To our knowledge this is the first study that shows that AP24534 Ma2 autoantibodies is a biomarker with diagnostic and prognostic relevance in the blood stream of SI-NET patients. Ma2 autoantibodies evaluation can identify recurrence of SI-NET patients with higher precision than measurements of plasma CgA levels. This novel finding can significantly improve the clinical management of SI-NET patients. Results Indirect ELISA detects Ma2 autoantibodies in primary SI-NET patients Significantly higher Ma2 autoantibody titer in SI-NET patients compared to healthy volunteers were detected by using the novel indirect ELISA. The reproducibility of the assay is expressed in intra- and inter-assay percent coefficient of variation (CV) as explained in Material & Methods.

The difference was clear both considering the diverse categories of malignancies, primary tumors, lymph node and liver metastasis (Figure 1A), and the whole SI-NETs cohort of patients (Figure 1B). The sensitivity of the ELISA is from 46% to 50%. The specificity threshold is 98% as described in Material and Methods. Receiving operating characteristic (ROC) curve analyses were used to evaluate the possible use of Ma2 autoantibodies as early blood marker for SI-NET. ROC analyses show areas under the curves (AUCs) from 0.734 to 0.816 indicating good accuracy as a diagnostic test. Figure 1C, 1D and 1E show the results of healthy controls (HC) versus (vs.) SI-NET primary tumors (P), vs. SI-NET lymph node metastases (LNM) and vs. SI-NET liver metastases (LM). Figure 1F shows the results of HC vs. SI-NET patients as a whole, for all stages of disease.

The results are summarized in the upper part of Table 1. Figure 1 A novel indirect ELISA detects Ma2 autoantibodies in serum from SI-NET patients. Table 1 Results of indirect ELISA assay for Ma2 autoantibodies. Detection of Ma2 autoantibodies in sera from HC and primary SI-NET patients The ELISA results inspired us to further characterize the presence and specificity of autoantibodies to Ma2 in serum of SI-NET patients with primary tumor. The presence was verified by using western blot analysis and the specificity by using sequential immunoprecipitation. We loaded purified GST-tagged PNMA2 recombinant protein on SDS-PAGE and blotted the gel. The Western blot membrane was immunoblotted either with commercial antibodies or serum samples.

We confirmed that our commercial goat antibody specifically detects Ma2 antigen (Figure S1). To confirm the purity of GST-tagged PNMA2 recombinant Carfilzomib protein anti-GST- and anti-PNMA2 antibodies were used as controls (Figure 2A, lanes I and II). Sera from healthy controls exhibit minimum titer of Ma2 autoantibodies, determined by the indirect ELISA and shown by the low recognition of GST-tagged PNMA2 recombinant protein (lanes III and IV).