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Introduction: The clinical diagnosis of myelodysplastic syndrome (MDS) relies on the presence of persistent cytopenias, not otherwise explained, and evidence of morphologic dysplasia in the bone marrow. Low grade MDS (bone marrow blasts Methods: We screened patients who presented for evaluation of MDS between 2002 and 2014 that had consented for sequencing studies and had banked samples. Patients were selected based on 1) World Health Organization defined cytopenia (WBC Results: Thirty-eight patients met the selection criteria, and 30 of these had bone marrow aspirates available for morphologic review and were included in the study. A mean unique coverage depth of 913x was achieved for targeted genes and all reported variants had >50x coverage, variant allele fractions (VAFs) >3%, and minor allele frequencies (MAFs) < 1% in any population. Of the 30 sequenced cases, 25 had a somatic mutation in at least one gene (mean 3.3 mutations/case, range 1-10 mutations/case). The most commonly mutated gene was TET2 (7 cases), followed by ASXL1 (5 cases), EZH2 (4 cases), SRSF2 (4 cases), and U2AF1 (4 cases). Of the 285 sequenced genes, 44 were mutated in at least one case, and 14 were mutated in 2 or more cases. The mean VAF (variant reads/total reads) of detected mutations was 27% (range 3-98%). Morphologic review demonstrated definitive dysplasia (≥10% of cells in least one lineage) made by two pathologists in 18 of 30 cases (supporting the clinical diagnosis of MDS), no dysplasia in 6 of 30 cases, and equivocal dysplasia (where hematopathologists did not agree that dysplasia was ≥10%) in 6 of 30 cases. Thirteen of 18 cases (72%) with definitive dysplasia had a mutation, 5/6 cases (83%) without dysplasia had mutations, and 6/6 (100%) cases with equivocal dysplasia harbored somatic mutations. The mean VAF of mutations was 17.5% in cases without dysplasia, 29% in cases with equivocal dysplasia, and 28% in cases with definitive dysplasia. All of these groups included mutations in canonical MDS genes such as TET2, DNMT3A, SRSF2, RUNX1, and EZH2. Conclusions: In this cohort of 30 cytopenic patients with normal cytogenetics, 80% harbored a somatic mutation in at least one myeloid-associated gene. Somatic mutations were detected in 5 of 6 cases without definitive dysplasia ( Disclosures Duncavage: DI&P Consulting: Consultancy; Cofactor Genomics: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy.

The Genomics Tumor Board at Washington University was established to increase knowledge, competence, and performance in the application of genomic testing in cancer care. Here we report the findings from a Tumor Board case of concurrent germ cell tumor and acute myeloid leukemia. A 33-year old male presented with generalized weakness, weight loss, and dyspnea on exertion. Initial workup was notable for a platelet count of 5,000 platelets/μL, hemoglobin of 13.1g/dl, and a white count of 9,200 cells/μL with a normal differential. His AFP was 237 (ULN 8.1 ng/ml), LDH was 6760 U/L (ULN 250 U/L) and β-hCG Citation Format: Charles Lu, Peter Riedell, Peter Westervelt, Christopher Miller, Ian S. Hagemann, Eric J. Duncavage, Elaine R. Mardis, Richard K. Wilson, Bradley A. Ozenberger, Lukas D. Wartman. Exome sequencing identifies common somatic mutations in an adult patient with a concurrent germ cell tumor (GCT) and acute myeloid leukemia (AML) suggesting a single clonal origin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2015-LB-109

The tumor suppressor p19(Arf) (p14(ARF) in humans), encoded by the Ink4a/Arf locus, is mutated, deleted, or silenced in many forms of cancer. p19(Arf) induces growth arrest by antagonizing the activity of the p53-negative regulator, Mdm2, thereby inducing a p53 transcriptional response. p19(Arf) can also inhibit cell cycle progression of mouse embryo fibroblasts lacking Cip1 or lacking both Mdm2 and p53, although in the absence of p53, arrest occurs more slowly. Profiling with high-density oligonucleotide GeneChips and cDNA microarrays was used to interrogate mouse genes, the expression of which was induced or suppressed by a conditionally regulated Arf gene. Cluster analysis of temporal gene expression patterns and validation of the results by RNA analysis identified Arf-responsive genes whose induction was both p53-dependent and -independent. The latter included four members of the B-cell translocation gene family (Btg1, Btg2, Btg3, and Tob1) that were demonstrated to inhibit cell proliferation in primary mouse embryo fibroblasts expressing or lacking functional p53. Together, the results indicate that p19(Arf) induces a broad spectrum of proteins that likely act in concert to arrest cell proliferation.

We present the case of a 30 year-old man who was referred for evaluation of diffuse lymphadenopathy. Six weeks prior, he noticed darkening of his urine associated with pale stools, nausea and an eventual 30 lb weight loss within a month. The initial laboratory findings showed elevation of the liver enzymes. A CT scan showed mesenteric and periaortic lymphadenopathy with the largest lymph node measuring 2.8 cm. Other laboratory results were otherwise unremarkable (including a normal LDH) with the exception of positive serum antibodies against Epstein-Barr virus (EBV) associated antigens (IgM+ and IgG+). An excisional biopsy of 4 of the small neck lymph nodes showed a normal architecture with prominent follicles and an intact capsule. But, by immunohistochemistry two of the follicles showed aberrant coexpression of BCL-2, in addition to CD10 and BCL-6. In-situ hybridization for early Epstein-Barr virus mRNA (EBER) and immunohistochemistry for latent membrane protein-1 (LMP-1) stained both scattered positive cells, as well as BCL-2 positive B-cells. Although an original diagnosis of in-situ follicular lymphoma was favored at an outside facility, additional interphase fluorescence in situ hybridization (FISH) studies for t(14;18);(IGH-BCL2) rearrangement (performed on the BCL-2 + follicles microdissected from the tissue block; Abott probe dual colour fusion) and molecular studies (IGH gene rearrangement by PCR, also performed on the microdissected follicles) were negative. Serologic studies (positive EBV antibodies) and immunostains in conjunction with the molecular studies confirmed the reactive nature of the changes. Our case also shows direct immunopathogenic evidence of BCL-2 expression among the EBV-infected cells, which has to our knowledge not been previously documented in vivo. A diagnosis of EBV infection should, therefore, be considered when confronted with BCL-2 expression in germinal centers, particularly in younger individuals, as the diagnosis of FLIS may lead to extensive and invasive haematologic work-ups. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/ 1323656318940068 Case report A 30 year-old man was referred for evaluation of diffuse lymphadenopathy. 6 weeks prior, the patient noticed darkening of his urine associated with pale stools, nausea and an eventual 30 lb weight loss within a month. He also complained of fever, myalgias, joint pain, and fatigue, which occurred approximately 48 hours after the onset of the urine colour changes. The initial laboratory results showed elevation of liver enzymes (AST 278 Units/L, ALT 831 Units/L and total bilirubin of 1.9 mg/dl). The complete blood count (CBC) included the following results: WBC 8.4 (neutrophils 54.5%, lymphocytes 34.3%, monocytes 7.8%, eosinophils 2.5% and basophils 0.9%), Hgb 15.9, hematocrit 47.3, platelet count 151, LDH 179, RBC 5.12 MCV 92.5 and RDW 13.2. An abdominal ultrasound revealed a 2.9 cm mass within the pancreas and the liver. A follow-up CT scan showed mesenteric and periaortic lymphadenopathy with the largest lymph node measuring 2.8 cm. Two weeks later, the majority of the symptoms resolved, but the patient noticed new enlarged lymph nodes in the right neck and in the left groin, measuring less than 1 cm. No associated hepatosplenomegaly was identified. The patient's admission laboratory results were otherwise unremarkable (including a normal LDH) with the exception of positive serum

11099 Background: Next-generation sequencing (NGS) allows for simultaneous detection of numerous actionable somatic variants in cancer. We have implemented a clinical NGS panel to detect genetic alterations in 25 genes with established roles in cancer and report here the frequency of clinically actionable genetic variants in a variety of cancer types. Methods: NGS testing was performed in a CAP-certified, CLIA-licensed environment on DNA extracted from FFPE tissue in 209 cases spanning 41 histologic tumor types. DNA was enriched by hybrid capture and sequenced to >1,000x average coverage on Illumina sequencers with 2x101bp or 2x150bp reads. Variants were called using clinically validated parameters using the Genome Analysis Toolkit, Pindel, and the custom-written Clinical Genomicist Workstation. Results: Non-small cell lung cancer (45%), pancreatic cancer (10%), and colorectal cancer (8%) were the most common tumors sent for NGS analysis. An average of 3 (range 1- 16) non-synonymous, non-SNP sequence variants per case (SNVs and indels) were detected in the 130kb exonic target. Variants were most commonly seen in TP53, KRAS, and EGFR. 27% of cases (56/209) had one or more variants with therapeutic implications for the tumor type tested (e.g., EGFR mutation in NSCLC). 15% of cases (32/209) showed actionable variants not generally associated with the malignancy tested (e.g., detection of an activating KITvariant in thymic carcinoma). 10% of cases (21/209) had variants that were prognostically significant but not directly targetable. Some cases (9%) had variants that were prognostic/diagnostic and targetable. In 117 cases (56% of total), no therapeutically or prognostically significant variants were identified. Overall, in 92 cases (44%), NGS testing yielded information with therapeutic (majority), prognostic, or diagnostic ramifications. Conclusions: We found that 44% of unselected cancer cases have clinically relevant sequence variants in a set of 25 commonly mutated cancer genes. Our data suggest that clinical NGS testing may serve as an integral tool in realizing the potential of precision medicine in oncology.

We have implemented a clinical assay to perform next-generation sequencing (NGS) of twenty-five genes relevant to multiple cancer types. We report findings from 210 consecutive malignancies submitted to Genomics and Pathology Services at Washington University in St. Louis (GPS@WUSTL). DNA was extracted from formalin-fixed, paraffin-embedded tumor selected by histopathologic review. Up to 1 μg of DNA (minimum, 0.2 μg) was used for library preparation using liquid-phase cRNA capture probes targeting all exons of BRAF, CTNNB1, CHIC2, CSF1R, DNMT3A, EGFR, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MAPK1, MAP2K2, MET, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RET, RUNX1, TP53, and WT1 (302 kb). Libraries were sequenced by Illumina technology to obtain 101bp or 150bp paired-end reads. Mean coverage was 1596; 50x depth was achieved at >95% of positions. The analysis pipeline was validated for variant allele frequency >10%. Variants were called and interpreted using the Clinical Genomicist Workstation. A list of clinically actionable variants (“hot spots”) was curated by literature review. Of 210 cases, 94 (45%) were non-small cell lung cancer (NSCLC), 20 (9.5%) were pancreatic cancer (PCA), and 17 (8.1%) were colorectal cancer (CRC), the balance representing 38 diverse other malignancies. FFPE tissue cores yielded a median of 2.4 μg DNA (interquartile range 1.0-4.9 μg). Sequencing identified a mean of 5.4 non-synonymous coding and/or splice site variants per case (single-nucleotide variants and indels). Of these, a mean of 2.8/case were not known polymorphisms and likely represented somatic mutations. Somatic variants most commonly occurred in TP53 (233 variants in 107 cases), KRAS (52 variants in 51 cases), and EGFR (40 variants in 33 cases). Sequencing identified 74 clinically actionable (i.e., predictive or prognostic) mutations in 61/210 = 29% of cases. In NSCLC, actionable mutations were present in KRAS codon 12 (29/94 cases), EGFR exons 19 (5 cases), 20 (1 case) and 21 (5 cases), and the PIK3CA helical domain (4 cases). In PCA, there were no variants currently recognized as actionable, but one potentially targetable MET T1010I variant was found. In CRC, actionable mutations were present in KRAS codon 12 or 13 (6/17 cases), PIK3CA (3 cases) and KIT (1 case). Average turnaround time was 29 days. In conclusion, NGS-based diagnostics can provide clinically relevant information using readily available FFPE tissue. Actionable variants were identified in 29% of cases. The existence of targeted therapies suggests that such diagnostics have the potential to improve patient outcomes. Citation Format: Ian S. Hagemann, Hussam Al-Kateb, Catherine E. Cottrell, Christina Lockwood, TuDung Nguyen, David Spencer, Andrew Bredemeyer, Richard Head, Nobish Varghese, Rakesh Nagarajan, Karen Seibert, Eric J. Duncavage, Shashikant Kulkarni, John D. Pfeifer. Diagnostic yield of targeted next-generation sequencing for personalized cancer therapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 813. doi:10.1158/1538-7445.AM2013-813

Abstract 3547 Introduction: Recurrent somatic mutations are valuable prognostic markers in cytogenetically normal Acute Myeloid Leukemia (AML). The most common of these mutations is a 9 to ∼150 bp internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 (FLT3) gene, which is typically identified via PCR amplification and capillary electrophoresis. Since testing for individual mutations in this manner will become laborious and expensive as the number of clinically relevant mutations increases, we and others have proposed using targeted next-generation sequencing (NGS) for comprehensive detection of somatic mutations in multiple genes simultaneously. Successful application of this approach will require automated analysis methods capable of sensitive detection of a variety of mutation types, including single-base substitutions and insertions/deletions, with a low false-positive rate. However, the accuracy of current methods for identifying medium-sized insertions such as the FLT3 ITDs has not been established. Therefore, we sought to determine the ability of several common analysis tools to identify FLT3 ITDs from Illumina NGS sequence data. Methods: We performed targeted sequencing of 10 samples with known FLT3 ITDs ranging between 17 and 93 base-pairs (bp) as part of a larger test panel of 28 genes commonly mutated in AML and other malignancies. Nine of the FLT3 ITD-positive samples were from patients with newly diagnosed AML and were confirmed by PCR and capillary electrophoresis. A cancer cell line known to be heterozygous for a 30 bp FLT3 ITD, MV4-11, was also included. Indexed Illumina sequencing libraries were generated using automated library preparation and enriched for target regions using solution-phase hybridization-capture with biotinylated cRNA probes targeting exons +/− 200 bp plus 1 kb flanking the FLT3 gene and the 27 other genes in the panel. Enriched libraries were sequenced in multiplex on an Illumina HiSeq instrument using 2 × 101 bp reads. Demultiplexed reads were mapped to the hg19 reference sequence with novoalign, and indels were called in a 1 kilobase-pair region surrounding the FLT3 ITD with samtools, GATK, maq, CLC Genomics Workbench, PINDEL, and DINDEL using default parameters, in addition to de novo assembly of reads with partial similarity to the region using phrap. Insertion calls were then compared to results from PCR and capillary electrophoresis. Results: Multiplex sequencing resulted in 585 to 1,000-fold raw coverage of the FLT3 gene for the 10 study samples (Table 1). No FLT3 ITD insertions were detected in any sample using the common NGS analysis tools samtools, GATK, maq, DINDEL, and CLC Genomics Workbench. However, PINDEL identified insertions between 17 and 72 bp in 9 of 10 FLT3 ITD-positive samples. PINDEL failed to detect a 93 bp ITD insertion (the largest insertion in this set) in one patient sample, as well as an 84 bp insertion in a patient with two insertions (81 and 54 bp) detected by standard methods. De novo assembly of the FLT3 ITD region also resulted in detection of insertions in 9 of the 10 cases. No insertions were called in an additional set of 15 samples without known FLT3 ITDs. Conclusions: We evaluated the ability of several NGS analysis tools to detect previously known FLT3 ITDs in multi-gene targeted NGS data. Most of the general-purpose analysis tools we tested were unable to detect FLT3 ITD insertions. However, two approaches detected known FLT3 ITD insertions in 90% of the samples tested in this study, including the program PINDEL and de-novo assembly of the FLT3 ITD region using phrap. These results demonstrate that medium-sized FLT3 ITD insertions can be detected in clinical samples by high coverage NGS sequencing with the appropriate analysis pipeline. However, further methods for reliable detection of larger (>70bp) insertions must be developed before clinical NGS-based methods can be applied to the detection of the full spectrum of somatic mutations present in leukemias and other malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 581 Introduction: Acute Myeloid Leukemia (AML) is currently subclassified based on the presence of recurrent cytogenetic abnormalities. The 2008 WHO guidelines recognize seven such translocation events: t(8;21)(q22;q22), inv(16)(p13.1;q22), t(15;17)(q22;q12), t(9;11)(p22;q23), t(6;9)(p23;q34), inv(3)(q21q26.2), t(1;22)(p13;q13). Current clinical methods for the detection of translocations rely on low-resolution techniques such as conventional cytogenetics or fluorescence in-situ hybridization (FISH). Although FISH offers higher resolution compared with conventional cytogenetics, FISH is unable to detect novel translocation partners since break-apart probes detect only gene rearrangements of the target loci; this limitation is particularly problematic for genes known to undergo rearrangement with numerous partners, including the mixed myeloid leukemia gene (MLL) on 11q23 that is disrupted in up to 12% of AMLs and has over 80 known translocation partners. Here we describe a novel method based on hybrid capture enrichment, next generation sequencing, and bioinformatic analysis for identifying prognostically-significant translocations and gene mutations in AML. Methods: Biotinylated RNA capture probes were designed to 2X tile across introns and exons of all currently known genes implicated in AML risk assessment (table 1) for a total target region of 1.0Mb. Approximately 5μ g of patient genomic DNA, extracted from bone marrow samples of patients with AML and known recurrent cytogenetic abnormalities, was ligated with Illumina sequencing adapters, hybridized to the capture probes and enriched by the application of streptavidin-coated magnetic beads. Captured DNA was then eluted, amplified, and sequenced on an Illumina GAII genome analyzer using 60bp paired-end reads. The resulting paired-end reads were then aligned using MAQ to gene regions targeted by the capture probes. Aligned data were then used to identify chimeric single-end reads representing actual translocation boundaries via the SLOPE software package. These putative translocation sites were then validated by examining sequence pairs in which each of the two reads mapped near the chimeric sequence to construct contigs spanning the translocation boundaries. The contigs were aligned to build37 of the human genome to determine the identity and location of translocation partners. Results: As a proof of principle, we extracted DNA from the bone marrow of a patient with AML and a t(9:11) translocation detected by conventional cytogenetics. Approximately 18% of the sequence reads (9,302,232 reads) mapped to the regions targeted by the capture probes, with an average coverage of 1,100 fold. Without a priori knowledge of the actual translocation breakpoint, we identified a single MLLT3-MLL translocation (ch9:20345470-20345657; ch11:118,354,278-118,354,440 involving intron 9 of MLL and intron 7 of MLLT3). There was no evidence of mutations in other relevant genes targeted by the capture probes, including FLT3, NPM1, or CEBPA. Conclusion: We present a novel method for detection of prognostically-significant translocations in clinical AML specimens with single-base resolution, regardless of the translocation partner. While molecular methods such as inverse PCR, ligation-mediated PCR, and panhandle PCR are also capable of identifying translocations with single-base resolution, they are technically complex, time consuming, and generally not well-suited to clinical laboratory applications. By combining numerous capture probes spanning all currently known genes implicated in AML risk assessment in a single assay, the method has the capability of replacing multiple test methodologies including FISH, PCR, and Sanger sequencing. Compared with clinical full-genome sequencing, our targeted approach offers decreased cost, quicker turn-around time, and much simplified bioinformatic analysis. Furthermore, given the high sequence coverage, it will be possible to multiplex samples from different patients in a single assay, further reducing the cost of testing. Disclosures: Armstrong: Cofactor Genomics: Employment.

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel-based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.

Next-generation sequencing (NGS) is becoming a common approach for clinical testing of oncology specimens for mutations in cancer genes. Unlike inherited variants, cancer mutations may occur at low frequencies because of contamination from normal cells or tumor heterogeneity and can therefore be challenging to detect using common NGS analysis tools, which are often designed for constitutional genomic studies. We generated high-coverage (>1000×) NGS data from synthetic DNA mixtures with variant allele fractions (VAFs) of 25% to 2.5% to assess the performance of four variant callers, SAMtools, Genome Analysis Toolkit, VarScan2, and SPLINTER, in detecting low-frequency variants. SAMtools had the lowest sensitivity and detected only 49% of variants with VAFs of approximately 25%; whereas the Genome Analysis Toolkit, VarScan2, and SPLINTER detected at least 94% of variants with VAFs of approximately 10%. VarScan2 and SPLINTER achieved sensitivities of 97% and 89%, respectively, for variants with observed VAFs of 1% to 8%, with >98% sensitivity and >99% positive predictive value in coding regions. Coverage analysis demonstrated that >500× coverage was required for optimal performance. The specificity of SPLINTER improved with higher coverage, whereas VarScan2 yielded more false positive results at high coverage levels, although this effect was abrogated by removing low-quality reads before variant identification. Finally, we demonstrate the utility of high-sensitivity variant callers with data from 15 clinical lung cancers.