Johnson, Sarah H.

7568 Background: The molecular events in the initiation and progression of invasive lung adenocarcinoma remain poorly characterized. These tumors are thought to develop through an adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), invasive adenocarcinoma (AD) sequence. The goal of our study was to assess lineage relationships between in situ and invasive components within adenocarcinomas with prominent lepidic growth patterns. Methods: Frozen tissue from fourteen lung adenocarcinomas with mixtures of AD together with an adjacent in situ component (AIS), varying in ratio between 40 to 80%, were selected from our Lung Specimen Registry. Laser capture microdissection (LCM) of each component was performed separately for each tumor. Genomic DNA was isolated using a direct in situ whole genome amplification (WGA) methodology, and Next Generation Sequencing performed using an Illumina Mate Pair (MP) library protocol. MP sequence reads were mapped to the human genome and primers spanning the fusion junctions were used in validation PCRs. Results: Genomic break points identical and unique to a specific patient were identified between the AIS and AD components in 13 (of 14) cases. The total number of events per case ranged from 4-215, and the number of identical events shared between the AIS and AD components ranged from 30 to 90%. Recurrent genomic breakpoints between cases were also observed, with the 2 most common involving 8q24.3 in 5 cases and FAM19A2 on chromosome 12 in 4 cases. PCR validation of selected genomic alterations confirmed the genomic break points identified from sequencing in both the AIS and AD in all cases. Interestingly, we also observed a limited number of genomic alterations present in a zonal distribution of surrounding normal lung around the tumor mass. Conclusions: Our study demonstrates unique chromosomal alterations present in both the AIS and AD components of individual lung tumors with features of lepidic growth. These data provide evidence for lineage relationship and clonal relatedness between the two components, and provide genomic evidence for a model of stepwise progression from AIS to invasive AD in this subset of lung adenocarcinomas.

Purpose: B-cell acute lymphoblastic leukemia/lymphoma (B-ALL/LBL) represents the most common childhood malignancy. Classification of recurrent genetic abnormalities in B-ALL/LBL is essential for risk stratification and patient management. Observed in approximately 5% of B-ALL/LBL cases, hypodiploidy is considered a poor prognostic finding and can be further characterized as near-haploidy (24-31 chromosomes), low hypodiploidy (32-39 chromosomes) and high hypodiploidy (40-43 chromosomes). Importantly, masked hypodiploid clones that have undergone endoreduplication can be mischaracterized as hyperdiploidy, a favorable prognostic finding in B-ALL/LBL. These "doubled" hypodiploid clones are termed pseudo-hyperdiploid and their accurate detection is critical. Mate-pair sequencing (MPseq) is a next-generation sequencing (NGS) technology designed to sequence larger DNA fragments (2000-5000bp) than paired-end sequencing. This allows for the detection of large genetic rearrangements (e.g. translocations, inversions, etc.) and copy number variants (CNVs) at a high resolution with low base coverage. It has demonstrated particular clinical utility for genetic characterization of hematologic malignancies. To expand the role of MPseq in the evaluation of hematologic neoplasms, we aimed to improve the analysis of MPseq results to allow for the detection of copy-neutral loss of heterozygosity (cnLOH) that would enable the differentiation between hyperdiploid and pseudo-hyperdiploid clones. Methods: A new algorithm was applied that statistically analyzes common SNP locations in MPseq data in order to detect both cnLOH regions, and provides global heterozygosity levels at a resolution that allows for the detection of hypodiploidy. This method accounts for the low base-coverage of MPseq data (<10x) in order to provide the highest possible accuracy and precision without increasing cost by requiring changes to the input or sequencing requirements. The algorithm was applied to 17 B-ALL samples classified by conventional chromosome analysis, FISH, and/or chromosomal microarray as either pseudo-hyperdiploidy (n=8) or hyperdiploidy (n=9). The cases were sequenced using MPseq with 101bp read lengths across a range of base coverages (1-10x; 30-135 million fragments) and tumor percentages (50-100%). Results: We demonstrate the method's ability to differentiate between hyperdiploidy and pseudo-hyperdiploidy in all cases without any changes to the MPseq input or sequencing requirements. We detected an average of 11.5 whole-chromosome cnLOH (range 8-20) in the pseudo-hyperdiploidy cases, compared to an average of 0.67 whole-chromosome cnLOH (range 0-2) in the hyperdiploidy cases. Additionally, the method allows for the detection of cnLOH regions across the genome. For example, we observed a recurrent cnLOH of chromosome 9 in the hyperdiploid subtype (3/9 hyperdiploid cases), which is a recurrent abnormality in the disease. The method provides an easy to read visual output that compares favorably to the output of chromosomal microarray, increasing its utility for clinical application. Conclusions: This advancement in the analysis of low-coverage sequencing data makes MPseq available for the genetic characterization of B-ALL/LBL, where hypodiploidy is a primary cytogenetic abnormality and is sometimes missed by conventional cytogenetic techniques. Additionally, the algorithm allows for the detection of cnLOH regions across the genome, which is important across many disease types for diagnosis, prognosis, and the detection of potential therapeutic targets. The addition of the algorithm to the MPseq analysis pipeline makes it a more complete genetic characterization tool and brings it closer to its promise as a potential single assay replacement for conventional cytogenetic techniques. Disclosures No relevant conflicts of interest to declare.

Abstract Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88L265P-expressing B cells. A better understanding of the proteins involved in MYD88L265P signaling may lead to the development of more targeted and effective therapeutic approaches. In an attempt to identify MYD88L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88L265P. To assess if the formation of a MYD88L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88WT or MYD88L265P expression plasmids. Together, these studies suggest that MYD88L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB. To confirm the significance of TAK1-mediated MYD88L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism. In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88L265P signaling. Disclosures: Fonseca: millennium: Consultancy; amgen: Consultancy; Binding site: Consultancy; onyx: Consultancy; medtronic: Consultancy; Genzyme: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; lilly: Consultancy; Onyx: Research Funding; cylene: Research Funding.

Abstract Abstract 633 Background: To date, the only well characterized chromosomal translocations among peripheral T-cell lymphomas (PTCLs) are those involving ALK, which occur in about half of anaplastic large cell lymphomas (ALCLs). ALK-negative ALCLs remain poorly understood, despite morphology and phenotype similar to ALK-positive ALCLs. We recently described translocations near IRF4 on 6p25.3 in cutaneous ALK-negative ALCLs. In the present study, we utilized massively parallel (Next Generation) sequencing to answer three questions about these translocations: (1) Were they recurrent in the more lethal systemic form of ALK-negative ALCL? (2) What was the translocation partner? (3) What were the likely genetic consequences? Methods: We used a mate pair library approach to juxtapose DNA sequences originally lying ~5 kb apart: these paired sequences map to distinct areas of the genome if a translocation breakpoint lies between them. Because only the ends of the original DNA fragments are sequenced, this approach is more resource efficient than whole genome sequencing. Genomic DNA from a systemic ALK-negative ALCL with an uncharacterized 6p25.3 rearrangement was fragmented to 2–5 kb, biotinylated, and separated on an agarose gel. Fragments ~5-5.5 kb were excised, purified, circularized, and further fragmented to ~300-600 bp. Fragments containing biotin (representing the ends of the original 5 kb fragments) were immobilized on streptavidin beads and amplified using the Illumina library protocol. Paired end sequencing was performed on one lane of a flow cell on an Illumina GAIIx. Sequencing data were mapped to the genome using a binary indexing algorithm. Non-mapping and duplicate sequences were discarded. Candidate translocations suggested by >4 unique mate pairs were examined by BLAT to exclude false positives explained by mapping to homologous regions. PCR and Sanger sequencing of tumor DNA was used for validation. Fluorescence in situ hybridization (FISH) was performed on additional PTCLs. mRNA and miRNA expression was quantified using real-time PCR. Results: Of 28.90×106 paired sequences, 8.61×106 were non-duplicate pairs in which both ends mapped to the human genome (bridged coverage, ~7x). Only one candidate translocation involved 6p25.3, represented by 10 unique mate pairs. PCR and Sanger sequencing confirmed a balanced translocation: one breakpoint disrupted the dual-specificity phosphatase gene, DUSP22, telomeric to IRF4 on 6p25.3; the other was telomeric to the FRAH7 fragile site on 7q32.3. Additional ALK-negative ALCLs with known 6p25.3 rearrangements were tested using a 7q32.3 breakapart FISH probe, which showed a 7q32.3 rearrangement in 13/29 (45%; 7 systemic and 6 cutaneous). Dual fusion FISH confirmed t(6;7)(p25.3;q32.3) in 11/11 cases tested. None of 120 PCTLs (including 39 ALCLs) lacking 6p25.3 rearrangements had a 7q32.3 rearrangement. Levels of 5′ and 3′ DUSP22 mRNA in cases with 6p25.3 rearrangements were decreased relative to those in non-rearranged cases (5′: 0.02±0.01 vs 1.00±0.74, p=.002; 3′: 0.09±0.08 vs 1.00±0.55, p=.0002; mean±SD, t test). IRF4 mRNA levels were similar in both groups. Levels of MIR29A and MIR29B1 within FRAH7 in cases with 7q32.3 rearrangements were increased relative to those in non-rearranged cases (MIR29A: 2.44±2.20 vs 1.00±1.34, p=.15; MIR29B: 4.93±4.68 vs 1.00±1.05, p=.007). Conclusions: The t(6;7)(p25.3;q32.3) is the first recurrent translocation characterized in systemic and cutaneous ALK-negative ALCLs. This translocation was entirely specific for ALK-negative ALCLs, and was present in 45% of cases with 6p25.3 rearrangements. The t(6;7)(p25.3;q32.3) was associated with down-regulation of DUSP22 and up-regulation of MIR29, but not dysregulation of IRF4. DUSP22 inhibits mitogen activated protein kinase activity in T cells, and may represent a novel tumor suppressor in ALCLs. Increased MIR29 levels may additionally contribute to tumorigenesis, as recently shown in acute myeloid leukemias. Finally, clinical testing for t(6;7)(p25.3;q32.3) may aid in differentiating ALK-negative ALCL from other PTCLs. Our findings highlight the utility of mate-pair library sequencing to discover novel translocations in lymphoma and other cancers. Disclosures: No relevant conflicts of interest to declare.

AbstractObjectiveAcute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints.MethodWe evaluated whether an NGS‐based methodology that detects structural abnormalities and copy number changes using mate pair sequencing (MPseq) can enhance the diagnostic yield for patients with AML.ResultsUsing 68 known abnormal and 20 karyotypically normal AML samples, each recurrent primary AML‐specific abnormality previously identified in the abnormal samples was confirmed using MPseq. Importantly, in eight cases with abnormalities that could not be resolved by conventional cytogenetic studies, MPseq was utilized to molecularly define eight recurrent AML‐fusion events. In addition, MPseq uncovered two cryptic abnormalities that were missed by conventional cytogenetic studies. Thus, MPseq improved the diagnostic yield in the detection of AML‐specific structural rearrangements in 10/88 (11%) of cases analyzed.ConclusionUtilization of MPseq represents a precise, molecular‐based technique that can be used as an alternative to conventional cytogenetic studies for newly diagnosed AML patients with the potential to revolutionize the diagnosis of hematologic malignancies.

Abstract The mechanistic target of rapamycin (mTOR) is a central regulator of cellular proliferation and metabolism. Depending on its binding partners, mTOR is at the core of 2 complexes that either promote protein biosynthesis (mTOR complex 1; mTORC1) or provide survival and proliferation signals (mTORC2). Protein biosynthesis downstream of mTORC1 plays an important role in MYC-driven oncogenesis with translation inhibitors garnering increasing therapeutic attention. The germinal center B-cell oncogene UCHL1 encodes a deubiquitinating enzyme that regulates the balance between mTOR complexes by disrupting mTORC1 and promoting mTORC2 assembly. While supporting mTORC2-dependent growth and survival signals may contribute to its role in cancer, the suppression of mTORC1 activity is enigmatic, as its phosphorylation of its substrate 4EBP1 promotes protein biosynthesis. To address this, we used proximity-based proteomics to identify molecular complexes with which UCH-L1 associates in malignant B cells. We identified a novel association of UCH-L1 with the translation initiation complex eIF4F, the target of 4EBP1. UCH-L1 associates with and promotes the assembly of eIF4F and stimulates protein synthesis through a mechanism that requires its catalytic activity. Because of the importance of mTOR in MYC-driven oncogenesis, we used novel mutant Uchl1 transgenic mice and found that catalytic activity is required for its acceleration of lymphoma in the Eμ-myc model. Further, we demonstrate that mice lacking UCH-L1 are resistant to MYC-induced lymphomas. We conclude that UCH-L1 bypasses the need for mTORC1-dependent protein synthesis by directly promoting translation initiation, and that this mechanism may be essential for MYC in B-cell malignancy.

Abstract Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Consistent with this carcinogenic exposure, cytogenetic analyses have identified multiple recurrent structural chromosomal abnormalities in this malignancy, but more recent high-throughput sequencing evaluations of point mutations suggest that there is a low mutational burden in mesothelioma. Since tumor mutational burden has been correlated with responses to treatment with immune checkpoint inhibitors such as nivolumab, it was not consistent that patients with mesothelioma and low mutation burdens would have similar response rates in clinical trials with immune checkpoint inhibitors as patients with non-small cell lung cancer which is associated with a high mutation burden. In order to reconcile these differences, and given the potential for an improved understanding of the molecular pathogenesis of mesothelioma to improve therapeutic options, we used mate-pair sequencing (MPseq) and RNA sequencing (RNAseq) to understand how structural variants affect the transcriptome. MPseq differs from standard next generation sequencing approaches by tiling the whole genome with larger fragments (2-5kb) to reliably detect structural variants such as insertions, deletions and rearrangements. Amongst 22 mesothelioma specimens there were 1535 chromosomal rearrangements (median 41, range 3-298 per specimen), that resulted in junctions or novel fusions of non-coding DNA or genes. Six-hundred thirty-seven of these rearrangements (median 22, range 5-103 range per specimen) resulted in novel fusions of genes. Many of these inter- or intra-chromosomal rearrangements were consistent with a pattern of chromoanagesis such as chromoplexy or chromothripsis. Chromosomal rearrangements detected by MPseq were used to guide analysis of RNAseq data and revealed that these chromosomal junctions resulted in the expression of 179 novel amino acid sequences (median 5, 0-51 range per specimen). To determine whether transcription of chromosomal rearrangement-related junctions have neoantigenic potential, we used in silico tools to determine whether any of the expressed junctions contained peptides that could be presented by patient-specific HLA molecules. The top candidate rearrangement-related peptides with neoantigenic potential bound patient-specific HLA molecules nearly as well or as well as a positive control in competitive binding assays. Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel therapeutic strategies, the selection of patients to receive immunotherapy, and blood-based treatment monitoring strategies. Citation Format: Aaron S. Mansfield, Tobias Peikert, James B. Smadbeck, Julia B. Udell, Farhad Kosari, Stephen J. Murphy, Hongzheng Ren, Vishnu V. Serla, Janet L. Schaefer Klein, Giannoula Karagouga, Faye R. Harris, Carlos Sosa, Sarah H. Johnson, Wendy Nevala, Svetomir N. Markovic, Aaron O. Bungum, Eric S. Edell, Haidong Dong, John C. Cheville, Marie Christine Aubry, Jin Jen, George Vasmatzis. Rearrangement-related peptides with neoantigenic potential in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5726.