laboratories using NGS of the difficulty in accurately calling variants in these ...... have now integrated this system within a clinical reporting software application.
GENETICS G01. Characterization of 137 Genomic DNA Reference Materials for 27 Pharmacogenetic Genes: A GeT-RM Collaborative Project V.M. Pratt1, P. Aggarwal2, B.N. Beyer1, U. Broeckel2, R. Epstein-Baak3, R.E. Everts4, P. Hujsak3, R. Kornreich5, J. Liao5, R. Lorier2, S.A. Scott5, C. Huang Smith3, L.H. Toji6, A. Turner2, L.V. Kalman7 1Indiana University School of Medicine, Indianapolis, IN; 2Medical College of Wisconsin, Milwaukee, WI; 3Autogenomics Inc., Vista, CA; 4Sequenom Inc., San Diego, CA; 5Icahn School of Medicine at Mount Sinai, New York, NY; 6Coriell Cell Repository, Camden, NJ; 7Centers for Disease Control and Prevention, Atlanta, GA. Introduction: Pharmacogenetic (PGx) testing is increasingly available from clinical laboratories, however a limited number of quality control and other reference materials (RMs) are currently available. We previously published the characterization of 107 publicly available genomic DNA samples for 5 commonly tested PGx genes. In this study we have expanded the number of PGx genes and alleles characterized. The Centers for Disease Control and Prevention (CDC)-based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized 137 additional genomic DNA samples for 27 genes commonly genotyped by PGx testing assays and included proficiency testing surveys (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, VKORC1). Methods: To expand the number of characterized PGx variants and genes, 137 Coriell cell lines were selected based on partial genotype characterization from several sources. DNA samples were coded and distributed to volunteer testing laboratories for targeted allele genotyping. Participants used a number of commercially available and laboratory developed tests. Results: Through consensus verification of 137 DNA samples, we have increased the number of multiply-genotyped PGx genes from 5 to 27 and confirmed the presence of over 83 variant alleles. At least 13 different ethnic groups are represented among the samples. Similarly, we also expanded the number of verified alleles (from 23 to 42) for the 5 genes that had been previously characterized. One example is CYP2D6, where 7 additional alleles (*14, *15, *36, *40, *10XN, *17XN, *41XN) were verified. These samples are also being characterized with other PGx assays, including DNA sequence analysis, and the results will be reported separately. Conclusions: The results were consistent among laboratories, with the majority of differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6. The alleles included in the different assays varied, but many that are commonly tested were identified in the set of 137 DNA samples. These samples will be very useful for assay development and validation, quality control and proficiency testing, and should help to improve the accuracy of pharmacogenetic testing in clinical laboratories. These and other genomic DNA RMs developed by GeT-RM are publicly available from the Coriell Cell Repositories, and are described on the GeT-RM website: http://wwwn.cdc.gov/dls/genetics/qcmaterials/default.aspx. G02. Clinical Performance of Comprehensive Familial Cancer Screening Using ColoSeq and BROCA B.H. Shirts, S. Casadei, A. Jacobson, E. Turner, J.F. Tait, M. King, T. Walsh, C.C. Pritchard University of Washington, Seattle, WA. Introduction: Next-generation sequencing technologies have expanded the ability to evaluate patients and families for a wide range of hereditary cancer by clinical testing using targeted panels of many genes. We evaluated the clinical performance of BROCA and ColoSeq panels for cancer risk assessment. Methods: We evaluated findings from 1285 sequential cases clinically tested for cancer risk using either the 19 gene ColoSeq panel or 51 gene BROCA panel between November 2011 and February 2014. DNA samples from whole blood were sequenced using Illumina HiSeq after enrichment using Agilent SureSelect custom probes. We classified variants using an IARC based Bayesian framework with input from multiple genetics specialists. Results: Of 1285 cases 169 (13%) harbored pathogenic or likely pathogenic variants, and 136 (10%) carried either variants of uncertain significance in established cancer risk genes or clearly deleterious variants in emerging genes. Important mutations were identified in APC, ATM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, HOXB13, MLH1, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RET, SMAD4, STK11, and TP53. These included missense, nonsense, splice-site altering, and frameshift inducing small indel mutations. Larger mutations reported included single and multi-exon deletions and insertions, and one single-exon-spanning complex inversion. Several mutations identified were mosaic. Mutation carrier frequency varied by gene, clinical situation, and by ordering provider. Of patients with personal history of breast or colon cancer and no previous clinical sequence testing, 15% harbored a clinically actionable mutation. Of patients previously determined to have normal sequence of BRCA1 and BRCA2, 10% had an actionable mutation in another gene. In certain situations very selective providers had positive results at a rate substantially higher than average. Multi-specialist review of variants for all patients increased sensitivity for likely pathogenic mutations and decreased the number of variants of uncertain significance reported. The proportion of variants of uncertain significance reported also decreased with experience. Conclusions: Our data supports a comprehensive multi-gene testing approach for patients at high risk of an inherited cancer. Classifications of mutations
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that recognize the nature of uncertainty in genetic findings may be beneficial. Mutation detection and variant of uncertain significance classification rates depend on genes tested, patient population tested, provider selectivity, as well as cumulative and combined experience of medical directors reviewing variants. G03. Pharmacological Manipulation of Intracellular NAD+ Levels as a Treatment for Ataxia-Telangiectasia R.E. Shackelford1, H. Lederman2 1Louisiana State University Hospital Shreveport, Shreveport, LA; 2Johns Hopkins Hospital, Baltimore, MD Introduction: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by recurrent sinopulmonary infections, progressive ataxia, telangiectasias, and a ~1,000-fold elevated cancer incidence. In culture, A-T cells shown many defects, including rapid cellular senescence, attenuated checkpoint responses, radio-resistant DNA synthesis, and a high sensitivity to oxidant exposure. Nicotinamide adenine dinucleotide (NAD+) levels are low in A-T cells and Atm-deficient mice. Therefore we hypothesized that nicotinamide phosphoribosyltransferase (Nampt) which catalyzes the rate-limiting step in NAD+ synthesis, would be low in A-T and pharmacological NAD+ supplementation would increase the viability of A-T, but not wild-type cells. Methods: Western blotting and immunohistochemistry for Nampt was performed on wild-type and A-T cells, and 6 samples of wild-type and 6 A-T human cerebellar brains. Nampt levels were also determined in wild-type and A-T cells with and without t-butyl hydroperoxide exposure and the effect of recombinant ATM protein expression in A-T cells on Nampt protein levels was examined. Last, colony-efficiency forming assays were performed to measure A-T and wild-type cell viability following nicotinnamide or nicotinic acid pretreatment, followed by t-butyl hydroperoxide exposure. Results: Nampt levels were low in A-T cells and the cerebellar tissue from individuals with A-T compared the wild-type controls. Additionally, the expression of recombinant ATM in A-T cells increased basal Nampt levels, indicating that ATM protein expression increases Nampt protein levels. Last, Nampt proteins levels were increased by t-butyl hydroperoxide treatment in wild-type, but not A-T cells. Conclusions: Intracellular Nampt levels largely determine NAD+ levels within cells by converting nicotinamide into NAD+. NAD+ is required for cell growth and survival, and is low in A-T cells. The lower Nampt levels in A-T likely explain the low NAD+ levels in A-T and partially the lower cellular viability and enhanced susceptibility of these cells to oxidative stress. Interestingly, nicotinc acid, but not nicotinamide, increased A-T, but not wild-type, cell viability following oxidative stress. This result is interesting as nicotinc acid, but not nicotinamide, increases intracellular NAD+ levels, indicating that increased intracellular NAD+ increases A-T cell viability. Recombinant ATM expression in A-T cells also increased basal Nampt levels, thus ATM protein expression increases Nampt expression. Last, t-butyl hydroperoxide induced Nampt protein levels in wild-type, but not A-T cells. Thus ATM may play a role in increasing Nampt and intracellular NAD+ levels following oxidative stress. Our data indicates that pharmacologically increasing NAD+ in A-T increases A-T cell viability and be a possible treatment for A-T. G04. Traditional vs. Next-Generation Testing of Hereditary Breast and Ovarian Cancer Genes in a Large Clinical Population S. Lincoln1, A. Kurian2, A. Desmond3, M. Gabree3, M. Powers1, F. Monzon1, L. Ellisen3, J. Ford2 1Invitae, San Francisco, CA; 2Stanford University, Stanford, CA; 3Massachusetts General Hospital, Boston, MA. Introduction: Targeted next-generation sequencing (NGS) has gained acceptance in clinical laboratories, although medically important classes of variation remain challenging for NGS. Expanding on our recently published work (Kurian et al., J Clinical Oncology 2014) we considered whether NGS can replace traditional methods (e.g., Sanger, QPCR, arrays) for hereditary cancer testing. Methods: Using an NGS panel we tested over 1,000 patients who were indicated for hereditary breast and ovarian cancer testing under clinical guidelines. Most (90%) of these patients had traditional genetic testing results available for comparison, and we utilized an independent lab to validate NGS findings when corresponding data were not available or in the case of conflicts. Previously, we had developed a coordinated set of NGS protocols and algorithms intended to provide thorough coverage and to detect challenging classes of variation (large indels and small copy-number changes, for example). This study allowed us to validate these methods in a clinical setting. Results: Two hundred sixtyone (261) alterations (196 pathogenic and 65 others) were reported in the traditional clinical data and all were detected by NGS when the corresponding test was ordered. In this set are 141 insertions, deletions, delins events and copy number changes, with particularly challenging examples including indels up to 126 bp and both deletions and duplications as small as an exon. Unsurprisingly, most of the previously reported alterations were in BRCA1/2, and thus we supplemented the study with 112 reference samples containing a highly accurate set of over 2000 alterations in other genes covered by our assay, all of which were also detected. Specificity was also high. All NGS variants for which we sought orthogonal confirmation (n>2000) were confirmed, including 51 pathogenic variants not previously reported. No single calling algorithm achieved this performance but rather the combination (GATK, Freebayes, PolyMNP, CNVitae and split-read detection) did. Conclusions: NGS can be a viable replacement for traditional genetic testing in some applications, with the additional benefit of costeffectively increasing diagnostic yield (by over 50% in this study). Orthogonal confirmation of NGS results remains a strongly recommended practice, although the high concordance of traditional and NGS data suggests that the cost-benefit of
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AMP Abstracts confirmation merits careful consideration over time. Of course, further work is required to detect additional classes of variation by targeted NGS such as trinucleotide repeats, inversions, and variants in some highly conserved regions. All of these variants and their interpretations will be released to public databases by the time of the meeting. G05. 5.3 Megabase Chromosome 17q24.2q25.1 Deletion Associated with SOX9 Haploinsufficiency in a 46, XX Female Presenting with an Acampomelic Phenotype A. Pickart1,2, B. Chirempes2, J. Wittke2, E. Virlee2, G. Scharer1,2 1Children's Hospital of Wisconsin, Genetics Center, Milwaukee, WI; 2Advanced Genomics Laboratory, Milwaukee, WI. Introduction: Campomelic dysplasia (CD) is characterized by congenital bowing and angulation of long bones (campomelia), together with other skeletal and extra-skeletal defects. Up to two-thirds of affected XY males have a spectrum of genital defects or may develop as phenotypic females. Although classic CD is caused by heterozygous mutation in SOX9 or gene deletion (haploinsufficiency), the acampomelic phenotype can be found in patients with chromosomal rearrangements flanking the SOX9 locus, with breakpoints in the 5-prime region of the gene, as well as deletions that are upstream of SOX9. We report an 8 month old infant, who was diagnosed with congenital duodenal obstruction due to annular pancreas requiring duodenoduodenostomy. Genetic evaluation was requested based on coexisting mild hypotonia and few subtle dysmorphic features. Methods: High-resolution karyotyping and CMA analysis (CytoscanHD) revealed a 46, XX female with a 17q24.2q25.1 deletion (chr17: 66,843,381-72,120,303x1). The deletion encompasses both SOX9 (CD) and KCNJ2 (Andersen-Tawil syndrome, ATS); however, this patient’s clinical presentation appeared to fit neither of these two genetic disorders. Results: Skeletal survey showed mild clinodactyly of the toes and fingers, foreshortening of the radii and slight mesomelia of legs. At 3 months, the patient demonstrated mild delays in motor skills and she is receiving physical therapy. Somatic growth is borderline low for height (3rd percentile) and normal for weight/OFC (40th/70th percentiles). The subtle dysmorphic features include low-set ears and a depressed nasal bridge. Consequently, this patient’s clinical presentation is more consistent with acampomelic CD. ATS is an autosomal dominant multisystem channelopathy characterized by periodic paralysis, ventricular arrhythmias, valvular heart disease, dysmorphic facial features and skeletal anomalies. ATS shows incomplete penetrance and intrafamilial variability. Our patient underwent echocardiogram at 1 day of age and EKG at 6 months. Neither study detected the typical findings of ATS; a small PDA observed after birth had resolved at 6 months. Conclusions: In the absence of the characteristic physical features of CD in this patient we hypothesize that the chromosome deletion possibly affects a downstream target of SOX9 signaling (located either 5-prime of SOX9 or more likely in the 3-prime region); thus, ameliorating the effects of the haploinsufficiency. The phenotypic variability of ATS is well described and is consistent with this patient’s atypical phenotype. In addition, there is evidence (Circ Cardiovasc Genet 2014;7(1):1722) that the noncardiac features of ATS occur only with missense variants in the KCNJ2 gene, resulting in a dominant negative effect on Kir2.x channels. G06. Establishing a Catalog of Medically Relevant Genes with High Sequence Homology to Facilitate Accurate Exome and Genome Sequencing D. Mandelker1, A. Ankala2, K. Gibson3, H. Sharma4, R. Shakhbatyan4, A. Santani3, M. Lebo4, M. Hegde2, B. Funke1,4 1Harvard Medical School, Boston, MA; 2Emory Genetics Laboratory, Atlanta, GA; 3Children's Hospital of Philadelphia, Philadelphia, PA; 4Laboratory for Molecular Medicine, Cambridge, MA. Introduction: Pseudogenes and other sequences with high homology to functional genes can pose significant analytical challenges for medical next-generation sequencing, as unique read mapping may be impossible, increasing the risk for both false negative as well as false positive variant calls. Deep gene-specific knowledge including the presence of highly homologous regions historically resides within laboratories offering disease-targeted gene panels; however, a single laboratory cannot be expected to have a priori knowledge of all homologous genes in the exome/genome. It is therefore imperative to establish a catalog of medically relevant genes affected by pseudogene interference and create a resource for laboratories as exome/genome sequencing (ES/GS) is beginning to be widely adopted. Methods: We performed a multi-tiered exome wide analysis that integrated a 250 bp sliding window BLAT homology analysis and ES/GS based mapping quality data to identify genes with regions that preclude accurate interrogation by current NGS technologies. This list was further integrated with the “Medical Exome,” a list of 4631 genes with known or suspected disease associations and refined by incorporating additional measures of clinical importance including the confidence level of gene-disease association as well as medical annotations including disease prevalence, age of onset, and clinical actionability. Results: We present a clinically and technically annotated, ranked list of these genes. Exome wide analysis revealed 1998 genes with at least one exon of high homology (defined as >98% homology across sliding windows encompassing at least 250 contiguous base pairs.) Intersection with the Medical Exome narrowed this list to 286 genes with various lengths of homology that can be used for several downstream applications such as implementation of bioinformatics scripts to flag variants in such regions or exclude them altogether if the length of the region exceeds what can be confirmed by Sanger sequencing. Prominent examples of affected genes include an ACMG incidental findings list gene (PMS2) and well established disease genes such as SMN1 and SMN2 (Spinal Muscular Atrophy), GBA (Gaucher disease), VWF (Von
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Willebrand disease), HBA1 and HBA2 (Hemoglobinopathies, Alpha Thalassemia), and STRC (non-syndromic hearing loss). NGS technologies are likely to evolve to produce longer reads; however, genes such as STRC with 8.5 kb of uninterrupted 100% identity are likely to remain challenging. Conclusions: We have developed a resource of medically curated genes with homology issues that can be used to alert clinical laboratories using NGS of the difficulty in accurately calling variants in these regions. G07. A de Novo Korean Case of 18q Deletion Syndrome with Large Atrial Septal Defect Accompanying Cyanosis Confirmed by Microarray Study Y. Kim, H. Yoon, T. Park, Y. Choi School of Medicine, Kyung Hee University, Seoul, Korea. Introduction: 18q deletion syndromes show various clinical phenotypes including cardiac anomalies. However, the atrial septal defect accompanying cyanosis is uncommon. We present a case of female newborn with microarray confirmed de novo 18q deletion syndrome and large atrial septal defect (ASD) causing cyanosis. The patient was born after an unremarkable pregnancy by normal vaginal delivery at 40+4 weeks of gestation with a birth weight of 3350 g. This girl was second child of nonconsanguineous healthy parents. The patient was intubated and ventilated with 100% of oxygen due to initial severe cyanosis and respiratory distress after 2 hours of birth. Clinical and genetic investigations were performed. Methods: The echocardiography and routine peripheral blood karyotyping were performed. G-banded karyotype analysis (650 band level) was done on metaphase spread from cultured peripheral bloods from the patients and her mother. Genomic DNA of the patient was analyzed using CytoScan 750K Array (Affymetrix, Santa Clara, CA, US) according to the manufacturer’s instructions. Genomic coordinates were based on human reference sequence assembly (NCBI Build 37/hg19, February 2009). Result: The echocardiography revealed large to moderate tricuspid valve regurgitation, however the shunt direction through patent ductus arteriosus was left to right, and a huge ASD of 16 mm size, which resulted in large amount of mixed blood physiology and explained the infant’s cyanosis of heart origin. The G-banded karyotype analysis of patient showed 46,XX,del(18)(q22.1). The maternal karyotype was normal. The array analysis on patients’ genomic DNA revealed a 15Mb (63,244,135-78,013,728) deletion in 18q22.1q23. Deleted important OMIM genes were identified as RTTN, CYB5A, TSHZ1, NFATC1 and CTDP1. Conclusions: Our array identified 18q deletion syndrome in neonate showed relatively mild phenotype however, unusual large ASD causing cyanosis was remarkable among the previously reported 18q deletion syndromes. Our case supports the correlation between 18q terminal deletion and cardiac anomaly, and could be used for further study on genotype-phenotype correlation of 18q deletion syndrome. G08. Prenatal and Postnatal Detection of Low-Level Mosaic Mutations Causing Overgrowth Syndromes Using Next-Generation Sequencing F. Chang, L. Liu, E. Fang, G. Zhang, M.M. Li Baylor College of Medicine, Houston TX. Introduction: Overgrowth syndromes are genetically heterogeneous diseases caused by both germline and somatic mutations of different genes. Recent studies have showed that a group of overgrowth syndromes, such as congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES), Proteus syndromes, two overlapping disorders, megalencephaly-polymicrogyria-polydactylyhydrocephalus (MPPH) and megalencephaly-capillary malformation (MCAP), and isolated macrodactyly are caused by postzygotic activating mutations in the genes involved in PI3K-AKT signaling pathway. Due to the low-abundance nature of these mutations, routine Sanger sequencing often yields negative results. Methods: We developed a next-generation sequencing (NGS) test that targets all known mutations in multiple genes involved in PI3K-AKT pathway. Ten patients including two prenatal cases and eight postnatal cases suspected of overgrowth syndromes were tested using the NGS sequencing panel. Results: A somatic mutation in the PIK3CA gene was identified in 8 of the 10 cases including one G542K mutation, two H1047L mutations, two H1047R mutations, two G914R mutations and one E110del mutation with the mutant allele frequencies ranging from 4.65% to 38.36%. These PIK3CA mutations were only present in the affected tissues in the majority of the cases demonstrating causal role of the mutations in the development of the diseases and the importance of obtaining affected tissue for testing. Reviewing the history of two negative cases, one lacked the phenotype of overgrowth syndrome and the other may have sampling issues. Phenotype-genotype correlation analysis showed mutations at amino acid residue 1047 are often associated with CLOVES syndrome or isolated overgrowth of extremities or fatty mass, whereas the G914R mutation is preferentially related to syndromes with overgrowth of brain, such as MPPH/MCAP. In addition, in vitro cell culture showed significant enrichment of the cells harboring mutant alleles, indicating that the activating mutation may render growth advantages to the mutant cells. In a prenatal case, the PIK3CA G542K mutation was positive in the DNA extracted from cultured amniocytes but negative in uncultured cells of the same amniotic fluid specimen, suggesting that cultured amniocytes could be used for prenatal diagnosis of these syndromes. Conclusions: In summary, NGS technology demonstrates high accuracy and sensitivity for the detection of causal mutations in the overgrowth syndromes.
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G09. Diagnostic Evaluation of a Multiple-Gene Sequencing Panel for Hereditary Cancer Risk Assessment E. Cho, J. Jang, J. Lee, T. Lee, Y. Kim Green Cross Genome, Yongin, Kyunggi-do, Korea. Introduction: Clinical genetic testing for hereditary cancer risk assessment has become widespread and next-generation sequencing technology has enabled multiple gene panel testing at low cost. We evaluated the diagnostic performance of a multigene sequencing panel for hereditary cancer risk assessment in a representative clinical sample. Methods: DNA from 52 patients with germline pathogenic mutations (2 different mutations with BRCA1, 20 BRCA2, 2 TP53, 15 APC, 7 MLH1, 6 MSH2) which had been previously identified by Sanger sequencing and 20 normal volunteers were sequenced for all coding regions of 9 cancer susceptibility genes (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, CDH1, TP53, RET). Access array system (Fluidigm, USA) was designed to multiplex 48 samples simultaneously for 509 amplicons and the average sequencing depth per base was 3,200X using MiSeq sequencer (Illumina, USA). We developed analysis pipelines to avoid false negative results and detect all pathogenic mutations. The reads were aligned to a reference genome (NCBI human genome assembly build 37) using the BWA-MEM, then all candidate variants called with minimum filtering parameter. Results: All of the known pathogenic mutations (31 insertions/deletions of up to 13 nucleotides, 21 point mutations) were identified by NGS. In our configuration, NGS outperforms current diagnostic methods, providing a reduction in analysis time and in reagent costs compared with conventional sequencing. Conclusions: There was no false negative of pathogenic mutations from NGS. This approach enables widespread genetic testing and personalized risk assessment for hereditary cancer. G10. SERPINA1 Full Gene Sequencing Identifies Rare Mutations Not Detected by Conventional Testing and Should Be Part of the A1AT Deficiency Diagnostic Algorithm R.P. Graham, M.A. Dina, S.C. Howe, K.S. Willkomm, D.L. Murray, M. Snyder, K. Rumilla, K.C. Halling, W.E. Highsmith Mayo Clinic, Rochester, MN. Introduction: Alpha-1 antitrypsin (A1AT) deficiency is a hereditary condition responsible for chronic obstructive pulmonary disease and in some cases hepatic disease. The most common mutations are the Z allele and S allele. The combination of S and Z genotyping and isoelectric focusing (IEF) has been used clinically to resolve most clinical cases of A1AT deficiency. This approach, however, does not detect null mutations or several other deleterious alleles. Thus, in some patients with low serum A1AT levels, no deleterious alterations are identified preventing definitive diagnosis. We sought to assess the utility of full gene sequencing of SERPINA1 in the testing cascade of patients with low serum A1AT levels in the absence of identifiable deleterious alterations by S/Z genotyping and IEF. Methods: Patient samples with serum A1AT levels A (p.V1188E, rs17222723), c.4544G>A (p.C1515Y, rs8187710) Variants Can Be in Cis in the ABCC2 Gene V.M. Pratt, B.N. Beyer, G.H. Vance, T.C. Skaar, D.A. Flockhart Indiana University School of Medicine, Indianapolis, IN. Introduction: The ABCC2 (ATP-binding cassette, sub-family C [CFTR/MRP], member 2) gene is a member of the ATP-binding cassette transporters and is involved in the transport of molecules across the cellular membranes. Substrates that are transported by ABCC2 include: antiepileptics, statins, tenofovir, cisplatin, irinotecan, carbamazepine (http://www.pharmgkb.org/gene/PA116, accessed March 13, 2014). Due to the pharmacogenomics implications, we designed and validated a clinical laboratory-developed procedure to test for seven variants in the ABCC2 gene: c.3563T>A (p.V1188E, rs17222723), c.1249G>A (p.V417I, rs2273697), c.3972C>T (p.I1324I, rs3740066), c.2302C>T (p.R768W, rs56199535), c.2366C>T (p.S789F, rs56220353), c.-24C>T (5'UTR, rs717620), and c.4544G>A (p.C1515Y, rs8187710). During the validation process, we noted several DNAs, obtained from the Coriell Cell Repository (Camden, NJ), that contained both c.3563T>A, c.4544G>A and a third variant, suggesting that c.3563T>A and c.4544G>A are in cis. We obtained DNA samples from a trio (father, mother, and offspring) and tested the ABCC2 variants and
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confirmed that c.3563T>A and c.4544G>A were on the same chromosome. Here we report a novel haplotype in ABCC2. Methods: DNA was amplified by real-time PCR on the LifeTech QuantStudio 12K Flex (software v1.2.2; Grand Island NY) and subjected to Taqman allele discrimination using LifeTech (Grand Island, NY) reagents and software (Genotyper software, v1.3) in a custom designed open array. Seven variants were genotyped: c.1249G>A (p.V417I; rs2273697), c.3972C>T (p.I1324I; rs3740066), c.24C>T (5'UTR; rs717620), c.3563T>A (p.V1188E; rs17222723), c.4544G>A (p.C1515Y; rs8187710), c.2302C>T (p.R768W; rs56199535), and c.2366C>T (p.S789F; rs56220353). Results: One hundred four (104) anonymous DNAs were genotyped for 7 variants in the ABCC2 gene. We observed 5 samples where c.3563T>A and c.4544G>A occurred with a third variant (ie, c.3972C>T, c.2302C>T, c.1249G>A). We noted that c.3563T>A and c.4544G>A could also occur independently. One (1%) sample was heterozygous for c.3563T>A, 6 (6%) samples were heterozygous for c.4544G>A, and 11 (11%) samples were compound heterozygous. A trio was obtained. The father of the trio was compound homozygous for c.3563T>A and c.4544G>A. The mother was compound heterozygous for c.3563T>A and c.4544G>A. The offspring of the trio was compound heterozygous for c.3563T>A and c.4544G>A. Conclusions: During the validation studies, unexpected triple variants where c.3563T>A and c.4544G>A occured with a third variant were identified in ABCC2. A literature search was performed and the relationship of c.3563T>A and c.4544G>A in cis had not been previously reported. A trio was obtained (mother, father, offspring), genotyped for ABCC2 and confirmed that c.3563T>A (p.V1188E, rs17222723), c.4544G>A (p.C1515Y, rs8187710) can occur in cis in ABCC2. G12. Understanding the Phenotype of Patients with Heterozygous Deletions at the MCS-R2 (HS-40) Region of the Alpha-Globin Gene U. Aypar, P.A. Lundquist, W.E. Highsmith Mayo Clinic, Rochester, MN. Introduction: The alpha-thalassemias are a group of recessively inherited disorders caused by reduced or absent synthesis of the alpha-globin chain of hemoglobin. The phenotype of patients with alpha-thalassemia is variable depending on the number of dysfunctional alpha-globin genes. Alpha-thalassemia trait results from deletion or dysfunction of two of the four alpha-globin genes. These patients have reduced hemoglobin, hematocrit, MCV, and MCH; however, these changes are less severe than those in individuals with Hemoglobin H (HbH) or Hemoglobin Bart (Hb Bart). Upstream of the alpha-globin genes are four highly conserved, noncoding sequences called multispecies conserved sequences (MCS-R1 to MCS-R4). Although the role(s) of the MCS-R1, -R3, and -R4 are unclear, the MCS-R2 (HS-40) has been shown to be essential for alpha-globin expression. Although rare, deletions of HS-40 have been described in patients with alpha-thalassemia. However, the importance of this deletion is not very well understood and this deletion is typically interpreted as a VUS (variant of uncertain significance) in clinical laboratories. Methods: At the Mayo Clinic Molecular Genetics Laboratory, we have identified 5 patients with heterozygous HS-40 deletion between November 2007 and February 2014 as detected by a laboratory developed multiplex ligation-dependent probe amplification assay. Results: We were able to obtain the CBC reports for 4 out of these 5 patients. These 4 patients with heterozygous HS-40 deletion had low MCV (mean = 74.2; range 68 to 78) and low MCH (mean = 23.8; range 22 to 25.1), in the range typically associated with alpha thalassemia trait (71.6±4.1 and 22.9±1.3, respectively) with high red cell size distribution values. Three of the 4 had low hemoglobin values and 2 of 4 had low hematocrit values. All 4 of the patients had normal RBC and WBC values. Conclusions: This study suggests that the heterozygous deletion of HS-40 is a pathogenic mutation that leads to an alpha thalassemia trait (in cis conformation) phenotype. Based on this study, we recommend that the detection of the HS-40 deletion should be routinely studied in alphathalassemia testing and that these findings should be interpreted as pathogenic. Identification of this deletion has an impact not only on the patient being tested but also the future generation, which is at risk for HbH disease or Hb Bart syndrome. Molecular studies on the patient’s reproductive partner are also recommended to further clarify their reproductive risk. G13. Genetic Puzzle of the Role for the MTHFR c.677C>T Variant and c.1298A>C Y. Xie1,4, J. Cui1,4, S. Li1, Y. Xu2, E. Randell1, H. Zheng3, H. Wang2, M. Godlewski4, F. Han1 1Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada; 2Zhengzhou University First Affiliated Hospital, Zhengzhou, Henan, China; 3Zhengzhou University, Zhengzhou, Henan, China; 4Saint John Regional Hospital, Saint John, New Brunswick, Canada. Introduction: The MTHFR c.677C>T and c.1298A>C variants have been extensively studied in a large number of genetic association studies with Cardiovascular diseases including myocardial infarction (MI) and stroke. These studies have also yielded highly discordant results which may be due to variable genetic modifiers in complex populations and a wrong hypothesis for their biological roles in the studied clinical conditions. Methods: Using Taq Man SNP genotyping technology on real-time PCR, genotyping of the MTHFR c.677C>T and c.1298A>C variants were performed in two studied populations including 1,032 myocardial infarction (MI) patients and 1,014 healthy controls from a Canadian genetically isolated Newfoundland population, and 1429 patients with ischemic stroke and 1171 healthy controls from the Chinese Han population. Results: Interestingly, the two studies showed a contradiction in the results for the two studied variants in gene frequencies and genetic association between the two studied populations. Both of the 677T and the 1298C alleles in Newfoundland
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AMP Abstracts population were presented as rare alleles with allele frequencies 34.6% and 31.5% respectively. Compared to the controls, the 677T showed a significantly reduced prevalence in patients (OR=0.700, PC). Parental studies showed that only the father had this mutation. SNP microarray analysis detected a 247.2 Mb region of homozygosity on chromosome 1: 1p36.33p44 (37,714-247,195,072)x2 hmz and the GBA gene maps to this region confirming a region of paternal isodisomy for this region. Case 2. A newborn presented with complex congenital heart disease, heterotaxy, and anomalous pulmonary venous drainage. Microarray results showed a 14 Mb region of homozygosity on chromosome 7: 7p21.1p15.1 (16,974,692-30,970,344)x2 hmz. There are several genes located within this region including DNAH11, which is associated with Primary Ciliary Dyskinesia (PCD). Further Sanger sequencing of DNAH11 revealed a homozygous mutation in exon 24 (c.4348C>T), resulting in a premature protein termination (p.Arg1450Stop), thus confirming the diagnosis of PCD. Case 3. A 3 year old boy presented with muscle weakness, hypotonia, developmental delay, macrocrania and short stature. Chromosome analysis revealed an abnormal Y chromosome. Microarray results showed a triplication of the X chromosome (Xp22.33 to Xp22.2) and a duplication of the Y chromosome (Yp11.32 to Yq12). Subsequent FISH analyses determined that this patient has two SRY signals and deletions of SHOX gene on both Yp arms. The karyotype was: 46,X,ider(Y)(Yqter->Yp11.32::Xp22.33->Xp22::Xp22>Xp22.33::Yp11.32->Yqter).ish (SRY++). The copy number gains of parts of the X and Y chromosomes and deletions of the SHOX genes were essential in understanding this patient’s phenotype. Conclusions: In summary, our observations show that many genetic disorders have complex etiologies, and the utilization of several genetic
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diagnostic technologies can assist in the diagnosis and understanding of both known and rare disorders with atypical modes of presentation or inheritance. G20. Quantitative Analysis of DNA Degradation in Whole Blood under Different Storage Conditions A. Ishwar1, A. Rounsavall1, M. Smith1, M. Alfaro1, J. Permenter1, C. Sailey2 1Arkansas Children's Hospital, Little Rock, AR; 2University of Arkansas for Medical Sciences, Little Rock, AR. Introduction: Proper storage of whole blood is crucial for isolating nucleic acids from leukocytes and to ensure adequate performance of downstream assays in the laboratory. Newer automated genomic DNA extraction methods allow for heparin and EDTA-stored blood to be used, but recommended storage methods are lacking. Degradation of DNA in leukocytes is caused by two forms of cell death - necrosis and apoptosis. Apoptosis displays as a ladder pattern of DNA on agarose gels through caspase-activated DNase activity. Necrosis causes a smear-like pattern through random fragmentation. Gel electrophoresis enables only qualitative estimation of DNA degradation. We attempted to quantify the extent of DNA degradation in heparinized and EDTA blood stored at room temperature (RT) and 4°C. Methods: Whole blood was obtained from 42 patients. 11 were EDTA-4°C, 10 were EDTA-RT, 10 were heparin-4°C, and 11 were heparin-RT. DNA was extracted using the Promega Maxwell16 automated platform at 7 time-points over 4 months. All extracted DNA were normalized to 50ng/µL. Integrity of DNA was analyzed on Agilent 2200 TapeStation system using TapeStation Analysis application. As a measure of degradation, the percentage of the integrated area (Ti) from 150-10,000bp was calculated. The concentration and region molarity (MR) were also monitored. Results: Average Ti for heparin-4°C samples increased from 12% to 74%, whereas it stayed consistent for EDTA-4°C samples. Ti for heparin-RT samples increased the greatest from 16% to 96%. Ti for EDTA-RT samples plateaued off at ~42% from day 8. The concentration and MR showed similar trends. At least 5 distinct peaks, characteristic of apoptotic laddering, appeared on day 6 in heparin-RT and EDTA-RT samples; none were detected with heparin-4°C, despite the increase in Ti. Instead, traces were distributed across the defined range, consistent with smear-like appearance in the gel. EDTA-4°C samples showed no significant variation in peak distributions (one peak at ~55,000bp), consistent with intact DNA. Conclusions: DNA degradation was most prominent for heparin-RT and least for EDTA-4°C samples. Apoptotic laddering occurred in both EDTA and heparin samples stored at RT, suggesting that apoptosis is characteristic of RT storage conditions. The extent of DNA degradation for EDTA-RT increased during the first week then plateaued, suggesting that EDTA may be countering the effects of RT-induced apoptosis. The patterns for heparin-4°C samples suggest that DNA may have degraded through necrosis. More studies are needed to determine the effects on downstream assays. EDTA-4°C samples were the most stable, showing almost no degradation after 3 months. G21. Clinical Validation and Initial Analysis of the PGRN-Seq Capture Reagent for Use in the RIGHT Protocol J.M. Skierka, B.E. Moore, J.H. Blommel, J.K. Bruflat, L.M. Peterson, T.L. Veldhuizen, N. Fadra, S.A. Lagerstedt, L.J. Train, L.M. Baudhuin, E.W. Klee, M.J. Ferber, S.J. Bielinski, J.L. Black Mayo Clinic, Rochester, MN. Introduction: The Right drug, Right dose, Right time- Using Genomic Data to Individualize Treatment(Right Protocol) is a study of 1013 individuals where preemptive genotyping will be performed utilizing PGRN-Seq, a next-generation sequencing (NGS) capture reagent. PGRN-Seq t includes a custom capture of 84 genes with known drug phenotype associations. Initially, pharmacogenomics data for a subset of genes (CYP2C9, CYP2C19, SLCO1B1, and VKORC1), will be reported in the patients’ electronic medical record (EMR), thus allowing the physician to utilize the pharmacogenomics information to help guide therapy if needed. Methods: A total of 117 unique samples were assessed for the clinical validation of the PGRN-Seq. Ninetysix of the samples were purchased DNA from Coriell, which were previously characterized by Hapmap or 1000 Genomes studies. The other 21 samples were deidentified anonymous donor EDTA whole blood samples. The PGRN-Seq capture reagent was optimized for automated high through-put sample preparation. Each sequencing run was performed on 48 samples multiplexed on the Illumina HiSeq 2500 in rapid run mode. Regions of interest containing known functional star alleles or known variants from the four genes, CYP2C9, CYP2C19, SLCO1B1 and VKORC1, were validated. Results were compared from the PGRN-Seq reagent to those obtained from the current clinical (confirmatory) assays and/or previously published results. Results: Utilizing 2012 guidelines from New York State on NGS for somatic genetic variant detection, the accuracy of the reagent was verified by assessing each star allele or variant of interest. Once a specific variant was encountered twenty times (consisting of at least ten wild-type calls and ten variant calls), and confirmed by an existing clinical-grade alternative method, further verification was not required. The PGRN-Seq and confirmatory assays were 98.7% concordant. One false negative result was observed in our confirmatory Sanger sequencing assay due to allelic drop-out. When comparing published data to results obtained with the PGRN-Seq, only two out of 1063 calls were discordant, resulting in 99.9% concordance. Furthermore, the PGRNSeq was evaluated for reproducibility (inter- and intra- assay) and analytical sensitivity, thereby establishing the analytical measurement range and analytical range of our method. Conclusions: We have clinically validated the PGRN-Seq for use in the RIGHT Protocol at Mayo Clinic. Allowing for preemptive genotype results to be utilized
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AMP Abstracts real-time with clinical decision support and EMR integration; optimizing drug therapy for each of the 1,013 individuals. G22. Allele Sizing from Abbott/Celera’s FMR1 Triplet Repeat Primed PCR Assay M.A. Jama, E. Lyon ARUP Laboratories, Salt Lake City, UT. Introduction: Fragile X (FX) syndrome is the most common cause of inherited mental retardation resulting from a dynamic expansion of a trinucleotide (CGG) repeat in the fragile X mental retardation gene (FMR1). Testing for FX and FX-related symptoms constitutes a significant workload in many genetic laboratories and requires a rapid and inexpensive test to identify normal, grey and premutation alleles and distinguish them from full mutations that will need additional testing for methylation status. The triplet primed PCR is a chimeric PCR that generates a stuttering peak pattern used to screen for expanded alleles in FX sample. The stuttering pattern represents PCR products differing in size by one triplet CGG repeat. The stutters and prominent peak (representing the full length product) can be used for counting the number of CGG repeats in a sample. The aim of the present study was to evaluate Abbott/ Celera’s Triplet Repeat Primed PCR (TRP-FX-PCR) General Purpose Reagents for its reliability to size CGG repeat alleles for normal, intermediate and premutation alleles. Methods: Thirty fragile X samples genotyped previously (ten normal alleles, ten intermediate alleles, and ten premutation alleles) were analyzed in this study. The genotypes ranged from 20 to 123 CGG repeats. PCR was performed according to the manufacturer’s recommendations. Six of the 30 samples representing two normal alleles, two intermediate alleles, and two premutation alleles were amplified in triplicate and assayed in three separate runs for within run and between run reproducibility studies, respectively. Results: All 30 fragile samples were 100% concordant with previously obtained results from PCR designed for sizing by amplifying over the CGG repeat. Sizing was reproducible with no variation either within or between runs. Conclusions: This FX screening assay was found to be efficient and rapid test for screening for expanded alleles and sizing the CGG repeats for FX samples in the normal, grey and premutation ranges. Since many of the FX testing is to “rule out” FXs this assay is an economical and reliable method in reducing the number of samples needing methylation analysis, yet still providing sizing information to clinicians. G23. Selective Enrichment of Genomic Loci for the Noninvasive Detection of Fetal Aneuploidies T.J. Jensen1, S.K. Kim1, H. Tao1, J. Geis1, C. Deciu1, D. van den Boom2, M. Ehrich2 1Sequenom Laboratories, San Diego, CA; 2Sequenom, Inc., San Diego, CA. Introduction: Current commercial offerings for non-invasive prenatal testing (NIPT) differ in the breadth of their coverage (whole genome or reduced content). Although whole genome methods provide an unbiased view of the entire genome and thus the potential detection of a broad array of genomic aberrations, reduced content or targeted methods provide efficiency for detecting a limited set of copy number variations. Previous evaluations of reduced complexity NIPT assays have proposed a number of methodologies including the use of single nucleotide polymorphisms (SNPs), target enrichment and universal amplification, the use of repetitive regions, and leveraging differences in DNA methylation between maternal and fetal circulating cell free (ccf) DNA. The objective of this study was to develop and evaluate a NIPT assay which is focused upon the selective enrichment and analysis of a subset of the genome. Methods: Regions for targeted evaluation were selected using a combination of bioinformatic and experimental methods. Ccf DNA was extracted from the plasma of pregnant female donors and amplified using multiplexed amplification in a single well targeting more than 50 genomic loci. Amplified products were then evaluated using multiple methods including MALDI-TOF mass spectrometry and massively parallel sequencing. Results: We evaluated the performance of the developed assay in an unblinded set of 288 ccf DNA samples from pregnant donors with known fetal karyotypes including 258 euploid and 30 aneuploid samples. These samples were measured using multiple technologies including MALDI-TOF mass spectrometry and massively parallel sequencing. Using these methods, 28 of the 30 aneuploid samples were detected with a specificity ranging from 0.95 to 0.99, depending on the chromosome. Taken together, these data demonstrate the feasibility of using this region selective method for fetal aneuploidy detection. Conclusions: Overall, this method leverages specifically selected genomic loci to enable a proof of concept for a low cost, platform flexible method for the non-invasive detection of fetal autosomal aneuploidies. G24. Capture-Based Next-Generation Sequencing (NGS) Is Less Susceptible to Allelic Drop-Out Compared to Sanger Sequencing B.E. Moore, J.M. Skierka, J.K. Bruflat, S.A. Lagerstedt, J.H. Blommel, L.M. Peterson, T.L. Veldhuizen, N. Fadra, L.M. Baudhuin, E.W. Klee, M.J. Ferber, S.J. Bielinski Mayo Clinic, Rochester, MN. Introduction: Clinical Next-generation sequencing (NGS) processes often involve secondary confirmation of results by previously established testing methods such as Sanger sequencing. Sanger sequencing has been long considered the ‘gold standard’ for genetic analysis in spite of known limitations. Sanger confirmation of NGS results is both costly and time consuming. Methods: Clinical validation of the Pharmacogenomic Research Network’s NGS capture reagent (PGRN-Seq) was completed on a total of 117 unique samples. PGRN-Seq includes a custom capture of 84 genes with known drug phenotype associations and is being deployed in The Right drug, Right dose, Right time- Using Genomic Data to Individualize Treatment (Right Protocol) to provide pre-emptive pharmacogenomic genotyping on the 1,013 individuals enrolled.
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Confirmatory testing of NGS results for regions of interest was done utilizing validated clinical testing assays, including Sanger sequencing (CYP2C9, CYP2C19, and VKORC1) and a real-time Taq-Man assay (SLCO1B1). Samples run on clinically validated assays and/or previously published results were compared to the PGRN-Seq results. Each specific variation was confirmed twenty times by clinical testing, consisting of at least ten wild-type and ten variant calls of either homozygous or heterozygous results. Results: Confirmation of the PGRN-Seq resulted in 98.7% concordance; four of 298 confirmatory results were discordant between NGS and Sanger methodologies.The discordant results were from four samples containing a heterozygous CYP2C9*8 (c.449G>A) variant by NGS, but indicated as homozygous normal, by Sanger. A second independent Sanger sequencing test using alternate primers for PCR amplification confirmed that the CYP2C9*8 allele was indeed heterozygous for all four samples. Review of the sample specific DNA sequence using both Sanger and NGS data revealed a novel variant under the priming region of the original PCR. This variant was preventing the amplification of the CYP2C9*8 allele, leading to a false negative genotype. Conclusions: This abstract shows that NGS is superior to Sanger sequencing in some instances. This is interesting since validation of laboratory developed genetic tests such as NGS often requires Sanger sequencing to confirm the presence or absence of gene variants. Our observations confirm that Sanger sequencing has its own limitations that the laboratorian must be aware of. These results suggest that for some types of variants, NGS alone could be sufficient for clinical reporting. G25. Development of a Clinical Next-Generation Sequencing Assay for Lysosomal Storage Disorders Mutation Screening: Utilization of Low Quantity DNA from Dried Blood Spot R. Majumdar, A. McDonald, B. Dukek, N. Fadra, D. Gavrilov, K. Raymond, S. Tortorelli, P. Rinaldo, D. Matern, D. Oglesbee Mayo Clinic, Rochester, MN. Introduction: With over 50 different disorders and a combined incidence of up to 1/7000 births, the diagnosis of lysosomal storage disorders (LSD) is difficult due to overlapping clinical profiles, genetic heterogeneity and problems inherent to biochemical diagnosis. Next-Generation Sequencing technology (NGS) is appealing in a clinical setting due to lower sequencing costs and better molecular diagnosis compared to current Sanger sequencing. Little is known to date about the application of NGS to low amounts of DNA obtained from dried blood spots (DBS), the most widely used sample type for LSD screening. Here we evaluate the usefulness of DNA obtained from DBS and other sources for generating NGS data for LSD diagnosis. Methods: 200 ng of DNA from each of 11 samples (5 DBS, 3 peripheral blood, 3 Coriell fibroblast cell lines) was used. Library preparation, including indexing and enrichment, was performed according to the Agilent SureSelectXT Target Enrichment v1.6 protocol. Paired end sequencing of the libraries was completed on the Illumina MiSeq and included a total of 1392 exons, exon-intron-boundaries, and specific intronic regions (target size 350 kb) of the genes known to be associated with LSD. Bioinformatics analysis was performed using a commercial (CLC Bio Genomics Server, Qiagen) and an in-house developed resulting software. Results: The MiSeq run generated ~5.2 million reads with >65% of reads mapped to the target region, and an average read depth of 1100. On average, a total of 99.9% of bases of the region of interest were covered by at least 100 reads in all samples including DBS. Interestingly, all previously known mutations associated with LSD in Coriell cell lines were confirmed by NGS. We consistently discovered five poor regions with read depth A; 1330G>C] (p.[A171T; D444H]) (14%). In addition, one Hispanic newborn carried a novel pathogenic mutation (c.587delC (p.Thr196Lysfs*68)), and two patients (one Caucasian and one Hispanic/Caucasian) had mutations that did not account for their profound deficiency, suggesting the presence of additional undetected genetic alterations. Among the newborns with partial BD, the most common mutation, as expected, was c.1330G>C (p.D444H) (44% allele frequency). This mutation was most frequently accompanied by the complex allele c.[511G>A; 1330G>C] (p.[A171T; D444H]) (15% allele frequency). Three novel missense variants were detected in three different patients (one Hispanic and two Asians): c.329T>C (p.F110S), c.1250T>A (p.V417D), and c.866C>T, p.(A289V). Also, sequencing failed to identify any mutations in one patient of Asian descent. Conclusions: Molecular analysis of the BTD gene, particularly sequencing, is a useful confirmatory approach in newborns with BD. However, occasionally, sequencing of exons is insufficient to explain the BD phenotype, especially in nonCaucasian newborns. Moreover, novel variants are more common among nonCaucasian newborns given that those populations have been understudied. G27. Evaluation of a Novel PCR Methodology That Can Quantify >150 Hexanucleotide Repeats at the C9orf72 Locus G.J. Latham, J.A. Kemppainen Asuragen, Austin, TX. Introduction: (GGGGCC)n hexanucleotide repeat expansions in the noncoding region of the C9orf72 gene represent the first genetic link between Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), and are represented in ~10% of all clinical cases. These repeat expansions are also associated with other neurodegenerative disorders, including Alzheimer’s disease. Although the most common repeat numbers are 2-8, expansions often include hundreds to thousands of repeat units and have been observed with a frequency of ~1/600 in population studies. Thus, the repeat region is of considerable interest for both diagnostic and screening applications. The GC-rich nature of the repeat poses formidable challenges to PCR, and, consequently, Southern blot (SB) analysis is currently required to identify the molecular features of expanded alleles. Here we describe an innovative PCR assay as a potential alternative to SB that leverages AmplideX FMR1 reagent technology. Methods: AmplideX PCR reagents were optimized for the amplification of C9orf72 hexanucleotide repeats. Amplicons were sized on a 3500xL Genetic Analyzer (Thermo Fisher) and/or NuSieve agarose gels (Lonza). A total of 101 DNA samples were evaluated, including 9 previously characterized genomic DNA samples from the Coriell ALS sample repository (4 normal and 5 expanded) and 92 specimens collected from healthy volunteers. Results: In repeatability studies, the repeat number was resolved within a single unit for 24/24 normal samples (range: 2 to 28 repeats). All 5 expanded Coriell samples could be quantified to >150 repeats using CE; the upper limit was constrained only by the intrinsic resolution limitations of the sizing platform. On agarose gels, higher molecular weight bands consistent with >200 repeats were detected. Significant size mosaicism for expanded alleles was also observed consistent with SB analysis. Evaluations of analytical sensitivity revealed that a 5% expanded allele in background of 95% of a normal allele could be detected. Repeat sizes for 92 samples sourced from healthy volunteers ranged from 2 to 16 repeats with a median of 2 repeats. Conclusions: Molecular characterization of C9orf72 hexanucleotide repeat expansions represents a promising yet challenging opportunity to support the clinical diagnosis of ALS and FTD and other age-onset neurodegenerative disorders. We have developed a prototype PCR assay that can reliably quantify >150 hexanucleotide repeats, a value that is 3- to 5-fold greater than reports from the scientific literature. The availability of a fully optimized PCR assay may advance clinical research and emerging diagnostic, therapeutic, and screening applications. G28. Targeted DNA Library Construction Methods Dramatically Affect NextGeneration Sequencing Results on GC-Rich Regions and Repetitive Regions in the Human Genome W. Li, J.L. Buckley, T. Triche Children’s Hospital Los Angeles, Los Angeles, CA. Introduction: Next-generation sequencing (NGS) technology has transformed the molecular diagnostic field, and it holds great promise for personalized and precise medical practice. However, to obtain good quality NGS data remains challenging. For example, GC-rich regions usually could not be covered at reasonable depth, and repetitive regions could not be sequenced well. Sequence uniformity sometimes is poor, with some areas over-sequenced whereas others under-sequenced. All these technical problems pose a challenge for NGS methods to interrogate the entire human genome. In this study, we compared different targeted DNA library construction methodologies and showed that DNA library construction methods could dramatically affect the sequencing results on these challenging regions in the genome. Methods: RB1 gene was chosen for sequencing because it contains 180,000 bp with several GC-rich regions and numerous repetitive regions. Three normal samples were used for different library constructions. Long-range PCR amplicon-based library building method was established inhouse using custom designed PCR primers. Ion TargetSeq libraries were constructed using Ion TargetSeq Custom Enrichment kits. Agilent Haloplex libraries were constructed using Agilent Haloplex Target Enrichment System. For all the DNA libraries, further template amplification and enrichment were performed on the Ion OneTouch 2 system followed by sequencing on PGM using Ion PGM 200bp sequencing kit. Sequence alignments were done in Torrent Suite Software v. 4.0. The sequencing data was also viewed and further analyzed by Genetrix software that was
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developed inhouse. Results: RB1 targeted DNA libraries were built using long-range PCR method, Ion TargetSeq method, as well as Agilent Haloplex method. We analyzed sequencing coverage depth, quality score and uniformity on RB1 entire gene, and GC rich regions, as well as repetitive regions in RB1. Agilent Haloplex method performed the best among the three methods. It gave good quality sequences and sufficient coverage depth on GC rich regions, as well as repetitive regions in RB1 gene. Longrang PCR amplicon-based method had trouble sequencing repetitive regions and sequencing uniformity was poor. Ion Torrent TargetSeq method was comparable to Haloplex method except it could not sequence through GC-rich regions. Conclusions: Different DNA library building methods can dramatically influence the quality of NGS data on the GC-rich regions as well as on the repetitive regions in the genome. This study shed light on how to build robust NGS-based diagnostic and research assays to comprehensively interrogate the genomic regions, especially GC-rich and repetitive regions in the human genome. G29. Comprehensive Evaluation of Congenital Immunodeficiency by NextGeneration Sequencing H. Yu1, E. Gorman1, S.M. Chen1, A.E. Mangubat1, K. Bagley1, J. Wang1, K.E. Berge2, H.S. Sorte2, J.R. Lupski1, J.S. Orange1, L.F. Forbes1, I.C. Hanson1, A. Stray-Pedersen1, V.W. Zhang1, L. Wong1 1Baylor College of Medicine, Houston, TX; 2Oslo University Hospital, Oslo, Norway. Introduction: Congenital immunodeficiency is an inherited disorder of the immune system. Severe combined immunodeficiency (SCID), characterized by profound deficiencies of T cells and/or B cells at the time of birth, is the most severe form of congenital immunodeficiency. If not treated promptly, affected patients usually do not live beyond infancy due to severe, recurrent infections. Genetic heterogeneity of SCID frequently delays the diagnosis, which is crucial for life-saving treatment and optimal management. Methods: All targeted exons and at least 20 bp of flanking genomic sequences of 46 genes involved in SCID are enriched using SeqCap EZ solution-based capture and are subjected to Massively Parallel Sequencing (MPS) on Illumina HiSeq2000. Results: The target gene capture/MPS provides an average coverage of 1000X. Exons with insufficient coverage (T at position 430 and A>C at position 1075, described as 2C9 *2 and *3 respectively. The VKORC1 kit targets 2 sites that display linkage disequilibrium, which are the G>A substitution at -1639 and the C>T polymorphism at position 1173. Results: Only 34.4% of tested subjects either had no mutation for all 3 alleles or bore only one heterozygous polymorphism that had superficial phenotypic consequence. The majority of the remaining 65.6% were predominantly of Chinese and Malay descent, manifesting a homozygous polymorphism for the VKORC1 gene. Subjects of Indian descent displayed the most variability in genotype polymorphism with representation in either one or both genes implying significant phenotypic consequence. One third of this Indian cohort carry the heterozygous *2 variant either singly or with a VKORC1 mutation. Conclusions: This data suggests that a majority of our patients’, should they require warfarin therapy, may benefit from pharmacogenetic testing of the Cytochrome P450 2C9 and VKORC1 genes. In the Chinese and Malay subjects tested, 70.7% and 35.8% respectively manifest intermediate metabolism of this drug. In totality, 65.6% of the population has a single or dual gene polymorphisms that implies highly modify drug metabolism from normal. Additionally, only 1 out every 2 subjects of Indian descent has ‘normal’ warfarin metabolism. Our data shows considerable warfarin heterogeneity in the local population and suggests that warfarin genotyping is a useful adjunct for patients’ warfarin prescription so as to achieve optimal therapeutic outcomes. G36. BAP1 Missense Mutation c.2054 A>T (p.E685V) Completely Disrupts Normal Splicing through Activation of Cryptic Splicing Sites A. Morrison1, Y. Chekaluk2, R. Bacares2, M. Ladanyi2, L. Zhang2 1Wake Forest University, Winston Salem, NC; 2Memorial Sloan Kettering Cancer Center, New York, NY. Introduction: BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal melanoma, malignant pleural mesothelioma (MPM), clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germlinemutations have been reported in families with combinations of these same cancers. It has been proposed that BAP1 germline mutations define a new familial cancer syndrome. Mutation screening of BAP1 is ongoing at MSKCC and classification of variants in BAP1 is critical for patient management. A particular challenge is the classification of rare non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Methods: A homozygous substitution mutation, BAP1 c.2054 A>T (p.Glu685Val) was identified in an MPM cell line, HMeso01A (derived from a mesothelioma patient). RT-PCR was performed using primers in exon 14 and 17 and products were gel purified then cloned into the TOPO vector. Subsequent sequencing was performed on individual clones. Results: Upon separation of the RT-PCR products, the HMeso01A cell line displayed several aberrant splicing products not observed in the controls. The cloning results indicated that all clones derived from the major splicing product in the HMeso01A cell line contained a 4 bp deletion compared with the full length transcript. The BAP1 c.2054 A>T mutation at the third nucleotide from the 3’ end of exon 16 introduced a cryptic 5’ splice site (GU). This utilization of this cryptic splicing site resulted in the deletion of 4 base pairs and presumably protein truncation. In addition, we observed a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16. We also observed partial intron 14 and 15 retentions. The partial intronic retentions of introns 14 and 15 are caused by activation of alternative 3’ splice acceptor sites (AG) in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing in the mutant allele. Conclusions: These results support the conclusion that BAP1 c.2054 A>T (p.E685V) variant is a deleterious mutation and contributes to MPM through disruption of normal splicing. G37. Library Pooling of Multiple Assays for Clinical Next-Generation Sequencing X. Wu, Z. Tu, L.M. Peterson, B.A. Dukek, J.H. Blommel, S.A. Henke, W.C. Thompson, E.W. Klee, M.J. Ferber Mayo Clinic, Rochester, MN. Introduction: Next-generation sequencing (NGS) is widely used in research because it produces large amounts of sequencing data. Clinically, it is a very powerful tool for detecting multiple-gene related disorders in a single sequencing run. There are different sequencing platforms which can generate significantly different amounts of sequencing data. Currently, clinical NGS is performed based on library preparation chemistry and typically one assay per instrument run. These single assay runs typically produce more sequencing data than needed, so for the clinical laboratory it becomes important to define a way to more efficiently utilize the sequencing instruments. The ability to pool various assays together on multiple sequencing platforms offers significant advantages to a clinical laboratory including reduced staffing needs, reduced reagent costs, and reduced total instruments needed. We have developed a tool to more efficiently run the instruments and laboratory whereas producing consistent results regardless of target size or library preparation chemistry. Methods: Previously prepared sample libraries from different clinically validated panel assays were re-quantitated for use in this study. A library pooling formula for single test was evaluated: (samples to be pooled = reads/lane x sequencing cycles)/(target sequencing size (bp) x average coverage). Based on this formula, multiple samples from various clinically validated assays were pooled together on one instrument run and data was evaluated for minimal coverage across all targeted regions (100x to 500x, depending on the assay). Results: Sixteen
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sample libraries from four clinically validated NGS panels were pooled at equimolar concentrations for sequencing at the same time on one Illumina MiSeq instrument. A similar number of reads were generated for each library. Variants from the pooled libraries were compared with previously generated sequence data from individually performed assays. All variants, including large deletions, were 100% concordant with the previous data. Conclusions: We have developed a tool and have demonstrated the library pooling formula successfully determines the number of samples from assays with variable target sizes that can be pooled on the same instrument run. Once implemented clinically, this strategy will enable us to maximize sequencing time on our instruments by running them at full capacity. Other advantages to the clinical laboratory include reducing reagent and labor costs, as well as reducing costs for patients. G38. Assessment of the Clinical Utility of a Custom Exon-Targeted Microarray for the Detection on Deletions and Duplications as a Component of a Hereditary Colon Cancer Panel T.J. Gliem, P. Brodersen, B. Thomas, E. Thorland, M. Ferber, W.E. Highsmith, B. Dawson Mayo Clinic, Rochester MN. Introduction: A multi-gene panel for hereditary colon cancer was designed to identify germline mutations associated with an increased risk for colon cancer. Genes that had definitive evidence to support clear disease risk, a substantial body of research to support clear disease-risk, and sufficient evidence to suggest associated disease-risk were included in the panel. A total of 17 genes/regions are investigated clinically, of which 15 are interrogated by array comparative genomic hybridization (aCGH). Methods: The methods used to analyze the clinical samples include next-generation sequencing (NGS), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and exon-targeted aCGH. The aCGH probes were selected using Agilent’s eArray web-based software for designing custom microarrays. The array was created on an 8x60,000 probe format, allowing 8 samples to be run concurrently on a single chip. Design parameters included: 1) a minimum of 5 probes covering each exon; 2) inclusion of all available catalog probes within the introns; and 3) inclusion of all available catalog probes within 5 kilobases, 5’ and 3’ of the gene. Microarray experiments were performed using standard Agilent protocols. aCGH results for samples reported from May 2013 to May 2014 (n=456) were reviewed. A total of 62 panel tests and 394 single gene analyses are included in the review. Results: A total of 9 arrays were interpreted as positive for a 2.0% detection rate. The positives included 7 deletions; CDH1 (1), SMAD4 (1), STK11 (1), and TP53 (4) and 2 duplications of the SCG5-GREM1 region. All were ordered as single gene analysis. The aCGH test is more cost effective than MLPA when multiple genes are analyzed. The array also has the advantage of interrogating multiple probes per exon. However, this means that a copy number change (CNC) of only 2-3 probes within an exon may be significant. SNPs were also shown to impact the binding of multiple overlapping probes causing the probes to shift toward deletion or duplication. Regions of homology are problematic for any probe-based assay and impacts targeted aCGH. Currently, PMS2 is investigated by MLPA and not by aCGH due to pseudogene homology. Conclusions: The detection rate of 2.0% demonstrates that a targeted aCGH analysis as a component of an NGS targeted gene-panel for hereditary colorectal cancer has clinical utility. The ability of SNPs to impact multiple probes demonstrates the benefit of analyzing the aCGH data in conjunction with the sequencing data. G39. Using Chromosomal Microarrays to Further Delineate Traditional Chromosome Studies: A Case Report K.A. Lebel1, S.A. Marconi1, A.M. Tyropolis1, C.M. Bissaillon1, J. Smith1, C. Phillips1, K. Casavant1, G.J. Tsongalis2, S.M. Pflueger1 1Baystate Health, Springfield, MA; 2Dartmouth-Hitchcock Medical Center, Lebanon, NH. Introduction: The use of chromosomal microarray (CMA) in combination with traditional karyotype analysis has become standard of practice in the workup of patients with suspected genetic diseases. Karyotyping allows for identification of balanced translocations as well as large deletions and duplications. CMA can identify small deletions and duplications not discernible by standard methods. Here we discuss a case where CMA was used to resolve a previously reported abnormality identified by karyotype analysis. Methods: A twenty-five year old female with a personal history of seizures, developmental delay, and learning disability presented to the clinical genetics department because of her previously known abnormal karyotype, with an inversion and insertion of chromosome 17q. This finding was seen in other maternal family members with similar developmental delays and learning disabilities. Because of the strong family history, the patient was seeking information about this abnormality and how it might affect her current pregnancy. After genetic counseling and chart review, the patient’s peripheral blood was submitted for testing. DNA extraction was performed utilizing the automated EZ 1 DNA tissue kit (Qiagen, Germantown, MD). DNA concentration and purity was assessed by the NanoDrop 2000. SNP array analysis was performed and data analyzed per protocol utilizing the CytoScan HD kit and CHAZ software (Affymetrix, Santa Clara, CA). Data parameters included regions of homozygosity, number of allele tracks and median copy number state. For karyotype analysis, peripheral blood was cultured in PB Max (Life Technologies, Grand Island, NY) and harvested for chromosome analysis. GTW banded chromosomes were analyzed at a band level of 850. FISH studies were performed per protocol utilizing WCP 17 (Cytocell, Cambridge, United Kingdom). Results: Repeat karyotype analysis confirmed the presence of an inverted 17q. CMA identified 5 regions of duplication across chromosome 17 including a clinically significant gain at 17p13.3. Whole
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AMP Abstracts chromosome paint was used to determine the location of the duplications with regard to the entire genome. All duplications were found on chromosome 17. Conclusions: The previously reported inversion of 17q was thought to be the reason for this family’s history of developmental delay and learning disability. Through CMA testing, a clinically significant gain of material on 17p has provided a potential diagnosis for this patient and her family members. CMA analysis of affected family members as well as this patient’s current pregnancy may provide additional information. G40. Comprehensive Characterization of FMR1 Controls Using CGG Repeat Sizing, AGG Mapping, and CpG Methylation Analysis by Both Methylation PCR and Next-Generation Sequencing A.G. Hadd1, S. Filipovic-Sadic1, R. Shroff1, D. Budamirovic2, G.J. Latham1 1Asuragen, Inc., Austin, TX; 2Kennedy Krieger Institute, Baltimore, MD. Introduction: CGG repeat expansions in the 5’ untranslated region of the fragile X mental retardation (FMR1) gene are associated with fragile X syndrome and multiple fragile X-related disorders. Testing paradigms for FMR1 gene alterations, including phenotype correlation studies, expansion risk assessments as well as carrier screening and expansion assays, can benefit from access to well-characterized controls. In this study, we used advanced PCR techniques and next-generation sequencing (NGS)based methylation analysis to comprehensively characterize the FMR1 5’ UTR for a set of fragile X blood specimens and matching lymphoblastoid cell lines. Methods: DNA from 11 whole blood specimens, 10 matched cell lines, and 4 clinical samples were obtained following IRB approval. Each sample was characterized using AmplideX FMR1 PCR, mPCR and Xpansion Interpreter assays, as well as Southern blot (SB) analysis and Sanger sequencing to reveal the CGG size, AGG interruption pattern, and the methylation status. Methylation sequencing of 1226 bases upstream and 424 bases downstream of the CGG repeat region was achieved using 10 primer pairs and PCR of bisulfite treated DNA followed by sequencing on an Illumina MiSeq. Unmethylated and methylated genomic DNA controls were mixed at various ratios to create analytical standards. Results: Cell line DNA controls (AmplideX FMR1 Controls) represented alleles ranging from 20 CGG to ~1200 CGG in both males and females. The number of CGG repeats was determined by Sanger sequencing for up to 223 repeats using novel GC-rich amplification reagents. AGG interruption patterns for all samples were concordant between the immortalized cell lines and the original blood specimen. The CGG repeat lengths varied following clonal selection in the cell lines but the methylation status was preserved. In all cases, samples analyzed by mPCR and SB analysis were in 100% agreement for both genotype and methylation status. Methyl-Seq analysis across 80 CpG islands upstream of the repeat region demonstrated equivalency for the methylation fraction at EagI and NruI restriction sites commonly used in SB, and both HpaII sites used in mPCR. Conclusions: A combination of genotyping and epityping techniques was used to characterize whole blood and matching cell line controls that represented normal, intermediate, premutation, and full mutation genotypes in males and females. The controls may be used as assay controls, in proficiency studies, or for comparing FMR1 repeat technologies. In addition, the approaches described are amenable to broader cohort studies for genotyping and methylation analysis that advance the field of fragile X research. G41. Genetic Heterogeneity in Familial Hypocalciuric Hypercalcemia (FHH): Preliminary Screening of an At-Risk Cohort for FHH3 (AP2S1) B.Y. Wong1, B. Lee1, S. Kittanakom2, G.N. Hendy3, D.E. Cole1 1Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada; 2University of Toronto, Toronto, Ontario, Canada; 3McGill University, Montreal, Quebec, Canada. Introduction: About 65% of familial hypocalciuric hypercalcemia (FHH) is caused by inactivating mutations of the CASR gene, now classified as FHH1. Recently, FHH2 and FHH3 have been defined by inactivating mutations in the GNA11 gene and specific missense mutations in the AP2S1 gene, respectively. AP2S1 encodes the s1 subunit of adaptor-related protein complex 2 that appears to be important for clathrin-mediated endocytosis of the calcium-sensing receptor. So far all the AP2S1 mutations identified in FHH3 have been found to occur at codon 15 in exon 2 (p.R15L, p.R15C and p.R15H). These mutations are reported to be responsible for about 15% to 20% of FHH cases without CASR mutation. Methods: This study aims to determine the frequency of AP2S1 mutations in a cohort of individuals suspected of having FHH, and negative for CASR mutation. A total of 104 archived genomic DNA samples were subjected to Sanger sequencing of an exon 2 amplicon that included the codon 15 sequence of AP2S1. Positive samples were confirmed by bidirectional sequencing using a different primer set. Results: In 104 FHH cases without CASR mutation, 3 cases (2.9%) were found to carry AP2S1 mutations: two with p.R15L, and one with p.R15C. Conclusions: In our intermediate risk cohort (suspected of having FHH but unconfirmed and negative for CASR), we found some with AP2S1 codon 15 mutations. Although the numbers are not large, our results tend to affirm the view that clinical testing for FHH-causing mutations should begin with the CASR gene, and if negative, be followed by screening for codon 15 mutations in AP2S1. Similar studies of the GNA11 gene for mutations causing FHH2 are needed before recommending its inclusion in the clinical testing algorithm.
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G42. Findings from Chromosomal Microarray Analysis in Clinical Practice: A Study of 177 Patients K.A. Lebel1, S.A. Marconi1, A.M. Tyropolis1, C.M. Bissaillon1, G.J. Tsongalis2, S.M. Pflueger1 1Baystate Health, Springfield, MA; 2Geisel School of Medicine, Lebanon, NH. Introduction: The ACMG Practice Guidelines recommend Chromosomal Microarray Analysis (CMA) for copy number variation as the first-line test in postnatal evaluation of individuals with multiple anomalies not otherwise specified to well-delineated genetic syndromes; as well as other developmental, growth and speech delays. Variants identified by this testing range from likely benign to likely pathogenic findings. In this study, we describe the CMA hit rate of findings at a large, inner city, tertiary medical center. Methods: One hundred seventy-seven patients were tested utilizing the Affymetrix CytoScan HD array according to the manufacturer’s protocol and analyzed using Chromosome Analysis Suite (ChAS) software (Affymetrix, Santa Clara, CA). Copy number changes were analyzed using standard settings: 50 markers and 100 kb for gains and 50 markers and 50 kb for losses. Cases were subsequently analyzed for Regions of Homozygosity (ROH) greater than 3000 kb. A clinical review of the patient’s history was provided by the requesting physician. Findings were compared to the patient’s clinical history/presentation and categorized into four categories: likely benign, unclear clinical significance, likely significant, and diagnostic for a specific disease state or syndrome. Results: One hundred seven cases (60%) showed no significant gains, losses, or regions of heterozygosity. Thirty-six cases (21%) had findings diagnostic for a specific disease or syndrome. Eighteen cases (10%) had gains or losses of unclear clinical significance whereas 7 cases (4%) had ROH greater than 2% reported as significant for expression of a recessive trait. Nine cases (5%) had significantly sized gains or losses characterized as likely benign. Conclusions: Our CMA testing service has a hit rate of findings of 21% where the cases presented to the laboratory for CMA analysis produced diagnostic results whereas another 14% were identified as having likely significant findings. This study supports the use of CMA as a useful clinical tool for first line assessment of individuals with multiple anomalies or varied developmental delays. G43. Clinical Advantages of High Coverage Comprehensive NGS Panels in the Molecular Diagnosis of Hereditary Cancer Mutations G. Douglas, E. Gorman, S. Chen, X. Tian, Y. Feng, F. Li, J. Wang, L. Wong, V.W. Zhang Baylor College of Medicine, Houston, TX. Introduction: Accurate assessment of hereditary cancer risk by molecular diagnosis of cancer predisposition genes is essential for disease management and genetic counseling. When ordering genetic testing for hereditary cancer evaluation, it may be challenging to single out relevant genes, whereas still aiming to obtain a comprehensive evaluation. High coverage capture-based next-generation sequencing (NGS) panels comprised of multiple hereditary cancer-associated genes, sequenced simultaneously, provide a promising solution to meet these clinical demands in a cost-efficient way. Methods: A total of 61 hereditary cancer-associated genes, grouped into 12 disease-specific panels and one comprehensive panel, were targeted with in solution capture followed by next-generation sequencing. Exons with coverage G and c.1145G>A, VHL c.592_607del16, PTEN c.547_550delinsTTT and APC c.1081_1087dup7, were successfully detected. Furthermore, pathogenic CNVs ranging from single/few exon deletions in VHL, MLH1, TP53, PTEN and EPCAM to a whole gene deletion of the APC gene were also identified successfully and confirmed. As an example of highlighting the advantage of our assay, we were able to detect both a point mutation, c.65C>A, and an exon 14 deletion in the PMS2 gene in one sample simultaneously, despite the well-known PMS2 pseudogene interference. Conclusions: This capture based deep coverage NGS panel provide accurate and comprehensive analysis for hereditary cancers genes. The combination strategies of both deep exonic base-to-base coverage of all coding regions and Sanger confirmation/complementation allows accurate identification of a wide spectrum of mutation types, including point mutations, small indels, mosaicism. In addition, copy number changes at the exon level can be analyzed simultaneously. Our data underscore the important clinical utility of NGS-based analysis in the molecular diagnosis of hereditary cancer.
jmd.amjpathol.org ■ The Journal of Molecular Diagnostics
G44. The Use of an Artificial Pseudo-Heterozygous Mitochondrial Genome as an Internal Control for WES and WGS Diagnostic Tests J.L. Wendt Andrae, M.R. Tschannen, D.S. Helbing, A.A. Buzzell, D.P. Dimmock, H.J. Jacob Medical College of Wisconsin, Milwaukee, WI. Introduction: Next-generation sequencing tests such as Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) are rapidly becoming valuable diagnostic tools, aiding in the diagnosis of rare diseases. The Human and Molecular Genetics Center at the Medical College of Wisconsin became one of the first laboratories in the world to use WES and WGS as routine diagnostic tests. These tests are complex in nature and must adhere to strict Clinical Laboratory Improvement Amendment (CLIA) requirements. One such requirement is that the laboratory must monitor all steps of the clinical assay to evaluate the quality, accuracy, and precision of the test. To accomplish this, control materials are typically run in parallel with all patient samples. The ideal control should resemble patient specimens to accurately monitor the test. Methods: In the case of WES or WGS, the control material must contain multiple heterozygous variants which are spread across the entire genome or exome. It is too cost prohibitive to sequence an entire human genome or exome during each patient sequencing run to a depth of coverage necessary to accurately call heterozygous variants. The mitochondrial genome, however, is small enough to be sequenced at a high depth to determine heterozygous variants. Our solution was to create an artificial pseudo-heterozygous mitochondrial genome, Mito 50-50, by pooling equi-molar quantities of mtDNA from two unrelated male individuals. Sanger sequencing of the mixed sample revealed 30 heterozygous and 23 homozygous variants. To use Mito 5050 as control during a WES or WGS test, a small concentration of uniquely indexed Mito 50-50 is spiked into each pool of prepared patient sequencing libraries. Results: To determine the quality of each sequencing run, 28 heterozygous and 22 homozygous variants in Mito 50-50 must be correctly identified on a per-Illumina flowcell basis as well as 13 heterozygous and 10 homozygous variants on a per-lane basis. We do not expect to see 100% concordance on a per-lane basis due the lower depth of coverage of Mito 50-50 in each lane. If either of these criteria are not met, the run is determined to be of low quality and the patient samples are re-sequenced. Conclusions: The use of Mito 50-50 as a control material in our WES and WGS diagnostic tests allows us to monitor quality, accuracy, and precision, thereby meeting CLIA requirements and maintaining the highest standards for our extremely complex diagnostic tests. G45. Adventitious Detection of Maternal Neoplasia During Noninvasive Prenatal Testing (NIPT) N. Dharajiya1, T. Monroe2, D.H. Farkas3, T. Boomer1, J. Wardrop1, J. Jesiolowski2, E. Almasri1, C. Zhao1, C. Deciu1, J. Saldivar1 1Sequenom Laboratories, San Diego, CA; 2Sequenom Laboratories, Morrisville, NC; 3Sequenom Laboratories, Grand Rapids, MI. Introduction: Noninvasive prenatal testing has become a part of the standard of care in pregnant women with increased risk for fetal aneuploidies. Overrepresentation of chromosomes 21, 18 or 13 is detected by comparison with normal euploid genome and mathematically calculated into Z score. In rare circumstances of multiple trisomies or significant negative Z score, there is a possibility of global genomic changes. These cases, when explored by detailed bioinformatics analysis, revealed multiple aneuploidies and subchromosomal changes spanning the genome. We postulated the presence of “extra genetic material” in such otherwise euploid samples and that the source of this material could be neoplastic cells. Out of 32 such “non-reportable” cases, clinical follow-up identified maternal neoplasm in 19 cases. Methods: Maternal blood samples submitted to Sequenom Laboratories for MaterniT21 PLUS laboratorydeveloped test were subjected to DNA extraction from fractionated plasma, library preparation, whole genome massively parallel sequencing and bioinformatics analysis. Results: We identified 32 cases with multiple trisomies and a variety of microdeletions and microduplications, the results of which could not be explained by any particular syndrome or aneuploidy. We postulated that extraneous cell free DNA was present, and that the origin of this DNA could be neoplastic cells given the presence of global genomic abnormalities. These cases were resulted as “non-reportable” for the detection of fetal aneuploidies, however, the incidental findings were discussed with the ordering providers. Subsequent detailed clinical follow-up identified a variety of benign and malignant maternal neoplasms: uterine leiomyoma (10 cases), uterine leiomyosarcoma (1), Hodgkin’s lymphoma (1), follicular lymphoma (1), infiltrating ductal carcinoma of the breast (2), angiosarcoma (1), colon carcinoma (1), teratomy of the ovary (1) and multiple myeloma (1). For 13 other cases with similar abnormal global genomic changes, clinical follow-up is pending. Conclusions: Initially developed for detecting an over-representation of chromosome 21, the MaterniT21 PLUS test has advanced to include detecting other aneuploidies for chromosomes 13, 16, 22, fetal sex chromosomes, and select subchromosomal microdeletions. Here, we report 32 cases with incidental findings, 19 of which were subsequently confirmed to have maternal malignancies. For eight cases, these incidental findings precipitated detailed workups resulting in a primary diagnosis. These adventitious results of NIPT warrant further exploration for clinical and medical utilization of cell free fetal DNA analysis.
The Journal of Molecular Diagnostics ■ jmd.amjpathol.org
AMP Abstracts
G46. A Clinically-Validated Next-Generation Sequencing Diagnostic Platform for Germline BRCA1 and BRCA2 Variants Detected A. Qu, J. Eskdale, C. Davis, T. Do, A. Shurshalina, U. Muhammad, C. Pascual, J. Cohen, E. Mordechai, M. Adelson Medical Diagnostic Laboraties, L.L.C./Institute for Biomarker Reserach/Genesis Biotechnology Group, Hamilton, NJ. Introduction: BRCA1 and BRCA2 are human genes that produce tumor suppressor proteins. Specific inherited variants in BRCA1 and BRCA2 increase the risk of female breast and ovarian cancers; associations with an increased risk for several additional types of cancer have also been reported. However, identifying variants in these two large genes by traditional Sanger sequencing method in a clinical testing laboratory setting is both time consuming and costly. Next-generation sequencing (NGS) technology, together with Sanger sequencing confirmation of variants, has the potential to improve the efficiency and throughput of clinical diagnostic sequencing. Methods: We have developed a NGS pipeline in our clinical laboratory for the detection of BRCA1 and BRCA2 variants. One hundred sixty seven small amplicons covering all of the coding exons and intron-exon boundaries of both genes were amplified using the Ion AmpliSeq BRCA1 and BRCA2 Community Panel followed by specimen-specific barcoding and pooling. The NGS template was constructed and enriched by using the Ion Torrent OneTouch 2 system and then sequenced using the Ion Torrent PGM platform. All resulting sequencing data was processed and filtered by Torrent Suite and variants were identified by the Torrent Variant Caller with the Germline low stringency setting. All variants were interpreted, annotated and reported with our in-house developed variant database based on NCBI ClinVar and Breast Information Core databases. To demonstrate the suitability of this pipeline for clinical diagnostic testing, we validated the pipeline in two individual studies. In the first study, 32 patient samples with known variants were sequenced. In the second study, mouth wash samples from 58 volunteers were collected. Genomic DNA was extracted, sequenced and analyzed end-to-end. All variants identified from both studies were confirmed by Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA) technology was also used to examine any possible large BRCA gene deletion/duplications. Repeatability and intra- and inter-assay reproducibility were also tested. Results: The average coverage was 150-fold for each sample which was processed in pools of 16. Validation of 32 previously characterized samples resulted in 98% detection of the clinically significant known variants without any false-positive results. More than 99% of the variants identified in both studies were concordant with Sanger sequencing confirmation. The optimized pipeline accomplished excellent analytical specificity and sensitivity of 99.99%. Conclusions: The results demonstrate that our NGS BRCA1 and BRCA2 variant detection pipeline is suitable for sensitive, high-throughput diagnostic testing of these important tumor suppressor genes in a clinical laboratory setting. G47. Implementation of a Highly-Sensitive and Inexpensive Laboratory Developed Test for 22q11.2 Deletion Syndrome T. Sander1,2, A. Mitchell2, M. Mitchell1, L. Noll1, D. Mahnke2, J. Routes1, P. North1 1Children's Hospital of Wisconsin, Milwaukee, WI; 2Medical College of Wisconsin, Milwaukee, WI. Introduction: Chromosome 22q11.2 deletion syndrome is characterized by a hemizygous deletion of chromosome 22q11.2, often found in patients diagnosed with DiGeorge syndrome, velocardiofacial syndrome, and a variety of other clinical syndromes. It is estimated to be the most prevalent inheritable genetic deletion syndrome, occurring in ~1 in 3,000 to 4,000 live births. The typical deleted region on chromosome 22q11.2 is approximately 3.0 Mb and contains more than 35 genes. FISH is the gold-standard for diagnosis, but is expensive and can miss smaller microdeletions within the proximal 22q region. Our goal was to offer a more sensitive, specific, and cost-effective screening test for early clinical intervention in the treatment of children with 22q11.2 deletion syndrome. Methods: A multiplexed quantitative real-time PCR assay to detect 22q11.2 deletion was translated from research within the Children’s Research Institute to performance as a clinical diagnostic test in the Children’s Hospital of Wisconsin main clinical lab. This assay specifically tests for deletion of the T-box gene 1 (TBX1) and v-crk sarcoma virus CT10 oncogene homolog (avian)-like gene (CRKL). TBX1 is considered a major genetic determinant of the chromosome 22q11.2 deletion syndrome and CRKL is a candidate for causing congenital heart disease within the deleted region. Testing is performed on genomic DNA isolated from whole blood specimens under standards established by the College of American Pathologists. Specimens are analyzed on a QuantStudio real-time PCR instrument using Applied Biosystems Taqman Copy Number assays to TBX1, CRKL, and an internal reference (RPPH1). Results: Analytical specificity and sensitivity are 100%, with 95% confidence intervals ranging from 88%-100%. Clinical sensitivity is 100%, with 95% confidence intervals of 91% to 100%. Turn-around-time is 72 hours. As part of our quality management program within the clinical lab, mutation detection frequency is monitored and correlation studies performed. Our observed mutation detection rate is ~10%, which falls well within the expected range for FISH testing. There is also 100% concordance with duplication and deletion microarray results from the same patient, confirming the validity and accuracy of the test. Recent studies demonstrate that this test can be performed using DNA isolated from dried blood spot cards and is easily adapted to a droplet digital PCR (ddPCR) platform for increased precision and decreased cost. Conclusions: In summary, we have implemented a fast, highlysensitive, and inexpensive laboratory developed test for 22q11.2 deletion syndrome that can be expanded to provide outside reference testing at a low cost.
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AMP Abstracts G48. Processed Pseudogene Confounding Deletion/Duplication Assays for SMAD4 A.S. Millson1, T.B. Lewis1, T. Pesaran2, D.J. Salvador2, K. Gillespie2, C. Gau2, E. Lyon1, P. Bayrak-Toydemir1 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT; 2Ambry Genetics, Aliso Viejo, CA. Introduction: SMAD4, mothers against DPP homologue 4, located on 18q21.1, is a member of the Smad family of proteins. These proteins are phosphorylated through action of the TGFbeta pathway and accumulate in the nucleus. Their role is to regulate transcription of target genes by incorporating various DNA-binding co-factors which confer the target selectivity. Mutations detected by sequencing or large deletions/duplications in the SMAD4 gene have been associated with predisposition to juvenile polyposis syndrome (JPS) and/or hereditary hemorrhagic telangiectasia (HHT) syndrome. Methods: Months apart, two samples sent in for SMAD4 duplication/ deletion testing (one to Ambry Genetics and the other to ARUP Laboratories) resulted in the same unusual duplication pattern. Using multiplex ligation dependent probe amplification (MLPA) technology, a duplication of exons 2-3 and 5-12 was found. Additional workup was performed at ARUP, including quantitative real-time PCR, custom comparative hybridization array (CGH) and sequencing. Results: Quantitative PCR failed to confirm the duplication. Upon closer inspection it was noted that in each of the SMAD4 primer pairs used, the reverse primer was in the 3’ intronic sequence immediately after the exon. Array CGH showed duplication of only exonic probes, all intronic probes showed two copies. These results led to the hypothesis of the existence of a processed pseudogene. To test this hypothesis, PCR was performed using primers located completely in exonic sequence with the expectation of getting PCR products from the processed pseudogene. Two products were generated, one correlating with genomic sequence and one correlating with the expected size of only exonic sequence. The smaller of the two products was Sanger sequenced, confirming only exonic sequence. Conclusions: The presence of a processed pseudogene, as demonstrated by the exonic duplication of SMAD4, was confirmed in our patients. This case illustrates the potential of a processed pseudogene to confound quantitative results. This information is important for molecular diagnosis of SMAD4 large exonic deletions/duplications, so as not to misinterpret a duplication. G49. Allele Frequencies of Two Previously Unreported Cytochrome P450 3A Haplotypes D.L. Pierce, J. Forcellini, M.P. Borgman, M.W. Linder, K.K. Reynolds PGXL Laboratories, Louisville, KY. Introduction: Cytochrome P450 3A4 (CYP3A4) is central to the phase I metabolism of up to 60% of all pharmaceuticals. It is the most abundantly expressed cytochrome P450 in the liver and intestines. Genetic variations in CYP3A4 result in metabolic differences of up to 10-fold. Additionally, most people have variants in CYP3A5 that reduce enzyme activity that is typically compensated for by the 80% metabolic overlap with CYP3A4. Instances where CYP3A4 and CYP3A5 are both deficient or both active have important clinical implications and highlight the importance of genotyping patients for both enzymes. Our laboratory routinely tests for common variants including, but not limited to, CYP3A4*3 and CYP3A4*22, and CYP3A5*2 and CYP3A5*3, which all result in decreased enzymatic activity. We identified samples heterozygous (HET) for CYP3A4*3 and homozygous variant (MUT) for CYP3A4*22. We also identified samples HET for CYP3A5*2 and MUT for CYP3A5*3. These observations suggested the existence of previously unreported CYP3A4 and CYP3A5 haplotypes. Methods: SNP genotyping was performed using real-time PCR with TaqMan chemistry. Dideoxy sequencing was performed using an ABI Prism. DNA extraction was performed using Life Technologies MagMAX or Qiagen EZ-1 reagents. Results: We identified seven CYP3A4*3 HET CYP3A4*22 MUT samples and an additional six CYP3A4*3 HET CYP3A4*22 HET with a known third SNP, leading us to hypothesize that the CYP3A4*3 and CYP3A4*22 are on the same chromosome, with the third SNP on the other. We identified 344 CYP3A5*2 HET CYP3A5*3 MUT samples. These SNP combinations did not match known haplotypes on the CYP alleles database (http://www.cypalleles.ki.se/). Three CYP3A4*3 HET CYP3A4*22 MUT samples were sequenced by capillary electrophoresis confirming the genotype was not an analytical artifact. We found the overall incidence of CYP3A4*3 and CYP3A4*22 occurring on the same allele to be approximately 0.03%. Ten CYP3A5*2 HET CYP3A5*3 MUT samples were confirmed by sequencing with an overall incidence of 0.8%. Both CYP3A4 and CYP3A5 genotypes were evenly distributed between males and females, and were found among Caucasian, Hispanic, and African-American patients. Conclusions: The unexpected CYP3A4 and CYP3A5 genotypes were confirmed by sequencing and provided evidence for a new CYP3A4 haplotype including both the CYP3A4*3 and CYP3A4*22 SNPs as well as a new CYP3A5 haplotype which includes both the CYP3A5*2 and CYP3A5*3 SNPs. Each of these cases were reported as decreased metabolizer status based on confirmation of homozygous variant CYP3A4*22 or CYP3A5*3 genotype.
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G50. Expanded Genetic Screening Panel for the Ashkenazi Jewish Population B.W. Baskovich1, S. Hiraki2, K. Upadhyay2, P. Meyer2, S. Carmi3, N. Barzilai2, A. Darvasi4, K. Offit5, S. Bressman2, L. Ozelius6, I. Peter6, J. Cho6, G. Atzmon2, L. Clark3, T. Lencz2, I. Pe'er3, H. Ostrer2 1Montefiore Hospital, New York, NY; 2Albert Einstein College of Medicine, Bronx NY; 3Columbia University, Manhattan, NY; 4Hebrew University of Jerusalem, Givat Ram, Jerusalem; 5Memorial Sloan-Kettering Cancer Center, Manhattan, NY; 6Mount Sinai School of Medicine, Manhattan, NY. Introduction: Carrier screening programs that identify the presence of known founder or recurrent mutations have been effective in reducing the incidence of autosomal recessive conditions in the Ashkenazi Jewish population and other populations. Yet, many known autosomal recessive, dominant, and X-linked conditions are not screened for and the mutational basis of other conditions for which screening might be offered is unknown. We sought to expand the knowledge base of Ashkenazi Jewish mutations and propose an expanded screening panel as a result. Methods: Samples for 128 disease-free Ashkenazi Jews had whole genome sequencing performed by Complete Genomics. The average raw sequencing depth was 56X. Variants identified were referenced with the Online Mendelian Inheritance in Man and ClinVar databases for known pathogenicity. Of 13,768,157 variants, 201 were called pathogenic in the databases. These variants were then manually curated through literature review and scored for pathogenicity. These results were combined with other previously reported mutations among Ashkenazi Jews, then prioritized based on clinical utility and expected population prevalence. Results: A panel of 168 significant mutations was established for 52 autosomal recessive, 34 autosomal dominant, and 5 X-linked disorders adding 69 conditions to the 22 that appear on at least one currently offered Ashkenazi Jewish panel. Diseases that inform risk to offspring are mostly autosomal recessive and range from very severe, such as glycine encephalopathy and Leigh syndrome, to milder, such as hyperoxaluria and ichthyosis. Some conditions that confer different phenotypes in the monoallelic and biallelic states offer information pertaining to both personal risk as well as risk to offspring. Screening for recessive disorders may also be diagnostic for conditions in adults that have atypical presentations or later onset. Most presymptomatic screening conditions confer future risk of cancer, such as BRCA1/2 and mismatch repair gene testing, but this group also includes diseases such as obesity and macular degeneration. Prevalent, benign biochemical traits, such as cystathianuria and pentosuria, were not included. Conclusions: With our increasing ability to identify prevalent disease variants in the Ashkenazi Jewish (and other) populations, we need to reframe our goals of population screening and genetic counseling. Screening for a broader range of disorders could not only further reduce the incidence of autosomal recessive disorders, but could also offer the benefits of early or presymptomatic diagnosis, including reduction of morbidity and mortality, satisfy a need for information and plan for potential future life events. The number of conditions identified for testing is feasible, more cost-effective than sequencing and individually selectable following genetic counseling. G51. The ACE Clinical Exome: An Augmented Exome Providing Accurate Structural Variant Detection A. Patwardhan, S. Chervitz, M. Li, J. Harris, G. Bartha, D. Newburger, M. Pratt, S. Garcia, J. Tirch, N. Leng, C. Haudenschild, S. Luo, D.M. Church, J. West, R. Chen Personalis, Inc., Menlo Park, CA. Introduction: Next-generation sequencing (NGS) technologies have revealed important discoveries in the genetic basis of disease and are increasingly being used to provide definitive diagnoses to patients affected by Mendelian disorders. Whole-exome sequencing (WES), which aims to comprehensively target the entire protein-coding region of the genome, has proven to be a valuable tool in identifying common and rare disease-causing variants, even in cases where traditional diagnostic tests have failed. Structural variations (SVs), a class of large (>50bp) variants known to be important for Mendelian disease, are not reliably detected using standard exome-based approaches. Methods: We describe an augmented Exome (ACE Exome) that combines an augmented exome assay and novel informatics approaches to enable more accurate genome wide SV detection. The ACE-Exome approach improves coverage over all biomedically interpretable genes compared to standard WES assays and augments coverage to detect large structural variants genome wide. Downstream algorithms utilize the augmented exome data to call disease associated SVs in combination with other smaller variants typically identified in an exome (SNVs and InDels). Results: Using a set of over 40 samples known to harbor pathogenic SVs (deletions/duplications), an internally developed "Gold-Set" of high-confident SV calls, and an internally developed simulation dataset, we demonstrate high sensitivity and specificity over a wide range of SV sizes (1KB to >1MB). We illustrate how improved accuracy in SV detection translates into improved diagnostic yield, by presenting recent cases in which pathogenic SVs were identified using these methods. Conclusions: This single NGS-based clinical test provides a level of SV detection accuracy and characterization necessary for improved diagnostic yield and clinical use, whereas still providing the efficiencies and enhanced coverage associated with exome-based tests.
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G52. Assessing the Impact of False Positive and False Negative Variant Calls on Clinical Genome Interpretation J.M. Zook1, R. Goldfeder2, J. Priest2, M. Grove2, M. Wheeler2, E. Ashley2, M. Salit1 1National Institute of Standards and Technology, Gaithersburg, MD; 2Stanford University, Stanford, CA. Introduction: As genome sequencing comes to impact clinical care, geneticists and pathologists have become appropriately focused on accuracy of next-generation sequencing platforms. The Genome in a Bottle Consortium has recently published a set of benchmark SNP, indel, and homozygous reference genotypes for the first candidate whole genome NIST Reference Material. These benchmark calls are currently being used to assess false positive and false negative rates of sequencing pipelines. In this work, we examine whether the false positive and false negative variant calls influence the clinical interpretation of the genome or primarily fall in regions of the genome for which no clinical interpretation is available. Methods: For our pilot whole genome NIST Reference Material, we have annotated all potential SNP and indel sites as truncating, splice site, or nonsynonymous mutations. To determine the evidence for uncertain potentially functional variants in ACMG, ClinVar, or OMIM genes, we have manually curated alignments from multiple short and long read sequencing technologies. Then, for a few common pipelines, we have identified false positive and false negative variants that are potentially clinically significant. Results: In our high confidence calls (~77% of the genome), we have identified 2,110 high confidence nonsynonymous, 48 truncating/frameshift, and 18 splicing mutations in OMIM/ClinVar genes. In addition, so far we have manually inspected short and long read alignments around 41 uncertain splicing variants, which include 23 likely variants, 8 likely non-variant, and 10 unclear sites. With a low calling threshold, both Illumina and complete genomics call all true truncating SNPs but miss one and four frameshifting indels, respectively. In addition, Complete Genomics called but the Illumina pipeline missed a true 71 bp splicing deletion. We have also identified 54 false positive nonsynonymous, truncating/frameshift, and splicing mutations in OMIM/ClinVar genes caused by systematic errors in Illumina sequencing before filtering, which should be suspect any genome sequenced with Illumina. Conclusions: We are performing the most comprehensive characterization of potentially clinically significant variants in a benchmark genome. We have identified potentially functional false positive and negative variants from various pipelines, as well as potentially clinically relevant sites that are difficult to assess with any current technologies due to ambiguous mapping. We are investigating additional sites, including manual inspection of long and short read alignments and manual clinical interpretation of variants. G53. Carrier Screening Using Whole Genome Sequencing: Basic Genetic Concepts Revisited Y.M. Akkari1, P. Jain1, M.O. Dorschner2, L. Amendola2, G.P. Jarvik2, D.A. Nickerson2, B. Wilfond2, K.A. Goddard3, C.S. Richards1 1Oregon Health & Science University, Portland, OR; 2University of Washington, Seattle, WA; 3Kaiser Permanente Northwest, Portland, OR. Introduction: As part of the NHGRI CSER Consortium, our laboratory collaborated on the classification and Sanger confirmation of variants detected through carrier screening of healthy women presenting for preconception genetic testing using whole genome sequencing (WGS). Recruitment is ongoing; to date we have clinically reported results from four consented individuals. We present these findings, and demonstrate the importance of considering basic genetic concepts when analyzing WGS data. Methods: WGS was performed in the Illumina CLIA-certified laboratory. Resulting sequences were aligned against the reference human genome (version hg19), and a modified version of the SeattleSeq tool was used to annotate variants found within a defined set of approximately 500 genes. We analyzed the resulting variants for pathogenicity using a laboratory-developed classification algorithm. For confirmation studies, genomic DNA was extracted and pathogenic variants were confirmed by custom designed Sanger sequencing. Results: Three individuals were found to be carriers for both novel and known pathogenic variants in diseases included in our extensive gene list, whereas no such variants were detected in the fourth individual. These diseases ranged from severe infantile neurodegenerative to mild nephrolithiasiscausing disorders. Nonsense, missense, and splice-site variants were detected. They were classified using an algorithm that includes the interrogation of well-reputed control and mutation databases and computational protein prediction programs. We considered basic genetic concepts such as linkage of certain variants with benign polymorphisms for pathogenicity, modifying factors for expression of disease, and the ethical evaluation of variant reporting based on the possibility of offering future prenatal diagnosis. We discuss the extensive evaluation of these data in relation to the potential disease burden on each individual and compare this approach to the current state of preconception carrier screening. Conclusions: WGS is a new frontier in genetic screening and diagnosis, particularly in the preconception arena. We address issues that arise when classifying potentially pathogenic variants in a population of healthy individuals. G54. Quality Controls for AmpliSeq Hotspot NGS Panel Tests Using Plasmids and Cell Lines M. Chan1, M. Wu1, C. Lee1, W. Huang1, A. Smirnov1, C.E. Carmack2 1Vela Diagnostics, Singapore; 2Vela Diagnostics, Newark, NJ. Introduction: Next-generation sequencing (NGS) somatic mutation tests are quickly gaining acceptance in clinical diagnostics in oncology. We have designed an automated NGS workflow, starting from FFPE sections, to identify actionable mutations based on the Ion Torrent AmpliSeq sequencing protocol. Currently, no standardized quality
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AMP Abstracts
control (QC) materials exist for NGS clinical tests. Positive and negative controls are critical for any regulated clinical diagnostic. We have used custom plasmids and cell-lines to QC the entire workflow. We used an external control to monitor the workflow operations, spike-in controls to QC DNA/FFPE samples, and a negative control to detect DNA contamination, in every run. This test is run on multiplex patient samples (7) using molecular barcodes with 1000X depth of coverage to achieve 5% sensitivity LOD (Limit of Detection) on the Ion Torrent PGM 318 sequencing chip. Methods: Synthetic plasmids carrying wildtype-TMV (tobacco mosaic virus) sequence, as well as TMV sequence with three artificially designed mutations were designed. Mutant and wildtype plasmids were mixed. This constitutes the System Control (SC) such that TMV mutations were present at 5% variant frequency. Cell-lines with naturally occurring or genetically engineered mutations were blended to carry ten known characterized COSMIC mutations present at specific variant frequencies (4-33%) and were made into FFPE blocks to mimic clinical samples (Horizon Diagnostics). Additionally, genomic DNAs from a set of four cell-lines containing a total of 27 mutations, were used as confirmatory reference materials. Results: FFPE and genomic DNA reference materials were used to validate the performance of the workflow. The minimum DNA input required for accurate variant calling was 1ng. Samples were sequenced in barcoded runs comprising DNA from an FFPE-section spiked with 100 fg of SC at the start of extraction, and DNA containing known amounts of SC (0.1, 0.25, 0.5, 1, 5, 10 fg). In No Template Controls (NTCs) containing SC alone, all three mutations in SC were recovered as expected, which otherwise produced no other signal, confirming the usefulness of this sample as both NTC and external (positive) control. An inverse relationship between the coverage of SC and DNA input was observed, suggesting that the signal from SC may be employable as a QC parameter for DNA input. Finally, the feasibility of the systems control was evaluated using patient FFPE specimens. Conclusions: Plasmids and cell lines can and should be used to quality control NGS clinical diagnostic tests. G55. Cell Line Genomic DNAs for Molecular Diagnosis of Cancer F. Tian ATCC, Manassas, VA. Introduction: Large-scale cancer genome programs have generated rich data of genetic abnormalities observed in thousands of clinical patient tumors, which provides a major opportunity for molecular diagnosis of cancer. However, the lack of control materials for molecular tests has been a challenge. Because of the reproducible nature of the cell lines, genomic DNAs of the fully characterized and authenticated cell lines provide a solution. Methods: genomic DNAs were extracted from over 70 common used human cancer cell lines derived from breast, lung, colon, pancreas as well as haematopoietic and lymphoid tissue. Cancer gene mutations were identified by nextgeneration sequencing. Gene copy number changes were analyzed by qBiomarker copy number PCR assay kit. Moreover, the selected cell lines were analyzed by qPCR, western blot and IF staining to verify gene expressions and protein expressions. Results: Here, we present a large list of over 70 genomic DNAs isolated from authenticated cancer cell lines that contain the desired biomarkers for oncology molecular diagnosis. In addition to driver mutations such as BRAF V600, KRAS G12, PI3K E545 and EGFR T790, the gene copy number amplifications of AKT, FGFR, MET, ERBB2 and deletion of PTEN are presented in the cell lines that have been used to extract the genomic DNAs. In addition, we show systematic molecular characterization and clustering of those human tumor cell lines, which represent the most common human cancer types found in the clinic, such as lung, breast, colon, pancreatic, skin cancer and so on. By next-generation sequencing, the genomic DNAs of those tumor cell lines were fully analyzed to capture the driver gene mutations and allelic frequency. Gene DNA copy number variations were determined as well. Moreover, the gene expressions, protein expressions and relevant cell signaling pathway activations of the cell lines have also been profiled. To be paired with mutations, a set of wild-type controls that were derived from normal tissues were characterized in parallel. Conclusions: Overall, the genomic DNAs from authenticated cell lines provide useful control materials to molecular diagnostic labs for genetic testing. G56. The Utility of Cytogenomic SNP Array Testing in the Analysis of Product of Conception Specimens K. Petras, J. Keller-Ramey, C.A. Fitzpatrick University of Chicago, Chicago, IL. Introduction: Cytogenomic microarray analysis (CMA) is recommended as a first tier test for detecting copy number abnormalities in postnatal specimens. The benefits of this methodology have been widely described and include higher resolution whole genome abnormality detection, more objective assay, and success without active dividing cells. CMA testing has also been more recently recommended for the detection of copy number variants in prenatal, fetal demise and stillbirth specimens with many of the same benefits. The utility of CMA for the evaluation of early pregnancy loss is less clear due to a lack of data. Methods: Our laboratory has validated the Affymetrix CytoScan HD Single-Nucleotide Polymorphism (SNP) CMA platform for use in POC specimens. Here we summarize one year of our clinical experience using this platform highlighting select cases that illustrate utility. Results: We have performed CMA testing on 20 POC specimens to date from early and late gestation pregnancy losses in conjunction with routine chromosome analysis. Sixteen of these cases were reported as normal by both chromosome analysis and CMA. Two cases were culture failures secondary to contamination of the specimen at the time of collection and lack of cell attachment and growth. Both cases were successfully analyzed by CMA and reported
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AMP Abstracts as normal. The remaining two cases were also culture failures with no apparent specimen contamination but lack of cell attachment and growth. CMA results for both cases were consistent with monosomy 21. This abnormality is very rarely observed by routine chromosome analysis and likely explains the lack of cell growth in these cultures. Consequently, monosomy 21 could represent a cause of pregnancy loss that has previously been underreported. Conclusions: Together, these two cases represent a significant advantage of performing CMA testing on POC specimens: the ability to interrogate the entire genome for chromosomal imbalance in specimens that otherwise fail to grow in culture. Since validating CMA in our laboratory we have been able to report results in all instances of culture failure, which remains approximately 5-15% of all POC specimens. G57. Next-Generation Molecular Diagnostic Testing of Cystic Fibrosis Using Complementary Long Padlock Probes J.I. Odegaard1, M.I. Lefterova1, P. Shen2, E. Fung2, G. Peng3, K.M. Broady4, W. Gu2, J. Zehnder4, R.W. Davis2, W. Wang3, I. Schrijver4, C. Scharfe2 1Stanford Hospital & Clinics, Palo Alto, CA; 2Stanford University, Palo Alto, CA; 3The University of Texas MD Anderson Cancer Center, Houston, TX; 4Stanford University School of Medicine, Palo Alto, CA. Introduction: Cystic fibrosis (CF) is among the most common genetic disorders with an estimated 30,000 children and adults affected in the United States. More than 2,000 sequence changes have been identified in the CF transmembrane conductance regulator gene (CFTR) with a high level of allelic heterogeneity within different CF populations including single nucleotide variants (SNVs), short insertions and deletions (indels) and genomic copy number variations (CNVs). Here we describe the development and clinical validation of a highly sensitive, specific, and rapid clinical diagnostic assay capable of detecting both single nucleotide and structural variants using multiplex targeted next-generation sequencing (NGS). Methods: Our approach utilizes a novel DNA sequence capture technology based on complementary long padlock probes (cLPPs) that achieves exceedingly high specificity among available target enrichment technologies. cLPPs are physically tethered primers capable of amplifying only targets that have both primer binding sites present at a defined distance apart on the same nucleic acid molecule. cLPP single-tube capture of all CFTR exons, flanking intronic and regulatory regions followed by library preparation can be performed within four hours. The barcoded capture libraries of 96 patient samples were pooled and directly sequenced on the Illumina MiSeq instrument (mean sample coverage >2,000x). Results: We evaluated the performance of our method in comparison to known CFTR genotypes generated using bidirectional Sanger sequencing for 96 samples. Using a combination of software tools and manual data curations we were able to correctly identify all known pathogenic mutations and sequence variations in the 96 samples. In total, this represents 433 sample positions, 122 of which are unique, in addition to the IVS8 poly-thymidine and poly-thymidineguanine polymorphic tracts. Additionally, our assay quantitatively recovered target regions, allowing for the detection of two known CNVs (an exon 2-3 deletion and an exon 6-10 duplication) after target normalization. Conclusions: Our assay enables the accurate sequencing of the CFTR gene in the clinical molecular laboratory, whereas simultaneously reducing turnaround time. G58. Identification of Mutations in Korean Patients with Amyotrophic Lateral Sclerosis Using Multi-Gene Panel Testing C. Ki1, H. Kim2, Y. Kim1, M. Kwon3, K. Oh4, W. Choi4, S. Oh4, S. Kim4 1Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 2Green Cross Laboratories, Yongin, Korea; 3Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea; 4Hanyang University College of Medicine, Seoul, Korea. Introduction: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease involving motor neurons and mutations in a number of genes have been identified in patients with ALS. Recently, multi-gene panel testing by targeted capture and high-throughput sequencing enables rapid screening of mutations in multiple genes with relatively low costs. Therefore, we tried to apply this novel method to identify mutations in Korean patients with ALS. Methods: Multi-gene panel testing including 18 ALS-related genes (SOD1, SETX, FUS, ANG, TARDBP, TAF15, VCP, UBQLN2, SQSTM1, SIGMAR1, ALS2, FIG4, VAPB, OPTN, DAO, MAPT, SPG11 and GRN) except C9orf72 hexanucleotide repeats was developed and applied to simultaneously identify mutations in 4 familial (fALS) and 148 sporadic (sALS) patients with ALS. Results: One SOD1 mutation (p.N87S) was found in a fALS patient and a total of 32 nonsynonymous variants of unknown significance (VUS) in 10 different genes were found in 33 patients. After further validation by Sanger sequencing and in silico analysis, more than 6 VUS in 4 different genes including ALS2, SETX, SPG11, and TAF15 were considered to be possibly pathogenic and some patients had more than two variants in different gens. Conclusions: Multi-gene panel testing could identify mutations and VUSs in 18 ALS-related genes, which seems better approach for molecular diagnosis of ALS than conventional gene-by-gene approach. It is of note that relatively low frequency of mutations in known ALS genes implies marked genetic heterogeneity in Korean patients with ALS.
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G59. A Practical Approach to Genotyping CYP2D6-CYP2D7 Hybrid Alleles Using TaqMan B. Ramey Hartung, J. Forcellini, M.W. Linder, K. Reynolds PGXL Laboratories, Louisville, KY. Introduction: The CYP2D6 region is highly polymorphic and its homology with the neighboring CYP2D7 pseudogene predisposes it to recombination events such as deletions, duplications, multiplications, and gene conversions. Most commercially available genotyping methods have been designed to detect the most common CYP2D6 alleles, although each has limited ability to detect hybrid alleles. Each method identifies the determinant SNPs for the most common star alleles as well as allele duplications and deletions, although by different techniques. These differences can lead to discordances and indeterminate results, particularly in samples with gene conversions. We have developed a method for identifying patients with common CYP2D6-CYP2D7 hybrid alleles that produce no enzymatic activity and are not readily identified by standard techniques. Methods: CYP2D6 genotypes were determined by 16 TaqMan SNP assays and one TaqMan copy number assay (Exon 9) for approximately 25000 buccal or blood samples submitted for routine CYP2D6 testing. DNA extraction was performed using Life Technologies MagMAX or Qiagen EZ-1 Tissue or Blood DNA reagents. When SNP data and copy number results conflicted, Intron 2 copy number was interrogated, as several CYP2D6-CYP2D7 hybrid conversions obliterate the Exon 9 target sequence but do not affect Intron 2. We used these copy number assays to identify a subset of samples with CYP2D6-CYP2D7 hybrid alleles. SNP and copy number data were used to estimate the results generated by a variety of common analytical platforms. Results: Of 25,000 samples tested for CYP2D6, 530 required additional testing for presence of the CYP2D6-CYP2D7 hybrid alleles. We identified CYP2D6*36, *63, *68, and *83 hybrids in 29 of these samples (5.5%). The Luminex and Roche Amplichip methodologies would, according to their user manuals, yield indeterminate results for 11 of these 29 samples (37.9%); the remaining 18 samples would yield genotype calls, but 66.7% would fail to detect the inactive hybrid allele would not be detected. The Autogenomics method would produce star allele calls in all cases; however, 23 of these (79.3%) would likely be misgenotyped and yield overestimates of CYP2D6 activity. Conclusions: CYP2D6 genotyping is difficult due to recombination events such as gene conversions that create CYP2D6CYP2D7 hybrid alleles. The prevalence of such hybrid alleles is low but the importance of detection is vital in obtaining the correct patient phenotype. Although our TaqMan methods enable identification of some hybrid alleles, other methods are in development for detection of additional hybrids such as CYP2D6-CYP2D7*4N, *57, and *10+*36 tandem. G60. Next-Generation Sequencing Technology Detects Deletions in the RB1 Gene, Ranging from a Single Base Pair to Whole Gene (~170 kb) with a Higher Sensitivity than FISH A. Mannan1, S. Aggarwal1, J. Pandian2, R. Gadkari1, R. Ramalingam1, S. Sankaran1, V. Veeramachaneni1, P. Ramamoorthy3, K. Subramanian1, R. Hariharan1 1Strand Center for Genomics and Personalized Medicine, Bangalore, Karnataka, India; 2Sankara Nethralaya, Chennai, Tamil Nadu, India; 3Strand Center for Genomics and Personalized Medicine, San Francisco, CA. Introduction: Next-generation sequencing (NGS) based tests provide clinical diagnostic labs with an attractive alternative to the traditionally employed sequential testing strategies. With the right combination of test design and analysis tools a single NGS test can identify genetic abnormalities arising from single nucleotide variations (SNVs) and structural variations (SVs) including small and large indels and copy number variations (CNVs). At Strand Center for Genomics and Personalized Medicine, we have run hundreds of diagnostic tests ranging from whole exome to disease specific panel based NGS tests. We illustrate the clinical utility of combining efficient panel based tests with the right bioinformatics analysis tools in providing quick and economic diagnosis in a representative set of retinoblastoma cases. Methods: The samples were tested using either Illumina’s TruSight Cancer panel (consisting 94 genes) or our custom designed Eye Disorders panel. In both panels, the RB1 gene is included. Paired end sequencing was done to an average coverage of >100X and the analyses were done using AVADIS NGS software. Results: We were able to provide a positive diagnosis in all retinoblastoma cases with the causative variants ranging from SNVs to small deletion to whole gene deletion. Performing CNV analysis using normalized read depths and z-scores from all samples previously run on the panel, we are able to pick up statistically significant changes causing heterozygous deletion of the whole RB1 gene as well as a small deletion of the gene. This NGS based method was more sensitive than FISH in identifying deletions. As in one case, previous analysis by FISH (fluorescence in situ hybridization) had identified a 13q14 deletion, however NGS test revealed a heterozygous splice site variation. Further analysis revealed that the genetic markers (risk haplotype) spanning the RB1 gene were intact and co-segregating with the disease. Conclusions: Our study suggests that NGS based panel testing is quick, efficient and highly sensitive method to detect deletion ranging from a single base-pair to over 100 kb. We propose that NGS based testing should be used for detection of both SNVs and CNVs in the RB1 gene. To expand the utility of NGS tests in potentially replacing FISH testing additional validation studies are warranted.
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AMP Abstracts
G61. Premutation Carrier Frequency of Fragile X Syndrome in Korean Women Undergoing Prenatal Population-Based Screening J. Cho, G. Oh Eone Laboratories, Incheon, Korea. Introduction: Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability, affecting approximately 1 in 4,000 males and 1 in 8,000 females. The disease is caused by the expansion of a CGG repeat in the FMR1 5’-untranslated region. Fragile X carrier testing may be important in that premutation alleles range in size from 55-200 CGG repeat resulting in an expansion to full mutation when passed from mother to child. In this study, we estimate the prevalence of permutation carriers in Korean women undergoing prenatal population-based screening. Methods: The blood samples were obtained from 14,183 women undergoing prenatal population-based screening. Carrier screening was performed using commercialized CGG repeat primed PCR (Asuragen Inc., Austin, TX). Results: Among the 14,183 who underwent fragile X carrier testing, we identified 15 premutation carriers (1 in 946, 0.11%) and 117 intermediate allele carriers (1 in 121, 0.82%). None of the women was found to carry the fully mutated allele. The normal alleles were found in 14,051 of 14,183 (99.07%) women. The CGG repeat in all samples was ranged from 9 to 108, and the CGG repeat in normal alleles represents bimodal distribution with most common alleles having 29 (62.91%) and 30 (21.60%) CGG repeats. Conclusions: The permutation carrier frequency of FXS in Korean women is 1:946, which is lower than in western populations, but relatively higher than in other Asian populations. This study provides the permutation carrier frequency of FXS in a large Korean samples and offers foundation for further investigations of FXS.
medication and performed drug interaction searches using Micromedex database. Final genotypes were correlated to medications and pharmacist comments regarding dose adjustment or alternative considerations were included. Medications with unknown clearance mechanisms were not assessed further. Results: The pilot included 180 patients and a total of 2325 prescriptions, of which 856 had a possible genetic interaction. The average patient was prescribed 12 medications, with 5 having a genetic link. 353 (41%) medications had a genotype-drug conflict: 42 (12%) showed severe gene-drug contraindications indicating discontinuation per guidelines published on pharmgkb.org and/or drug monographs; 50 (14%) were major gene-drug interactions indicating ≥50% dose adjustment; 230 (65%) were moderate indicating 20% to 50% dose adjustment; and 31 (9%) were minor indicating no adjustments. 27/85 (32%) opioids were prescribed to patients with CYP2D6 gene variants, predicting inefficacy risk. 47/66 (71%) SSRIs were prescribed to patients with gene variants in SLC6A4, correlating with inefficacy, or in CYP2D6/CYP2C19, correlating with alternative dosages. Another 27 (41%) patients taking SSRIs had gene conflicts in both SLC6A4 and the relevant CYP, requiring alternative medications. Micromedex identified 762 drug-drug interactions: 1% contraindicated; 44% major, 55% moderate. Conclusions: It is feasible to incorporate patient medications into pharmacogenetic test reports and to provide pharmacist- and publication-based alternative considerations. Databases are now under development to launch a larger clinical program. Although genotyping contributes significantly to risk and sets the medication baseline for a given patient, inclusion of both gene-drug and drug-drug interactions are critical to guide better therapeutic decisions as part of the overall patient management strategy.
G62. Phenotypic Filtering Strategies and Importance of Clinical Information for Successful WES Analysis P. Vitazka, J. Scuffins, D. Copenheaver, J. Neidich, K. Retterer, J. Juusola, S. Bale, G. Richard, N. Smaoui, E. Haverfield GeneDx, Gaithersburg, MD. Introduction: Whole Exome Sequencing (WES) has recently gained wide spread popularity among clinicians as an effective diagnostic tool for identification of molecular defects responsible for Mendelian genetic disorders. WES, on one hand, allows comprehensive analysis of translated portion of the genome, and on the other hand, it yields thousands of variants that need to be analyzed and interpreted. Different bioinformatics analysis and filtering strategies have been developed to decrease the large number of variants to a more manageable number that can be then manually curated and correlated to patient’s clinical presentation. Most commonly used strategies include population based filtering, cross-species conservation based filtering, filtering using in silico prediction algorithms, or filtering by inheritance patterns. Here we described use of phenotype based filtering strategies facilitated by data from the Human Phenotype Ontology (HPO) and Human Gene Mutation (HGMD) databases. Methods: We have completed over 2000 exome cases out of which 31.3% (n=648) were reported as positive with identified definitive disease causing mutation. Clinical information received for each patient was abstracted using standard HPO or HGMD terms which were entered into our exome analysis tool – Xome Analyzer. Each HPO or HGMD term is linked to a list of genes assembled based on information from Online Mendelian Inheritance in Man (OMIM) or other sources. Results: From all available terms, 43.3% (3246/7678) of HPO terms and 17.4% (1462/8432) of HGMD terms were used during clinical abstracting. The most commonly used HPO terms were: developmental delay, generalized hypotonia, speech delay, seizures, and facial dysmorphism. Most commonly used HGMD terms included: mitochondrial disease, developmental delay, microcephaly, Ehlers-Danlos syndrome, and autism. Most commonly reported genes linked to either an HPO or HGMD term include: ARID1B, SCN1A, FBN1, FOXG1, and STXBP1. Conclusions: Including all reported positive cases, 68% (440/648) of mutations could be identified using either HPO or HGMD filtering. Combination of bioinformatics filtering strategies and filtering using patient’s clinical data is the optimal strategy for analysis of the thousands of variants generated by exome sequencing. Good clinical information remains, however, paramount for successful exome analysis.
HEMATOPATHOLOGY
G63. Correlation of Pharmacogenetic Results with Patient Medication Regimens: A Pilot Study for Reporting Gene-Drug and Drug-Drug Interactions with Pharmacy Support K.K. Reynolds1,2, F. Weitendorf1,2, M.W. Linder1 1PGXL Laboratories, Louisville, KY; 2University of Louisville School of Medicine, Louisville, KY. Introduction: As clinical pharmacogenetic (PGx) testing gains more acceptance for optimal medication management, standardization of results into clear, patient-focused reports are needed. Adding the patient’s active medication list into the genotype report can help identify gene-drug conflicts in the current regimen and any significant drugdrug interactions. This requires the support of PGx-trained pharmacists, and represents a unique collaborative opportunity. Logistics are complicated by many factors: polypharmacy, discontinued drugs, multigene panels, and informatics necessary to build clear and actionable reports. This pilot study assessed the feasibility of reconciling patient medications with genotyping results and developing an enhanced interpretive report. Methods: Fourteen physicians participated by providing current medication lists for each patient. CYP450 genotyping was performed using TaqMan real-time PCR. SLC6A4 genotyping was performed using PCR-RLFP. Clinical scientists and a clinical pharmacist identified metabolic pathways and receptor targets for each
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H01. Sensitive and Rapid Detection of CALR Somatic Mutations in Myeloproliferative Neoplasms (MPN) Using PCR and Fragment Analysis R. He, M. Mai, A.J. Wood, S.A. Lassila, T.G. Lokken, D.S. Viswanatha Mayo Clinic, Rochester, MN. Introduction: Somatic mutations (insertions and/or deletions) in exon 9 of the CALR gene were recently discovered to be the second most frequent somatic mutation in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients, and are mutually exclusive of JAK2 and MPL mutations. CALR mutations have a reported frequency of approximately 50% to 90% in JAK2 and MPL-wild-type ET and PMF, and is not found in polycythemia vera (PV). Therefore, a CALR mutation serves as a critical diagnostic molecular marker in ET and PMF. In PMF, a CALR mutation carries a favorable survival impact and a triple-negative mutation status (JAK2, MPL and CALRnegative) was recently identified as a high-risk molecular signature. CALR impact on survival in ET is unclear; however, it is associated with lower risk of thrombosis. We sought to develop an efficient and sensitive clinical assay for CALR mutation for diagnosis and prognostication of MPN. Methods: As the somatic CALR mutations involved in MPN are insertion and/or deletions inexon 9, we developed a rapid and sensitive assay using PCR spanning exon 9, followed by capillary fragment analysis (ABI 3130 xl, Applied Biosystems, Foster City, CA). Analytical and clinical performance parameters were established and validated on peripheral blood (PB) and bone marrow (BM) samples. Results: 43 samples (20 positive and 23 negative, 21 PB and 22 BM) were analyzed by both ABI fragment analysis and Sanger sequencing with 100% concordant results. Inter-run and intra-run precision was 100%. The analytical sensitivity was 5% to 6% for most mutation types except for a 20% sensitivity for the rare del1bp type. No assay interference was identified in EDTA, heparin or ACD anticoagulated samples. Of 31 BCR-ABL-negative MPN patients tested, 5 were positive for JAK2V617F. In the 26 JAK2V617F-negative patients, 20 were positive for CALR mutation. None were diagnosed with PV, excluding the necessity for JAK2 exon 12 evaluation. MPL exon 10 mutation status was not assessed, however it would not have significant impact on the clinical sensitivity due to its low frequency in ET and PMF. Therefore the clinical sensitivity was approximately 77%. All 20 normal donors and 5 JAK2V617F–positive MPN patients were negative for CALR mutation. The clinical specificity was 100%. Conclusions: We have successfully developed and validated a clinical assay for efficient and sensitive detection of CALR somatic mutations. H02. High Prevalence of Somatic MAP2K1 Mutations in Langerhans Cell Histiocytosis N.A. Brown1, L.V. Furtado2, B.L. Betz1, M.J. Kield1, H.C. Weigelin1, M.S. Lim1, K.S. Elenitoba-Johnson1 1University of Michigan, Ann Arbor, MI; 2University of Chicago, Chicago, IL. Introduction: Langerhans cell histiocytosis (LCH) is a clonal proliferation of specialized cells with characteristics resembling antigen presenting cells that reside in the skin and mucosa. BRAF V600E mutations are present in approximately half of LCH cases. The additional observation that ERK1 is activated in BRAF wild-type cases suggests an alternative mechanism for MAPK activation. Methods: To discover other genetic mechanisms that might explain ERK1 activation, we screened 8 LCH cases using both BRAF V600E allele-specific PCR and the Ion AmpliSeq Comprehensive Cancer Panel. After identifying a MAP2K1 mutation using the screening approach, an additional 32 cases of LCH were evaluated using bidirectional Sanger sequencing of MAP2K1 exons 2 and 3 and BRAF V600E allele-specific PCR. Results: Initial screening of 8 LCH cases using targeted next-generation sequencing demonstrated an E102_I103del mutation in MAP2K1 in a BRAF wild-type case of LCH. Analysis of 32 additional LCH
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AMP Abstracts cases using BRAF V600E allele-specific PCR and Sanger sequencing of MAP2K1 exons 2 and 3 revealed mutually exclusive BRAF and MAP2K1 mutations in a total of 18/40 (45.0%) and 11/40 (27.5%) cases, respectively. All MAP2K1 mutations were somatic based on sequencing of matched constitutional DNA. MAP2K1 mutations identified in this study involved the negative regulatory region encoded by exon 2 and the catalytic core encoded by exon 3. In contrast with the predominance of missense mutations observed in other neoplasms, the majority of MAP2K1 mutations in LCH were in-frame deletions. Six deletions involved exon 3 including residues E102 and I103. Another two deletions occurred in exon 2 affecting the helix A regulatory region. Five missense mutations were also identified. Of note, two cases demonstrated two separate missense mutations at similar allele frequencies – one case with C121S and G128V and one case with R49C and A106T. Conclusions: This is the first report of somatic MAP2K1 mutations in LCH which occur in 50% of BRAF wild-type cases. The mutually exclusive nature of MAP2K1 and BRAF mutations implicates a critical role of oncogenic MAPK signaling in the pathogenesis of LCH. This finding may also have implications for the use of BRAF and MEK inhibitor therapy. H03. Standardization of BCR-ABL1 Quantitation: Successes and Challenges, the Stanford Experience M.D. Ewalt, L. Gojenola, J.L. Zehnder, I. Schrijver Stanford University School of Medicine, Stanford, CA. Introduction: The BCR-ABL1 translocation is the driving genetic alteration underlying chronic myelogeneous leukemia (CML), and targeting of this aberration with tyrosine kinase inhibitors, such as imatinib mesylate has led to dramatic outcome improvements. Since the IRIS trial, quantitation of BCR-ABL1 transcript levels has become an integral part of monitoring response to therapy with various thresholds defining response and resistance to therapy. A major challenge with this testing is the variability in methodology and reporting from different laboratories across the world, which led to the development of an International Scale (IS) for the reporting of BCR-ABL1 transcript levels. Methods: RNA from ten peripheral blood specimens from patients with CML harboring BCR-ABL1 p210 b2a2 (e13a2) or b3a2 (e14a2) was prepared. BCR-ABL1 transcripts were quantified in-house and on RNA sent to another reference laboratory. Real-time Quantitative PCR (RQ-PCR) was performed at Stanford using the Qiagen ipsogen BCR-ABL1 Mbcr IS-MMR kit on a Roche LC480 real-time PCR instrument according to the manufacturer’s instructions and using ABL as a reference gene. The reference laboratory used an RQ-PCR assay on the ABI 7000 using GUS as a reference gene. The values were compared using the IS and discrepant results were repeated at both laboratories. Statistics were performed using Stata/IC version 11.2 for Mac. Results: Of the 10 samples, 7 showed good correlation in BCR-ABL1 transcript quantitation following conversion to the IS. Three of these 7 specimens were below the reportable range of the respective assay; however, maintained good correlation. Overall, the results showed strong inter-laboratory correlation (Spearman's rank correlation coefficient = 0.9515, p < 0.0001); however, the results were statistically different (Wilcoxon signed-rank test, z = 0.0218). In the 3 specimens that showed significant inter-laboratory variability, RQ-PCR was repeated in both sites and each obtained results similar to their original findings. Conclusions: Whereas the International Scale allows reproducible quantitation across laboratories using different methodologies in the majority of cases, our analysis suggests that inter-laboratory variability persists. Repeated testing shows good intra-laboratory reproducibility. These results indicate that caution should be used when comparing serial results from laboratories that use different reference genes. To this end, assessment of additional samples with a second reference laboratory that uses ABL as a reference gene is ongoing. H04. Successful Red Blood Cell Genotyping from Leukoreduced Packed Red Blood Cell Tubing Segments E. Azzato, C. Gentile, H. Carpenter, D. Siegel, C. Watt Hospital of the University of Pennsylvania, Philadelphia, PA. Introduction: Patients with clinically significant alloantibodies to red cell antigens require packed red blood cell (pRBC) units lacking the corresponding antigen(s) to prevent hemolysis. Determining RBC phenotype is routinely performed on heat-sealed segments made from tubing attached to the pRBC unit (~300 μl to 600 μl of anticoagulant-diluted packed cells per segment). Depending on antigen incidence, immunophenotyping of random pRBC units to find those compatible for transfusion is time consuming and expensive; having a subset of pRBC units with known genotypes can improve efficiency and cost. Most molecular immunohematology methods leverage the fact that many blood group antigens result from single nucleotide polymorphisms; for example, the JKA and JKB alleles encode for the common clinically-significant Kidd blood group antigens, Jka and Jkb, respectively, with JKA and JKB differing at nucleotide 838 (G vs. A) in SLC14A1. Genotyping methods have been developed primarily for large blood collection centers, where separate tubes of whole blood can be drawn from donors to meet their DNA input requirements (≥15 ng/μL). Implementation of RBC genotyping in a hospital blood bank would require optimization for the available specimen, i.e., segments from leukoreduced (LR) pRBCs that contain only limited numbers of WBCs (1.10%) in 25 of the 41 samples (60.9%). Conclusions: Urine is an easy, self-obtainable sample that could become a useful diagnostic tool for detecting HPV in high-risk asymptomatic males who are partners of women with HR-HPV related lesions. This may be beneficial in preventing re-infection in females by helping eradicate the HPV infection and regressing cervical disease. Using assays that combine ISH and flow cytometry seem more reliable than PCR-based methods for analyzing HPV in urine since the presence of proteins and nitrates in urine influence the amplification process. ID27. Performance Comparison of the Lyra Multiplex HSV1/2 + VZV PCR with a Laboratory Developed Single-Plex HSV-1 and HSV-2 PCR from Retrospectively Tested Samples J. Mancini, C. Svetcharnik, K. Rocklin, D. Payne American Pathology Partners-Unipath, Denver, CO. Introduction: Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) infections can cause lesions at a variety of sites, i.e., genital, cutaneous, etc. VZV infection results in chickenpox and shingles, and can be misdiagnosed as HSV-1 or 2. A 2003 study demonstrated that 3% of genital lesions may have detectable VZV DNA. In this study, the Lyra HSV1/2 +VZV PCR assay was used to retrospectively determine the prevalence of VZV in genital samples. In addition, the performance characteristics of the multiplex Quidel assay was compared with the performance characteristics of two single-plex laboratory developed tests (LDT) for HSV-1 and HSV-2 and a consensus result. Methods: Over 400 samples, that were either positive or negative for HSV-1 and HSV-2 using an LDT assay, were tested with the Lyra assay. Discordant results were evaluated using a third method. Statistical analysis for agreement used Cohen’s Kappa analysis. Results: For HSV-1, 329 samples were negative, and 59 were positive by both tests. Eight discordant samples were identified as being positive by the LDT test. Discordant analysis confirmed 7 positive LDT results. For HSV-1, the agreement between the Lyra method and the consensus (LDT) data are: kappa= 0.934 (0.925), SE of kappa = 0.025 (0.026) and 95% confidence interval: From 0.886 to 0.982 (0.873 to 0.976). Observed HSV-1 agreements were 389 to 388 (98.23% to 97.98% of the observations) versus the number of agreements expected by chance which were 289 to 290.0 (73.06% to 73.22%). For HSV-2, 318 samples were negative, and 70 samples were positive by both tests. Eight discordant samples were identified (i.e., 3 were positive by Lyra assay and 5 were positive by the LDT assay). All discordant HSV-2 samples were positive by a third method. The HSV-2 data analyses for the consensus (LDT) are: kappa= 0.959 (0.934), SE of kappa = 0.018 (0.023) and 95% confidence interval: From 0.924 to 0.995 (0.888 to 0.979). Observed HSV-2 agreements were 391 to 388 (98.74% to 97.98% of the observations) versus the number of agreements expected by chance which were 273.8 to 275.7 (69.13% to 69.61%). Three samples were positive by VZV and confirmed by a third method as being positive. Conclusions: The multiplex Lyra HSV1/2 +VZV PCR assay has very good agreement with the consensus data and the singlex-plex LDTs. The VZV frequency was approximately 1% in this population. VZV should be considered as a potential cause for genital lesions. ID28. Detection of Herpes Simplex Virus 1 and 2 from Lesions with a Rapid Loop Mediated Isothermal Amplification (LAMP) Method R.V. Ponaka, C. Curioso, V. Elagin, V. Slepnev Meridian Bioscience, Inc., Cincinnati, OH. Introduction: Genital and non-genital Herpes is caused by the Herpes Simplex viruses type 1 (HSV-1) or type 2 (HSV-2), which belong to the Herpesviridae family. Although these infections are very common, the majority of them remain underdiagnosed because they are asymptomatic or unrecognized. A clinical diagnosis of herpes should be confirmed by laboratory testing through the use of direct tests for viral isolation or detection of HSV DNA using molecular techniques. Loop mediated isothermal amplification (LAMP) is a rapid nucleic acid amplification method which can be used to amplify and differentiate HSV type 1 and type 2 DNA under isothermal conditions without the need for thermal cycling. Methods: The illumigene HSV assay (Research Use Only; Not FDA cleared) is an isothermal LAMP amplification method uses Bst DNA Polymerase for both strand-displacement and target DNA amplification and differentiation of HSV. Following DNA amplification, magnesium-pyrophosphate is produced (by-product of LAMP) resulting in a turbid solution. The absorbance characteristics are measured to determine the reaction performance. Quantified stocks in Viral transport medium (VTM) or the clinical samples previously collected in VTM and tested by either culture (ELVIS) or real-time PCR, were mixed with illumigene HSV reaction buffer containing internal control and heat treated. The heat treated sample mixture added to the illumigene HSV-1 and HSV-2 devices containing the lyophilized HSV test and control reagents before loading on to the illumipro-10 instrument. Results: The analytical Limit of Detection (LoD) of HSV-1 strainsin different viral transport media was determined to be 100 TCID50/mL, to 300 TCID50/mL (0.3
The Journal of Molecular Diagnostics ■ jmd.amjpathol.org
AMP Abstracts
TCID50 to 0.9 TCID50/reaction), HSV-2 strains ranged from 100 TCID50/mL to 3000 TCID50/ mL (0.3 TCID50 to 9.0) TCID50/reaction) depending on the strain and medium used. Preliminary data of 282 retrospective swab specimens collected from various anatomical regions of the body in VTM including 62 HSV-1 and 70 HSV-2 positives samples prior to discordant resolution revealed the sensitivity and specificity of 100% for HSV-1; 95.7% and 99% respectively for HSV-2. Conclusions: The LAMP based illumigene HSV assayis capable of detecting and differentiating the HSV samples without the need for thermal cycling in less than one hour. No cross-reactivity was observed with the 47 bacteria, viruses, or Human genomic DNA tested. ID29. Ambient Temperature Shipping and Storage for Molecular Infectious Disease Testing: ViveST A.M. McClernon1, G.A. Cloherty2, D.R. McClernon1 1bioMONTR Labs, Research Triangle Park, NC; 2Abbott Molecular, Des Plaines, IL. Introduction: A key component of molecular infectious disease testing centers around the preservation of biological samples during shipping and storage. Samples collected at one site are shipped to a central laboratory for testing. Time elapses between sample collection and analysis; therefore, it is imperative to have a shipping and storage process that is efficient and preserves sample integrity over time. The current standards require dry ice, special packaging and ultra-cold storage, all of which is expensive and laborious. This study evaluated the stability of HIV-1 plasma samples shipped and stored on ViveST, a transformational dried ambient temperature storage and transportation device (ViveBio, Alpharetta, GA, US). Methods: For each study listed below, 1.15mL HIV-1 plasma was loaded on ViveST, dried overnight (RT) and capped. All samples were recovered using 1.15mL of molecular grade water and analyzed with Abbott’s RealTime HIV-1 assay (0.6 mL protocol, Abbott Laboratories, Illinois, US). For each study, identical aliquots were prepared, stored frozen and analyzed simultaneously for comparative purposes. To evaluate ViveST for shipping, 3 sets of HIV-1 plasma samples (4 levels, 5 replicates each) were prepared and shipped via FedEx to 3 distinct geographic locations across the continental United States. The ViveST were returned to bioMONTR Labs and analyzed. To evaluate stability of plasma stored on ViveST, 21 sets of plasma samples were prepared. Each set contained twenty 1.15mL aliquots of HIV-1 plasma (4 levels, 5 replicates each) and 1 negative control. Seven sets were stored at each of three different conditions (RT, 4°C and 40°C/75% RH). One set was removed from each storage condition at Days 1, 3, 7, 10, 14, 21 and 62 and analyzed. Results: For HIV-1 plasma shipped on ViveST, the average reduction recorded across all levels/all sites was 1.07 log c/mL (SD=0.10) with linear fit (R2>0.99) retained across all levels/all sites. For HIV-1 plasma stored on ViveST for a 62-day period, maximum reduction recorded when compared to frozen plasma was 0.91 log c/mL (ambient temperature), 0.84 log c/mL (4°C) and 1.69 log c/mL (40°C/75%RH). A linear fit (R2>0.98) was retained over the course of the 62-day study across all test points/all storage conditions. Conclusions: These studies confirm that ViveST Sample Storage and Transport Device has unmatched stability, reproducibility and accuracy for viral load testing. ViveST provides significant cost savings as compared to dry ice shipments and can enhance access to healthcare globally whereas significantly reducing the cost burden associated with frozen samples. ID30. Comparative Study of ElitechGroup’s MRSA/SA ELITe MGB and BioRad’s MRSA Select Media P. Ruiz1,2, J. McCue1,2, F. Amole1, F. Salman1, L. Diaz2, S. Medina2, E. Donhert2, M. Centeno2 1University of Miami Health System, Miami, FL; 2Auxilio Mutuo Hospital, San Juan, Puerto Rico. Introduction: Staphylococcus aureus is a major cause of infection among solid-organ transplant recipients. MRSA accounts for more than 25% of transplant bacteremias caused by S. aureus in many North American and European countries. Among central venous catheter-associated bloodstream infections caused by S. aureus in United States intensive care units, more than 50% are caused by MRSA. A sensitive and specific method for MRSA detection has significant value in a strategy for the eradication of MRSA. Methods: Four hundred and two (402) inpatient (non-transplant) nasal swabs were collected by Auxilio Mutuo and cultured on selective media (BioRad MRSA Select, ref. 63747) and incubated a minimum of 24 hours. The swabs were transported to the University of Miami and inoculated into Trypticase Soy Broth and then analyzed using the MRSA/SA ELITe MGB test according to package insert specifications. Discordant samples were retested with MIC/Susceptibility when possible. Results: Three hundred sixty-four (364) specimens were negative by both culture and the MRSA/SA ELITe MGB test for a specificity of 100% and twelve (12) specimens were positive by both. Twenty-six (26) specimens were culture negative and positive with the MRSA ELITe MGB test. Based on these results, culture performance against the MRSA/SA ELITe was 31.6% sensitive. The positive predictive value was 100% and the negative predictive value was 93.3%. Two PCR discordant samples were initially identified as MRSA culture positive but identified as S. aureus by the MRSA/SA ELITe MGB test. After subsequent analysis, it was determined that both were correctly identified by the MRSA/SA ELITe MGB test. Conclusions: The MRSA/SA ELITe MGB test appears to be highly sensitive when compared with the BioRad MRSA Select media with a significantly better turnaround time.
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AMP Abstracts ID31. Survival of Bacterial DNA in Human Blood: Relevance to Direct Molecular Detection of Bacteremia K.H. Rand, E.H. Boslet, H.J. Houck University of Florida, Gainesville, FL. Introduction: Detection of bacteria directly from patient blood within hours is possible by current molecular technology, but faces significant technical barriers. Commercial methods (e.g., SeptiFast, Roche; SepsiTest, Molzym) often detect bacterial DNA when blood cultures are negative, but conversely have lower sensitivity ranging from 60% to 90% (Liesenfeld, 2014). In adults, 30 to 40 ml blood is usually obtained for blood cultures, whereas molecular methods use only 1.5 to 2 ml/patient. Testing a larger volume should improve molecular methods, but extracting even 5 to10 ml whole blood is a daunting prospect. If significant amounts of free bacterial DNA circulate, then extraction of the larger volume of blood may be important, whereas if living or dead bacteria constitute the majority of the molecular target, then larger volumes could be concentrated by centrifugation. Methods: We spiked EDTA anticoagulated blood with P. aeruginosa DNA to give ≈300 copies/ml, and held the samples at 23°C for 48h to determine “survival” of free bacterial DNA. A 16 S DNA Taqman PCR for P. aeruginosa was used to measure “survival” of PCR amplifiable DNA. For patient samples, we developed 16S based broad range primers and used Taqman probes with exact sequence matches for Enterobacteriaceae, P. aeruginosa and Staphylococcus sp. EDTA blood tubes were obtained from the clinical laboratory after testing for blood counts. We chose specimens drawn as close as possible to the time blood was obtained for blood cultures that had become positive (2 gram negative rods, 8 gram positive cocci). After centrifugation at 10,000g for 10 min to pellet intact bacteria and cells, we extracted 0.8 ml plasma with the MagnaPure compact in 100 μl and then further concentrated the extracts by isopropanol precipitation and resuspension in 15 μl of which 3 μl was assayed. Results: Using 5 replicates per time point, at time 0, Cts averaged 32.9 ± .36, at 24 h 33.4 ± .33 and at 48 h 34.1 ± .36. Two more experiments gave similar results. All 10 patient plasma specimens were negative after 40 cycles. The limit of detection of the patient plasma assay was ~5 copies/ml. Conclusions: Bacterial DNA survives sufficiently well in whole blood to potentially contribute to molecular detection. Further study is needed before one can conclude that free bacterial DNA in blood is not useful diagnostically, but we could not detect any in a limited number of samples with limited plasma volumes. ID32. Comparison of Whole Blood and Plasma for CMV Viral Load Measurement Using a Commercial, Automated PCR System S. Suganda, J. Carr, L. Tang, S. Pounds, R.T. Hayden St. Jude Children's Research Hospital, Memphis, TN. Introduction: Quantitative PCR testing for Cytomegalovirus (CMV) viral load is standard of care for hematopoietic stem cell transplant (HSCT) recipients. However, a lack of commercially available tests has contributed to widely variable assay results. An FDA cleared quantitative real-time assay has recently become available for testing plasma samples; however, its performance using whole blood samples is not well established. Methods: One hundred eighty three fresh whole blood (EDTA) samples were assayed for CMV viral load using a laboratory developed quantitative PCR assay. Remaining samples were separated into aliquots of whole blood and plasma, frozen at 80°C and subsequently thawed and tested using COBAS AmpliPrep/COBAS TaqMan CMV system (Roche Diagnostics, Indianapolis, IN). Whole blood samples were split into two aliquots, each undergoing dilution with a different pre-extraction reagent. Samples were diluted 1:11 with SPEX (Roche Diagnostics, Indianapolis, IN) or 1:5 with DNAgard Blood (Biomatrica, San Diego, CA) prior to extraction and processing. Results were compared both qualitatively and quantitatively, the latter using McNemar’s test. Results: A higher number of positive results was obtained using plasma (43), compared to whole blood in SPEX (34) or whole blood DNAgard (33). All discrepant samples showed viral loads of less than 3.63 Log IU/mL in the positive test. Quantitative comparison showed a fairly strong correlation between the results of the SPEX treated whole blood or DNAgard treated whole blood (p-value=0.01, r2=0.87). Results of plasma did not correlate as well with the results in whole blood treated with either SPEX or DNAgard (r2= 0.47 and 0.36, respectively). Plasma tended yield a lower viral load than whole blood samples. Conclusions: Quantitative PCR using a commercialized, automated system requires a pre-dilution step when testing whole blood samples, resulting in diminished sensitivity at the low end of the detection range compared to plasma samples. Viral loads from whole blood pre-treated with either of two diluents were highly correlated with each other, but showed weaker correlations with plasma and tended to produce higher viral loads. Quantitative testing using a commercialized automated system may be used for either plasma or whole blood samples; the clinical correlation of the results obtained with these different blood compartments requires further study. ID33. Performance of the artus QS-RGQ MDx Kits on the QIAsymphony RGQ MDx System for the Detection of Clostridium difficile, Group B Streptococci, Vancomycin-Resistant Enterococci and Herpes Simplex Viruses Compared to Culture Methods P.A. Patel, A. Grayes, D. Schora, L.R. Peterson NorthShore University HealthSystem, Evanston, IL. Introduction: Molecular detection of pathogens has provided rapid diagnosis that is helpful for patient treatment and infection prevention. The artus QS-RGQ MDx Kits (artus assay) are in vitro polymerase chain reaction tests used on the QIAsymphony RGQ MDx System (QIAsymphony) for the direct detection of Clostridium difficile (Cdiff)
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toxin A and B genes, Group B Streptococci (GBS)-specific neuA gene, vancomycinresistance vanA/vanB genes (VRE) and Herpese simplex virus (HSV) from clinical specimens. We tested these artus assays on the QIAsymphony system. Methods: Excess, de-identified specimens collected for routine testing from patients suspected of Cdiff/GBS/ VRE or HSV associated disease were enrolled for this study. Samples and reagents were loaded into the QIAsymphony following a study specific protocol. The automated sample preparation (extraction/purification) and assay setup steps are performed in the combined QIAsymphony SP/AS instrument. Amplification and detection are performed in the Rotor-Gene Q MDx analyzer. For Cdiff, enriched toxigenic culture was performed by inoculating stool into chopped meat carbohydrate broth, and toxicity was confirmed by Bartels Cytotoxicity assay following manufacturer’s directions. For GBS culture, a vaginal/rectal sample was inoculated into Lim or Todd Hewitt broth and incubated for 24 hours. The broth was subcultured to blood and Columbia Naladixic Acid agars. Suspect colonies were confirmed by Gram stain, catalase and Lancefield serological typing. For VRE, a rectal or perianal swab sample was inoculated to BBL Enterococcosel agar and broth. Presumptive colonies were identified by Gram stain, catalase and PYR. Confirmed enterococci were identified to the species level using the Vitek 2 system (bioMérieux) and vancomycin MIC was determined by E-test for resistant strains. Viral cultures for detecting HSV were performed using the Enzyme-Linked Virus Inducible System (ELVIS, Diagnostics Hybrids,) shell vial assay according to the package insert. Results: A total of 896specimens were enrolled for this study. Of those, 175 were tested for Cdiff, 200 for GBS, 214 for VRE and 307 for HSV. The sensitivity/specificity for all assays compared to their reference standard method were: C. diff 86%/99%; GBS 100%/92%; VRE 100%/61%; HSV-1 95%/91% and HSV-2 100%/94% respectively, prior to discordant resolution. Conclusions: Our results show that excellent sensitivity (95% to 100%) was observed with GBS, VRE and HSV-2 assays and excellent specificity (99%) was observed with the Cdiff assay. Our data suggests that the artus QS-RGQ MDx assay is useful in a diagnostic setting. This fully-automated platform reduces hands-on time and improves standardized sample preparation and PCR assay setup. ID34. Validation of a Molecular Identification Assay for Fusarium species in a CLIA Certified Molecular Diagnostics Laboratory J.R. Lindner, D.A. Sutton, N.P. Wiederhold, A.W. Fothergill, B.L. Wickes, C. Sanders, D.I. McCarthy, Y. Wang, K. Vadlamudi, J. Fu, W. Furmaga, H. Fan University of Texas Health Science Center at San Antonio, San Antonio, TX. Introduction: Fusarium species are a potential human pathogen, causing local or disseminated infections as well as mycotoxicosis. Fusaria predominantly infect immunocompromised patients, but may also afflict immunocompetent humans through trauma and injury. Due to their medical significance, fast and accurate identification of Fusaria is essential. We report results of a validation using Sanger sequencing to identify Fusarium species through a multi-locus approach: sequences from translation elongation factor (EF-1a), internal transcribed spacer (ITS), and RNA polymerase II second largest subunit (RPB2) gene regions were used. This assay was developed according to the Clinical and Laboratory Standards Institute (CLSI) Guidelines MM18-A. Methods: DNA was extracted from 40 clinical strains of various Fusarium spp. using cetyl trimethyl-ammonium bromide (CTAB) buffer and physical disruption by bead beating, followed by manual chloroform/ ethanol extraction. The EF-1a, ITS, and RPB2 regions were then amplified using M13 tagged EF1/EF2, ITS1/ITS4 and 5f2/7cr primers, respectively. Takara Ex Taq (Takara Biotechnology Co., LTD) and 4% DMSO was used in the PCR reactions. Amplicons were sequenced bi-directionally with M13F and M13R primers, BigDye sequencing reagents, and the ABI3130xl Genetic Analyzer (Applied Biosystems). Raw sequence data were aligned and edited using Lasergene software (DNASTAR, Inc). Sequences were analyzed by searching the CBS-KNAW database. The assay’s accuracy, specificity, sensitivity, and reproducibility were then assessed. Results: 39/40 Fusarium isolates amplified and sequenced successfully for at least two of the three target regions, and 36/40 isolates had sequence data for all three regions. Clean sequences with average read lengths of ~500 to 900 base pairs were obtained. Most cases had BLAST similarities between 99% to 100%. Several isolates did not produce high-similarity BLAST matches for the RPB2 region, as this target is used most frequently for certain subsets of Fusaria, such as F. solani and F. chlamydosporum species complexes. Accuracy was confirmed using sequence data from the UTHSCSA Nucleic Acid Core Facility and morphologic/phenotypic characteristics. The specificity was confirmed by no cross reactivity to human, viral, and bacterial DNA. The assay sensitivity allows for successful sequencing and identification from 1.5 nanograms of starting DNA. The precision and reproducibility was confirmed by analyzing two Fusarium isolates in four runs on four different days by different technicians. Conclusions: The results of this validation study for the molecular identification of Fusarium species using Sanger sequencing targeting EF-1a, ITS, and RPB2 was satisfactory by diagnostic standards. This assay can be performed routinely on clinical isolates of various Fusarium species. ID35. Molecular Identification of Rhizopus arrhizus and Rhizopus delemar by Internal Transcribed Spacer (ITS) Sequence J.R. Lindner, N.P. Wiederhold, D.A. Sutton, C. Sanders, K. Vadlamudi, H. Fan University of Texas Health Science Center at San Antonio, San Antonio, TX. Introduction: Mucormycosis is a severe invasive mycosis, and infections may involve the lungs, brain, sinuses, gastrointestinal tract, and cutaneous/subcutaneous tissues. R. arrhizus (R. oryzae) is a common cause of mucormycosis, capable of causing infections in different hosts. Due to the severity of mucormycosis, early and accurate identification
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of the causative species is critical. R. delemar is closely related to and morphologically indistinguishable from R. arrhizus. The pathogenesis and antifungal susceptibility of these two species may be dissimilar, although any differences are not well established. The internal transcribed spacer (ITS) has been utilized as the universal target for molecular fungal identification. We report the ability of the ITS DNA sequence to distinguish between R. delemar and R. arrhizus. Methods: DNA sequencing of ITS and D1/D2 (domains 1 and 2 of the large ribosomal subunit) for fungal identification has been validated and routinely offered in our CLIA certified molecular diagnostics laboratory. Fungal DNA was extracted from clinical isolates, and the ITS and D1/D2 target regions were amplified using the panfungal ITS1/NL4 primer pair. PCR products were sequenced bi-directionally. The obtained sequences were queried in the CBSKNAW database. Final identification was achieved by combining morphologic/physiologic characteristics with molecular sequence data. Among 1179 isolates analyzed during August 2012 through April 2014, 18 isolates were identified as R. arrhizus based on morphologic/physiologic characteristics and ITS-D1/D2 sequence analysis. Alignment of the ITS sequences of these 18 samples was then performed using MacVector (Cary, NC). Results: ITS DNA sequences ranging from 629-633 base pairs were aligned. After alignment to reference sequences (R. arrhizus CBS 387.34 and R. delemar UTHSCSA 99-880), a nucleotide similarity of 99% was observed among the 18 isolates. Using differences detected in 4 distinct areas, the 18 isolates can be divided into 2 groups: R. arrhizus (N=12) versus R. delemar (N=6). When compared to the R. arrhizus group, the R. delemar group carries 2 bp (CT) insertion at nucleotide position 53-54, as well as single nucleotide polymorphisms T>C at positions 517 and 559, and G>A at position 414. Conclusions: Our study suggests the ITS sequence is informative in distinguishing between R. arrhizus and R. delemar. Since ITS DNA sequencing is routinely performed in our clinical laboratory, it provides a simple, rapid and accurate method for discrimination between these species. Further studies on virulence and antifungal drug susceptibility of these species are needed to confirm the clinical significance of these findings. ID36. Genital Swab Sample-to-Answer Verification Studies Using Focus Diagnostics’ Simplexa HSV 1 & 2 Direct Assay J. Chen, Y. Xie, Y. Parocua, H. Mai, M. Tabb Focus Diagnostics, Cypress, CA. Introduction: Simplexa HSV 1 & 2 Direct assay is a sample-to-answer multi-analyte detection system performed on the 3M Integrated Cycler. Genital swab specimens are loaded directly onto a Direct Amplification Disc without any separate specimen preparation or extraction steps. Results are generated in approximately 60 minutes. Simplexa HSV 1 & 2 Direct detects and differentiates herpes simplex viruses (HSV) 1 and 2. The objective of this study was to evaluate and compare the performance of the Simplexa HSV 1 & 2 Direct assay with results from clinical molecular laboratory tests that utilize conventional nucleic acid extraction procedures. Methods: Limit of detection (LoD) studies were performed to determine the analytical sensitivity of the assay. A panel of bacteria and viruses was tested to evaluate cross reactivity. A panel of potentially interfering substances was tested to determine whether any inhibition was observed. A reproducibility study was performed with medium and low positive panels. Clinical performance was determined by testing blinded genital swab clinical specimen panels and comparing to molecular assays requiring extraction. Results: LoD studies showed that Simplexa HSV 1 & 2 Direct detected HSV-1 strains at less than 45 TCID50/mL and HSV-2 strains at less than 15 TCID50/mL. None of the organisms tested for cross reactivity showed positive detection. No inhibition or interference was observed from any of the substances tested. Inter- and intra-assay reproducibility assays yielded 0.6 for all conditions with qualitative agreements of “good” and “very good” among specimen types. Conclusions: These results highlight the utility of the UDF system for qualitative and quantitative T. vaginalis detection from female urogenital specimens. In addition, residual cobas 4800
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AMP Abstracts eluates appear to be stable upon frozen storage and amendable for downstream PCR applications. ID57. Performance Evaluation of Commercially Available Multi-analyte Positive Control Materials for the Quantitation of CMV, EBV and BKV in the Management Transplant Patients M.F. Sabato, C.I. Dumur, M.H. Voelkner, A. Ferreira-Gonzalez Virginia Commonwealth University, Richmond, VA. Introduction: Real-time PCR (QPCR) assays for the quantitation of CMV, EBV and BKV are widely used in the management of transplant patients. Most of these assays are Laboratory Developed Procedures (LDP) for single analytes. Development of control materials for QPCR for each assay/ analyte has proven challenging. Recently, multi-analyte controls specifically developed for QPCR assays relevant for transplant patients have been developed. These QC materials are highly standardized assayindependent controls. Here we present the performance evaluation of such multianalyte materials for quality control purposes for our CMV, EBV and BKV quantitative QPCR LDP assays. Methods: Two different lots of High and Low Positive Control materials from AcroMetrix Multi-Analyte Controls—Transplant (Life Technologies, Inc.) were tested over a period of 9 months. The Low and High controls were extracted in single aliquots in the QIAsymphony RGQ system in an integrated mode for our CMV, EBV and BKV LDP assays using artus reagents (Qiagen). Each lot of reagents were used over the 9 month period. Results: Each lot of Low and High control material was tested 75 times in different runs for each of the different assays. The mean log10 values for CMV Low controls were 3.4 and 3.17 with coefficient of variations (CV) of 4.1% and 8.8%, respectively. The mean log10 values for CMV High controls were 4.61 and 4.54 with CV of 1.3% and 1.98%, respectively. The mean log10 values for EBV Low controls were 3.00 and 2.91 with and CV of 5.3% and 5.15%, respectively. The mean log10 values for EBV High controls were 4.47 and 4.54 with and CV of 2.0% and 1.1%, respectively. The mean log10 values for BKV Low controls were 3.4 and 3.31 with CV of 10.2% and 17.5%, repectively. The mean log10 value for BKV High controls were 4.79 and 4.67 with SD of 0.25 and 0.22 and VC of 5.5% and 4.7%, respectively. The Low and High commercial controls outperformed our laboratory developed High and Low controls for all analytes as the commercial controls showed consistently lower CV for all levels and analytes. There was no statistical difference in the performance between the two different lots for High and Low controls for all analytes tested. Conclusions: In summary the performance of both lots of High and Low Positive Control materials from AcroMetrix Multi-Analyte Controls—Transplant perform consistently and are a good alternative for laboratory developed controls for clinical testing. ID58. Evaluation of Luminex NxTAG Respiratory Pathogen Panel (NxTAG RPP) on Nasopharyngeal Swabs S. Chong1, S. Gonsalves2, M. Axford2, C. Yip2, C. Dey2, H. Zhang2, J. Mahony1 1St. Joseph’s Healthcare, Hamilton, Ontario, Canada; 2Luminex Toronto, Toronto, Ontario, Canada. Introduction: The Luminex NxTAG Respiratory Pathogen Panel (NxTAG RPP) prototype assay for investigational use is designed to simultaneously detect and discriminate nucleic acids from 18 viruses and 3 atypical bacteria extracted from respiratory samples. NxTAG RPP is a ready to use system. Nucleic acid from 200 ul of raw sample is extracted using commercially available methods and an aliquot of the eluate is directly added to pre-plated lyophilized reagents. Multiplexed RT-PCR and bead hybridization occurs in a closed PCR vessel under a single cycling program, eliminating the need for post-PCR transfer steps. Results are generated on the Luminex MAGPIX in 2 NAA ___ Method Sensitivity ___ Specificity ___ SBA 42/76 (55%) 115/115 (100%) SSA 55/76 (72%) 115/115 (100%) Igene 72/75 (96%)* 110/114 (96%)* Max 76/76 (100%) 106/114 (93%)* Xpert 75/76 (100%) 110/115 (96%) *There was one invalid result in each of these categories for this assay. Conclusions: NAA tests each represent a substantial improvement compared to the detection of GBS recovered from SBA or SSA. The CDC guidelines recommend enrichment broth followed by subculture to SBA, selective agars or NAA for detection of GBS. Although GBS rates have fallen dramatically after implementation of universal screening, Stoll et al. (Pediatrics: 127(7); 817-826, 2011) have shown that 80% of infants with early onset disease were born to women who had been screened and were negative for GBS. She and her colleagues suggested this could be due to suboptimal collection, delays in processing, suboptimal laboratory technique or a change in colonization prior to delivery. The data from our study would support the notion that a substantial portion of GBS colonization is not detected by subculture screening methods and consideration should be given to adoption of NAA detection from Lim broth. Acknowledgement: Cepheid, BD and Meridian each provided training, assay test kits and evaluation instruments for the nucleic acid amplification assays for this study. ID66. Aptima assay for High-Risk Human Papillomavirus Shows Higher Specificity and Better Correlation to Pap Smears Compared to Cervista Assay T.L. Lindeman, K.L. Edwards, J. Sizemore, L. Eskildsen, S.S. Talwalkar CPA Lab, Norton Healthcare, Louisville, KY. Introduction: Testing for high-risk human papillomavirus (hrHPV) in cervical specimens by PCR based assays has become the cornerstone of cervical cancer screening and management of the patients. Several assays are available for this testing. Two of the more commonly used assays are, Aptima (Hologic/Gen-Probe) which detects hrHPV E6/E7 mRNA using transcription-mediated amplification and Cervista (Hologic/Gen-Probe) which detects hrHPV genomic DNA using cleavase/Invader technology. We compared the two assays with each other and in turn with Pap smear results which is still considered the gold standard for cervical cancer screening. Methods: One hundred eighty-nine (189) cervical cytology specimens were used for this comparison. Representative aliquots were tested, initially by Cervista assay on a fully automated HTA platform, followed by Aptima assay on the Panther instrument. Raw data from the instruments was reviewed for all the discordant cases along with the DNA input concentrations. Corresponding PAP smears were also reviewed, separately by a pathologist and a cytotechnologist, who were blinded to the hrHPV results. Results: One hundred seventy-three (173) out of the 189 cases showed concordance between the two assays (91.4%) with 16 samples showing discrepancy. hrHPV was detected by both assays in 18 specimens, by Aptima alone in 2 and by Cervista alone in 14. Both assays were negative in 155 specimens. Of the 16 discrepant samples, 14 were positive by Cervista and 2 were positive by Aptima. Of the 14 specimens positive by Cervista, 5 were positive in all 3 mastermixes (triple positive). No significant correlation was seen between the amount of DNA input and likelihood of a triple positive result. PAP smears were positive for ASCUS in both the samples that were positive by Aptima and only 2 out of the 14 samples that were positive by Cervista. CIN was not detected in any of the discrepant samples. Conclusions: Aptima assay showed higher specificity and accuracy to Pap smear results when compared to Cervista assay. This may be due to the fact that it detects active infection following viral integration in host cellular DNA. ID67. A Novel Transduction Particle-Based Reporter Assay Enables a Simple-toUse Culture-Free MRSA Screening Test N. de Forest, S. Shukla, H. Cox, B. Ghavami, N. Clute-Reining, J. Chuc, B. Scofield, K. Dunphy, X. Liu, D. Rey, W. Frei GeneWeave Biosciences, Inc., Los Gatos CA Introduction: For active multidrug-resistant organism surveillance programs, the time required to identify carriers of drug-resistant bacteria as well as the ease of use of detection systems, are key factors for program success. GeneWeave Biosciences, Inc. is developing the vivoDx methicillin-resistant Staphylococcus aureus screening assay (vivoDx MRSA Screen) for the determination of the presence or absence of MRSA directly from nasal swab specimens within 4 hours, without the need for sample preparation or culture. The test is based on a transduction particle-based live-cell luminescence reporter assay that enables a homogeneous assay for phenotypic detection of MRSA. In this study, we investigated the analytical performance of a prototype vivoDx MRSA Screen and its performance on nasal swab samples. Methods: The analytical reactivity, cross-reactivity, and microbial interference characteristics of the vivoDx MRSA Screen were evaluated on a panel of 75 clinical isolates of MRSA, 30 clinical isolates of methicillin-sensitive S. aureus, coagulase negative Staphylococcus species, other coagulase-positive Staphylococcus species, and various Gram-negative organisms, Gram-positive rods, and yeast. The performance of the vivoDx MRSA Screen was evaluated by testing remnant nasal swab samples collected for the purpose of MRSA screening with reference to enriched culture. Remnant nasal swab samples were first eluted into Amies media and portions of the eluted sample were used for testing with the reference method and the vivoDx MRSA Screen by directly adding the crude eluted sample to a reaction container without any subsequent sample
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preparation. All studies were designed based on Clinical and Laboratory Standards Institute guidelines utilizing an investigational use only prototype of the vivoDx MRSA Screen. Results: The vivoDx MRSA Screen exhibited 100% reactivity to the panel of MRSA, no cross-reactivity, and no interference from all other non-MRSA organisms tested. In testing remnant nasal swabs 671 samples were tested of which 21 samples were determined to be positive for MRSA based on the reference enriched culture method. The vivoDx MRSA Screen exhibited 95% (20/21) sensitivity and 98.3% (639/650) specificity. Conclusions: The vivoDx MRSA Screen demonstrated excellent analytical performance. The assay required no sample preparation and no culture. It provided results in 4 hours with excellent concordance with the reference enriched culture method. The elimination of the need for sample preparation and culture will enable a simple to operate system for detecting drug resistant bacteria. ID68. An Assessment of Automated Qiagen artus and Modified BD GeneOhm Protocols for Detection of Toxin Producing Clostridium difficile D.D. Walton, N.L. Tisdel, T. Lasco, A.J. Chen Baylor CHI St Luke's Medical Center, Houston, TX. Introdution: Clostridium difficile (C. diff) is the most common infectious cause of diarrhea in hospitalized patients. Considering test performance, technologist hands-on (HoT), time turn-around-time (TAT) and cost, the optimal formula for the detection of toxigenic C. diff remains a challenge. However, we sought to compare our existing methodology of a modified BD GeneOhm CDiff real-time PCR assay (BD Assay) side by side with the automated Qiagen artus C. difficile QS-RGQ MDx Kit (artus Assay). Methods: Daily, several stool specimens are received in lab from various sites within the CHI St. Luke's Health System. These specimens are processed for tcdB DNA using the manual extraction of the FDA approved BD Assay. Over a 1 week period of testing by our current method, 22 tcdB positive and 22 randomly selected tcdB negative stool samples were blinded. Samples were then processed using automated extraction and set up on the QiaSymphony SP and AS based on the the artus protocol. Both assays were tested by real-time PCR and analyzed on the Qiagen Rotor-Gene Q (RGQ). Results: Of all samples, 40 results were included in calculating a concordance of 95%. Compared to the modified BD Assay, the artus Assay proved to be 100% sensitive and 93.33% specific, with a NPV of 100% and PPV of 83.33%. Efficiency proved to be 95.00%. TAT was approximately one hour faster for the BD Assay, whereas the artus Assay had a reduced HoT of about 45 minutes. Four samples were flagged as invalid and need further investigation to be resolved. Henceforth more samples will be tested to further evaluate the issue. Conclusions: In conclusion, HoT of the BD Assay was faster, but is variable depending on the proficiency of the technologist performing the assay. Alternatively, the artus Assay standardized the extraction and set up across all users and significantly frees technologist HoT. It is worth noting that the artus Assay has the capability of tcdA detection and, at this point, performs tcdB detection as sufficiently as our current modified BD Assay. ID69. Detection of Clostridium difficile with the Cobas Cdiff Test in Patients Suspected of CDI from France, Germany, and the United Kingdom* J. Osiecki1, A. Hiergeist2, M. Lewinski1, U. Reischl2 1Roche Molecular Systems, Pleasanton, CA; 2University Hospital Regensburg, Regensburg Germany. Introduction: Clostridium difficile is an anaerobic, toxin producing microorganism known to cause severe diarrhea in patients recently treated with antibiotics. In the absence of normal flora, C. difficile become well established, releasing potent toxins that can result in severe diarrhea and colitis. Nucleic acid amplification tests provide sensitive and timely identification of C. difficile infection and can be used as a tool for identifying C. difficile associated disease. This study was conducted to evaluate performance characteristics of the newly developed Cobas Cdiff Test using clinical specimens collected from patients in France, Germany, and the United Kingdom during routine diagnostic testing. Methods: Patients suspected with C. difficile associated disease, those who have received antibiotics within the last two months or have developed loose stools that take the shape of their container 72 hours after hospitalization, were included in the study. Residual clinical stool samples were transferred to sample vials that were frozen (Germany) or frozen and shipped (France and United Kingdom) then tested directly with the Cobas Cdiff Test on the cobas 4800 system for processing, PCR setup, amplification and detection. Samples were evaluated in parallel with an alternate molecular platform, the Xpert C.difficile/Epi Test. Results: Of the 883 clinical samples evaluated, 102 were concordant positive, and 768 were concordant negative by both molecular methods. Six specimens were found to be negative by the Xpert C.difficile/Epi Test, and positive by the Cobas Cdiff Test, where seven specimens were positive by Xpert and negative by the Cobas Cdiff Test. The positive percent agreement and negative percent agreement between the methods was 93.6%, and 99.2%, respectively. Conclusions: The Cobas Cdiff Test, run on the automated cobas 4800 system, demonstrated excellent performance for detecting C. difficile in unformed stool specimens. *This test is not available for use in the United States.
jmd.amjpathol.org ■ The Journal of Molecular Diagnostics
ID70. Converting to the eSensor HCVg Direct Test: An HCV Genotyping Validation H.B. Steinmetz1,2, L.J. Cook1, B.J. Dokus1, T.L. Wilson1, L.J. Tafe1, G.J. Tsongalis1, J.A. Lefferts1,2 1Dartmouth-Hitchcock Medical Center, Lebanon, NH; 2Geisel School of Medicine at Dartmouth, Hanover, NH. Introduction: HCV genotyping is an important test in the management of HCV infected individuals. Genotyping results can help predict response to therapy and general disease progression. Traditional genotyping methods are time-consuming and can be difficult to interpret. Newer methods are available that offer a simplified workflow and shorter turnaround times. Our laboratory previously validated the eSensor HCV Test v1.2 which uses PCR amplicon from a specific FDA-cleared test as template in a genotyping assay. This test is being replaced by the eSensor HCVg Direct Test which used viral RNA as a template and offers improved accuracy. We report on our laboratory’s validation study for conversion to the eSensor HCVg Direct Test. Methods: Total nucleic acid was isolated from plasma specimens with the EZ1 Virus Mini Kit v2.0 (QIAGEN). Testing was performed using 41 residual plasma specimens previously submitted for HCV genotyping using the eSensor HCV Test v1.2 (GenMark). Additional testing was performed with plasma obtained from Life Technologies and SeraCare representing genotypes 1-6. Limit of detection studies were performed using patient or control plasma diluted with EDTA dilution matrix (Life Technologies) and viral concentrations were determined using the COBAS TaqMan HCV Test (Roche). Results: Concordance between the two versions of the eSensor HCV genotyping test was observed in 36 of 41 patient samples tested. Three samples previously genotyped as 6a/b and one sample previously genotyped as 3a were all genotyped as 1a in the HCVg Direct assay. Three of these discordant results were resolved by Sanger sequencing in favor of the HCVg Direct results and confirmatory testing is in process for one sample previously identified as genotype 6a/b. One sample that was previously identified as genotype 2b initially failed to produce a genotype with the HCVg Direct assay but was confirmed as 2b with repeat testing and sequencing. Dilutions of plasma samples of several known genotypes were tested in 10-15 replicates over multiple days to assess reproducibility/repeatability and the limit of detection. The limit of detection for the assay was established at 1600 IU/mL although some genotypes could be reliably detected at 500 IU/mL. Conclusions: The eSensor HCVg Direct Test demonstrated improved accuracy in its ability to distinguish between the various HCV genotypes. Additionally, the limit of detection was found to be suitable for the majority of HCV genotyping testing performed in our laboratory. ID71. Accessory Gene Regulator Group II Polymorphism and Vancomycin Susceptibility Testing in Methicillin-Resistant Staphylococcus aureus H. Kim Halllym University, Anyang, Korea. Introduction: A relationship between the accessory gene regulator (agr) group II polymorphism in methicillin-resistant Staphylococcus aureus (MRSA) and clinical efficacy of vancomycin was described. Vacomycin treatment failure in MRSA bacteremia was described when the strain had a vancomycin minimum inhibitory concentration (MIC) of 2 mg/L. In this study, we studied the correlation between the agr group II polymorphism and vancomycin MICs in MRSA isolates. Methods: A total of 118 MRSA bloodstream isolates were analyzed by agr II-specific PCR. Vancomycin susceptibility tests were performed by E-test (AB bioMerieux), MicroScan (Siemens) and CLSI broth microdilution (BMD). Etest GRD (AB bioMerieux) was performed to detect heterogeneous vancomycin-intermediate S. aureus (hVISA). Results: Fortythree (36.4%) of the 118 isolates had the agr II polymorphism. Twenty-six isolates showed vancomycin MIC of 2 mg/L, 73 isolates showed vancomycin MIC of 1 mg/L, and 19 isolates showed vancomycin MIC of 0.5 mg/L by BMD.In general Microscan and E test MIC results were higer than BMD MIC results. Fourteen (53.8%) of 26 isolates with vancomycin MIC of 2 mg/L, 26 (35.6%) of 73 isolates with vancomycin MIC of 1 mg/L, and 3 (15.8%) of 19 isolates with vancomycin MIC of 0.5 mg/L had the agr II polymorphism. Four isolates were positive for hVISA by Etest GRD, and 3 of 4 isolates had the agr II polymorphism. Conclusions: The agr II polymorphism is more common among MRSA isolates vancomycin MIC of 2 mg/L than among MRSA isolates vancomycin MIC of 1 or 0.5mg/L. There is a possible relationship between the agr II polymorphism and hVISA. ID72. Clinical Utility of GeneXpert MTB/RIF Assay on AFB-smear Positive and Negative Respiratory and Non-Respiratory Samples Collected from Patients Seen in a Large Urban Hospital Y.F. Wang1,2, S. Merritt2, T. Drake2 1Emory University School of Medicine, Atlanta, GA; 2Grady Memorial Hospital, Atlanta, GA. Introduction: The GeneXpert MTB/RIF Assay is a FDA cleared real-time PCR assay that detects the Mycobacterium tuberculosis complex (MTB) DNA and Rifampicinresistance (RIF) associated mutations of the rpoB gene. Clinical performance of the assay on patient samples for diagnosis of MTB was evaluated on the acid-fast bacilli (AFB) – smear positive and smear negative samples, as well as sputum and nonsputum samples. Methods: Before replacingMTB Direct (MTD) assay, 33 processed clinical samples collected from patients seen in a large inner city hospital were used for early evaluation of the assay. The sediments after routine digestion and concentration of the samples were used for AFB smear, AFB culture, the MTD assay, and the MTB/RIF assay. After replacing MTD, 62 clinical samples were tested in routine clinical setting, AFB culture and MTB/RIF results were used for data analysis. Results: Before
The Journal of Molecular Diagnostics ■ jmd.amjpathol.org
AMP Abstracts
replacing MTD, 33 processed samples including 7 non-sputum samples (bronchial wash, fine needle aspiration, pleural fluid, urine and lymph node), 1 urine sample and 7 sputum samples were positive for MTB by MTB/RIF, MTD, and culture. MTB/RIF were later performed routinely in the clinical laboratory to replace MTD. Of 62 clinical samples tested, 15 were non-sputum samples. Of non-sputum samples, 5 were AFB smear positive with 2 positive for MTB (1 BAL and 1 tissue). Of 47 processed sputum samples, 28 were AFB smear positive with 12 positive for MTB, and 19 were AFB smear negative with 2 positive for MTB. Of 95 samples with 22 non-sputum tested, MTB/RIF provided results consistent with cultures, with exception for only one sample that was positive for MTB by MTB/RIF but grew M. avium complex (MAC). Possible dual infection will be studied. Conclusions: The GeneXpert MTB/RIF assay has been shown to have excellent performance for clinical diagnosis of MTB on processed clinical samples. ID73. Clinical Utility of the Luminex xTAG GPP Assay in a Characterized Culture Negative High-Risk Population Cohort Suggests Enhanced Detection of Infectious GI Associated Illness Compared to Conventional Culture Testing and Hospital Ordering E. Herding1, G. Hansen1,2 1Hennepin County Medical Center, Minneapolis, MN; 2University of Minnesota, Minneapolis MN Introduction: “Syndromic” testing for infectious disease has become common among clinical laboratories. The FDA approved Luminex xTAG GPP (GPP) assay have been amongst the first to offer GI panel based testing. Although data comparing the GPP assay to culture has been published, few series have attempted to establish the clinical utility of the assay in comparison to culture in characterized high-risk patient populations. In an attempt to define the clinical utility of the GPP assay we examined assay performance in a group of high-risk GI patients in whom culture results were initially reported as negative by the laboratory. Methods: Retrospective chart review identified 292 sequential non-duplicate cases meeting a case definition for “high-risk” infectious GI illness. High-risk was defined based one or more of the following high risk parameters i) GI illness listed as primary admitting diagnosis ii) abnormal/elevated laboratory finding related to serum lactate, albumin, creatinine, WBC >15, fever, iii) diarrheal episodes >3/24hrs. All samples were culture negative by standard stool culture on blood agar, MacConkey, Campylobacter, Chomogenic 0157, Hektoen enteric agar, selenite enrichment broth. Patient samples were cohorted into 1 of 3 groupings: 1) samples submitted only for the hospitals’ in-house C. difficile PCR assay; 2) samples which were submitted for culture requests only; and 3) cases submitted for C. difficile PCR + culture. Results: For patients in which C. difficile PCR and stool culture were co-requested, GPP assay detected an unrecognized/reported GI target in 16% (23/123) cases. For cases submitted with culture requests only, GPP assay detected an unrecognized/unreported GI target in 18% (13/61) cases. For cases in which C. difficile PCR was the only test ordered GPP assay detected an unrecognized/unreported GI pathogen in 14% (10/62) of the cases. In 66% of the cases, an unrecognized virus was detected, unrecognized cases of Campylobacter spp, Salmonella spp, E.histolytica, C.difficile and Shigella were also noted. Conclusions: In a patients presenting to inpatient/outpatient/ED settings with compatible clinical symptoms associated with a GI illness, the GPP assay detected an unrecognized/unreported GI associated pathogen in 16%, 18%, and 14% of patient cohorts tested. The inability to routinely detect GIassociated virus tested combined with uncertainly in convention test request orders contributes to under-diagnosis and recognition of potential GI pathogens in symptomatic patients. The GPP assay represents a comprehensive approach for the detection of GI pathogens in hospital settings. In this defined patient cohort the detection of GI pathogens by GPP was superior to culture based testing ID74. WITHDRAWN ID75. Evaluation of Simplexa MRSA Direct (Focus Diagnostics) on a Large Collection of mecA- and mecC-Positive S. aureus Representing the Major MRSA Clones Circulating throughout the World F. Laurent, H. Meugnier, A. Garriga, J. Tasse, A. Tristan, M. Bes, F. Vandenesch French National Reference Centre for Staphylococci, Hospices Civils de Lyon, Lyon, France. Introduction: Methicillin resistant Staphylococcus aureus (MRSA) is one of the major pathogen responsible for nosocomial infections. Early screening for MRSA carriage in patients is pivotal to limit infections and transmissions. Several PCR assays have been designed to ensure rapid and reliable detection of MRSA in nasal swabs. They classically targeted i) the junction between orfX and SCCmec cassette, ii) a gene specific to S. aureus (SA), and iii) for some assays the mecA gene. Recent identification of a mecA variant, named mecC, with less than 70% DNA homology to mecA, further complicates MRSA detection due to misdiagnosis as a result of mismatch in primers targeting the mecA gene. In this context, using a very diverse collection of MRSA clinical isolates, we evaluated a new commercial real-time PCR (Simplexa MRSA Direct) assay that is able to detect both mecA and mecC genes. Methods: A panel of 148 MRSA clinical isolates and 4 methicillin susceptible S. aureus (MSSA), used as controls, were included. MRSA strains have been previously extensively characterized (mecA/mecC PCR, agr typing, DNA microarray (Alere/Clondiag)) and selected to be representative of the major worldwide-circulating MRSA clones: they covered 35 clonal complexes and more than 70 spa-types, and included 25 mecCpositive strains. The Simplexa MRSA Direct assay is a real-time PCR that detects
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AMP Abstracts conserved regions of S. aureus genome (spa gene) and methicillin-resistant genes (mecA and mecC). The software automatically compares the results (including Ct) of both targets to identify if MRSA or SA is present. The 1-hour assay was performed using the Direct Amplification Disc (8 tests) and the 3M Integrated Cycler with Integrated Cycler Studio Software version 5.0. Results: All clinical isolates were accurately classified as SA (n=4) or MRSA (n=148). All, except one, isolates were identified as S. aureus. A 99.34 % agreement was reported for the Simplexa MRSA Direct assay in comparison to reference results. Conclusions: The Simplexa MRSA Direct assay proved to be very fast and simple to detect methicillin susceptible and methicillin resistant S. aureus from isolated colonies. Using a large collection of strains representing the major MRSA clones circulating throughout the world, we obtained a 99.34% agreement with reference results. A single discrepancy has been observed with a MRSA isolate for which spa gene was not detected; this is likely related to point mutations in the spa gene. Sequencing of the gene is underway for this strain. ID76. Protein Disulfide Isomerase A2 Is a Candidate Host Susceptibility Gene for Chlamydia trachomatis Infection K.M. Bennett, A. Balogun, E. Boone, G. Sanchez, E.C. Hendrix Texas Tech University Health Sciences Center, Lubbock, TX. Introduction: Chlamydia trachomatis infection is the most commonly reported sexually transmitted infection in the United States. There are many differences between patients in the persistence and progression of infection that are believed to be strongly influenced by the genetics of the host. The objective of this study is to identify genetic differences that may confer susceptibility to genital Chlamydia trachomatis infection. Protein disulfide isomerase-A1 (PDIA1/P4HB) was previously shown to play a role in the attachment and entry of C. trachomatis bacteria. In this study, single nucleotide polymorphisms (SNPs) in protein disulfide isomerase-A2 (PDIA2) were examined in patients with and without C. trachomatis infection. Methods: Single nucleotide polymorphism (SNP) genotyping was performed with a sample size of 167 subjects, which was 75% female with a median age of 22 years (18-33 years). The C. trachomatis infection status was determined by the Roche Amplicor CT/NG assay. DNA was isolated from de-identified urethral or endocervical swab specimens, using the Qiagen QIAmp DNA isolation kit, and genotyping was performed using TaqMan allelic discrimination assays (Applied Biosystems). A percentage of samples were also genotyped by pyrosequencing (Qiagen Pyromark Q24) to confirm original results. The rs400037 polymorphism (R388Q) was studied in 167 subjects, and the rs419949 polymorphism (E185K) has been examined in 42 subjects, thus far. Results: Chisquare analysis indicated that all genotypes were in Hardy-Weinberg equilibrium. The genotype frequencies were then analyzed by Fisher’s Exact Test to determine if there was an association between genotype and C. trachomatis infection status. The odds ratio for the PDIA2 rs400037 GG genotype was 2.13 (95% CI: 1.12 to 4.06) in controls vs. positives (p=0.023), indicating a greater likelihood of finding this genotype in individuals not infected with C. trachomatis. The less common A allele was nearly twice as likely to be found in patients that were positive for C. trachomatis (p=0.005). An increased frequency of the rs419949 GG genotype was also observed in controls (OR 1.73, 95% CI: 0.51-5.91), although this did not reach statistical significance. A greater sample size is needed for rs419949 to better show the genotype distribution of this locus. Conclusions: Results show that the PDIA2 rs400037 SNP is associated with C. trachomatis infection. Further studies will determine whether the rs419949 SNP also shows an association. This is evidence that PDIA2 is a susceptibility gene for infection, although a direct role for PDIA2 in C. trachomatis infection has not yet been determined.
OTHER (EDUCATION, ETC.) OTH01. Implementation of a Molecular Tumor Board L.J. Tafe, M.D. Chamberlin, T.W. Miller, F.B. de Abreu, J.A. Lefferts, X. Liu, J.R. Pettus, J.D. Marotti, V.A. Memoli, A.R. Schned, A.A. Suriawinata, G.J. Tsongalis Geisel School of Medicine at Dartmouth and the Dartmouth Hitchcock Medical Center, Lebanon, NH. Introduction: Over the past year, clinical laboratories have begun using nextgeneration sequencing technologies in targeted approaches for somatic mutation detection in human cancers. The vast amount of data produced for each patient sample is unprecedented in molecular diagnostic testing. Identified variants must then be curated to determine if they may be clinically actionable. In addition, this information must be communicated to the oncologist in a comprehensive and useful manner. We established a Molecular Tumor Board (MTB) to analyze and discuss cases of interest with mutational profiles within a 50-gene panel. Methods: Given the increasing demand for and production of genomic sequencing data, we first created a steering committee to establish the feasibility of a MTB. In selecting the most convenient day and time, it was important that representatives from basic science, clinical medicine and laboratory medicine be available to participate. Several meetings were held to discuss the logistics and implementation of such Board. Results: The MTB is a monthly meeting of clinicians, scientists, and pathologists. Case presentations include clinical, laboratory, and scientific information pertinent to patient management. Genomic information specific for identified variants is discussed that encompasses diagnosis, ethical considerations, prognosis, and therapeutic selection, as well as the potential for germline mutations and, eligibility and relevance of participation in clinical trials. Challenges
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include the interpretation of variants of uncertain clinical or pathological significance. Since the first meeting in February 2014, 12 cases have been presented with a range of malignancies including leiomyosarcoma, metastatic colorectal cancer, metastatic melanoma, pilocytic astrocytoma and metastatic nasopharyngeal carcinoma; mutations were identified in 9/12 cases. Conclusions: This multidisciplinary approach to interpretation of genomic data is critical for the proper utilization of next-generation sequencing data. Our MTB is able to discuss and disseminate patient-centric genomic information that can impact patient management. OTH02. Implementing Lean Methods to Improve the HER2 Fluorescence in Situ Hybridization Process in the Diagnostic Molecular Pathology Laboratory H. Steinmetz, O. Perry, W. Wells, L.J. Tafe Dartmouth-Hitchcock Medical Center, Lebanon, NH. Introduction: Lean and Six Sigma process improvement methods have been utilized in the manufacturing industry for decades, and are now being applied to healthcare settings. HER2 FISH result turnaround times (TAT) were not routinely meeting some customers’ needs. To provide better service to our providers the current HER2 FISH processes in the Molecular Pathology Laboratory were evaluated using Lean Six Sigma analysis tools. Opportunity to improve the workflow was identified. Methods: The evaluation of the HER2 FISH assay had multiple phases: documentation of the current state to establish a baseline measurement of TAT; workflow observations to determine areas for improvement; and, pilot studies to test theories to decrease overall TAT. A flow chart was constructed to record the baseline processes from the time of order to result reporting and current TAT was generated from the LIS. In the analysis phase, time points for all exchanges that occurred for each HER2 case were collected over the course of 10 consecutive clinical runs. Following 10 cycle analysis, multiple user to user variations in processing times were identified. A single location for pick up and drop off of all HER2 slides was implemented within this phase. From the analysis phase, it was evident that the time of day that the technologist read the slides impacted when the Pathologist could complete a case review. The improvement phase of the project included a pilot study in which two technologists analyzed and drafted reports for all HER2 slides by 12pm daily. This allowed sufficient time for slide review, report entry by a technologist and verification of the results, thus decreasing the possibility of an overnight delay. Results: The baseline TAT for the HER2 FISH assay was measured at 7 business days. In the analysis phase, with a centralized slide pick-up location, the TAT improved to 5 business days and the number of hand-offs, misplaced slides and recuts decreased (waste). After the improvement phase, the early read pilot was implemented as standard procedure resulting in overall TAT decreasing to 3 business days, fully satisfying customer expectations. Conclusions: Within six months, the implementation of Lean processes to improve the workflow of HER2 FISH testing in our molecular pathology laboratory resulted in improved overall patient care and reduced assay TAT and waste. Process improvement strategies, like Lean, should be embedded into all workflow analyses in a clinical laboratory. OTH03. Cost Feasibility of High Risk HPV (hrHPV) Based Population Screening for Cervical Cancer Is Variable among Low and Middle Income Countries (LMIC) and Depends on Demographics and Availability of Locally Trained Cytopathology Manpower R. Dewar1,4, R. Khadapkar2,4, T. McDonald1, B.R. Das2, V. Parkash3 1Beth Israel Deaconess Medical Center, Boston, MA; 2SRL Limited, Mumbai, Maharashtra, India; 3Yale University School of Medicine, New Haven, CT; 4South Asia Institute, Cambridge, MA. Introduction: The three commonly used technique to screen for cervical cancer include VIA (promoted in LMICs), hrHPV, with liquid based (LBC) or conventional cytology (Pap). We evaluated the applicability of these methods in rural India. Methods: Three hundred eighty-six (386) patients participated in 4 screening camps conducted by Hindu Mission Hospitals. The costs of primary screening with hrHPV and triaging to LBC for positive cases was compared to primary LBC with reflex hrHPV in cases with ASCUS. Projected costs associated with population screening in this district and 5 LMICs with high prevalence of cervical cancer were estimated. Results: Fifty-eight (58) of the 386 patients were hrHPV positive and 33 had a squamous intra-epithelial lesion. Visual inspection with acetic acid had an unacceptably low sensitivity and specificity (>30% FP/FN rate), although it was the most cost-effective technique pathology expenses. Conventional Pap smear screening was most cost effective (~$1/patient). Liquid based cytology used either as a primary screening measure or a secondary screening measure in conjuction with hrHPV testing was relatively expensive. The difference between primary screening with hrHPV testing with reflex LBC vs reflex hrHPV with primary LBC was marginal (20%). At the population level, in the district of Trichy (one of 15 districts in TamilNadu, one of 25 states in India), with an estimated ~500,000 women, hrHPV testing is projected to impose a cost of $5 million (marked down cost of hrHPV testing to US$10), in comparison to Pap (~$1 per test) making this an impractical choice. Conclusions: Laboratory based (hrHPV and/or cytology) are best screening strategies compared to VIA. hrHPV followed by LBC is economically impractical for population based screening in a populous LMIC country like India. Conventional Pap smears followed by hrHPV for ASCUS, and identifying areas with high prevalence are two applications of hrHPV in these settings. However, a strong quality assurance program is essential to maintaining high standards for interpretation of conventional pap smears. Therefore, in less populous countries with poor pathology laboratory infrastructure and insufficiently trained cytopathologists, hrHPV followed by cytology may be more economically feasible and a viable primary screening strategy.
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For cervical cancer screening, many World Organizations, governments and NGOs develop guidelines, based on an assumed global applicability. We strongly advocate a region/country specific screening strategy that is dependent on target population, available manpower and infra-structure. OTH04. An Error-Based Approach to Validation of RUO Reagents M. Yu, J. Czech, T. Catduong, L. Jennings Lurie Children’s Hospital of Chicago, Chicago, IL. Introduction: On November 25, 2013, the US Food and Drug Administration (FDA) had finalized the guidance document for the distribution of products for research use only (RUO). Therein, the regulatory requirements reiterate the labeling of such products "For Research Use Only. Not for use in diagnostic procedures." However, given the limited number of FDA-approved molecular diagnostic tests, many clinical laboratories are using RUO instruments, software and reagents for their clinical tests. Validation of clinical tests using unregulated RUO products requires careful consideration of potential errors that may impact patient safety. Here we demonstrate an error-based approach to assay validation using RUO reagents. Methods: RUO probe sets for multiplex ligationdependent probe amplification (MLPA) were obtained from MRC-Holland. The P065 and P066 probe mixes containing 79 target-specific probes targeting all exons of FBN1 and TGFBR1. To validate probe-specificity, blocking oligos were designed and synthesized to correspond to the same targeted regions. These oligos were pooled into probe mixes of 8-13 and added to the RUO probe sets to competitively block the amplification of all target-specific probes. Samples with blocking oligos were normalized using the same samples without blocking oligos to verify the specificity. Results: Having previously performed an extensive method-based validation of MLPA, this validation focused on probe validation. Probe validation is first performed in silico by comparing probe sequence to the known human reference genome (build hg19), and then by comparing performance across a normal population. For these probe sets, blocking oligos were also added to verify the sequence specificity of all target probes. Addition of blocking oligos reduced the signal from corresponding target probes in a concentration-dependent manner without affecting the performance of other probes in the probe set. Conclusions: For unregulated RUO reagents, there is no assurance that primer or probe sequence or concentrations are as reported. Therefore, error-based validation of such reagents should include documented evidence of the sequence and concentrations. Given the lack of sufficient well-characterized samples, we chose to validate the RUO probe sets using blocking oligos. These oligos can also be used for quality assurance as controls in every run. OTH05. Knowledge Improvement and Learners’ Feedback from an Online Education Module on Quality Practices for Molecular Genetic Testing T. Brunson, L.A. Gjeltema, J. Zhong, B. Chen Centers for Disease Control and Prevention, Atlanta, GA. Introduction: In September 2012, the Centers for Disease Control and Prevention (CDC) launched a multimedia, interactive online course hosted by the Association of Public Health Laboratories (APHL) to help laboratory professionals understand recommended good laboratory practices and enhance competencies for molecular genetic testing. The course presents a realistic scenario in which a laboratory plans to introduce its first molecular genetic test and uses a team approach to addressing the quality management needs. Participants may earn the American Society for Clinical Laboratory Science (ASCLS) Professional Acknowledgment for Continuing Education (PACE) credits directly from the course portal, or the continuing medical education (CME) credits from CDC. Methods: Participants registered from September 1, 2012 through June 15, 2013 and were asked to complete all course components (including the pre- and post-test, the instructional content, and the course evaluation) by December 31, 2013. Knowledge improvements were calculated by determining the difference between the pre- and post-test score of each participant. Evaluation feedback was assessed with a combination of quantitative and qualitative analyses. Results: By December 31, 2013, 265 of 636 PACE and 39 of 157 CME registrants completed the course. Participants included laboratory and healthcare professionals at doctoral, master, baccalaureate, and other education levels from diverse work settings. The average pre-test score was 59.5% for PACE participants and 59.1% for CME participants. Upon course completion, knowledge improvement averaged 28.2% for the PACE participants (ranging 5% to 58%) and 30.4% for the CME participants (ranging 10% to 48%). More than 90% of the PACE participants indicated that the course successfully met the stated learning objectives and would help them perform their jobs better. The learners commented that the interactive format with the simulated laboratory activities was much more engaging than traditional CE modules; the knowledge check questions after each section effectively reinforced the learning; and the downloadable documents and resources were especially helpful. Among the CME participants, greater than 80% indicated that the course effectively met their educational needs and they would apply the knowledge gained if given an opportunity. However, many CME participants felt that the additional step to obtain CE credits from CDC was burdensome. Conclusions: These results support the educational value and effectiveness of this learning module for diverse laboratory and healthcare professionals. Further analyses are ongoing to explore the extent of implementation of the learning in practice and the correlation with the learners’ demographic characteristics.
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AMP Abstracts
SOLID TUMORS ST01. A Real-Time PCR Assay for Detecting and Distinguishing BRAF V600 E/K/D Mutations in Melanoma and Colorectal Cancer FFPE Specimens H. Su1, B. Erickson1, D. Murray1, W. Mak1, J. Voss2, S. Brankley2, J. Winters2, E.B. Fritcher2, B. Kipp2, K. Halling2, K. Abravaya1, S. Huang1 1Abbott Molecular, Inc., Des Plaines, IL; 2Mayo Clinic and Foundation, Rochester, MN. Introduction: BRAF, a key component of the RAS/RAF/MEK/ERK signaling pathway that regulates cell growth, is mutated in several types of cancers. Detection of BRAF mutations is useful for predicting therapy responses in melanoma and colorectal cancer (CRC) patients. A real-time PCR assay has been developed to detect and distinguish three BRAF mutations, V600E (T1799A; TG1799-1800AA), V600K (GT1798-1799AA; GTG1798-1800AAA) and V600D (TG1799-1800AT; TG1799-1800AC) in formalin-fixed, paraffin-embedded (FFPE) specimens from melanoma and CRC patients. Methods: Genomic DNA was extracted from FFPE slides and curls using the TargetPrep DNA FFPE Sample Preparation Kit and tested with the Abbott RealTime BRAF V600 E/K/D Mutations assay on the m2000rt system, which reported individual detection status for V600E, V600K and V600D. Analytical assay performance and concordance with clinically validated tests were evaluated. Results: The Abbott BRAF assay detected each targeted mutation (V600E, V600K, V600D) at 1% or lower in 5.0 ng or more of genomic DNA. No cross-reactivity was observed with BRAF homologues (aRAF, cRAF, RAF pseudogene), non-targeted BRAF mutations (V600R, D594G, G596R, L597Q, L597S, K601E) or wild-type genomic background. The Abbott assay was compared with the cobas BRAF V600 Mutation test in 142 melanomas and with the Mayo BRAF LDT in 20 melanomas. The overall agreement with the cobas test was 97.2% (138/142), with positive agreement of 100% (65/65) and negative agreement of 94.8% (73/77). Among specimens positive by both tests, eight were identified as V600K+ by the Abbott assay. Among the 4 discordants positive by the Abbott assay and negative by cobas assay, 3 were confirmed to be positive by a third test (one V600K+ by Sanger, one V600E+ and one V600K+ by next-generation sequencing) using the residual DNA and one did not have sufficient DNA for retest. Concordant results were generated for specimens containing high levels of melanin. The Abbott assay had 100% concordance with the Mayo LDT for the 20 melanomas. In addition to melanoma, 99 CRC specimens were tested by the Abbott assay as well as Mayo LDT and Sanger sequencing. The overall agreement between the Abbott and the Mayo LDT tests was 100%. The overall agreement between the Abbott assay and Sanger sequencing was 98.0% (97/99), with positive agreement of 100% (9/9) and negative agreement of 97.8% (88/90). Conclusions: The Abbott RealTime BRAF V600 E/K/D Mutations assay is highly sensitive and specific in detecting and distinguishing V600E, V600K and V600D mutations in melanoma and CRC FFPE specimens. ST02. Gastric Adenocarcinoma MicroRNA Profiles in Fixed Tissue and in Plasma D.L. Duncan1, A.L. Treece2, W. Tang2, S. Elmore2, D.R. Morgan2, M.O. Meyers2, R.L. Dominguez3, O. Speck2, M.L. Gulley2 1University of North Carolina Hospitals at Chapel Hill, Chapel Hill, NC; 2 University of North Carolina at Chapel Hill, Chapel Hill, NC; 3Western Regional Hospital, Santa Rosa de Copan, Honduras. Introduction: Modern molecular technology facilitates identification of cancer-related biochemical effects to assist in diagnosis and classification of tumors using readily accessible specimen types such as formalin-fixed, paraffin-embedded tissue (FFPE) and plasma. Methods: In this study, microRNA expression profiling was performed by rtPCR on 100 gastric cancer FFPEs macrodissected to enrich for adenocarcinoma or adjacent non-malignant mucosa, and on 32 matched plasma specimens or control plasmas. The GastroGenus miR Panel panel was developed and applied to measure 56 analytes including cancer-specific human microRNAs, Epstein-Barr virus (EBV) encoded microRNAs, and controls. EBV status was determined using EBER in situ hybridization, and EBV viral load was measured using Q-PCR. Results: In FFPE specimens with adequate housekeeper microRNAs, 13 human microRNAs were significantly dysregulated in gastric cancer versus benign gastric mucosa. The microRNAs upregulated in gastric cancer were hsa-miR-185, hsa-let-7i, hsa-miR-21, hsa-miR-196a, hsa-miR-196b and hsa-miR-155, whereas the downregulated microRNAs were hsa-miR-423-3p, hsa-miR-34a, hsa-miR-484, hsa-miR-744, hsa-miR187, hsa-miR-200a and hsa-miR-18a (all adjusted p 3.0; Illumina FFPE quality control assay) or low quantity (< 5 ng/mL) FFPE tumor tissues on the Proton. Samples >5 years old had a greater likelihood of sequencing artifact than samples 64 million AQ20 captured reads/sample. The average coverage, fraction covered at >10X and
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strand bias of the ES assay were 93%, 92% and 52%, respectively, with complete concordance in the variant QC metrics between the FFPE and FF samples. Accuracy assessment using HapMap DNA (Coriell NA12878) demonstrated >99% agreement between the WES results and the NA12878 benchmark dataset (Zook et al., 2014, Nature Biotechnology). The false positive rate for HapMap replicates was 90% coverage of BRCA1/2 coding regions at a minimum of 100x read depth. All BRCA mutations identified were confirmed, where possible by Sanger sequencing or replication, to eliminate the risk of false positive results due to FFPE artefacts. We also determined that BRCA1/2 mutations could be detected if present in >10% of the sample DNA. To cover the missing BRCA1/2 regions, such as the 11th coding BRCA2 exon, a more robust primer set (V2) was utilised. Re-testing of 12 of the FFPE samples using the enhanced V2 primer set resulted in similar performance characteristics and provided coverage of the regions missed previously. Conclusions: Comprehensive coverage of BRCA1/2 and routine analysis of FFPE tumors for BRCA1/2 mutations are feasible and could be performed prospectively to ensure optimum treatment for ovarian or breast cancer patients. ST32. Impact of 2013 HER2 Testing Guideline Update on FISH Interpretation and HER2-Targeted Therapy Eligibility M. Singh1, A. Sophian2, I. Almanaseer2, M. Pins2, C. August2, M. Mihalov2 1University of Illinois Chicago, Chicago, IL; 2ACL Laboratories, Rosemont, IL. Introduction: Breast cancer kills nearly 40,000 women annually in the United States. Targeted therapies are available to eligible patients with HER2-amplified tumors and have significantly improved clinical outcomes. Unfortunately, HER2 amplification occurs in only 15% to 20% of breast cancers. Assays utilizing fluorescent in situ hybridization (FISH) probes are a proven clinical methodology to detect amplification. In 2007, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) published interpretation guidelines to improve the accuracy of HER2 testing. These guidelines were, however, modified in 2013, at which time the recommended FISH scoring criteria changed, potentially altering the HER2 status and eligibility of previously tested patients for HER2-targeted therapy. To investigate the potential impact on HER2 FISH interpretation, we retrospectively analyzed a subset of HER2-negative and HER2equivocal invasive breast cancer cases reported prior to the release of the 2013 guidelines. Methods: Our analysis included 332, formalin-fixed, paraffin-embedded invasive breast carcinoma cases from August 2013 to October 2013, applying the new guidelines for reporting and comparing it to the documented interpretation to determine if there was a change in HER2 status. We utilized a modified version of the Abbott, FDA-approved, HER2-fluorescence-in-situ-hybridization assay validated in our laboratory. Results: HER2 status changed in 13 cases (3.9%). By 2007 criteria, 329 cases were non-amplified and 3 cases were equivocal; by 2013 criteria, 320 cases were non-amplified, 9 cases were equivocal and 3 cases were amplified. Specifically, 1 case which was non-amplified and 2 cases which were equivocal changed to amplified
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(0.9%), 9 cases which were non-amplified changed to equivocal (2.7%), and 1 case which was equivocal changed to non-amplified. Conclusions: Our data suggest that: 1) the eligibility for HER2-targeted therapy of a small number of previously tested patients will be affected by the implementation of the 2013 ASCO/CAP HER2 testing guidelines (previously ineligible patients will become eligible); 2) in the majority of patients, the interpretation will not change; and 3) the most common change will be from nonamplified to equivocal. ST33. A Non-Bisulfite Based DNA Methylation Assay to Detect MGMT Promoter Methylation L.M. Sabatini1, J.A. Nowak1, M. Bouma1, M. Krispin2, J.E. Petrisko2 1NorthShore University HealthSystem, Evanston, IL; 2Zymo Research, Irvine, CA. Introduction: MGMT encodes the DNA repair protein, O6-methylguaninemethyltansferase, which plays a critical role in genome stability. MGMT promoter methylation has recently been established as an important biomarker in patients diagnosed with glioma. Gliomas with inactivation of MGMT are less capable of repairing DNA leading to increased sensitivity to DNA alkylating agents. MGMT promoter methylation also correlates with prolonged overall survival and is a predictor of radiographic tumor pseudoprogression following treatment. Current strategies to evaluate methylation status typically rely upon bisulfite conversion of tumor DNA, which can be highly challenging. We describe a qPCR assay to investigate MGMT promoter methylation without the need for bisulfite conversion. Methods: The assay targets the first exon and enhancer of MGMT. This region, critical for loss of MGMT expression, is commonly interrogated in a variety of clinical assays. FFPE tissue sections are reviewed by a pathologist before microdissection and DNA extraction (Pinpoint Slide DNA Isolation System, Zymo Research). The assay relies on the digestion of unmethylated DNA with methylation-sensitive restriction enzymes (MSRE) leaving methylated DNA intact. The digestion is immediately followed by qPCR (OneStep qMethyl-Lite, Zymo Research). The level of DNA methylation is calculated based on the ∆Ct between test reactions (containing MRSEs) and reference reactions (without digestion). Results: DNA was isolated from 23 previously tested GBM (14 unmethylated, 9 methylated) and 8 non-neoplastic brain specimens. Non-methylated MGMT ranged from 0.1% to 10.1%, whereas methylated MGMT ranged from 32.6% to 58.4%; two samples reported as methylated gave methylation values of 5.8% and 7.4% and were considered non-methylated by this assay. The methylation status of the 8 non-neoplastic samples averaged 6.9% +/-4.5. One sample was unusually high (15.3%) and upon review showed evidence of marked hypoxia. Omitting this specimen yielded an average methylation of 4.5% +/-2.5 in normal brain tissue. Based on reproducibility studies (CV=20%) that calculated methylation levels will be affected by admixtures of tumor and normal tissues, and that the clinical significance of intermediate levels is currently unclear, a “gray zone” was established for this assay. Cut-off values were set as: 20% = methylated. Conclusions: As evaluation of MGMT promoter methylation status has become routine in the management of patients with glioma, a standardized assay format, which is simple, robust, and adaptable for low to high throughput laboratories is needed. We have developed and validated a non-bisulfite based qPCR assay to detect MGMT promoter methylation. ST34. Whole Exome Sequencing of Angiosarcoma Identifies Novel Variants J.N. Rosenbaum1, M.A. Accola1, D.M. Pavelec2, J.J. Laffin3, K.A. Johnson1, J. Hasenstein1, K.R. Kozak1, W. Rehrauer1, D. Buehler1 1University of Wisconsin Hospital and Clinics, Madison, WI; 2University of Wisconsin, Madison, WI; 3University of Wisconsin School of Medicine and Public Health, Madison, WI. Introduction: Angiosarcoma is an aggressive sarcoma with vascular differentiation, the main risk factors for which include prior radiation therapy (RT) and lymphedema. Surgery remains the mainstay of treatment, as RT and conventional chemotherapy are only mildly effective. Given the very poor prognosis of angiosarcoma, particularly in metastatic disease, novel systemic therapeutic targets would be extremely valuable. Genetically, angiosarcoma is complex with molecular mechanisms of pathogenesis remaining poorly understood. Whole exome and subsequent targeted sequencing permits identification of novel somatic mutations in angiosarcoma, comprehensive exploration of known or presumptive “actionable” targets, and identification of potential new therapeutic targets. Methods: Thirteen cases of angiosarcoma were obtained from surgical pathology tissue archives at the University of Wisconsin Hospital and Clinics (UWHC). DNA was isolated from both tumor and non-neoplastic deparaffinized tissue for each case after microdissection. Whole exome sequencing was performed on a Illumina HiSeq2000 following exome capture using Agilent Sure Select reagents. Briefly, somatic genetic variants were annotated and filtered using Cartagenia 1) relative to the paired non-neoplastic exome, 2) polymorphism databases (dbSNP, 1000 genomes, ESP 6500) 3) cancer gene databases (COSMIC, MyCancerGenome, BRCA database, Biomarkers with known Drug Association), 4) HGMD public and ClinVar and 5) potential coding effect. Results: Whole-exome sequencing wasperformed on 13 angiosarcomas (8M; 5F; median age 69 years, range 55 to 82 years), including 9 primary, 3 radiation-associated and one lymphedema-associated. Myc amplification was present in four cases. Sequencing generated 69,336,308/71,912,236 reads from thirteen normal/tumor pairs, with average depths of exome coverage of 48.54/51.25, respectively. We identified the following gene mutations related to tumorigenesis in two or more angiosarcomas: HCLS1 (6/13), PRB1 (4/13), TP53 (3/13), PRSS1 (3/13), and YBX2 (3/13), CYP2A6 (2/13), TNNT2 (2/13), and OPN1LW (2/13). HCLS1, the most
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AMP Abstracts
commonly mutated gene, has been previously associated with hematologic malignancies and malignant mesothelioma but has not been previously reported in angiosarcoma. We examine the significance of the identified mutations, as well as variant genes involved in angiogenic pathways implicated in tumorigenesis for angiosarcoma. Conclusions: Whole exome sequencing of angiosarcoma specimens offers a powerful tool for elucidating the biology of these clinically challenging tumors. The identification of previously undescribed mutations and tumorigenic pathways will expand our ability to diagnose, prognosticate, and treat angiosarcoma. ST35. A Novel Approach to Translocation Detection with Next-Generation Sequencing Using Formalin-Fixed, Paraffin-Embedded Sarcomas C.L. Baker1, D. Nix2, D. Baker1, D. Fisher1, C.P. Vaughn1, W.S. Samowitz1, A.H. Grossmann1 1ARUP Laboratories, Salt Lake City, UT; 2Huntsman Cancer Institute, Salt Lake City, UT. Introduction: Detection of diagnostic translocations in sarcomas is significantly limited by a lack of reagents and tools to query the wide variety of gene rearrangements that occur within these tumors. Using a next-generation sequencing (NGS) capture enrichment assay that included probes for most of the known sarcoma translocation genes, we asked whether we could develop, using a blinded approach, a bioinformatics pipeline to detect known translocations in which either both or only one of the gene partners was targeted. Methods: We designed a 2.7 Mb Agilent SureSelect hybrid capture array targeting exons and introns of 38 genes involved in various sarcoma translocations. DNA was extracted from two non-tumor samples, one Ewing sarcoma, and one Low Grade Fibromyxoid Sarcoma (LGFMS). Each were formalin-fixed, paraffin-embedded (FFPE). The sarcoma translocations were previously defined with RT-PCR followed by Sanger sequencing. The capture array contained probes for both known translocation partners in the Ewing’s sarcoma whereas only one gene partner was represented for the LGFMS. The library preps were performed in triplicate and sequenced with Illumina HiSeq 2000, 2 X 100 paired-end reads. Sequences were aligned with Novoalign2 twice; both with the full 101bp reads and with reads trimmed to 30bp. The trimmed reads render alignments that both span and flank translocation breakpoints, facilitating junction assembly. The sequences were then parsed by SamSVFilter from USeq into categories of varying significance to structural variation. DELLY was employed for split-read analysis, translocation detection, and final interpretation. Results: The t(21;22)(q22:q12) EWSR1-ERG translocation was detected in all three Ewing sarcoma replicates. Additionally the t(7; 16)(q33:p11) FUS-CREB3L2 translocation was detected in all three LGFMS replicates. The correct translocation in each of the replicas was the only translocation found to pass DELLY’s default thresholds. No significant translocations were found in the normal controls. Thus, the filtered alignments and DELLY pipeline operated with a 100% true positive rate and 0% false discovery rate. Conclusions: We have designed a unique approach to translocation detection by NGS that reliably and specifically predicts diagnostic gene rearrangements, without false positives, in a small sample set. Importantly, this approach was able to detect rearrangements when only one of the known translocation partners was queried, suggesting the potential to detect alternative or unknown gene partners. Moving forward, it will be important to challenge this approach with a larger number and variety of tumors with translocations, including tumors other than sarcoma. ST36. Validation of a Precision Next-Generation Sequencing Assay Designed for Oncology Specimens with Low Input DNA E. Azzato1, D. DeSloover1, S. Sukhadia1, A. Fox1, D. Roth1, J. Morrissette2, R. Daber1 1Hospital of the University of Pennsylvania, Philadelphia, PA; 2University of Pennsylvania, Philadelphia, PA. Introduction: Most next-generation sequencing (NGS)-based clinical oncological testing has focused on sequencing all or most of the exons of clinically-relevant and actionable genes; however, the major drawback is high DNA input requirements, which may not be met when only scant tumor material is available, such as with fine needle aspirate specimens. For example, with our current testing methodology (Illumina TruSeq Amplicon Cancer Panel (TSACP)), approximately 5% of all specimens that come to our laboratory have insufficient amplifiable DNA quantities to meet quality control requirements. Given the importance of molecular profiling on all specimens that enter the clinical laboratory, we sought to develop a focused NGS panel that captures hotspot amplicons and tumor suppressor exons, while requiring ≤10 ng input DNA. Methods: The precision panel targets 20 cancer-associated genes containing clinically significant variants involved in solid tumor prognostication and/or therapy selection. Thirty-four solid tumor specimens derived from formalin-fixed, paraffin-embedded samples (≥20% tumor) were chosen for validation. 0.5ng to 10ng of DNA was amplified with content-specific PCR primer pools then subsequently sequenced on a MiSeq with paired end 2 by 185 base pair reads to an average read depth of >6500x (Illumina, Inc.). Variants were detected using an in-house analysis pipeline. Sensitivity and specificity were assessed using results from the previously validated TSACP as the standard. Results: DNA extraction, NGS library prep and sequencing were successful on all specimens. Compared to TSACP, sensitivity of the precision panel was 99.3% (149/150) and specificity was 99.9% (8427/8428). One false negative was detected, secondary to a rare germline variant located under a primer site; the single discrepant positive was due to a low level variant detected below the reporting threshold of TSACP. Variant allele frequency (VAF) was highly concordant across both assays (r=0.99, p5% was 100%. In a theoretical comparison of our laboratory’s first year experience of routine
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AMP Abstracts clinical testing using TSACP (955 solid tumor specimens) with regions covered by the targeted assay, the precision panel would have captured 88% of pathological variants reported by TSACP. Conclusions: We developed a precision panel optimized for solid tumor specimens with ≤10 ng DNA that captures a high percentage of clinically relevant variants covered by the 48 to 50 gene commercially available NGS panels. The clinical utility for our precision panel lies in the ability to reflex test specimens with insufficient input DNA for larger panels. ST37. Validation of TruSight Tumor Panel Next-Generation Sequencing (NGS) Assay in Formalin-Fixed, Paraffin-Embedded (FFPE) Specimens Q. Ren, J. Haney, k. Jones, K. Gowan, P.H. O'Neill, G. Bocsi, D. Merrick, M. Seager, B. Siegele, W.A. Franklin, D.L. Aisner University of Colorado, Aurora, CO. Introduction: Characterizing the mutational spectrum of clinical tumor samples provides critical information for targeted therapy selection. Single gene approaches can be effective, but often require high DNA input, exhaust available tissue, and provide answers for a limited number of analytes. NGS provides an alternate approach to simultaneously detect alterations in multiple analytes. Here we present data on the validation of TruSight Tumor (Illumina, Inc.), a targeted sequencing panel examining selected regions of 26 tumor-related genes. Methods: Validation studies were performed using cell line dilution studies, commercially available reference material, and previously tested clinical samples. Sample assessment was performed using qPCR to determine sample integrity. Library preparation was performed with modifications to enhance ability to utilize low input quantities and sequencing was performed on the Illumina MiSeq. Data analysis was performed using custom-designed bioinformatic pipeline enabled by a web interface. Quality metrics were established utilizing Q30 scores, % reads mapped, and other quality metrics. Inter-run, inter-operator and intraoperator variability was assessed. Custom bioinformatics adjustments were implemented to reliably detect insertions and deletions and changes adjacent to priming regions. Results: Dilutional studies of well characterized cell lines demonstrated strong concordance between expected and observed allelic frequency based on somatic mutations, and single nucleotide polymorphisms. Evaluations utilizing commercially available reference material confirmed these observations. Inter- and intra- operator variability was evaluated and determined to be negligible. Optimization of input quantity demonstrated the ability to perform the assay on input quantities as low as 60ng, depending on DNA quality. For previously evaluated clinical samples, comparison with previous results was performed. Out of 51 total samples (22 lung, 3 GIST, 2 thyroid, 14 melanomas and 10 colon) 93 total mutations were identified, 76 of which were point mutations, 9 of which were insertions and 8 of which were deletions. Of these 93 total mutations identified, 79 had been previously determined from prior clinical analysis. The remaining 14 mutations were confirmed using an orthogonal technique (Sanger sequencing) for a final concordance of 100%. A custom web-based interface facilitated analysis, database generation, and curation of identified mutations once assay validation was completed. Conclusions: Targeted sequencing using next-generation sequencing is feasible, accessible and paradigms exist for validation in the clinical laboratory for routine reporting. Validation of the Illumina TruSight Tumor indicates the feasibility on moderate quantities of nucleic acid, and future studies will aim to evaluate a larger panel of target analytes. ST38. Incidental Detection of Myelodysplastic Syndrome by Germline NextGeneration Sequencing Cancer-Risk Panel Testing E.Q. Konnick1, M. Miller2, M. King1, T.D. Walsh1, A.L. Jacobson1, L.I. Thomas1, D. Wu1, J.F. Tait1, C.C. Pritchard1, B.H. Shirts1 1University of Washington, Seattle, WA; 2Providence Alaska Medical Center, Anchorage, AK. Introduction: Recent attention has been drawn to the reporting of incidental findings that are discovered during germline testing for a separate indication. This discussion has gained prominence due to the expansion of panel, exome, transcriptome and whole genome testing facilitated by next-generation sequencing (NGS) technology. However, the possibility of uncovering a previously unidentified neoplastic process as an incidental finding in germline testing has not been well-addressed. Methods: NGS testing was performed using the 51-gene BROCA comprehensive cancer risk panel to sequence DNA isolated from peripheral blood of a 74-year-old woman with a personal history of ovarian cancer and a family history of breast, stomach, pancreatic, liver, and hematopoietic cancers. The sample tested was DNA extracted from whole peripheral blood. Targets of interest were enriched using the Agilent SureSelect custom BROCA array, and sequenced on an Illumina HiSeq to an average depth of 463x. The data analysis pipeline included detection of single nucleotide variants, small insertion/deletions, copy number variants (CNV), and structural variants. CNV calling was accomplished by determining copy number states for individual targets, then comparing read-depth for each exon against a reference sample. CNVs were called on an exon-by-exon basis and visualized using the R package ggplot2. Results: BROCA testing revealed a deleterious TP53 mutation (p.R273H) at an allelic fraction of 64% (297/461 sequencing reads) and partial copy loss at six loci: APC, RAD50, and CTNNA1 on chromosome 5q; SMAD4 on chromosome18; and POLD1 and STK11 on chromosome19. This combination of findings was suspicious for a neoplastic hematopoietic process. Subsequent investigation confirmed a diagnosis of myelodysplastic syndrome (MDS) with cytogenetic results including 5q-, concordant with the findings from BROCA sequencing. Conclusions: To our knowledge this is the first description of MDS identified as an incidental finding in the setting of germline
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cancer risk assessment. Our findings demonstrate that cancer risk testing can reveal hematopoietic neoplasms, and that the possibility of somatic mutations should be considered in test interpretation, especially in older patients. Careful attention to variant allele fraction and CNV analysis can provide helpful clues. When a hematopoietic neoplasm is suspected, recommendations for appropriate diagnostic workup should be communicated by the laboratories providing these results. ST39. ROS1 Immunohistochemistry among Major Genotypes of Lung Adenocarcinoma: A Practical Method to Detect ROS1 Gene Rearrangements T.A. Boyle1, K. Masago2, K.E. Ellison1, C. Rivard1, Y. Yatabe3, F.R. Hirsch1 1University of Colorado-Denver, Aurora, CO; 2Institute of Biomedical Research and Innovation, Kobe, Japan; 3Aichi Cancer Center, Nagoya, Japan. Introduction: ROS1 gene fusions cause several cancers by constitutively activating the ROS1 tyrosine kinase receptor. ROS1-targeted inhibitor therapy improves survival in the approximately 1-2% of patients with lung adenocarcinomas with ROS1 gene fusions. Although fluorescence in-situ hybridization (FISH) is the standard diagnostic procedure for detecting ROS1 rearrangements, we aimed to study immunohistochemistry. Methods: ROS1 immunohistochemistry was initially performed on a “selected cohort” of 33 lung adenocarcinoma whole tissue specimens with alterations in the EGFR (N=5), KRAS (N=5), ERBB2 (HER2) (N=3), ROS1 (N=6), ALK (N=5), and RET (N=3) genes and pan-negative (N=6) detected by reverse transcriptase-polymerase chain (RT-PCR) reaction and FISH. We next used immunohistochemistry to screen 355 microarray lung cancer specimens for ROS1 expression. Results: In the selected cohort of 33 specimens, both ROS1 gene fusions and high ROS1 protein expression were detected in 6 specimens. Five of these 6 specimens were also positive by FISH for ROS1 gene rearrangements. All 27 lung cancer specimens negative for ROS1 rearrangements by genetic testing had no to low ROS1 protein expression. In screening cohort of 355 microarray specimens, 19 of 349 evaluable specimens demonstrated low ROS1 protein expression (H-score between 10 and 100) and 4 had high expression. Of the 4 specimens with high expression, 1 was positive and 2 were negative for ROS1 rearrangements by RT-PCR and FISH, and 1 was positive for an EGFR mutation so was not tested by RT-PCR or FISH. Conclusions: We have optimized ROS1 immunohistochemistry and scoring to provide high sensitivity and specificity for detecting ROS1 gene rearrangements in whole tissue. Tissue preparation should be optimized to maintain antigenicity and reduce falsenegative results. ROS1 immunohistochemistry is a practical and cost-effective method to screen for ROS1 gene rearrangements. ST40. Barrett’s Esophagus Has a Mutation Frequency Comparable to Invasive Cancers but Lacks Oncogene Activation M.D. Stachler1, S. Carter2, A. Taylor-Weiner2, S. Peng3, G. Getz2, A. Bass3 1Brigham and Women's Hospital, Boston, MA; 2Broad Institute, Cambridge, MA; 3Dana Farber Cancer Institute, Boston, MA. Introduction: Esophageal adenocarcinoma (EAC) is a deadly disease that emerges out of a premalignant condition called Barrett’s Esophagus (BE). As most patients with EAC are diagnosed with incurable disease that has already disseminated, there is great importance to understanding how EAC develops from BE and what markers could allow the diagnosis of patients with the highest risk of progression to cancer. Methods: The genomic status of 25 paired samples of BE and EAC were systematically characterized using whole exome sequencing. Results: The average mutation frequency was 2.82, 4.88, and 4.14 mutations/MB for non-dysplastic BE(ndBE), dysplastic BE(dysBE), and EAC respectively (P=NS). The high mutation rate seen even in ndBE is comparable to many invasive cancers. Interestingly, only 14 out of the 25 pairs shared mutations between the BE and paired EAC. The unrelated BE contained a mutational profile which was very similar to cases in which the BE and EAC was highly related. Copy number analysis revealed frequent deletions in BE which tended to increase with increasing diagnosis (9.9, 12.3, 15.4 mean deletions/sample for ndBE, dysBE, and EAC respectively, P=0.03 for ndBE vs EAC). BE harbored frequent loss of CDKN2A and TP53 as well as other tumor suppressor genes. Given that EAC frequently contains amplifications of common oncogenes instead of mutations, we also analyzed the BE for amplifications. Compared to EAC, BE had significantly less mean number of amplified regions than EAC (0.36 and 1.27 for ndBE and dysBE vs. 10.96 for EAC, P500x coverage. Variants were identified using the Ion Torrent Variant Caller Plugin (v4.0) and reference genome hg19. Golden Helix's SVS software (v7.7.8) was used for annotation of the variants, as well as prediction of the significance of the variants. Results: All variant calls were initially filtered to remove non-coding and synonymous mutations. Golden Helix was then used to annotate variants and help predict pathogenicity. All reported variants were covered at greater than 500X, and had an allelic frequency >5.0%. Only 17 cytology samples failed QC due to low DNA concentrations. Of the remaining samples, 97 had clinically actionable mutations (EGFR, KRAS, BRAF, ERBB2 or PIK3CA), 63 had variants of unknown significance and 26 were wild type. The most frequent mutations identified were in the following genes, TP53 (32%), KRAS (30%), EGFR (12%), and STK11 (10%). Conclusions: The Ion Torrent Ampliseq technology can be successfully utilized on small biopsy and cytology specimens, requires very little input DNA, and can be readily applied in clinical laboratories for genotyping of NSCLC. ST50. Towards Realizing the Dream of Precision Medicine for Meningiomas Using A Molecular MicroRNA Approach S. Sethi1, S.K. Michelhaugh2, A. Ahmad2, R. Ali-Fehmi2, W. Chen2, S. Sethi2, S. Mittal2, F. Sarkar2 1Wayne State University School of Medicine, Troy, MI; 2Wayne State University, Detroit, MI. Introduction: Meningiomas are among the most common primary intracranial tumors. High-grade meningiomas are particularly aggressive tumors with significant recurrence rates (up to 80%) following surgery and radiation therapy, and have a dismal prognosis. One of the largest obstacles in the development of novel targeted therapies for meningiomas is our limited understanding of its molecular pathogenesis. Recently described microRNAs (miRNAs) are novel tiny endogenous molecules which critically regulate tumor invasion, progression and metastasis. Identifying altered miRNAs in meningiomas can assist in early detection and determining prognosis. Modulating altered miRNAs would be a novel therapeutic strategy to achieve the goal of personalized medicine for meningiomas. Methods: Cases of meningioma (n=60) diagnosed at our hospital were retrieved from the computerized hospital database. These included 22 WHO grade I benign meningiomas, 19 grade II atypical tumors and 18 grade III malignant meningiomas. Additionally 20 normal brain tissues were included as normal controls. RNA was extracted from representative tissue blocks. Profiling was done for a total of 1474 miRNAs using microfluidic biochip assays (LC Sciences). Validation was done using quantitative real-time polymerase chain reaction (qRT-PCR). Data was statistically analyzed using the Anova test. Each miRNA from meningioma grade I to III was analyzed using linear regression analysis. Results: Expession profiling revealed significant altered expression of 21 miRNAs in meningiomas compared to normal brain tissues (p500x coverage/exon. 75/91 unmatched samples (83.3%) had 180bp copy number >400, 87bp >2500. 96.3% exons sequenced successfully (1.6 failed exons/sample); those with Q30 and 134.5 Gb at 70.4% >Q30. Whole genome data filtered by exome bed file coordinates was intersected with NIST NA12878 data. 66,975 variants were detected in whole genome data, with 66,492 >4-fold (99.3%) and 65,232 >8-fold coverage (98.1%), average 21-fold coverage. The NIST high confidence data contains 55,176 variants in these regions, where 53,958 (97.8%) were concordant between whole genome data and NIST data, and 49,526 (89.8%) were concordant between the exome data and NIST data. The copy number gain/loss results were concordant with high density SNP array. Conclusions: Preliminary analysis demonstrates an approximate 98% concordance between variant
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calls derived from NextSeq500 whole genome sequencing of NA12878 and high confidence NIST NA12878 variant data. More sophisticated analysis comparing different alignment and variant calling algorithms to improve alignment and variant call accuracy and speeds will be presented. Further analysis is underway to determine the cause of discordants between NA12878 NIST and whole genome data. Improvements in the speed of analysis and quality metrics will improve turn around time, and may preclude the requirement of Sanger verification for a subset of variants. TT65. Milling Microdissection Ensures Reliable Tumor Content for Molecular Testing without Sacrificing Yield K. Geiersbach1,3, N. Adey2, N. Welker3, D. Elsberry3, E. Downs-Kelly1, M. Salama1, M. Bronner1 1University of Utah School of Medicine, Salt Lake City, UT; 2AvanSci Bio, Salt Lake City, UT; 3ARUP Laboratories, Salt Lake City, UT. Introduction: Molecular genetic testing on formalin-fixed, paraffin-embedded (FFPE) tumors frequently requires dissection of regions of tumor from tissue sections mounted on glass slides. Traditional manual dissection may be inadequate for ensuring adequate tumor content in specimens with small foci of tumor cells surrounded by abundant stromal and inflammatory cells. We compared traditional manual dissection with a new method, milling microdissection, on a series of 32 FFPE pancreatic cancer samples. Methods: An H&E stained slide from each block was marked with a pen to guide manual dissection. For milling microdissection, regions of tumor measuring 200 microns or greater in width were marked digitally on the same slide within the areas marked for manual dissection. Neoplastic cellularity was estimated independently by three pathologists. Sequential slides from the same block were used for manual dissection and milling dissection. Manual dissection was performed using a scalpel and using the marked H&E slide as a guide. Milling microdissection was performed with the MilliSect instrument (AvanSci Bio). Dissected sample pairs (manual / milling) were compared for KRAS genotype and mutant allele fraction. KRAS hotspot mutation testing was performed using Sequenom MassArray technology. Results: Milling microdissection was sufficient to increase the neoplastic cellularity to 50% or higher for all specimens. KRAS mutations were detected in 26/32 sample pairs; seven KRAS mutated samples showed a detectable mutation with milling microdissection but not with traditional manual dissection. DNA yield and quality were comparable for both dissection methods. Conclusions: We have demonstrated a significant improvement in tumor content achievable by milling microdissection. Increased precision of tumor dissection allows for testing of samples that would otherwise be unacceptable for testing using traditional manual dissection. TT66. Silicon Bead-Based DNA Extraction Method Rescues Paraffin-Embedded Tissue Samples Containing Inhibitory Melanin for Use in Down-Stream DNA Polymerase-Based Applications M. Rutherford, A. Marolt, N. Laidley, S. Reid, R. McClure Health Sciences North, Sudbury, Ontario, Canada. Introduction: It is known that DNA and RNA extracted from tissue containing melanocytes (skin, malignant melanoma, hair) can contain sufficient melanin to inhibit down-stream applications. Melanin is a heterogeneous, pigmented polymer that is produced by epidermal melanocytes and is normally stored in melanosomes in the cytoplasm, such that it does not come into contact with nucleic acids or the majority of intracellular proteins. However, free melanin binds and inhibits the function of a variety of proteins, including DNA polymerase, and also intercalates into dsDNA. During DNA extraction, melanosomes are disrupted with release of melanin, which can be present in the final product in sufficient quantity to inhibit polymerase-based reactions. This is a relatively common clinical problem, particularly for surgical pathologists working with paraffin-embedded tissue samples containing pigmented lesions. This study evaluated 2 common DNA extraction methods for their ability to remove inhibitory melanin from paraffin-embedded skin samples containing malignant melanoma. Methods: DNA from 5 samples (2 heavily pigmented, 1 moderately pigmented, 2 non-pigmented) was extracted using a modified “salting out” method (Puregene from Gentra) and a siliconbead adherence method (EZ1 biorobot from Qiagen). Extracted DNA was tested in a shifted termination primer-extension assay (TrimGen) for detection of BRAF V600 mutations. DNA from the Puregene method was tested undiluted and at 1:10 and 1:20 dilutions; DNA from the EZ1 was tested without dilution. Results: DNA extracted using the Puregene method performed as follows: heavily pigmented samples: unsuccessful in all challenges; moderately pigmented sample: unsuccessful at full strength and 1:20 dilution, successful at 1:10 dilution; unpigmented samples: both successful in all challenges. DNA extracted from all samples using the EZ1 biorobot performed successfully in the BRAF assay. DNA solutions that were unsuccessful in the challenge assay had a brown color, assumed to be due to retained melanin. Conclusions: Regarding DNA extraction from paraffin-embedded samples containing melanin pigment: Use of a silicon-bead based method (EZ1 biorobot) produced DNA that performed successfully in a DNA polymerase-based assay challenge (BRAF V600 mutation assay) without further dilution; use of the “salting out” precipitation method (Puregene) produced DNA that retained sufficient melanin to be inhibitory. Thus, selection of a silicon bead-based DNA extraction method for samples containing melanin can rescue the DNA for successful use in down-stream, polymerase-based applications.
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AMP Abstracts
TT67. Estimating Contamination in Multiplexed Clinical Sequencing Assays Using Read Haplotypes at Highly Informative SNP Pairs D.H. Spencer, H. Abel, J.K. Sehn, M. Tyagi, A.J. Bredemeyer Washington University, St. Louis, MO. Introduction: Clinical next-generation sequencing assays are typically performed in multiplex, where several samples are processed in parallel and pooled together for sequencing, which creates the potential for cross-contamination at multiple steps. CAP guidelines require appropriate controls to assess for contamination, but effective methods have not been established. Here we describe haplotect, a novel method for detecting and quantifying cross-contamination that is based on analyzing single nucleotide polymorphism (SNP) haplotypes observed in individual sequencing reads, which can be applied to existing sequencing assays without additional controls or modifications. Methods: Haplotect assesses contamination using closely spaced SNP pairs with low linkage disequilibrium that exist in >2 haplotypes in the population. Contamination is suggested if >2 haplotypes are observed in the sequencing reads from a single sample, which is incompatible with having been derived from one diploid individual. We identified informative SNPs in the 1000 genomes database that overlap an existing 26-gene targeted sequencing assay performed at our institution. Contamination analysis was retrospectively performed on 41 samples, including 7 Hapmap samples, 16 laboratory-derived Hapmap mixes with defined proportions (5%, 10%, 20%, and 50%), and 17 lung cancer samples. Read haplotypes were obtained and contamination was estimated from the haplotype frequencies using haplotect. The verifyBAMID program for estimating contamination from variant allele fractions at known SNP positions was also run on each sample using parameters appropriate for standalone contamination assessment. Results: We identified 288 SNP pairs that met the criteria for read haplotype analysis. The mean number of SNP pairs with sufficient coverage for analysis was 170 (+/- 36 s.d.) across all 41 samples, and >2 haplotypes were identified at a mean of 24.5 (+/- 7.3 s.d.) loci in the 16 mixed samples. Contamination estimates were >5.8% for the mixed samples and agreed with the expected mix proportions (r-squared: 0.85); the mean contamination for pure Hapmap and lung cancer samples was 0.5% (range: 0% to 1.8%). Accurate contamination estimates were also obtained for the mixed samples using verifyBAMID (r-squared: 0.81), but estimates were inflated for lung cancer samples (mean: 25.6%), likely due to skewed allele fractions in regions with copy number changes. Conclusions: In this proof-of-concept study we show that contamination can be estimated using haplotype analysis of highly informative SNPs that occur by chance within regions targeted by an existing sequencing assay. This method performed favorably compared to verifyBAMID, and appears to be better suited for analysis of cancer samples with somatic copy number mutations. TT68. Examination of HER2 Overexpression and Amplification in Urothelial Carcinoma with Micropapillary Features Using ASCO/CAP 2013 Guideline H. Chen, R. Broaddus, L. Huo, M. Zhang, C. Guo, M. Robinson, S. Trivedi, R. Fernandez, N. Navai, X. Lu University of Texas M.D. Anderson Cancer Center, Houston TX. Introduction: Human epidermal growth factor receptor 2 (HER2) overexpression/amplification is known to predict a better response to HER2-targeted therapy. HER2 overexpression and amplification have been reported in bladder cancer, although less frequent than in breast cancer. The guideline for HER2 interpretation in breast cancer established by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) in 2007 and other variable interpretation criteria have been used for most urothelial carcinoma HER2 studies, although limited studies in bladder cancer have been reported. We examined HER2 overexpression and HER2 amplification status in bladder cancer using the 2013 ASCO guideline for breast cancer to assess the concordance between HER2 overexpression and HER2 amplification. Methods: We included twenty archived urothelial carcinomas with micropapillary features for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) studies to evaluate HER2 overexpression and HER2 amplification respectively. FISH was performed with PathVysion HER2 and CEP17 dual-probe (Abbott Molecular, Des Plaines, IL) following standard procedures provided. The ASCO/CAP 2013 recommendation for breast cancer and CAP 2009 guidelines for genetic heterogeneity in breast cancer were utilized for data analysis and interpretation on both IHC and FISH testing. Results: All twenty cases were successful in IHC study (100%) and eighteen cases were successful in FISH study (90%). HER2 amplification (HER2/CEP17 ratio≥2.0) was observed in six cases (30%). Among these six HER2 amplified cases, four cases were positive for HER2 overexpression (3+) and two cases were equivocal for HER2 overexpression (2+). Four cases (20%) showed equivocal HER2 amplification (HER2/CEP17 ratio
