Bait Set Research Articles

Driver Mutation in Waldenstrom's Macroglobullinemia and Their Clonal Heterogeneity during Progression and Relapse

Background: Whole genome sequencing has recently shown the presence of recurrent mutations, such as MYD88, CXCR4 and ARID1A, in patients with Waldenstrom's Macroglobulinemia (WM) at diagnosis. Nevertheless, the contribution of each genomic aberration within the clonal evolution of the tumor during WM progression has not been reported. We therefore aimed to investigate whether sequential genomic events sustain WM disease progression by using whole exome sequencing (WES) and targeted sequencing of serial samples. In addition, we investigated if specific genomic alterations can change in response to treatment with samples before and after proteasome or BTK inhibitor treatments.Methods: We have sequenced 74 samples from 32 patients using WES or targeted sequencing technology. DNA was collected from bone marrow CD19-selected cells that were isolated from 6 patients with WM at different stages of disease (2-3 serial samples per patient, total 15 samples), and was subjected to library construction, followed by Agilent Sure-Select Human All Exon v2.0-based hybrid selection. Germline DNA was isolated from matched CD19-depleted peripheral blood samples. All libraries were sequenced with Illumina Hiseq 2500 instrument (New York Genome Center, Rockefeller University, New York, NY). Reads were aligned to GRCh37, and quality control, mutation calling, insertion and deletion identification, copy number variation detection, coverage calculations were accomplished via Firehose at Broad. Somatic Single Nucleotide Variations (SSNVs) were identified and annotated using MuTect and Oncotator, respectively. Insertions and deletions were detected using Strelka. In addition, the deep targeted sequencing was done with a customized bait set in 59 independent serial samples obtained from 26 patients who were treated with either proteasome inhibitor (Pi)- or BTK inhibitor (BTKi)-based therapies in 12 and 14 cases, respectively.Results: Whole exome sequencing, performed at a total average depth of 83X for germline and 85X for tumor samples, led to the identification of average of 14 non-silent mutations per sample (range 2-60). Base conversion signature showed dominant A>C/G transitions. Copy number analysis showed 6q deletion being the most prevalent one. MYD88 was the most recurrent somatic variants in WM patients (>93%), followed by several genes including IRS4, VCAN, CXCR4 and ALDH2, being detected in ~20-30% of the samples. Specifically, changes in the Cancer Cell Fraction (CCF) of these mutations, such as MYD88 and CXCR4, occurred in the serial samples depending on progression and response to therapy.The mutated genes mentioned above, including MYD88, CXCR4, LRG1 and VCAN, were also observed in targeted sequencing of 59 independent serial samples (mean target coverage 434X). CXCR4 was linked to disease progression exposed to Pi or BTK; but not detected in patients responding to therapies. MYD88 was present in patients with either progressive disease or PR/VGPRs. Specifically, different frequencies of MYD88 mutations were identified in patients with progression or PR/VGPR to BTKi-based therapies compared to Pi-based therapies. LRG1 was detected in a patient in response to Pi, while VCAN was observed in both patients showing progression or response to Pi regimens only.Conclusion: These findings reveal the occurrence of clonal variations in patients with WM during disease progression and response to therapies. MYD88 was confirmed to be the most prevalent somatic aberration, and was present in post-treatment samples of progressors and responders to Pi- or BTKi-based regimens. In contrast, mutations in CXCR4 were enriched in patients with WM progressing to either Pi or BTKi therapies. This study demonstrates that WES and targeted sequencing of serial samples of WM patients can detect clonal variations during disease progression. DisclosuresGhobrial:Celgene: Honoraria, Research Funding; Noxxon: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

Whole Exome Sequencing and Targeted Sequencing Reveal the Heterogeneity of Genomic Evolution and Mutational Profile in Smoldering Multiple Myeloma

Introduction: Recent data shows that multiple myeloma (MM) almost always arises from precursor states called Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), but not all patients with MGUS or SMM develop MM. Risk factors of progression for SMM patients are largely based on tumor load as represented by an M-protein ≥ 3 g/dL, a free light chain (FLC) ratio outside the range of 0.125 to 8, and ≥ 10% plasma cells in the BM. However, the genetic lesions that underlie progression, the molecular factors that cause rapid versus slow progression, and the factors that distinguish the relatively indolent MGUS from SMM are not well known. Further, the genomic landscape of SMM is not well characterized. One potential factor is MYC overexpression. Bergsagel et al. have found that MYC levels increase when comparing MGUS, SMM and overt MM. Other frequently altered pathways in MM are NF-kB, MAPK and DNA damage. In addition, limited studies of paired SMM and MM samples show that in many cases, the aggressive subclones can already be detected, in small cell fractions, before overt MM develops. However, the cause of progression to MM is unclear, in large part because sequential genomic studies of MGUS/SMM progression have yet to be undertaken. To address these questions, in this study we examine clinically-annotated samples from patients with SMM.Methods: We performed whole exome sequencing (WES) (mean target coverage 50X/100X) on 49 germline-tumor matched samples from patients with SMM (DNA from bone marrow CD138+ plasma cells matched with germline DNA from peripheral blood mononuclear cells). Libraries were constructed using Agilent SureSelect XT2 library prep kit, and hybridized to Agilent's whole exome V5+UTR capture probes and then sequenced on HiSeq 2500 (Illumina). We also performed targeted deep sequencing using a custom enrichment bait set on 25 samples of progressor (n=12) and non-progressor (n=13) SMM samples. Libraries were also constructed with Agilent SureSelect XT2 library prep kit and enriched by hybridizing to an in-house designed customized target bait, then sequenced on HiSeq 2500. Sequencing data were analyzed using previously established analytic pipelines including MuTect, RecapSeg, GISTIC, MutSig, and ABSOLUTE.Results: The number of Somatic Single Nucleotide Variants (SSNVs) seen in SMM ranged from 1 to 98 nonsilent mutations with an average of 1.14 mutations/Mb, which is slightly lower than MM (1.6 mutations/Mb) from previous studies (p-value=0.05). This large and varying range of mutational load among samples suggests that SMM is likely a heterogenous entity where some patients are closer to MGUS and others closer to MM. We identified likely drivers in SMM in about ~32% of the samples, including mutations in MM candidate driver genes such as NRAS, KRAS and PTPN11(overall 36 events were present in COSMIC). SMM also had somatic CNAs in about ~50% of SMM samples, such as hyperdiploidy, gain of chromosome 1q, deletion of 13p and 17p, which match the hallmark chromosome changes seen in MM.Comparing deep targeted sequencing of 100 genes (mean target coverage 361X) in samples from 12 SMM patients who progressed to myeloma vs. 13 SMM patients who did not, we found non-synonymous mutations exclusive to progressors, suggesting that with more samples we may find genetic alterations that predict progression in SMM.Conclusion: This study demonstrates that WES and targeted sequencing of SMM patients can detect MM candidate driver genes as well as hallmark CNAs seen in MM patients. Further, there may be potential different mutational features between progressors and non-progressors. Thus, this approach can be used to identify genetic drivers of clonal progression from MGUS/SMM to MM that may present opportunities for early therapeutic intervention and prevention of disease progression. DisclosuresRoccaro:Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria; Novartis: Honoraria; BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Diffuse large B-cell lymphoma patient-derived xenograft models capture the molecular and biological heterogeneity of the disease

AbstractDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

Comprehensive Genomic Profiling (CGP) of Angioimmunoblastic T-Cell Lymphoma (AITL) to Prospectively Inform Diagnosis and Clinical Management

Background: AITL is a rare mature T-cell lymphoma with an aggressive clinical course. Accurate diagnosis is confounded by low tumor content limiting molecular characterization, histologic resemblance to peripheral T-cell lymphoma (PTCL), and a scarcity of sensitive diagnostic tools for routine sample analysis. Also, to date, no confirmed standard of care exists; most patients receive combination chemotherapy, but this approach is largely ineffective. Several research studies report RhoAG17V as a genomic hallmark of AITL that often co-occurs with somatic mutations in epigenetic regulators (TET2, IDH2, or DNMT3A); however these findings have yet to be translated in the clinic. The detection of clinically-relevant genomic alterations in AITL has the potential to improve outcome for this patient population by identifying targeted therapeutic agents. CGP provides an opportunity to address these collective unmet needs; herein, we report on our experience using this methodology in AITL.Methods: To assess RhoAG17V specificity as a diagnostic marker of AITL, RhoA mutation was explored in an unbiased fashion across the Foundation Medicine database. This led to inclusion in the study of 21 patient specimens with diagnoses of suspected AITL (4), confirmed AITL (12), PTCL (2), or other neoplasms (3). Prior autologous stem cell transplant was noted in 2 of 12 patients with confirmed AITL and in 1 with PTCL. Four patients with AITL were HHV-4 positive. Median age of the cohort was 64y (range 38-88y). Using the FoundationOne® Heme platform, DNA and RNA were extracted from samples submitted as either FFPE material (lymph nodes, skin, soft tissue) or blood. Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait set targeting 405 cancer-related genes, and 265 genes involved in cancer-related fusions. All captured libraries were sequenced in a CLIA-certified, CAP-accredited, NYS-approved laboratory (Foundation Medicine, Inc).Results: RhoAG17V was found in 69% (11/16) of AITL samples; none harbored RhoA mutation at sites other than G17. In cases of suspected AITL, RhoAG17V was found in 3/4 samples, and in 8/12 of those confirmed. TET2 mutations, predominantly prematurely truncating and inactivating, were identified in 100% (16/16) of AITL cases, exceeding reported frequencies. Mutation of IDH2, affecting exclusively R172, was observed at a frequency of 31% (5/16), while DNMT3A was observed at a frequency of 25% (4/16) and was most commonly inactivating. In all 5 AITL samples with IDH2 mutation, RhoA and TET2 mutation also co-occurred. In PTCL samples lacking an AITL diagnosis, mutation in RhoA, TET2, IDH2, and DNMT3A was detected in 1/2, 2/2, 0/2, and 1/2 cases, respectively. An unbiased query of the Foundation Medicine database identified RhoAG17V mutation in 1 sample each of pleomorphic myxofibrosarcoma (MFS), marginal zone lymphoma (MZL), and cutaneous T-cell lymphoma (CTCL); in contrast to cases of AITL, these samples were devoid of TET2 and IDH2 mutations.Conclusion: Using CGP, AITL samples harbored co-occurring RhoA/TET2 mutations at high frequency. Importantly, RhoAG17V has been proposed as a diagnostic marker for this aggressive PTCL subtype. Our findings are suggestive that assessing mutations in TET2 simultaneously with that of RhoA may have greater diagnostic utility than RhoA alone. All samples with concurrent RhoAG17V/TET2 mutation were from patients with suspected or confirmed AITL, with one exception. A single case was diagnosed as PTCL containing immunoblastic proliferations reminiscent of AITL morphology. In contrast, while RhoAG17V was detected in other malignancies (MFS, MZL, CTCL), these lacked TET2 and IDH2 mutation, suggesting the specificity of the co-occurring mutations for AITL. Co-occurrence of RhoAG17V/TET2 mutation, identified by CGP, may, therefore, enable genomic reclassification to AITL from other mature T-cell neoplasms when used in conjunction with other clinical criteria.CGP may furthermore inform therapeutic approaches for AITL. Detection of frequent and co-occurring alterations in epigenetic regulators suggests DNA hypermethylation is common in this lymphoma and implicates hypomethylating agents as relevant. Studies are currently under way to examine clinical responses of AITL patients with IDH2 or TET2 mutations to IDH2 inhibitors and hypomethylating agents, respectively. DisclosuresMorley:Foundation Medicine, Inc.: Employment, Equity Ownership. He:Foundation Medicine, Inc.: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc.: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc.: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine, Inc.: Employment, Equity Ownership. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Erlich:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Vergilio:Foundation Medicine, Inc.: Employment, Equity Ownership. Mughal:Foundation Medicine, Inc.: Employment, Equity Ownership.

Comprehensive Analyses of Genetic Features Identify Coordinate Signatures in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease characterized by multiple low-frequency alterations including somatic mutations, copy number alterations (CNAs) and chromosomal rearrangements. We sought to identify previously unrecognized low-frequency genetic events, integrate recurrent alterations into comprehensive signatures and associate these signatures with clinical parameters. For these reasons, our multi-institutional international group assembled a cohort of 304 primary DLBCLs from newly diagnosed patients, 87% of whom were uniformly treated with state-of-the-art therapy (rituximab-containing CHOP regimen) and had long term followup.Tumors were subjected to whole exome sequencing with an extended bait set that included custom probes designed to capture recurrent chromosomal rearrangements. In this cohort, 47% of samples had available transcriptional profiling and assignment to associated disease subtypes. Analytical pipelines developed at the Broad Institute were used to detect mutations (MuTect), CNAs (Recapseq+Allelic Capseq) and chromosomal rearrangements (dRanger+Breakpointer) and assess clonality (Absolute). To analyze formalin-fixed paraffin-embedded tumors without paired normals we developed a method which utilized 8334 unrelated normal samples to stringently filter recurrent germline events and artifacts. Significant mutational drivers were identified using the MutSig2CV algorithm and recurrent CNAs were assessed with GISTIC2.0. In addition, we utilized a recently developed algorithm, CLUMPS2, to prioritize somatic mutations which cluster in 3-dimensional protein structure. With this approach, we identified > 90 recurrently mutated genes, 34 focal amplifications and 41 focal deletions, 20 arm-level events and > 200 chromosomal rearrangements in the DLBCL series. Of note, 33% of the mutational drivers were also perturbed by chromosomal rearrangements or CNAs, underscoring the importance of a comprehensive genetic analysis.In the large DLBCL series, we identified several previously unrecognized but potentially targetable alterations including mutations in NOTCH2 (8%) and TET2 (5%). The majority of identified chromosomal rearrangements involved translocations of potent regulatory regions to intact gene coding sequences. The most frequently rearrangements involved Ig regulatory elements which were translocated to BCL2, MYC, BCL6 and several additional genes with known roles in germinal center B-cell biology.After identifying recurrent somatic mutations, CNAs and chromosomal rearrangements, we performed hierarchical clustering and identified subsets of DLBCLs with comprehensive signatures comprised of specific alterations. A large subset of tumors shared recurrent alterations previously associated with follicular lymphoma including mutations of chromatin modifiers such as CREBBP, MLL2, and EZH2 in association with alterations of TNFRSF14 and GNA13 and translocations of BCL2. This cluster was enriched in GCB-type DLBCLs and contained a subset with select genetic alterations associated with an unfavorable outcome.An additional cohort of tumors was characterized by alterations perturbing B-cell differentiation including recurrent BCL6 translocations or alterations of PRDM1. A subset of these DLBCLs had alterations of NOTCH2 and additional pathway components or mutations of MYD88 in association with TNFAIP3, CD70 and EBF1, a master regulator of B-cell differentiation.An additional group of DLBCLs exhibited frequent MYD88 mutations in association with alterations of CD79B, PIM1, TBL1XR1 and ETV6 and BCL2 copy gain; these tumors were highly enriched for ABC-type DLBCLs. This coordinate signature and additional alterations of p53 pathway components were associated with outcome.We explored bases for the identified genetic alterations in DLBCL by performing an in silico mutational signature analysis. The most frequent mutational signatures were those of spontaneous deamination (aging) and AID with rare cases of microsatellite instability. We also assessed the clonality of identified genetic features to define cancer cell fraction and establish the timing of specific genetic events.The comprehensive genetic signatures of clinically annotated DLBCLs provide new insights regarding approaches to targeted therapy. DisclosuresLink:Kite Pharma: Research Funding; Genentech: Consultancy, Research Funding. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Pfreundschuh:Boehringer Ingelheim, Celegene, Roche, Spectrum: Other: Advisory board; Roche: Honoraria; Amgen, Roche, Spectrum: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Diffuse Large B-Cell Lymphoma Patient-Derived Xenograft Models Capture Molecular and Biologic Heterogeneity and Inform Therapy

Diffuse large B-cell lymphoma (DLBCL) is a clinically and biologically heterogeneous disease defined by different transcriptional classifications, associated signaling and survival pathways and additional recurrent genetic alterations. In the cell-of-origin (COO) scheme, DLBCLs subtypes share certain features with normal germinal center B-cells (GCB) and activated B-cells (ABC). In comparison to GCB DLBCLs, ABC tumors have increased baseline NFκB activity and more frequent genetic alterations of NFκB pathway components. DLBCLs with shared functional features are also defined by the consensus clustering classification (CCC) which delineates B-cell receptor (BCR), Oxidative Phosphorylation (OxPhos) and Host Response (HR) tumors. BCR DLBCLs have increased reliance on BCR-signaling and survival pathways and aerobic glycolysis. BCR-dependent DLBCLs with high or low baseline NFκB activity (which largely correspond to ABC or GCB tumors, respectively) have distinct SYK-PI3K-dependent survival pathways and shared sensitivity to proximal BCR pathway inhibitors. Although DLBCLs have infrequent inactivating somatic mutations of TP53, these tumors commonly have copy number alterations (CNAs) of TP53 and genes encoding cell cycle pathway components.Given the clinical and molecular heterogeneity of DLBCL, we sought to develop faithful subtype-specific model systems to assess targeted therapies. Fresh tumor biopsies from 27 primary LBCLs were implanted under the renal capsule of immune compromised NSG mice. Nine of 27 tumors were successfully expanded in vivo, serially propagated for > 5 generations and considered stable LBCL PDX models. All models were EBV- and had clonal IgH rearrangements. Morphological and immunohistochemical signatures defined 8 PDX models as DLBCL and 1 as EBV- plasmablastic lymphoma (PBL).All LBCL PDX models were subjected to RNA-Seq and classified with respect to COO and CCC subtypes. Models were also evaluated by whole exome sequencing with a modified bait set which captured coding mutations and selected chromosomal rearrangements. Six of 9 DLBCL PDX models were ABC type. These models exhibited mutations of MYD88 alone or in association with PIM1 or CD79B with other alterations, as reported in primary ABC DLBCLs. The remaining 2 DLBCL PDX models were GCB type, with characteristic alterations of GNA13 and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC. Of note, BCL2 and MYC translocations are known adverse prognostic features of primary GCB DLBCL. Certain PDX models had additional mutations including B2M, MLL2, TNFAIP3, MEF2B and TP53.Only 25% (2/8) of the DLBCL PDX models harbored inactivating TP53 mutations whereas 75% (6/8) of tumors exhibited CNAs of TP53 or its upstream modifier, CDKN2A. These data are consistent with the reported incidence and type of TP53 pathway alterations in primary DLBCLs and contrast sharply with the near-uniform presence of TP53 mutations in DLBCL cell lines.Using the CCC classification, 6/8 DLBCL PDX models (both GCBs and 4 of 6 ABCs) were defined as BCR-subtype and 2 models as non-BCR type. To assess the utility of the DLBCL PDX models for functional analysis of BCR signaling, we first assessed cell surface immunoglobulin (sIg) expression by flow cytometry. All 6 BCR-type DLBCLs expressed sIgM whereas the 2 non-BCR DLBCL models and the PBL model lacked sIg. Next, we treated viable tumor cell suspensions with a selective SYK inhibitor, entospletinib (GS-9973). SYK inhibition significantly decreased the proliferation of all 6 BCR-type DLBCLs, but had no effect on the non-BCR-type DLBCLs or the PBL PDX. Given the distinctive SYK/PI3K-dependent signaling and survival pathways in DLBCLs with low or high baseline NFκB, we also assessed selective apoptotic pathway readouts in entospletinib-treated PDX cell suspensions. SYK inhibition selectively upregulated the pro-apoptotic BH3 family member, HRK, in BCR-dependent GCB-type DLBCL PDX samples and significantly downregulated the anti-apoptotic BCL2 family member, BCL2A1, in BCR- dependent ABC-type DLBCL PDX tumors, effects consistent with those previously observed in primary DLBCL samples.In summary, we have established and molecularly characterized faithful PDX models of DLBCL and PBL and demonstrated their usefulness in evaluating novel BCR pathway inhibitors. DisclosuresRodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding. Shipp:Gilead: Consultancy; Sanofi: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Node sampling for protein complex estimation in bait-prey graphs.

In cellular biology, node-and-edge graph or "network" data collection often uses bait-prey technologies such as co-immunoprecipitation (CoIP). Bait-prey technologies assay relationships or "interactions" between protein pairs, with CoIP specifically measuring protein complex co-membership. Analyses of CoIP data frequently focus on estimating protein complex membership. Due to budgetary and other constraints, exhaustive assay of the entire network using CoIP is not always possible. We describe a stratified sampling scheme to select baits for CoIP experiments when protein complex estimation is the main goal. Expanding upon the classic framework in which nodes represent proteins and edges represent pairwise interactions, we define generalized nodes as sets of adjacent nodes with identical adjacency outside the set and use these as strata from which to select the next set of baits. Strata are redefined at each round of sampling to incorporate accumulating data. This scheme maintains user-specified quality thresholds for protein complex estimates and, relative to simple random sampling, leads to a marked increase in the number of correctly estimated complexes at each round of sampling. The R package seqSample contains all source code and is available at http://vault.northwestern.edu/~dms877/Rpacks/.

Characterization of the Mutational Landscape of Multiple Myeloma Using Comprehensive Genomic Profiling

Introduction:Multiple myeloma (MM) is a neoplastic disease of the bone marrow characterized by a malignant transformation of plasma cells. Many patients relapse after initial treatment and require additional therapies. Impaired cell cycle regulation and DNA repair mechanisms as well as exposure to genotoxic drugs leads to accumulation of genomic alterations with progressive disease. Pressure from antineoplastic agents, including novel agents, eventually leads to the selection of resistant clones. Assessing acquired somatic mutations in MM patients can identify key genomic drivers and guide the development of a rational, individualized therapy plan for each patient with advanced disease. Here we report on the mutational landscape of cancer-associated genes in 214 patients who underwent comprehensive genomic profiling.Methods:Review of this data was approved by the UAMS institutional review board. DNA and RNA were extracted from CD138+ selected cells from bone marrow aspirates. Adaptor ligated sequencing libraries from extracted nucleic acids were captured by solution hybridization using bait sets targeting 405 cancer-related and 265 frequently rearranged genes (FoundationOne Heme®; Foundation Medicine ). For samples with low cell yield only the DNA portion was performed. All samples were sequenced in a CLIA-certified, CAP-accredited laboratory to an average depth >500x.ResultsWe identified 147 clinically relevant alterations with an average of 3 alterations per patient ranging from 1 to 8. The most frequently altered genes were KRAS (29% of cases), NRAS (23%), TP53 (19%), RB1 (10%), BRAF (8%), TRAF3(8%), CDKN2C (7%), DNMT3A (5%), NF1, FAF1 and TET2 (4% each). While RAS, RAF, RB1 and TP53 mutations are also found in previously untreated patients, albeit in lower frequencies, mutations of DNTM3A and TET2 are rarely reported in the early phase of the disease, arguing for the accumulation of genomic alterations over time. We found concomitant alterations in KRAS and BRAF in 5, KRAS and NRAS in 3, and NRAS and BRAF in 2 patients. The vast majority of RAS alterations occurred at hotspots resulting in activating alterations at codons 12, 13 or 61 with mutant allele frequencies ranging from 0.01 to 0.92 with an average of 0.30. In the 17 patients with BRAF alterations the hotspot mutation V600E was found in 7 with mutant allele frequencies ranging from 0.01 to 0.48 with an average of 0.32. Overall the MAPK pathway was affected in 128 of 214 patients. 61 patients had alterations of genes associated with DNA damage repair. Among the 10 patients with DNMT3A alterations 2 also had alterations of TET2 suggesting significant epigenetic deregulation in a subset of patients. Data on subclonal structure and correlation of mutation status with paired gene expression profiles will be presented as well, as will be selected responses of patients treated on the basis of these results.ConclusionSubjecting CD138 selected bone marrow cells to comprehensive genomic profiling allows for the identification of clinically relevant alterations, which deregulate critical pathways in multiple myeloma. Small molecule inhibitors that target key genes in these affected pathways (MEK, BRAF) have recently been approved for therapy in other cancers or are being actively developed (PI3K, AKT, PARP). This comprehensive genomic characterization allows rational development of individualized clinical strategies using molecular targets for MM patients who are refractory to standard of care therapies. DisclosuresWalker:Onyx Pharmaceuticals: Consultancy, Honoraria. van Rhee:Senesco: PI Other. Zangari:Norvartis: Membership on an entity’s Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Morgan:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Janssen: Membership on an entity’s Board of Directors or advisory committees; Myeloma UK: Membership on an entity’s Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity’s Board of Directors or advisory committees; The Binding Site: Membership on an entity’s Board of Directors or advisory committees; MMRF: Membership on an entity’s Board of Directors or advisory committees. Barlogie:Celgene: Consultancy, Patents & Royalties, Research Funding; Millenium: Consultancy, Patents & Royalties, Research Funding.

Whole-genome enrichment and sequencing of Chlamydia trachomatisdirectly from clinical samples

BackgroundChlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.MethodsC. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.ResultsFull length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.ConclusionsThe combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0591-3) contains supplementary material, which is available to authorized users.

Target enrichment of ultraconserved elements from arthropods provides a genomic perspective on relationships among Hymenoptera.

Gaining a genomic perspective on phylogeny requires the collection of data from many putatively independent loci across the genome. Among insects, an increasingly common approach to collecting this class of data involves transcriptome sequencing, because few insects have high-quality genome sequences available; assembling new genomes remains a limiting factor; the transcribed portion of the genome is a reasonable, reduced subset of the genome to target; and the data collected from transcribed portions of the genome are similar in composition to the types of data with which biologists have traditionally worked (e.g. exons). However, molecular techniques requiring RNA as a template, including transcriptome sequencing, are limited to using very high-quality source materials, which are often unavailable from a large proportion of biologically important insect samples. Recent research suggests that DNA-based target enrichment of conserved genomic elements offers another path to collecting phylogenomic data across insect taxa, provided that conserved elements are present in and can be collected from insect genomes. Here, we identify a large set (n = 1510) of ultraconserved elements (UCEs) shared among the insect order Hymenoptera. We used in silico analyses to show that these loci accurately reconstruct relationships among genome-enabled hymenoptera, and we designed a set of RNA baits (n = 2749) for enriching these loci that researchers can use with DNA templates extracted from a variety of sources. We used our UCE bait set to enrich an average of 721 UCE loci from 30 hymenopteran taxa, and we used these UCE loci to reconstruct phylogenetic relationships spanning very old (≥220 Ma) to very young (≤1 Ma) divergences among hymenopteran lineages. In contrast to a recent study addressing hymenopteran phylogeny using transcriptome data, we found ants to be sister to all remaining aculeate lineages with complete support, although this result could be explained by factors such as taxon sampling. We discuss this approach and our results in the context of elucidating the evolutionary history of one of the most diverse and speciose animal orders.

Profiling Genomic Alterations Of Diffuse Large B-Cell Lymphoma (DLBCL) At Diagnosis, Relapse, and Transformation, Using a Novel Clinical Diagnostic Targeted Sequencing Platform

Whole genome and exome sequencing studies have identified numerous genomic alterations in DLBCL, but these methods have limited applicability for the clinical care of lymphoma patients due to cost, specific tissue requirements, and laborious bioinformatic analysis. FoundationOne-Heme (FOH) is a novel next-generation sequencing platform designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of mutations within specific genes, copy number changes, and translocations. FOH can be performed on small quantities of formalin-fixed paraffin-embedded (FFPE) tissue, detect rare variants due to extensive depth of sequencing coverage, and rapidly provide results via streamlined bioinformatic interpretation. Here we report the first experience using this novel platform to evaluate the genetic landscape of DLBCL.Genomic DNA and total RNA were isolated from FFPE tissue on a cohort of 53 cases of DLBCL, including de novo (n=30), relapsed/refractory (n=12), and large cell transformation from low-grade lymphoma (n=11). The cohort included 25 cases with combined MYC and BCL2 overexpression by IHC (criteria for positivity: >40% MYC, >70% BCL2), of which only one had a known translocation involving MYC. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom bait sets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 frequently-rearranged genes for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >658x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors.The DNA sequencing component of FOH detected translocations in BCL2, BCL6, and MYC, while the RNA sequencing component detected fusion transcripts involving BLC6 and MYC, in agreement with independent cytogenetic analysis via karyotype and FISH where available. The assay detected copy number alterations of 44 different genes, most commonly amplification of REL (15%) or loss of CDKN2A/CDKN2B (17%). The most frequent alterations of known significance are detailed in Figure 1. The most commonly altered gene was CDKN2A, exhibiting either homozygous deletion or loss of function mutation in 28% of cases. Chromatin modifying factors (e.g. MLL2, CREBBP, EZH2) represented the most frequently altered biologic category with alterations occurring in >50% of cases. Recurrent alterations in components of the Notch pathway (NOTCH1/2/4, FBXW7, SPEN), each predicted to activate the pathway, were identified in 23% of cases. Cell-of-origin was determined as per the Hans model using IHC for CD10, BCL6, and IRF4/MUM1; CD79B mutations were detected exclusively in non-GCB and EZH2 mutations were found exclusively in GCB-phenotype cases. Furthermore, IHC MYC+/BCL2+ de novo DLBCL cases (n=11) exhibited more frequent hypermutation of PIM1 (46%) compared with the 19 cases of IHC MYC-/BCL2- de novo DLBCL (11%). When comparing the various clinical categories, we found that mutations in tumor suppressors were significantly more common in relapsed/refractory than de novo DLBCL (47% vs 75%, p=0.02). Alterations in TP53 were most frequently observed in transformed lymphoma (55%).Our results demonstrate the feasibility of using a targeted next-generation sequencing platform on FFPE clinical specimens from patients with DLBCL as a means of providing an integrated analysis of gene mutations, copy number alterations, and translocations. This streamlined approach combines multiple molecular and cytogenetic tests into a single platform and uses a small amount of tissue to perform a multifaceted assessment of genomic alterations with potential diagnostic, prognostic, and therapeutic implications. Future efforts will be directed at analyzing additional cases of DLBCL to better establish the biologic and clinical significance of the observed genetic alterations, and to prospectively incorporate this novel platform to select patients for mechanism-based targeted therapy. Disclosures:Intlekofer:Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.▪

Capturing Protein-Coding Genes Across Highly Divergent Species

DNA hybridization capture combined with next generation sequencing can be used to determine the sequences of hundreds of target genes across hundreds of individuals in a single experiment. However, the approach has thus far only been successfully applied to capture targets that are highly similar in sequence to the bait molecules. Here we introduce modifications that extend the reach of the method to allow efficient capture of highly divergent homologous target sequences using a single set of baits. These modifications have important implications for comparative biology.

Abstract 819: Targeted sequencing to detect somatic mutations, translocations and copy-number variation in human tumors simultaneously.

Abstract The ability to harness the power of next generation sequencing for characterizing the cancer genome would be extremely valuable to the cancer research community and ultimately improve diagnosis and individualized therapy. We present a targeted approach using massively parallel sequencing to simultaneously detect mutations, translocations and copy-number variations in archived clinical tumor specimen. Targeted sequencing was achieved by designing RNA baits to capture the exons of 504 genes with relevance to cancer. The bait set was augmented with specific intronic sequences to detect translocations often involved in cancer. The overall performance of the hybrid capture was improved by optimizing the tiling strategy of the baits. These improvements significantly shifted fragments with low or no-coverage closer to the overall mean, resulting in a more uniform coverage distribution. To validate the targeted gene panel we sequenced tumor samples harboring mutations, translocations and copy-number alterations that were previously identified by clinically approved assays. All the known alterations were confirmed with additional potentially actionable mutations. The ultimate goal of the targeted panel is to screen cancer patients quickly and economically allowing simultaneous detection of all common genomic alterations in the cancer genome. Citation Format: Paul Van Hummelen, Matthew Ducar, Robert T. Jones, Alina Raza, Ashwini Sunkavalli, Megan Hanna, Adri Mills, Ravali Adusumilli, Prateek Kumar, Laura Schubert, Marc Breneiser, Anna C. Cooley, Elizabeth Garcia, Lynette M. Scholl, Neal I. Lindeman, Nikhil Wagle, Levi Garraway, Kristian Cibulskis, Scott L. Carter, Michael Lawrence, Gad Getz, Matthew L. Meyerson, William C. Hahn, Laura E. MacConaill. Targeted sequencing to detect somatic mutations, translocations and copy-number variation in human tumors simultaneously. [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 819. doi:10.1158/1538-7445.AM2013-819

Identification of protein complexes from co-immunoprecipitation data

Motivation: Advanced technologies are producing large-scale protein–protein interaction data at an ever increasing pace. A fundamental challenge in analyzing these data is the inference of protein machineries. Previous methods for detecting protein complexes have been mainly based on analyzing binary protein–protein interaction data, ignoring the more involved co-complex relations obtained from co-immunoprecipitation experiments.Results: Here, we devise a novel framework for protein complex detection from co-immunoprecipitation data. The framework aims at identifying sets of preys that significantly co-associate with the same set of baits. In application to an array of datasets from yeast, our method identifies thousands of protein complexes. Comparing these complexes to manually curated ones, we show that our method attains very high specificity and sensitivity levels (∼ 80%), outperforming current approaches for protein complex inference.Availability: Supplementary information and the program are available at http://www.cs.tau.ac.il/~roded/CODEC/main.html.Contact: roded@post.tau.ac.ilSupplementary information: Supplementary data are available at Bioinformatics online.

Construction and Characterization of a Normalized Yeast Two-Hybrid Library Derived from a Human Protein-Coding Clone Collection

The nuclear yeast two-hybrid (Y2H) system is the most widely used technology for detecting interactions between proteins. A common approach is to screen specific test proteins (baits) against large compilations of randomly cloned proteins (prey libraries). For eukaryotic organisms, libraries have traditionally been generated using messenger RNA (mRNA) extracted from various tissues and cells. Here we present a library construction strategy made possible by ongoing public efforts to establish collections of full-length protein encoding clones. Our approach generates libraries that are essentially normalized and contain both randomly fragmented as well as full-length inserts. We refer to this type of protein-coding clone-derived library as random and full-length (RAFL) Y2H library. The library described here is based on clones from the Mammalian Gene Collection, but our strategy is compatible with the use of any protein-coding clone collection from any organism in any vector and does not require inserts to be devoid of untranslated regions. We tested our prototype human RAFL library against a set of baits that had previously been searched against multiple cDNA libraries. These Y2H searches yielded a combination of novel as well as expected interactions, indicating that the RAFL library constitutes a valuable complement to Y2H cDNA libraries.

Control of Vespula germanica (Hym. Vespidae) populations using toxic baits: bait attractiveness and pesticide efficacy

Abstract: Our aim was to test a set of baits and pesticides for the control of yellowjacket populations in NW Patagonia (Argentina), through field trials. We tested the attractiveness of protein (fresh and freeze‐dried beef) and carbohydrate (corn syrup and honey) baits (alone and mixed) and three pesticides commonly employed to control terrestrial domestic arthropods (hydramethylnon 2%, permethrin 0.3% and chlorpyrifos 0.25%). Our results show that beef proved to be the most attractive bait tested, for Vespula germanica wasps in NW Patagonia. Honey and corn syrup alone or mixed with beef did not attract foraging wasps as did beef‐only baits throughout the wasp season. Additionally, we show that the attractiveness of lyophilized beef baits is similar to that of fresh beef. The efficacy of the insecticides tested was limited. In fact, only baiting with hydramethylnon 2% reduced wasp populations (54%) and this occurred after 72 h since poisoning. However, given the potential of insects to develop resistance to a consistent use of a single product (e.g. fipronil 0.1%), the use of hydramethylnon may allow the alternating of insecticides in specific situations. The information provided here contributes to the existent knowledge on baits and insecticides for the control of yellowjackets using toxic baits.

Geographic and Seasonal Variation in Fragrance Choices and Preferences of Male Euglossine Bees

Variation in choices and preferences for fragrances are examined for a large portion of the euglossine bee assemblages of Barro Colorado Island (BCI) and Cerro Campana, Panama. Census data were obtained from a year-long baiting program utilizing 16 chemical attractants. Interand intraspecific variation in fragrance choices occurred. Within sites, fragrance choices of species overlapped considerably, but each species was attracted to a unique set of baits. For some species, choices of infrequently visited chemicals differed among sites. The five baits preferred by most species were the same for both sites. Geographic variation in intraspecific fragrance preferences occurred for seven of eleven species that were abundant at both BCI and Cerro Campana. Seasonal variation in fragrance preferences occurred for 16 of 21 species on BCI. The lack of seasonal shifts was not related to the number of fragrances to which a species was attracted. Indirect evidence exists for age-dependent shifts in chemical preferences by bees. Substantial intraspecific variation in fragrance preferences occurs and is related to some combination of season, age, and geography. The degree to which natural fragrances are available and genetic differences among individuals affect variation in choices and preferences for fragrances remains unknown. THE NATURAL HISTORY OF THE NEOTROPICAL Euglossini (Apidae), commonly known as orchid bees, is unusual and male behavior is both perplexing and intriguing. Male bees utilize the same nectar sources as females (Ackerman 1985), and their foraging behavior at these flowers is similar (Ackerman et al. 1982). Males differ from females in that they have no role in nest construction or in cell provisioning. Furthermore, male bees collect fragrances from a variety of sources, including rotting fruits and wood, tree bark, exposed roots, and flowers (Ackerman