P048 Gene expression profiling of transplant formalin-fixed paraffin-embedded biopsies: comparison of a custom TaqMan® low density array and quantitative nuclease-protection assay

We developed a custom TLDA to monitor changes of gene expression in FFPE renal and small bowel allografts. Here we carried out a parallel comparison of expression profiles of marker genes obtained with TaqMan® technology and with the automated qNPA system. Kidney, small bowel and heart transplant FFPE biopsies with different levels of rejection were selected. 5–6 curls of 10μm were cut from each biopsy for analysis in TLDA (47 markers including immune, inflammatory and apoptosis genes), and in HTG Edge System using the Immuno-Oncology Expression assay (HTGIOE, 26 markers), or HTG EdgeSeq Oncology Biomarker assay (HTGESOB, 2558 genes, format for NGS). Data was presented as differential expression. There are 10 overlapping markers between our TLDA and the HTGIOE assay. Analysis of 30 kidney biopsies with both technologies gave similar results for all 10 markers. Several markers were not detected in some samples analyzed with the HTG Edge System, mostly in normal donor and non-rejection samples. 11 unique markers of HTGIOE assay showed potential value for monitoring gene expression in kidney. 55 samples of heart biopsies were analyzed in TLDA and HTGIOE. Similar results were obtained with both technologies for all overlapping markers. 15 useful markers for monitoring gene expression in heart were found in HTGIOE assay. HTGIOE markers often did not express in non-rejection samples. We also analyzed 17 small bowel biopsies in HTGESOB, which contains 39 of the 47 markers in our TLDA (8 for SB, 17 for both kidney and small bowel, and 14 for kidney). 17.3% of the 2558 genes in the panel showed 2-fold increase expression relative to non-rejection. 15 markers in our TLDA (6 small bowel specific, 7 for both kidney and small bowel, and 2 kidney specific) were in the 5% of markers with the highest fold change. We showed that expression analysis carried out with TLDA and qNPA are comparable. The qNPA technology developed by HTG Molecular Diagnostics, Inc. is a good alternative to assess expression in biopsies. We found that the HTGIOE could be an excellent automated tool to evaluate panels with low number of markers in a clinical lab. The HTGESOB allows evaluation of a large number of genes, but it includes a NGS step that makes it costlier and time consuming. It is a good exploratory tool for searching new markers

P251 Testing HIV and HBV viral load in ffpe transplant biopsies with the cobas ®mpliprep/cobas®taqman®system

Determine if the COBAS® AmpliPrep/COBAS® TaqMan® System (COBAS® System) could be used to quantify viral loads of HIV and HBV in FFPE biopsies using as input sample purified RNA and DNA, respectively. Cell lines containing HIV (CRL-8993 producing multiple non-infective copies of the HIV genome), HBV (CRL-2235 containing the genome of the HBV) and negative (Jurkat Clone E6-1) were used. Formalin treated (fixed) and non-treated cells suspended in SPEX buffer were analyzed in COBAS® System. RNA from non-treated cells was purified using QIAamp RNA Blood Mini Kit (Qiagen). RNA or DNA of fixed cells and FFPE cells were purify in a Maxwell® 16 LEV (Promega) with the corresponding Maxwell® 16 LEV FFPE RNA or DNA extraction kit. RNA and DNA were diluted in SPEX buffer for analysis. HIV cp/ml obtained in the COBAS® System with fixed CRL-8993 cells were 100 times lower than with non-treated cells. HIV cp/ml obtained with non-treated cells was similar to counts obtained with RNA from the same number of cells. Fixed cells RNA gave about half the cp/ml obtained by non-treated cells RNA. The drop was due to lower fixed cells RNA recovery, as adjusted values to cp/ng of RNA added showed no difference between fixed and non-treated cells. FFPE cells were prepared from dilution series of CRL-8993 into jurkat cells, maintaining a constant cellular density. The cp/ml and cp/μg of RNA obtained with RNA from FFPE curls of each cellular dilution showed high degree of correlation with the number of FFPE CRL-8993 cells. Quantitation of HBV was possible in CRL-2235 cells suspended in SPEX buffer. However, the same was not achieved in fixed cells. Quantitation of HBV in DNA from fixed and non-treated cells was very similar, with superimpose best fit equations. The COBAS® System can be used in the clinical laboratory to quantitate HIV and HBV in purified RNA and DNA from FFPE samples, respectively. There was no detectable loss of input RNA. This was not directly tested for DNA. It was revealed that the origin of nucleic acid was not a factor in the test as long as the appropriate purification method was applied. The methodology of this study could be extended to quantify HIV and HBV in RNA and DNA from fresh tissue biopsies. The procedure can be extended to other RNA and DNA viruses like HCV and CMV, respectively, for which kits are available

P073 Multiplexing a variety of clinicaaximize capacity of ion torrent next generation sequencing

We have established the Ion-torrent based Next Generation Sequencing (NGS) system in our clinical laboratory. The chief use of the combination Ion-ChefTM and Ion-S5 is for high resolution HLA typing. However, the full sequencing capacity of the system is not always reached with the HLA typing workflow. In this study, we validated the practicality of combining library pools of different clinical tests into a single NGS run. HLA library pools of 24–32 patient samples were generated with NXType™ Ion-Torrent NGS for Class I (HLA-A, -B, and -C) and Class II (HLA-DR, HLA-DQ, and HLA-DP). These libraries were run alone and then separately combined with library pools of other clinical NGS-based assays. These included: (1) our lab-generated custom tests (APOL1 genotyping (single gene, three SNPs) and Cholestasis genotyping (15 genes, multiple SNPs and INDELS), and (2) commercial assays (16S-Metagenomics, Thermo Fisher Scientific, Inc.) and Lymphotrack, T and B cell clonality (Invivoscribe). All library pools were run on Ion-Chef™ and Ion-S5. HLA data was analyzed with TypeStream™ software v1.1.0.11. The concentration of HLA library pools was maintained at 100pM. Importantly, the concentration of library pools of other tests were adjusted between 5 and 20pM, depending on the test (number of genes and size of amplicons) and the number of samples in the pool. Exported data of APOL1, Cholestasis and 16S-Metagenomics were analyzed with Ion Reporter™ software. Lymphotrack data was exported and analyzed with Lymphotrack data analysis software. The number of reads covering HLA genes were slightly reduced when runs were combined with libraries of other tests, however, the results of HLA library pools run alone and combined were 100% concordant. The coverage and read numbers obtained for the other tests were sufficient or exceeded the minimal required. To our knowledge, this is the first report showing that multiplexing different assays in a single run of Ion-S5 was achievable. We demonstrated that mixing sequencing samples of different genotyping tests in a single NGS run generated accurate results of each individual test. The findings of this works have a positive impact reducing turnaround times and sequencing costs