TO006NON-INVASIVE DIAGNOSIS OF BK VIRUS-ASSOCIATED NEPHROPATHY USING URINARY PROTEOMICS IN KIDNEY ALLOGRAFT PATIENTS

Background and Aims BK virus-associated nephropathy (BKvN) is an important complication occurring after kidney transplantation. Its treatment mainly relies on lowering immune suppression. BKvN’s histological aspect can mimic T cell-mediated rejection (TCMR) and other inflammatory conditions featuring tubulitis and interstitial infiltrate. At present, the screening strategy consists of measuring urinary and blood viral loads of BKv. Upon rising BK viremia, BKvN diagnosis is established by a graft biopsy. Biopsies are invasive and are not suitable for repeated screening. Therefore, the aim of this case-control study was to establish a urinary proteome-based test for BKvN diagnosis. Method Urine was obtained from 700 allograft recipients prior to either protocol or „for cause“ biopsies during the first year of post-transplantation surveillance. Aside from normal biopsy findings (N=294), the histological diagnoses included BKvN (N=50), IFTA II-III (N=145), glomerulonephritis (N=17) and T cell-mediated, antibody-mediated, mixed or borderline rejection episodes (N=194). From all patients, relevant clinical and demographic data including Banff classification scores, HLA mismatches and the presence of donor-specific antibodies was collected at the date of sample collection and in further clinical follow-up. BKvN cases were defined as having SV40-positive immunostaining on allograft biopsy. BKvN patients were considered as cases and all other recipients were considered as controls. Patient’s urinary peptide profiles were generated using capillary electrophoresis coupled to mass spectrometry consisting of 9430 different peptides in the mass range of 0.8 to 20 kDa. Results The CE-MS peptide profiles of randomly selected 30 BKvN cases and 307 kidney allograft controls (one sample per patient) were statistically analyzed to identify the most discriminative peptide markers for BKvN. In total, there were 117 peptides detected to have significantly different urinary excretion rates between both patient groups after false discovery rate adjustment for multiple testing. Thirty-two of these peptides were selected by cross-validated variable selection to establish a Support Vector Machine-based multi-marker model. Applying the 32-peptide marker model to an independent validation cohort consisting of 20 BKvN cases and 343 controls resulted in an area under the receiver operating characteristic curve of 0.86 and a 95% confidence interval from 0.82 to 0.89 with the p-value below 0.0001 and sensitivities and specificities for BKvN diagnosis of 85 and 76 %, respectively. Most notably, distribution of the classification values (figure 1) in the different patient groups of the validation set indicated specificities of the peptide marker model for TCMR/borderline of 85% (17 of 20 true negative classifications) and for viruria/viremia of 73% (8 of 11 true negative classifications) Conclusion In conclusion, the established urinary peptide marker model might serve as a non-invasive diagnostic test for BKvN and its differentiation from TCMR and states of isolated viruria or viremia.

Urine Proteomic Analysis Reveals Disease-Specific Patterns in Pediatric Patients with Classical Hodgkin's Disease(HD). an Addon Study to the Euronet-PHL-C2 Trial

Background HD is a B-lineage lymphoma characterized, depending on subtype, by a prominent inflammatory infiltrate and fibrosis. Clinically, inflammatory symptoms like fever, weight loss and night sweats (B-symptoms) and increased blood biomarkers of inflammation, including ESR and CRP, are characteristic of more advanced disease. The clinical trial EURONET-PHL-C2 (Second International Inter-Group Study for Classical Hodgkin's Lymphoma in Children and Adolescents) is a randomized, prospective trial that compares chemo- and radiotherapy treatment concepts of different intensities in patients with intermediate and advanced HD. Patients are stratified by risk into 3 therapy groups (TL-1 to TL-3). An ESR> 30mm/h had been a risk factor for relapse in previous studies and leads to upstaging from the lowest (TL-1, Ann Arbor stage I and IIa without additional risk factors) to the intermediate risk group TL-2 in the current study. This addon pilot study tested urine proteomic patterns from pediatric patients with HD at diagnosis and compared them to the patterns of normal children. The questions were: Is there a HD-specific pattern, a pattern that identifies high risk or a pattern that correlates with inflammatory markers? Patients and methods Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to compare the peptide profiles in the mass range of 0.8 to 20 kDa of urine samples (N=34) from 16 children with pediatric Hodgkin lymphoma (PHL) as case and 32 age-matched children with no evidence of a disease (N=28) or with urinary tract infection (N=4) as control groups. Marker selection was based on a two-step strategy. First, a group-wise comparison of rank sum differences was performed on a set of 2418 annotated peptides with distribution frequencies above 30% in at least one of the groups with subsequent adjustment for multiple testing by the method of Bonferroni. In the second step marker candidates were further restricted to those demonstrating a significant positive or negative Spearman rho correlation coefficient (≥0.34 or ≤-0.34) to the Ann-Arbor classification criteria. From the resulting peptides a multivariate peptide marker classifier was established by support vector machine modeling and applied to an independent confirmation set of PHL (N=16, 31 urine samples) and control (N=18, 18 urine samples) patients to determine classification accuracy in receiver operating characteristics (ROC) analysis. Peptides included in the PHL classifier were resolved in their amino acid sequence by tandem mass spectrometry to identify the proteins from which the peptide markers are derived. Results The established multivariate peptide marker model consisting of 40 naturally occurring urinary peptides enabled absolute differentiation between PHL patients and children without signs of disease or urinary tract infection in independent validation as revealed by an area under the ROC curve value of 1.0 (95% confidence interval: 0.93 to 1.00, p<0.0001). Amino acid sequencing revealed that the majority of peptides are interstitial collagen fragments from specific hot-spot regions within the proteins linear sequence and in part with overlapping amino acid sequences indicative for the activity of specific extracellular matrix degrading proteases. Other PHL peptide markers are derived from the tumor associated proteins S100-A9, Prostaglandin-H2 D-isomerase and Cytokeratin-8. Conclusions and perspective We were able to identify a proteomic pattern characteristic for HD, markers for relapse risk group or HD-associated inflammation were not yet identified. Disclosures Metzger: Mosaiques Diagnostics: Employment.

Short Communication: Genetic diversity of the IGF2 gene as a source of genetic marker for halal authentication

Hamdani AD, Madihah, Suryohastari RB, Virgianti DP, Putrie RFW. 2018. Genetic diversity of the IGF2 gene as a source of genetic marker for halal authentication. Nusantara Bioscience 10: 203-209. The main issue of halal authenticity is the availability of reliable and rapid analytical methods to identify animal species in raw and processed food. The two most popular procedures for identifying the source of the meat are protein-based and DNA-based methods, including mass spectroscopy (MS) using a peptide marker or PCR based DNA marker, respectively. This study aims to investigate the genetic diversity of insulin-like growth factor 2 (IGF2) gene, mainly from porcine (Sus scrofa) and bovine (Bos taurus) as a source of genetic marker for halal authentication by in-silico analysis using bioinformatic tools. Multiple sequence alignment and phylogenetic tree construction of IGF2 protein sequences from representative species of mammals, birds, and fishes showed the paraphyletic relationship between the IGF2 protein of S. scrofa and B. taurus. Protein structure analysis revealed differences in helical structures near the carboxyl end of the protein, while gene analysis showed different number of exon and motifs. By in-silico analysis, we have designed a peptide marker from amino acids at position of 93-107 (S. scrofa) and 93-112 (B. taurus) from peptide preptin region that resulted in different pattern of mass spectrum between the two species. We have also identified two DNA markers that can be detected by PCR using two primers sets designed from the IGF2 transcript sequences, to examine the presence of porcine in the sample. Thus, this study provides new genetic predictive markers, derived from the IGF2 gene, to identify the source of meat for halal authentication.