Implementation and Routine Clinical Use of the TruSight Myeloid Sequencing Panel in Patients with Myeloid Malignancies

s 361 due to limited numbers of laboratories performing this type of testing and lower DNA yields from FFPE tissue. Additionally, use of DNA or tissue samples from other laboratories may not be ideal because of inter-laboratory differences in FFPE DNA extraction methods and tissue processing. Focusing on gliomas and dermatology specimens, we describe a strategy used in our laboratory to validate the OncoScan FFPE Assay Kit (Affymetrix) using specimens from our internal tissue archive. DNA was extracted from 16 glioma and 7 melanocytic lesions using modified protocols of the Gentra Puregene Tissue kit (QIAGEN) or QIAamp FFPE Tissue kit (QIAGEN). Select tissue specimens were extracted using both methods. DNA was subjected to CMA analysis using the OncoScan FFPE Assay kit (Affymetrix) according to the manufacturer’s protocol. CMA results were compared with previous 1p/19q FISH results (gliomas) or a combination of traditional histopathology, immunohistochemistry, and CMA testing in a reference laboratory (dermatopathology). CMA results from all 16 glioma samples were concordant with respect to 1p/19q co-deletion status (6 positive, 10 negative for the co-deletion) with additional concordance for other gains and losses involving chromosomes 1 and 19. All 7 melanocytic lesions were concordant with previous CMA (n Z 2), BAP-1 immunohistochemistry (3p loss; n Z 2), or histopathology (n Z 3). Validation of a Modified OncoScan Protocol for Use in a Clinical Laboratory Christian N. Paxton, Leslie R. Rowe, Sarah T. South ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA Introduction of new procedures into the clinical laboratory always poses potential challenges to the established workflow. In recent years microarray analysis has gained acceptance as a first tier test for detection of copy number changes (CNCs) and loss of heterozygosity (LOH) in the clinical laboratory. Affymetrix recently introduced the OncoScan array for the testing of formalin-fixed paraffin embedded samples. The primary challenge to adopting the OncoScan array into our lab was a difference in hybridization temperatures between the OncoScan and CytoScan arrays, preventing simultaneous hybridization. We also identified additional steps to improve efficiency in the clinical laboratory setting. The modified OncoScan procedure was validated for use in our laboratory. Nineteen samples processed according to the manufacturerrecommended protocol were also processed using our modified protocol. Modifications to the protocol included the elimination of a brief “chill and spin” step, adjustment to the overnight hybridization temperature, and minor reductions in consumables throughout the procedure to reduce material costs. A comparison of 19 paired samples processed using both protocols showed that our modified protocol performs similarly to the manufacturer-recommended protocol, yielding equivalent quality control metrics and calls. In addition, a recent review of over 200 samples shows continued retention of quality control metrics with our modified protocol. Implementation and Routine Clinical Use of the TruSight Myeloid Sequencing Panel in Patients with Myeloid Malignancies Jason D. Peterson, Francine de Abreu, Prabhjot Kaur, Deborah L. Ornstein, Gregory J. Tsongalis Pathology Department, Geisel School of Medicine, Hanover, NH, USA; Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH, USA Molecular testing in patients with myeloid diseases has important diagnostic, therapeutic, and prognostic implications. With the addition of next-generation sequencing to routine clinical practice, we have the ability to provide a much more comprehensive screening of clinically actionable mutations. This study describes the data obtained during both the validation process and routine clinical practice. Genomic DNA from 24 patients was extracted from either bone marrow aspirates or peripheral blood. Library preparation was performed using the 54-gene TruSight Myeloid Sequencing Panel according to manufacturer’s recommendations, and sequenced on the Illumina MiSeq System. Base-calling and sequence alignment were performed using the MiSeq Reporter Software, and variants were annotated using VariantStudio v2.1. The average cluster density per run was greater than 1,000 K/mm, and approximately 95.0% of the total bases were Q30. For the validation process, samples with known point mutations in the IDH2, NRAS, FLT3, and JAK2 genes, as well as INDELS in NPM1 and FLT3 genes were included. In the clinical samples, the most commonly identified mutations were present in the SRSF2 (2 samples), ASXL1 (2), STAG2 (2), TP53 (2), CDKN2A (3), and TET2 (5) genes. Isolated mutations were also observed in other genes (EZH2, CEBPA, CSF3R, GATA2, DNMT3A). Of the variants called, 100.0% were covered at >500 , while 92.0% were covered at >1,000 . The detection of mutations associated with myeloid malignancies may lead to an early diagnosis of these diseases, as well as to an appropriate treatment and better clinical outcomes. Evaluation of SNP Genomic Microarray Analysis as an Alternative to FISH Analysis of Pediatric Solid Tumors Anthony Arnoldo , James Stavropoulos , Paul Thorner , Cynthia Hawkins , Gino R. Somers , Mary Shago * Divisions of Genome Diagnostics and Pathology, University of Toronto, Toronto, Ontario; Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, University of Toronto, Toronto, Ontario; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario Central and peripheral nervous system tumors including medulloblastoma, astrocytoma and neuroblastoma are amongst the most common solid tumors in children. Recurrent gains or losses of distinct chromosomal regions are prognostic in these 3 tumors. Currently, we test these loci by FISH analysis, followed by karyotyping if there is sufficient material. We have validated the use of the genomic microarray platform