Multi-omics profiling reveals dysregulated ribosome biogenesis and impaired cell proliferation following knockout of CDR2L.

BACKGROUND: Cerebellar degeneration-related (CDR) proteins are associated with paraneoplastic cerebellar degeneration (PCD) - a rare, neurodegenerative disease caused by tumour-induced autoimmunity against neural antigens resulting in degeneration of Purkinje neurons in the cerebellum. The pathogenesis of PCD is unknown, in large part due to our limited understanding of the functions of CDR proteins. To this end, we performed an extensive, multi-omics analysis of CDR-knockout cells focusing on the CDR2L protein, to gain a deeper understanding of the properties of the CDR proteins in ovarian cancer. METHODS: Ovarian cancer cell lines lacking either CDR1, CDR2, or CDR2L were analysed using RNA sequencing and mass spectrometry-based proteomics to assess changes to the transcriptome, proteome and secretome in the absence of these proteins. RESULTS: For each knockout cell line, we identified sets of differentially expressed genes and proteins. CDR2L-knockout cells displayed a distinct expression profile compared to CDR1- and CDR2-knockout cells. Knockout of CDR2L caused dysregulation of genes involved in ribosome biogenesis, protein translation, and cell cycle progression, ultimately causing impaired cell proliferation in vitro. Several of these genes showed a concurrent upregulation at the transcript level and downregulation at the protein level. CONCLUSIONS: Our study provides the first integrative multi-omics analysis of the impact of knockout of the CDR genes, providing both new insights into the biological properties of the CDR proteins in ovarian cancer, and a valuable resource for future investigations into the CDR proteins.

A cerebellar degeneration-related protein 2-like cell-based assay for anti-Yo detection in patients with paraneoplastic cerebellar degeneration.

BACKGROUND AND PURPOSE: Commercially available tests for Yo antibody detection have low specificity for paraneoplastic cerebellar degeneration (PCD) because these assays use cerebellar degeneration-related protein 2 (CDR2) as the antigen, not CDR2-like (CDR2L). We aimed to test the hypothesis that use of a CDR2L cell-based assay (CBA), as an additional screening technique, would increase the accuracy of Yo-PCD diagnosis. METHODS: An in-house CBA to test for anti-CDR2L antibodies was developed and used to screen sera from 48 patients with confirmed anti-Yo-associated PCD. Fifteen non-Yo PCD patients, 22 patients with ovarian cancer without neurological syndromes, 50 healthy blood donors, 10 multiple sclerosis, 15 Parkinson's disease, and five non-paraneoplastic ataxic patients were included as controls. Sera were also tested by western blot analysis using recombinant CDR2 and CDR2L proteins developed in house, by the commercially available line immunoassays from Ravo Diagnostika and Euroimmun, and by the CDR2 CBA from Euroimmun. RESULTS: The CDR2L CBA identified all 48 patients with Yo-PCD. No CDR2L CBA reaction was observed in any of the control sera. The western blot technique had lower sensitivity and specificity as sera from eight and six of the 48 Yo-PCD patients did not react with recombinant CDR2 or CDR2L, respectively. CONCLUSIONS: The CDR2L CBA is highly reliable for identification of Yo-PCD. Although our findings indicate that, currently, the combination of CDR2 and CDR2L yields the most reliable test results, it remains to be evaluated if a test for single anti-CDR2L positivity will serve as a sufficient biomarker for Yo-PCD diagnosis.

Avidity of onconeural antibodies is of clinical relevance.

Onconeural antibodies are important in the detection of paraneoplastic neurological syndromes (PNS). The avidity of Hu, Yo, and CRMP5 antibodies from 100 patients was determined by immunoprecipitation (IP), and 13 of the Yo positive sera were also tested by surface plasmon resonance (SPR). There was a significant association between the results from IP and SPR. Yo antibodies had higher avidity than Hu and CRMP5 antibodies, and both high- and low-avidity antibodies were associated with tumors and PNS. High-avidity Yo antibodies were mainly associated with ovarian cancer, whereas high-avidity Hu and CRMP5 antibodies were mainly associated with small-cell lung cancer. Low-avidity CRMP5 and Yo antibodies were less often detected by a commercial line blot than high-avidity antibodies. The failure to detect low-avidity onconeural antibodies may result in under diagnosis of PNS.

CDR2 antigen and Yo antibodies.

Paraneoplastic cerebellar degeneration (PCD) is often associated with Yo antibodies that are directed against human cerebellar degeneration-related protein 2 (CDR2). Such antibodies may also be found in ovarian cancer patients without PCD. We studied if there was an association between Yo antibody production and differences in CDR2 cDNA sequence, mRNA or CDR2 expression in ovarian cancers. We found similar CDR2 cDNA sequence, mRNA and protein levels in primary ovarian cancers, with or without associated Yo antibodies. CDR2 was also present in other cancers, as well as in normal ovary tissue. The results suggest that Yo antibodies are not only related to the expression of CDR2 alone, but also to immune dysregulation.

CRMP5 Antibodies-Diagnostic Challenges.

CRMP5-associated paraneoplastic neurological syndromes (PNS) are rare, and only few studies describe larger cohorts of patients with CRMP5 antibodies. We have included 24 patients with CRMP5 antibodies and compared clinical findings with diagnostic findings from two different line assays (Ravo and Euroimmun), staining of cerebellar sections and results of a newly developed cell-based assay for detection of CRMP5 antibodies, CRMP5-CBA. We found that peripheral neuropathy and cerebellar ataxia together with lung cancer were the most common diagnoses associated with CRMP5 antibodies. CRMP5-CBA was easy to perform, identified all relevant cases for CRMP5-associated PNS and is therefore a valuable add-on for verification of CRMP5 positivity in diagnosis of PNS.

Paraneoplastic Cerebellar Degeneration: The Importance of Including CDR2L as a Diagnostic Marker.

OBJECTIVE: Investigate the value of including cerebellar degeneration-related protein 2-like (CDR2L) as a marker in commercial diagnostic tests for anti-Yo-associated paraneoplastic cerebellar degeneration (PCD). METHODS: We included sera and CSF samples from 24 patients with suspected PCD (6 of whom had PCD with underlying gynecologic or breast cancer), who were positive for Yo antibodies using the commercially available, paraneoplastic neurologic syndromes (PNS) 14 Line Assay from Ravo Diagnostika. The samples were further evaluated using the EUROLINE PNS 12 Ag Line Assay and a cell-based assay (CBA) from Euroimmun. For confirmation of positive lineblot results, we used indirect immunofluorescence of rat cerebellar sections. We also tested all samples in 2 assays developed in-house: a CBA for CDR2L and a Western blot analysis using recombinant cerebellar degeneration-related protein 2 (CDR2) and CDR2L proteins. RESULTS: In PNS 14 and PNS 12 Ag Line Assays, anti-CDR2 reactivity was observed for 24 (100%) and 20 (83%) of the 24 samples, respectively. Thirteen of 24 subjects (54%) were also positive using the Euroimmun CBA. Rat cerebellar immunofluorescence was the best confirmatory test. In our in-house CBA for CDR2L and Western blot for CDR2 and CDR2L, only the 6 patients with confirmed PCD reacted with CDR2L. CONCLUSIONS: Commercially available tests for Yo antibody detection have low specificity for PCD because these assays use CDR2 as antigen. By adding a test for CDR2L, which is the major Yo antigen, the accuracy of PCD diagnosis greatly improved. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that a CBA for CDR2L accurately identifies patients with PCD.

Antibody to CCDC104 is associated with a paraneoplastic antibody to CDR2 (anti-Yo).

Patients with cancer may develop paraneoplastic neurological syndromes (PNS) in which onconeural antibodies are important diagnostic findings. As the functional role of onconeural antibodies is largely unknown, insight gained by identifying associated antibodies may help to clarify the pathogenesis of the PNS. In this study, we identified patients with Yo antibodies who also had antibodies to an uncharacterized protein called coiled-coil domain-containing protein 104 (CCDC104). We found a significant association between CCDC104 and Yo antibodies (4 of 38, 10.5%), but not other onconeural antibodies (0 of 158) (P = 0.007, Fisher's exact test). The prevalence of CCDC104 antibodies was approximately similar in patients with cancer (8 of 756, 1.1%) and in healthy blood donors (2 of 300, 0.7%). CCDC104 antibodies were not associated with PNS, as this was found in only two of the ten CCDC104-positive patients. The CCDC104 protein, whose function is unknown, is expressed in various human tissues, including the brain, and is localized mainly to the nucleus, but is also found in the cytoplasm. The association between Yo and CCDC104 antibodies may indicate functional similarities.

Expression of the onconeural protein CDR1 in cerebellum and ovarian cancer.

Cerebellar degeneration related protein 1 (CDR1) is expressed in the cerebellum, and CDR1 antibodies have been associated with paraneoplastic cerebellar degeneration (PCD). In this study, we examined CDR1 expression in cerebellum and in ovarian and breast tumors, as well as the intracellular localization of CDR1 in cancer cells in culture. CDR1 was strongly expressed in the cytosol and dendrites of Purkinje cells and in interneurons of the molecular layer in cerebellum. CDR1 was also present in ovarian and breast tumors, as well as in ovarian and breast cancer cell lines, but was not present in normal breast or ovarian tissue. In cells overexpressing CDR1, CDR1 localized close to the plasma membrane in a polarized pattern at one edge. CDR1 was strongly expressed on the outer surface, apparently in filopodias or lamellipodias, in cells endogenously expressing CDR1. Overexpression of CDR1 showed a 37 and a 45 kDa band in western blot. The 37-kDa isoform was present in 16 ovarian cancer lysates, while the 45-kDa isoform was only found in three ovarian cancer patients. The presence of CDR1 in ovarian cancer was not associated with PCD. CDR1 antibodies were only found in serum from one patient with PCD and ovarian tumor with metastases. Therefore, CDR1 is probably not a marker for PCD. However, CDR1 may be associated with cell migration and differentiation.

Hu and Yo antibodies have heterogeneous avidity.

Onconeural antibodies such as anti-Hu and anti-Yo may be important in the pathogenesis of paraneoplastic neurological syndromes. The avidity of these antibodies is not known. In this study, we compared the avidity of Hu and Yo antibodies both at single time points and over a time range of 2 months to 6 years. The avidity of Yo and Hu antibodies differed among the patients, but anti-Yo generally had higher avidity than anti-Hu. Whether Yo antibodies are more pathogenic than Hu antibodies are presently unknown.

CDR2L Antibodies: A New Player in Paraneoplastic Cerebellar Degeneration.

OBJECTIVE: Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD). We have characterized Yo sera by measuring CDR2 and CDR2L antibodies and the localization of their antigens. METHODS: Forty-two Yo sera from patients with paraneoplastic neurological syndromes (PNS), 179 sera from ovarian and 114 sera from breast cancer patients without PNS and 100 blood donors were screened for CDR2 and CDR2L antibodies by radioactive immune assay (RIA). Fluorescence microscopy was also used to determine the presence of CDR2 or CDR2L antibodies by staining of HeLa cells transfected with CDR2 or CDR2L fused to green fluorescent protein (GFP). Confocal microscopy was further used to localize the CDR2 and CDR2L proteins. RESULTS: RIA showed that 36 of the 42 Yo positive sera contained CDR2 and CDR2L antibodies whereas 6 sera contained only CDR2 antibodies. Five of the ovarian cancer patients had CDR2L antibodies and 4 of the breast cancer patients had either CDR2 or CDR2L antibodies. Only patients with both antibodies had PCD. RIA and staining of transfected cells showed similar results. Yo antibodies were not present in the 100 blood donors. Confocal microscopy showed that CDR2 and CDR2L were localized to the cytoplasm, whereas CDR2L was also present on the cell membrane. INTERPRETATION: Yo sera usually contain CDR2 and CDR2L antibodies and both antibodies are associated with PCD. Since only CDR2L is localized to the cell membrane it is likely that CDR2L antibodies may be of primary pathogenic importance for the development of PCD.