery. Serum antibody concentrations' course might be useful to monitor response. In patients with low anti-GAD65 concentrations, especially in those without typical clinical phenotypes, diagnostic alternatives are more likely.

The expanded clinical spectrum of anti-GABABR encephalitis and added value of KCTD16 autoantibodies.

In this study we report the clinical features of 32 patients with gamma aminobutyric acid B receptor (GABABR) antibodies, identify additional autoantibodies in patients with anti-GABABR encephalitis that mark the presence of an underlying small cell lung carcinoma and optimize laboratory methods for the detection of GABABR antibodies. Patients (n = 3225) were tested for the presence of GABABR antibodies using cell-based assay, immunohistochemistry and live hippocampal neurons. Clinical data were obtained retrospectively. Potassium channel tetramerization domain-containing (KCTD)16 antibodies were identified by immunoprecipitation, mass spectrometry analysis and cell-based assays. KCTD16 antibodies were identified in 23/32 patients with anti-GABABR encephalitis, and in 1/26 patients with small cell lung carcinoma and Hu antibodies, but not in 329 healthy subjects and disease controls. Of the anti-GABABR encephalitis patients that were screened sufficiently, 18/19 (95%) patients with KCTD16 antibodies had a tumour versus 3/9 (33%) anti-GABABR encephalitis patients without KCTD16 antibodies (P = 0.001). In most cases this was a small cell lung carcinoma. Patients had cognitive or behavioural changes (97%) and prominent seizures (90%). Thirteen patients developed a refractory status epilepticus with intensive care unit admittance (42%). Strikingly, 4/32 patients had a rapidly progressive dementia. The addition of KCTD16 to the GABABR cell-based assay improved sensitivity of the in-house fixed cell-based assay, without loss of specificity. Twenty-two of 26 patients improved (partially) to immunotherapy or chemotherapy. Anti-GABABR encephalitis is a limbic encephalitis with prominent, severe seizures, but patients can also present with rapidly progressive dementia. The co-occurrence of KCTD16 antibodies points towards a paraneoplastic origin. The addition of KCTD16 improves the sensitivity of the cell-based assay.

Antibodies to TRIM46 are associated with paraneoplastic neurological syndromes.

Paraneoplastic neurological syndromes (PNS) are often characterized by the presence of antineuronal antibodies in patient serum or cerebrospinal fluid. The detection of antineuronal antibodies has proven to be a useful tool in PNS diagnosis and the search for an underlying tumor. Here, we describe three patients with autoantibodies to several epitopes of the axon initial segment protein tripartite motif 46 (TRIM46). We show that anti-TRIM46 antibodies are easy to detect in routine immunohistochemistry screening and can be confirmed by western blotting and cell-based assay. Anti-TRIM46 antibodies can occur in patients with diverse neurological syndromes and are associated with small-cell lung carcinoma.

The relevance of VGKC positivity in the absence of LGI1 and Caspr2 antibodies.

OBJECTIVE: To assess the clinical relevance of a positive voltage-gated potassium channel (VGKC) test in patients lacking antibodies to LGI1 and Caspr2. METHODS: VGKC-positive patients were tested for LGI1 and Caspr2 antibodies. Patients lacking both antibodies were matched (1:2) to VGKC-negative patients. Clinical and paraclinical criteria were used to blindly determine evidence for autoimmune inflammation in both groups. Patients with an inconclusive VGKC titer were analyzed in the same way. RESULTS: A total of 1,455 patients were tested by VGKC radioimmunoassay. Fifty-six patients tested positive, 50 of whom were available to be included. Twenty-five patients had antibodies to LGI1 (n = 19) or Caspr2 (n = 6) and 25 patients lacked both antibodies. Evidence for autoimmune inflammation was present in 7 (28%) of the VGKC-positive patients lacking LGI1 and Caspr2, compared to 9 (18%) of the VGKC-negative controls (p = 0.38). Evidence for autoimmune inflammation was mainly found in patients with limbic encephalitis/encephalomyelitis (57%), but not in other clinical phenotypes (5%, p < 0.01). VGKC titers were significantly higher in patients with antibodies to LGI1 or Caspr2 (p < 0.001). However, antibodies to Caspr2 could also be detected in patients with inconclusive low VGKC titer, while many VGKC-positive patients had no evidence for autoimmune inflammation. CONCLUSIONS: VGKC positivity in the absence of antibodies to LGI1 and Caspr2 is not a clear marker for autoimmune inflammation and seems not to contribute in clinical practice. No cutoff value for the VGKC titer was appropriate to discriminate between patients with and without autoimmune inflammation.

Cerebellar degeneration associated with mGluR1 autoantibodies as a paraneoplastic manifestation of prostate adenocarcinoma.

Subacute cerebellar degeneration associated with metabotropic glutamate receptor type 1 (mGluR1) autoantibodies is an uncommon syndrome known to be part of the spectrum of paraneoplastic cerebellar degenerations associated with neuronal autoantibodies. We describe a patient with prostate adenocarcinoma who developed a subacute cerebellar ataxia. Autoantibodies specific to mGluR1 were detected in patient's serum and cerebrospinal fluid (CSF). Immunohistochemistry analyses of patient's prostate adenocarcinoma revealed abundant mGluR1 expression in luminal acinar epithelial cells and binding of patient's IgGs to tumoral mGluR1. These findings suggest that cerebellar degeneration associated with mGluR1 antibodies can be a paraneoplastic accompaniment of prostate adenocarcinoma.

Multiplex serology of paraneoplastic antineuronal antibodies.

Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered 'well characterized' and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9-7.3%). The average between-run within-day CV was 5.1% (range 2.9-6.7%) while the average between-day CV was 8.1% (range 2.8-11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology.

Psychiatric phenomena as initial manifestation of encephalitis by anti-NMDAR antibodies.

OBJECTIVE: Autoimmune encephalitis associated with autoantibodies against the N-methyl-d-aspartate receptor (NMDAR) often presents with behavioural change. Our objective was to describe in detail the psychiatric presentation and pathways to care in order to aid the early diagnosis of NMDAR encephalitis. METHODS: Sera and cerebrospinal fluid (CSF) from patients with suspected NMDAR encephalitis were tested on HEK 293 cells transfected with the NR1 subunit of the NMDAR. Clinical information was obtained from the referring psychiatrists and neurologists and by review of the clinical records. RESULTS: Samples from 15 patients (13 female, 2 male, mean age 24 years, range 5-56 years) tested anti-NMDAR positive. Twelve of the 15 patients (80%) presented with prominent psychiatric symptoms and 8 were initially referred to a psychiatric service. The most prominent initial psychiatric symptoms were anxiety in seven (47%), behavioural change (often bizarre) in six (40%) and agitation in five (33%). All patients developed psychiatric symptoms in the first 6 weeks of illness. Thirteen patients received psychotropic medications: antipsychotics in 12 and benzodiazepines in 11. Treating physicians considered the psychotropic medication not effective in 11 patients resulting in many drug switches. At nadir, all patients were in a very poor condition. However, eight patients (53%) recovered (almost) completely. Outcome tended to be better in patients who had received early immunotherapy or tumour removal. CONCLUSIONS: Autoimmune encephalitis and anti-NMDAR testing in serum and CSF should be considered in patients, especially young females, presenting with atypical psychiatric phenomena. Early diagnosis and treatment will likely improve the prognosis of NMDAR encephalitis.

Identification of delta/notch-like epidermal growth factor-related receptor as the Tr antigen in paraneoplastic cerebellar degeneration.

OBJECTIVE: Anti-Tr is among the better described autoantibodies in paraneoplastic cerebellar degeneration (PCD) combined with Hodgkin lymphoma (HL); however, the Tr antigen remains unidentified. METHODS: We used immunoprecipitation of total rat brain extract followed by mass spectrometry to identify the antigen recognized by anti-Tr-positive sera. By Western blotting and cell-based assays, we tested a total of 12 anti-Tr-positive and 246 control sera and determined the region of the epitope recognized by the anti-Tr antibodies. Deletion and mutant constructs were generated to further map the antigenic region. RESULTS: Mass spectrometry analysis of immunopurified rat brain extract using 4 different anti-Tr-positive sera led to the identification of Delta/Notch-like epidermal growth factor-related receptor (DNER) as the Tr antigen. All but 1 of 246 control samples were negative in the HeLa cell-based screening assay, whereas 12 of the 12 anti-Tr-positive sera stained hemagglutinin-tagged DNER-expressing cells. Only 1 control subject with HL but no ataxia was found to be both DNER and Tr positive. Using deletion constructs, we pinpointed the main epitope to the extracellular domain. Knockdown of endogenous DNER in hippocampal and N-glycosylation mutations abolished the anti-Tr staining, indicating that glycosylation of DNER is required for it to be recognized by anti-Tr antibodies. INTERPRETATION: DNER is the antigen detected by anti-Tr-positive sera. Presence of anti-Tr antibodies in patients with PCD and HL or HL only can now be screened quickly and reliably by using a cell-based screening assay.

A new paraneoplastic encephalomyelitis autoantibody reactive with the axon initial segment.

Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.