Enrichment Panel Research Articles

Abstract 5362: Targeting BRCA1 and BRCA2 with NEBNext Direct™

Abstract The screening and detection of germline BRCA1 and BRCA2 mutations are critical for the effective management of patients with breast or ovarian cancer and for the identification of individuals with a high risk of developing these cancers. In addition, somatic detection of pathogenic variants in BRCA1/2 can influence treatment decisions due to the susceptibility of tumors with BRCA mutations to PARP inhibitors. Here we introduce the NEBNext DirectTM BRCA enrichment panel for the interrogation of BRCA1 and BRCA2 by Illumina sequencing. NEBNext DirectTM is a novel, hybridization-based method to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes. This approach includes features such as the incorporation of unique molecule identifiers (UMIs) and the ability to capture degraded DNA, enabling accurate detection of low-frequency variants from formalin-compromised DNA and other challenging sample types. We applied the NEBNext Direct BRCA1/2 panel to frozen tissue and achieved a high specificity for the BRCA targets (96% of the sequenced reads mapped to BRCA1 and BRCA2 and 80% of the sequenced bases were within the targeted regions). In addition, the resulting libraries were highly uniform in coverage, with 91% of the targeted bases covered at a value of at least 50% of the mean depth of coverage and 100% of the bases covered at 20% of the mean or greater. Some variability in the specificity was observed with formalin-fixed, paraffin embedded (FFPE) samples, and this effect was dependent on the quality of the FFPE DNA. The UMIs were used to identify PCR duplicates prior to variant calling, as well as for error correction, enabling the accurate detection of low-frequency variants. The library preparation for all samples was completed in one day, and the entire process of library preparation, sequencing on the Illumina Miseq, and data analysis was completed in a total of two days. In summary, we demonstrate that application of the NEBNext DirectTM method to the enrichment of BRCA1 and BRCA2 provides a tractable approach for the rapid and highly sensitive analysis of these cancer-associated genes. Citation Format: Scott M. Adams, Kruti M. Patel, Amy B. Emerman, Sarah K. Bowman, Charles D. Elfe, Noa Henig, Salvatore Russello, Andrew Barry, Theodore Davis, Cynthia L. Hendrickson. Targeting BRCA1 and BRCA2 with NEBNext Direct™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5362. doi:10.1158/1538-7445.AM2017-5362

Abstract 487: A comprehensive target enrichment panel for fusion detection

Abstract Growing interest in cancer classification and progression has accelerated the rate of novel gene fusions discovery with increasing recognition of their roles as biomarkers. RNA-Seq is an attractive method for expressed fusion discovery and detection because of its ability to provide unbiased fusion sequencing information. The ability to detect low expressing fusion transcripts, however, require high sequencing depth and represents a significant financial barrier and identification of clinically relevant fusion sequences from a large data set can be a bioinformatics challenge. To address these challenges we have tested the Ovation® Fusion Panel Target Enrichment System V2, a targeted RNA sequencing method using the Single Primer Enrichment Technology (SPET), with a number of control and clinical samples. Initial studies were performed using a comprehensive target enrichment panel targeting 502 genes with three samples from Horizon DX containing known fusions. Target enriched libraries were constructed with 10 ng and 100 ng inputs and the data was analyzed using the NuFuseD pipeline (available as a point and click BaseSpace application or downloadable linux package) which has been optimized for fusion analysis from this data. Expected fusions were identified at both input levels, even when down sampled to 500K reads, with fewer fusion calls compared to other publically available fusion detection software (Chimerascan and SOAPFuse), suggesting a lower false positive rate. NuFuseD fusion calls are provided with a P-value to help prioritize the identified fusions for subsequent validation. Additionally, NuFuseD detected novel fusions in the control samples demonstrating the advantage of a comprehensive panel compared to more restricted panels. We further validated the target panel using control RNA (UHR and Human Brain) and fresh or FFPE cell lines (NCI-H2228, HCC1937) to further demonstrate our ability to identify known fusions. Finally, the system was evaluated at an external site using patient FFPE samples. These samples (N=8) were from a set of breast, liver and ovarian cancers, containing a unique fusion in 4 of the samples based on DNA based sequencing. Only 1 of the 4 expected fusions were identified using whole transcriptome data (100 million reads) while 3 of the 4 fusions were detected with this assay (10 million reads) demonstrating its ability to generate targeted RNA sequencing libraries with increased sensitivity of gene fusion detection and reduced sequencing costs compared to standard RNA-Seq methods. Citation Format: Ashesh Saraiya, Brandon Young, Tobias Meißner, Brian L. Jones, Stephanie C. Huelga, Doug A. Amorese. A comprehensive target enrichment panel for fusion detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 487. doi:10.1158/1538-7445.AM2017-487

61 - The Application of a Hybridisation-Based Next-Generation Sequencing (NGS) Enrichment Panel for the Analysis of Key Genes Involved in Ovarian and Breast Tumours Using DNA From FFPE Samples

61 - The Application of a Hybridisation-Based Next-Generation Sequencing (NGS) Enrichment Panel for the Analysis of Key Genes Involved in Ovarian and Breast Tumours Using DNA From FFPE Samples

Estimating tumor mutation burden using next-generation sequencing assay.

e14529 Background: High tumor mutation burden is a promising biomarker shown in some cancer types to predict positive response to immune checkpoint inhibitors. We show the ability of a targeted cancer research panel to estimate tumor mutation burden per megabase. Methods: We present a single sample analysis workflow for estimating tumor mutation burden from FFPE research samples. Our assay utilizes a PCR-based target enrichment panel that interrogates 409 known key cancer genes covering ~1.7 megabase of genomic space. Our customized workflow requires only 20 ng of input DNA, and enables a 2 day turn-around time from sample to the result. The ease of our workflow enables less than 60 minutes of hands-on time for automated library preparation and templating on a batch of 8 samples. Next-generation Sequencing is performed using high throughput semiconductor sequencing platform to achieve sufficient depth (~500x coverage) and accuracy. Our custom analysis pipeline calls variants with optimized parameters on the tumor sample only, with no matched normal sample required, and applies filters to remove germ-line variants and background noise. Results: Through in silico analysis performed on The Cancer Genome Atlas (TCGA) data we demonstrate that the panel achieves high sensitivity ( > 90%) and specificity ( > 95%) necessary to separate high and low mutation burden samples. Our workflow provides clear separation between allele ratio of somatic and germ-line variants. Our filters consistently eliminate ~98% of germ-line variants from the set of all variants called in single sample analysis workflow. Evidence from tumor-normal analyses on matched tumor and normal samples suggests that our single sample analysis, on the tumor sample only, detects somatic mutations with high sensitivity and specificity with residual of < 3% germ-line variants. Our pipeline identifies mutational signatures consistent with specific mechanisms such as spontaneous deamination of 5-methyl-cytosine, as well as base-damage from FFPE processing. Conclusions: A simple workflow has been developed on the Ion Torrent sequencing platform to estimate per megabase somatic mutational burden from a single tumor FFPE sample. This solution can help advance research in immuno-oncology.

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies.

Abstract PR04: Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia

Abstract The WNT and MAPK signaling pathways are central to colorectal homeostasis. Mutation of the APC tumor suppressor gene activates the WNT pathway to initiate most conventional adenomas. By contrast, oncogenic BRAF mutation initiates development of sessile serrated adenomas (SSA) via activation of the MAPK pathway. The purpose of this study was to investigate the frequency, timing and mechanism of WNT pathway activation in serrated colorectal neoplasia. Methods: The WNT pathway was examined in a range of colorectal polyp and cancer subtypes. Aberrant nuclear immuno-localization of β-catenin was used as a surrogate for WNT pathway activation, compared to the normal membranous staining pattern. The entire coding region of the APC gene was sequenced using a custom enrichment panel that included 242 primer pairs (GeneRead DNAseq Targeted Panel, Qiagen) and the constructed library was sequenced using Illumina's HT TruSeq Kit. As another mechanism of activating WNT, mutations were also assessed in the E3 ubiquitin ligase genes RNF43 and ZNRF3 using Sanger sequencing. To test the interaction of the WNT and MAPK pathways in vivo, a conditional BRAF mutant mouse model was crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine at 2 weeks. The chemical carcinogen azoxymethane was administered weekly for 6 weeks to activate the WNT pathway from 3 weeks of age. The potential therapeutic implication of targeting these pathways was tested in vitro using the porcupine inhibitor LGK974 and MEK inhibitor PD0325901. Results: The majority of conventional pathway polyps and cancers had nuclear localization of β-catenin (22/28, 79% adenomas and 36/42, 86% BRAF wildtype cancers) and mutations in APC (17/20, 85% adenomas and 19/30, 63% BRAF wild type cancers). Sessile serrated adenomas infrequently showed nuclear staining of β-catenin and 0/20 SSA had mutations. By contrast, the WNT signal was increased in the dysplastic component of SSA with a focus of dysplasia (76/137, 55%), however APC mutations were uncommon (3/20, 15%). Similarly, BRAF mutant cancers had nuclear β-catenin in 36/92 (39%) cancers compared to APC mutation in only 9/80 (11%) samples. This did not significantly differ when samples were stratified by microsatellite instability status. RNF43 and ZNRF3 were commonly mutated in BRAF mutant / microsatellite unstable cancers (47/54, 87% and 16/54, 30%, respectively) as well as BRAF mutant / microsatellite stable cancers (8/33, 24% and 5/33, 15%) compared to only 3/79 (4%) RNF43 mutations and 0/27 ZNRF3 mutations in BRAF wildtype cancers. This was accompanied by lower transcript expression for both RNF43 and ZNRF3 in BRAF mutant compared to BRAF wildtype cancers (P<0.0001). We further demonstrated the importance of the WNT pathway in progression of serrated neoplasia by administering azoxymethane to mice with BRAF mutant-induced intestinal hyperplasia. This accelerated the serrated phenotype from 0.6 polyps per mouse to 5.9 per mouse (P<0.001). These all showed typical murine serrated adenoma morphology. To investigate potential therapeutic implications we treated colorectal cancer cell lines with the porcupine inhibitor LGK974 and showed a 50% reduction in growth only in RNF43 and/or ZNRF3 mutant cell lines. In the BRAF / RNF43 / ZNRF3 mutant cell line RKO, LGK974 synergized with MEK inhibition to dramatically reduce growth by approximately 95%. Conclusions: The WNT pathway is commonly activated in serrated neoplasia at the transition to dysplasia. This is not due APC mutation but rather mutation of the upstream RNF43 and/or ZNRF43 genes or other WNT pathway targets. The combination of WNT and MAPK pathway inhibition offers potential for improving therapy for patients with serrated pathway cancers. This abstract is also being presented as Poster A21. Citation Format: Vicki Whitehall, Jennifer Borowsky, Catherine Bond, Mark Bettington, Sally-Ann Pearson, Murugan Kalimutho, John Liu, Troy Dumenil, Diane McKeone, Saara Jamieson, Winnie Fernando, Lochlan Fennell, Andrew Clouston, Christophe Rosty, Ian Brown, Neal Walker, Barbara Leggett. Wnt and MAPK pathway activation in conventional and serrated colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR04.

Rare Pre-Existing MDS Subclones Contribute to Secondary AML Progression

Background: MDS is composed of a dominant founding clone and typically one or more subclones that descend from the founding clone. Using whole genome sequencing (WGS) of secondary AML patients who were initially diagnosed with MDS, we previously showed that AML progression always coincided with expansion of at least one subclone. However, this emergent subclone was not always detectable in the antecedent MDS samples using high-coverage sequencing. It is currently unclear whether these emerging secondary AML-specific clones represent new mutations acquired in cells during treatment or selection of a rare subclone that was below the level of detection in the diagnostic MDS sample. Identification of such pre-existing subclones at MDS diagnosis could provide important prognostic information. Using an ultra-sensitive, error-corrected sequencing approach coupled with high coverage depths, we sought to determine whether secondary AML-specific mutations could be detected in antecedent MDS samples.Methods: We studied 4 MDS patients who eventually progressed to secondary AML (mean 601 days to transformation; range 131-955) that had DNA from initial MDS banking, secondary AML, and skin (as a normal control). DNA from the secondary AML sample was whole genome (3 cases) or exome sequenced (1 case). Custom enrichment panels were designed to target somatic mutations identified in the secondary AML samples and used to sequence the prior MDS samples. All 4 MDS cases had at least one subclone present in the secondary AML sample that was not detected by standard panel-based re-sequencing of the initial MDS sample (539x average coverage). We designed custom molecular barcode-based reagents to perform high-sensitivity, error-corrected sequencing targeting mutations that were detected only at progression to secondary AML. These mutation sites were then sequenced to ultra-deep coverage in prior MDS samples. To determine the sensitivity and specificity of the assay, we first sequenced known dilutions of cell line DNA and showed a sensitivity of 75% to detect variant allele fractions (VAFs) of 0.06% (one mutant cell in 800 cells) while maintaining a specificity of >99.9%. Only reads with unique molecular barcodes that were seen at least three times and had >90% identical reads within a read family were considered. Samples were sequenced to an average total coverage depth of 187,000x (range 107k-474k fold) corresponding to 10,398 unique molecular barcodes of which 3,549 passed filtering (at least two unique read families were required to call a variant), resulting in a mean predicted maximum VAF sensitivity of 0.05% (one mutant cell in 1,000 cells).Results: Using error-corrected barcode sequencing all 4 MDS cases had a subset of mutations detected that were originally only detected in the secondary AML; the mean VAF of detected mutations was 0.25% (range 0.02% to 3.9%) in the antecedent MDS samples, corresponding to 1 mutant cell in 200 cells. However, not all of the ’secondary AML-specific’ mutations were detected in MDS samples. On average, only 17% (range 10-39%) of the ’secondary AML-specific’ subclone mutations were detected in each MDS sample. This suggests that while some mutations pre-exist in an ancestral subclone in the MDS sample, others are acquired during the intervening months to years between MDS and progression to secondary AML. As an example, in two patients who were given decitabine prior to secondary AML progression, mutations detected in the secondary AML subclone that were not detected in the antecedent MDS sample using error-corrected barcode sequencing were more frequently C->G transversions compared to commonly acquired C->T transitions seen in MDS samples (p=.005; Mantel-Haenszel test). Increased C->G transversions are associated with DNA damage induced by decitabine, suggesting that in these two patients an ancestral subclone pre-existed in MDS that acquired additional decitabine-induced mutations during treatment (i.e., C->G transversions).Conclusion: Collectively, the data indicate that very rare subclones pre-exist at MDS diagnosis. These pre-existing subclones can subsequently give rise to the dominant clone at progression to secondary AML. Additional mutations -- some induced by chemotherapy treatment --- are also acquired in ancestral subclones that pre-exist in MDS samples. The combination of subclonal mutations may ultimately contribute to disease progression. DisclosuresJacoby:Sunesis: Research Funding; Quintiles: Consultancy; Celgene: Speakers Bureau.

Patterns of Clonal Evolution Assessed By Whole Exome Sequencing during Progression from MDS to AML Are Related to Therapy

Introduction: Myelodysplastic syndromes (MDS) are hematological disorders at high risk of progression to acute myeloid leukemia (AML). Although, next-generation sequencing has increased our understanding of the pathogenesis of these disorders, the dynamics of these changes and clonal evolution during progression have just begun to be understood. This study aimed to identify the genetic abnormalities and study the clonal evolution during the progression from MDS to AML.Methods: A combination of whole exome (WES) and targeted-deep sequencing was performed on 40 serial samples (20 MDS/CMML patients evolving to AML) collected at two time-points: at diagnosis (disease presentation) and at AML transformation (disease evolution). Patients were divided in two different groups: those who received no disease modifying treatment before they transformed into AML (n=13), and those treated with lenalidomide (Lena, n=2) and azacytidine (AZA, n=5) and then progressed. Initially, WES was performed on the whole cohort at the MDS stage and at the leukemic phase (after AML progression). Driver mutations were identified, after variant calling by a standardized bioinformatics pipeline, by using the novel tool "Cancer Genome Interpreter" (https://www.cancergenomeinterpreter.org). Secondly, to validate WES results, 30 paired samples of the initial cohort were analyzed with a custom capture enrichment panel of 117 genes, previously related to myeloid neoplasms.Results: A total of 121 mutations in 70 different genes were identified at the AML stage, with mostly all of them (120 mutations) already present at the MDS stage. Only 5 mutations were only detected at the MDS phase and disappeared during progression (JAK2, KRAS, RUNX1, WT1, PARN). These results suggested that the majority of the molecular lesions occurring in MDS were already present at initial presentation of the disease, at clonal or subclonal levels, and were retained during AML evolution.To study the dynamics of these mutations during the evolution from MDS/CMML to AML, we compared the variant allele frequencies (VAFs) detected at the AML stage to that at the MDS stage in each patient. We identified different dynamics: mutations that were initially present but increased (clonal expansion; STAG2) or decreased (clonal reduction; TP53) during clinical course; mutations that were newly acquired (BCOR) or disappearing (JAK2, KRAS) over time; and mutations that remained stable (SRSF2, SF3B1) during the evolution of the disease.It should be noted that mutational burden of STAG2 were found frequently increased (3/4 patients), with clonal sizes increasing more than three times at the AML transformation (26>80%, 12>93%, 23>86%). Similarly, in 4/8 patients with TET2 mutations, their VAFs were double increased (22>42%, 15>61%, 50>96%, 17>100%), in 2/8 were decreased (60>37%, 51>31%), while in the remaining 2 stayed stable (53>48%, 47>48%) at the AML stage. On the other hand, mutations in SRSF2 (n=3/4), IDH2 (n=2/3), ASXL1 (n=2/3), and SF3B1 (n=3/3) showed no changes during progression to AML. This could be explained somehow because, in leukemic phase, disappearing clones could be suppressed by the clonal expansion of other clones with other mutations.Furthermore we analyzed clonal dynamics in patients who received treatment with Lena or AZA and after that evolved to AML, and compared to non-treated patients. We observed that disappearing clones, initially present at diagnosis, were more frequent in the "evolved after AZA" group vs. non-treated (80% vs. 38%). By contrast, increasing mutations were similar between "evolved after AZA" and non-treated patients (60% vs. 61%). These mutations involved KRAS, DNMT1, SMC3, TP53 and TET2among others. Therefore AZA treatment could remove some mutated clones. However, eventual transformation to AML would occur through persistent clones that acquire a growth advantage and expand during the course of the disease. By contrast, lenalidomide did not reduce the mutational burden in the two patients studied.Conclusions: Our study showed that the progression to AML could be explained by different mutational processes, as well as by the occurrence of unique and complex changes in the clonal architecture of the disease during the evolution. Mutations in STAG2, a gene of the cohesin complex, could play an important role in the progression of the disease.[FP7/2007-2013] nº306242-NGS-PTL; BIO/SA52/14; FEHH 2015-16 (MA) DisclosuresDel Cañizo:Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Intrapatient Functional Clonality Deconvoluted By Coupling Intracellular Flow Cytometry and Next Generation Sequencing in Human Leukemia

Leukemic clones within the same patient have unique sensitivities to drugs in vivo and may have unique capacities to engraft within xenograft models suggesting that there exist intra-patient functional clonal differences. However, methods to detect the functional differences assigned to genetically defined clones remain limited. Here, we describe a method to measure functional differences of clones within the same leukemia patient by coupling intracellular flow cytometry and next generation sequencing (NGS). We hypothesize that functionally distinct clones can be inferred by stimulating cells with cytokines, fluorescently staining with a relevant downstream marker of activation, and then isolating functionally distinct fractions by flow sorting. NGS can then digitally annotate somatic variants in sorted, functionally distinct samples that can be compared to the bulk leukemic cell population. To achieve this, we optimized a DNA isolation method that results in high-quality DNA despite originating from fixed/permeabilized cells. In this way, we annotate enrichment and de-enrichment of variants upon stimulation and infer the functional clonality of any given sample.To provide proof-of-concept, we explored the well-established relationship between Chronic Myelmonocytic Leukemia (CMML) and GMCSF. CMML cells are uniquely hypersensitive to GMCSF and simultaneously have a well-established mutational composition that can be annotated by sequencing only 49 genes. This allowed us to leverage an amplicon-based enrichment panel that employs emulsion PCR to achieve reproducible library preparation with 5 ng of input DNA hence bypassing the need for whole genome amplification.We tested our methodology by mixing two cell lines whose response to GMCSF was known and somatic variants had been annotated a priori. K562 (non-GMCSF responsive) and THP-1 (GMCSF responsive) leukemic cells were mixed at 1:1 and then stimulated with GMCSF. The resultant cells were stained with anti-pSTAT5 and cell sorting isolated positive and negative fractions. Unstimulated mixed cells and each positive and negative fraction was sequenced and variant allele fractions (VAF) was compared to unstimulated bulk (Figure 1A). To quantitate the change in VAF, we reasoned that summing the difference in VAF of unsorted/pSTAT5+ and unsorted/pSTAT5- populations would yield the most informative composite value as it would contain information about VAF difference for each mutation in both the GMCSF stimulated and GMCSF unstimulated fractions. Using this method, we found that 100% of THP1 and K562 variants were enriched in the pSTAT5+ and pSTAT5- fractions, respectively (Figure 1B). Receiver Operator Curves (ROC) demonstrated that the data functionally discriminated mixed THP-1 and K-562 cells with an AUC of 0.9868 (p<0.0001) (Figure1C). We additionally performed serial dilutions of THP-1 to K562 cell mixtures and demonstrated cell number dependent recovery of THP-1 variants upon stimulation with GMCSF.Next we reasoned that somatic mutations should be uniquely augmented by GMCSF stimulation making it possible for somatic variant identification. To test this, we sequenced 8 CMML bone marrow mononuclear cells evaluated in the same way as above. We additionally sorted CD3+ cells as germline controls to identify somatic mutations. Using our 49-gene panel, we identified a total of 35 somatic variants and 160 germline variants. ROC curve analysis confirmed that our assay was capable of discriminating germline from somatic mutations with an AUC of 0.9388 (p<0.001).In a representative CMML case, we counterstained with CD33 to isolate myeloid progenitors and inferred a functionally distinct clone that was hypersensitive to GMCSF and uniquely carried a JAK2 and PTPN11 mutation that was obscured by bulk leukemia sequence (Figure 2). This inference was orthogonally validated by sequencing individual colonies from the same case grown under GMCSF conditions (Figure 3). Further validation is ongoing comparing mutations identified at engraftment of cells differentially xenografted in NSG and NSGS mice that differ only in their production of human cytokines. Taken together, we describe a novel method to annotate functional differences among mutationally-defined populations and identify functionally distinct clones in primary leukemia samples. [Display omitted] [Display omitted] [Display omitted] DisclosuresKomrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau.

Abstract 5276: Impact of duplicate removal on low frequency NGS somatic variant calling

Abstract Cancer genomic profiles created by analysis of targeted Next Generation Sequencing (NGS) panels is emerging as a powerful tool for making informed clinical decisions. Of the critical informatics challenges to address, accurate mutation calls and allele frequency estimations after accounting for PCR-mediated artifacts are debated. The process of sample preparation for NGS sequencing involves amplification by PCR. While PCR is relatively error-free, mistakes early in DNA synthesis can be compounded, driving detection of spurious mutations and having an adverse impact on clinical reporting. Previous reports have addressed the utility of detecting and removing PCR duplicate reads in Mendelian applications but have rarely examined its use with targeted NGS panels. We performed deduplication with 3 widely used tools (Samtools, Samblaster, and Picard) to understand sensitivity to call low frequency alleles and any impact on false positive/negative rates. Furthermore, we evaluated effects of duplicate removal on targeted panels of varying sizes and effects of sample matrices using replicates of 7 verified reference samples with several digitally confirmed alleles with frequencies ranging from 1-5%. It is not practical to perform deduplication on PCR-enriched panels, therefore, we assessed 3 different hybrid enrichment panels of varying size (387kb, 1.3Mb, and 54Mb). Deduplication by Picard resulted in a greater decrease in the mean depth for the smaller panels (32-59%) compared to Exome (15%), showing that higher molecular diversity lowers duplication rates. Uniformity (percent of ROI with depth within 20% of the mean depth) improved 6-18% after deduplication for the smaller panels, but only 1% for the Exome. Independent of panel size, about 32% of the total reads were marked as duplicates, reducing the power to call low frequency variants by 18%. Importantly, after added sequencing 95-96% of onco-specific variants were detected post-deduplication with a lower limit of detection of 3% compared to 2.5% pre-removal. For low-quality DNA samples we find no benefit in added sequencing for any panel. We also generally observed higher sensitivity (0% to 10% for SNV and -3% to 3% for indels) post deduplication. Molecular diversity also varies by sample type. Intact DNA show higher molecular diversity and lower duplication rates than degraded FFPE samples. We profiled mixed-quality FFPE samples (n = 85), good-quality fresh frozen samples (n = 3), and NA12878 (n = 1) on our smallest panel and noted duplication rates of 64±14%, 40±5% and 30% respectively. On average, deduplication cut the number of SNV calls by 17.4%, with the FFPE samples affected the most (2-88%). From our analysis we recommend performing deduplication during analysis of targeted panels. While we observed the most benefit for smaller panels with low uniformity, improved variant sensitivity was seen regardless of panel size. During experimental design, we advise a worksheet to guide deduplication decisions. Citation Format: Jeran Stratford, Gunjan Hariani, Jeff Jasper, Chad Brown, Wendell Jones, Victor J. Weigman. Impact of duplicate removal on low frequency NGS somatic variant calling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5276.

Abstract 5217: Cancer target enrichment panels using advanced molecular inversion probes (MIPs) with ability to reduce amplification bias and detect low frequency variants

Abstract Next generation sequencing (NGS) of enriched targets is increasingly being used to discover and track mutations in genes implicated in cancer. Amplicon-based target enrichment approaches are frequently used with cancer samples because there is no sample library preparation needed and primer-based approaches are typically efficient with sequencing reads. However, traditional amplicon approaches don't scale up well and may have PCR and coverage uniformity bias. As the number of samples is increased, a fast, reliable, and cost-effective target enrichment method is a critical tool for mutation discovery and tracking. Existing target enrichment approaches based on molecular inversion probes (MIPs) have presented challenges with probe design and dependability. We have developed HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) using advanced versions of MIPs which enable library-free enrichment, improved scalability, and the ability to remove PCR duplicates. We have also developed companion command line software which trims primer sequences and removes PCR duplicates. This software uses community standard file formats (FASTQ, SAM/BAM) and is intended to be compatible with common “best practice” NGS analysis pipelines. Here we describe two HEAT-Seq cancer panels which have been optimized to improve coverage uniformity and probe reliability. The HEAT-Seq Oncology Panel targets both strands for all protein coding bases in 60 cancer-related genes where possible. The target for this design is 245 kb. With 2 million 2×76bp reads for each of 8 replicates, we observe an on-target read rate of &amp;gt;95%. After duplicate removal, the% of target bases with at least 20x coverage is &amp;gt;90%. We observe uniformity (percent of probes &amp;gt; = 20% of the target mean) of ∼90%. The HEAT-Seq Ultra Hot-Spot Panel is a mutation-focused design that provides ultra-deep sequencing coverage capability for sequencing of mutation hot-spots in 53 cancer-related genes to detect low frequency variants in heterogeneous samples. The target for this design is 30.5 kb. With 500,000 2×76bp reads for each of 24 replicates, we observe an on-target read rate of ∼80%. After duplicate removal, the% of target bases with at least 20x coverage is &amp;gt;95%. We observe uniformity (percent of probes &amp;gt; = 20% of the target mean) of ∼95%. We also observed detection of validated low frequency variants down to 1%. In summary, both panels have been shown to capture cancer-related genes and target regions at depths sufficient to identify and track genomic variants. Citation Format: Ryan Bannen, Michael Brockman, Mark D’Ascenzo, Keynttisha Jefferson, Dawn Green, Heather Halvensleben, Kurt Heilman, Todd Richmond, Daniel Burgess. Cancer target enrichment panels using advanced molecular inversion probes (MIPs) with ability to reduce amplification bias and detect low frequency variants. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5217.

Abstract 4530: EpiCapture: Benchmarking commercially available targeted bisulfite-sequencing platforms to gold-standard whole genome bisulfite sequencing

Abstract Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes necessary for cellular differentiation and plays a crucial role in gene regulation of many oncogenes and tumor suppressor genes. Aberrant DNA methylation at the level of both individual gene promoters and on a genome-wide scale has been heavily implicated in cancer initiation and progression. Whole genome bisulfite sequencing (WGBS) is the gold standard method for studying alterations in DNA methylation at a single base pare resolution. However, the high cost of WGBS is still prohibitive for high-throughput observational studies on large sample cohorts. Moreover, the WGBS data analysis requires substantial IT resources and a complex bioinformatics analysis. Targeted bisulfite sequencing enables a cost-effective and focused analysis of genomic regions of interest where most of DNA methylation alterations occur, such as promoters, CpG islands and shores, and enhancer regions. Currently, there are 2 main approaches for targeted bisulfite sequencing of medium-to-large genomic regions, reduced representation bisulfite sequencing (RRBS) and hybridization-based enrichment. We have performed a comparison of 5 commercially available platforms for RRBS and in-solution hybridization-based enrichment panels. We analyzed a panel of two cancer cell lines (Hela and Coriell NA12878) and a normal blood reference gDNA sample, in duplicate on each platform using recommended sample inputs, and compared the results to WGBS data. Given that sample input requirements differ significantly between RRBS (∼100ng or less), and hybridization based methods (500ng - 1 μg), we have analyzed the normal blood reference gDNA sample at 500ng input in duplicate across all platforms. All samples were sequenced to a ∼30x depth on an Illumina HiSeq 2500 sequencer. We compared the genomic coverage, precision and off-target effects and overall concordance in methylome profiles for each method. In summary, here we provide a comprehensive comparison of the performance of 5 different commercially available methods for medium to large scale targeted bisulfite sequencing, facilitating information to researchers for selection of appropriate method depending on required application. Citation Format: Miljana Tanic, Simon Rodney, James Barrett, Hannah Parker, Andrew Feber, Stephan Beck. EpiCapture: Benchmarking commercially available targeted bisulfite-sequencing platforms to gold-standard whole genome bisulfite sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4530.

Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma.

Follicular lymphoma (FL) is typically an indolent disease, but 30-40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies, and identified 45 recurrently mutated genes in the FL-tFL dataset and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D (MLL2), CREBBP, EZH2, BCL2, and MEF2B. Many recurrently mutated genes are involved in epigenetic regulation, the JAK-STAT or the NF-κB pathways, immune surveillance, and cell cycle regulation, or are transcription factors involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.

Rapid Capture Targeted Next Generation Sequencing (NGS) for Detection of Genomic Kinase- and Cytokine-Receptor Rearrangements in B-Precursor Acute Lymphoblastic Leukemia

Background: Next generation sequencing (NGS) applications have recently identified various recurrent kinase and cytokine receptor rearrangements in a subgroup of B-progenitor acute lymphoblastic leukemia (BPC-ALL) that is characterized by a Ph-like gene expression signature. Implementation of whole genome applications into diagnostics have so far been hampered by high costs and long turnover times. Therefore we developed a custom-made NGS based capture panel without the need for gene specific PCR amplification steps. With a rapid turnover time, pooling of multiple samples and standardized bioinformatics, this approach can be implemented in molecular diagnostics of BCP-ALL to identify patients who may benefit from an add-on therapy with specific tyrosine kinase inhibitors. Moreover, genomic breakpoints can be used as an additional patient specific target for minimal residual disease (MRD) monitoring. Here we report a feasible approach for the identification of kinase and kinase-dependent pathway aberrations in BPC-ALL utilizing a customized sequence enrichment capture panel for NGS.Patients and Methods: Ninety three samples from BCP-ALL patients with detectable minimal residual disease (MRD) levels (≥ 5x10-4) after induction therapy from COALL studies 07-03 or 08-09 were applied to the NGS custom panel. Genomic capture probes were specific for exonic and complete intronic regions of ABL1, JAK2, PDGFRB, CRLF2, EPOR, and for the IgH-JH region on chromosome 14. JAK1, JAK3, IKZF1 and SH2B3 were analysed for exonic mutations only. These predefined genomic regions from recurrently mutated or rearranged kinase and cytokine receptor genes were enriched from 50 ng of initial genomic DNA without gene specific PCR amplification steps, followed by paired end sequencing of the captured genomic fragments (Nextera Rapid Capture Custom enrichment protocol with paired end sequencing on a MiSeq benchtop sequencing platform; Illumina). Six positive controls with known rearrangements (BCR-ABL1 and ID4-JH) were included to verify this method.Results: All breakpoints from 6 positive controls were successfully identified and verified by patient specific amplification of the genomic fusion sites. ABL1 and JH specific capture probes allowed the "fishing" of the genomic fusion partner gene, here BCR and ID4, by paired end sequencing of overlapping, breakpoint spanning reads. In addition we identified a genomic breakpoint for CRLF2, PDGFRB, EPOR, ABL1 and JAK2 in 19/93 BCP-ALL patient samples. One additional patient showed a SH2B3 5 bp insertion in exon 5 that leads to a premature stop codon. The 11 patients with either EBF1-PDGFRB (4), EPOR-JH (4), or CRLF2-JH (3) rearrangements showed the highest levels of MRD (≥ 1x10-2) at the end of induction. MRD monitoring using the genomic breakpoint in 3 cases showed highly concordant data to our standardized IgH/TCR quantification assays. For the 3 patients tested so far, our breakpoint specific MRD assays clearly showed the association of these rearrangements with the dominant leukemic clone.Discussion: In summary, we identified kinase- or cytokine receptor rearrangements in 20/93 patients analyzed with our NGS based custom enrichment panel. The majority of patients had high MRD level at EOI therapy and an age of onset ≥ 10 years. Rapid identification of kinase- or cytokine receptor rearrangements in BCP-ALL is necessary for timely therapeutic intervention with target specific tyrosine kinase inhibitors. Short turnaround times of less than 2 weeks and processing of multiple samples followed by a standardized bioinformatical workflow allows the identification of unknown fusions partners from frequently rearranged kinase- and cytokine receptor genes, providing the exact breakpoint location, irrespective of the gene expression signature. MRD guided NGS panel analysis is a feasible strategy for identification of patients with a high risk of therapeutic failure or relapse suitable for targeted TKI intervention. Table 1Age at DxFusionMRD at EOI13EBF1-PDGFRB1,0E+009EBF1-PDGFRB9,0E-0113EBF1-PDGFRB3,0E-019EBF1-PDGFRB7,0E-0216EPOR-IGH6,0E-017EPOR-IGH3,0E-0116EPOR-IGH1,0E-0118EPOR-IGH9,0E-0211CRLF2-IGH9,0E-0211CRLF2-IGH4,7E-0216CRLF2-IGH4,0E-0215PAX5-JAK27,0E-0411RCSD1-ABL15,0E-0210SH2B3-INS3,0E-0310PAR15,0E-0110PAR17,0E-021PAR11,8E-025PAR17,0E-038PAR12,0E-032,5PAR11,5E-03 DisclosuresNo relevant conflicts of interest to declare.

Multiplexed High Resolution Melting Assay for Versatile Sample Tracking in a Diagnostic and Research Setting

Modern experimental procedures in molecular genetics, such as next-generation sequencing experiments, require that samples are taken along a whole series of wet- and dry-laboratory steps. It generally is accepted that by increasing the complexity and number of steps in the experimental pipeline, the risk of sample swaps increases. It therefore is recommended to confirm the identity of each individual sample at the end of any pipeline. Here, we present a versatile assay to determine the identity of samples rapidly and efficiently by genotyping 21 single-nucleotide polymorphisms (SNPs) using multiplex high resolution melting. The selected SNPs also are present in whole-exome sequencing data, and comparison of the differentially obtained genotypes allows reliable identification of individual samples. In this assay, we combined primers interrogating two to three SNPs per high resolution melting reaction, enabling the generation of the SNP genotype profile in only eight reactions per sample, limiting the hands-on time and minimizing the amount of reagents. This SNP profiling approach also can be used to track samples in custom next-generation sequencing enrichment panels by including these 21 SNPs in the target region, allowing for the often-required independent validation of sample identity in both clinical and research settings.

Dependable and Efficient Clinical Molecular Diagnosis of Chinese RP Patient with Targeted Exon Sequencing.

Retinitis pigmentosa (RP) is the most common inherited retinal disease. It is a clinically and genetically heterogeneous disorder, which is why it is particularly challenging to diagnose. The aim of this study was to establish a targeted next-generation sequencing (NGS) approach for the comprehensive, rapid, and cost-effective clinical molecular diagnosis of RP. A specific hereditary eye disease enrichment panel (HEDEP) based on exome capture technology was used to collect the protein coding regions of 371 targeted hereditary eye disease genes, followed by high-throughput sequencing on the Illumina HiSeq2000 platform. From a cohort of 34 Chinese RP families, 13 families were successfully diagnosed; thus, the method achieves a diagnostic rate of approximately 40%. Of 16 pathogenic mutations identified, 11 were novel. Our study demonstrates that targeted capture sequencing offers a rapid and effective method for the molecular diagnosis of RP, which helps to provide a more accurate clinical diagnosis and paves the way for genetic counseling, family planning, and future gene-targeted treatment.

Characterising the spectrum of autosomal recessive hereditary hearing loss in Iran

BackgroundCountries with culturally accepted consanguinity provide a unique resource for the study of rare recessively inherited genetic diseases. Although hereditary hearing loss (HHL) is not uncommon, it is genetically heterogeneous,...

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.

Abstract 3886: Comparing the mutational landscape of African American and Caucasian lung cancers

Abstract Background: The overall death rate from lung cancer is higher in African-Americans (AA) compared to Caucasians (CAU). Understanding differences in the prevalence and type of somatic alterations between races may illuminate differences in prognosis and lead to the reduction of outcome disparities by more precisely targeting patients’ treatment. Methods: Formalin-fixed paraffin embedded tumor samples were collected from Baptist Cancer Center, Memphis, TN. DNA was extracted and sonicated to 250 bp following Covaris FFPE DNA Extraction &amp; Purification protocol and further purified using Agencourt AMPure XP beads. The OncoPanel_v2 target enrichment panel (Agilent SureSelect) was used for hybrid capture of 502 cancer-related genes. Samples were pooled and sequenced on an Illumina HiSeq2500 to a mean depth of coverage of 210x. Tumors with &amp;gt;30x sequencing depth over &amp;gt;80% of targeted bases were considered for analysis. MuTect and SomaticIndelDetector were used to identify somatic single nucleotide variants (SNVs) and short insertions or deletions (indels), respectively. As matched normal DNA was not available for these samples, analysis was limited to variants previously observed in tumors as described in the Catalogue of Somatic Mutations in Cancer (COSMIC) database, and variants were excluded if found in the germline dataset of the Exome Sequencing Project (ESP). Results: 510 tumor specimens from 242 Black and 268 White patients (with self-reported race) were analyzed including 320 adenocarcinomas and 142 squamous cell carcinomas. Pathological classification was independently reviewed and confirmed for 374 of 472 cases for an overall concordance rate of 79%. Using principle component analysis (PCA) on germline SNVs, we observed that the biological ancestry was different than the self-reported race for 1.5% of patients. Mutational frequencies for genes with known roles in adenocarcinoma such as KRAS and EGFR were not significantly different between tumors from Black and White patients (Fisher's exact p-value &amp;gt; 0.05). Amplification rates for NKX2-1, MET, MDM2, and MYC and homozygous deletion rates for CDKN2A were also not significantly different between populations. Translocations involving ALK and ROS1 were detected in tumors from Black patients demonstrating that these events are present in both populations. Similarly, mutational frequencies for genes such as PIK3CA and PTEN were not significantly different across populations in squamous cell carcinomas. Conclusions: These results demonstrate that lung cancers from Black patients are more similar to Whites than East Asians with respect to genes such as EGFR, and suggest that clinical trials of targeted therapies could significantly benefit patients in both populations. Citation Format: Joshua Campbell, Christopher Lathan, Lynette Sholl, Matthew Ducar, Mikenah Vega, Ling Lin, Aaron Thorner, Nick Faris, Paul van Hummelen, Raymond Osarogiagbon, Matthew Meyerson, Laura MacConaill. Comparing the mutational landscape of African American and Caucasian lung cancers. [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 3886. doi:10.1158/1538-7445.AM2015-3886

Abstract 4813: High-depth sequencing of 800 cancer-associated genes in 48 matched tumor-normal tissues to identify somatic alterations including low-frequency variants in colorectal cancer

Abstract Purpose: Colorectal cancer is the 3rd most common cancer globally accounting for 10% of the global cancer burden. Catalogues of somatic mutations in cancer (e.g. The Cancer Genome Atlas (TCGA)) are based upon exome sequencing, typically performed at depths of ∼ 80x, which will miss sub-clonal low-frequency somatic variants. We performed high depth sequencing of 800 cancer-associated genes across 48 tumor/normal pairs of CRC to catalogue somatic variants including those present at low variant allele frequencies. Method: We used the Agilent SureSelect Target Enrichment system to design a customized enrichment panel comprising all coding exons of ∼800 cancer-associated genes. From genomic DNA extracted from frozen tissue specimens (matched primary tumor and adjacent normal), we performed library construction, target enrichment and illumina next generation sequencing. We used 3 variant-calling algorithms - a Genome Analyzer Toolkit (GATK) based pipeline, LoFreq and MuTect to identify somatic variants including sub-clonal variants present at low variant allele frequencies. Results: Sequencing was performed to a median of 302-fold and 310-fold coverage across target regions in the tumor tissues and normal tissues respectively. The use of 3 bioinformatics variant-calling algorithm allowed sensitive and comprehensive identification of variants down to a variant allele frequency of 2.5%; selected variants were validated by an orthogonal sequencing platform (Ion Torrent). The validation rate was &amp;gt; 97%. Each patient had a variant in a median of 19 genes (range 8-100) with a different complement of clonal and sub-clonal variants. Recurrent alterations (e.g. APC, KRAS, TP53) tended to be clonal. Broad regions of allelic imbalance (based on heterozygous SNPs identified in each patient's normal tissue) could also be mapped in each patient. Conclusion: High Depth sequencing of 800 cancer-associated genes in 48 matched tumor normal tissues permits identification of somatic alterations including low-frequency variants and sub-clonal alterations in patients with colorectal cancer. Citation Format: Clarinda Chua, Dan Liang Ho, Yuka Suzuki, Simeen Malik, John McPherson, Anna Gan, Dennis Koh, Choon Leong Tang, Sarah Boon Hsi Ng, Patrick Tan, Steve Rozen, Iain Beehuat Tan. High-depth sequencing of 800 cancer-associated genes in 48 matched tumor-normal tissues to identify somatic alterations including low-frequency variants in colorectal cancer. [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 4813. doi:10.1158/1538-7445.AM2015-4813