Background Metastasis is the primary cause of cancer mortality accounting for 90% of cancer deaths. Our understanding of the molecular mechanisms driving metastasis is rudimentary. Results We perform whole exome sequencing (WES), RNA sequencing, methylation microarray, and immunohistochemistry (IHC) on 8 pairs of non-small cell lung cancer (NSCLC) primary tumors and matched distant metastases. Furthermore, we analyze published WES data from 35 primary NSCLC and metastasis pairs, and transcriptomic data from 4 autopsy cases with metastatic NSCLC and one metastatic lung cancer mouse model. The majority of somatic mutations are shared between primary tumors and paired distant metastases although mutational signatures suggest different mutagenesis processes in play before and after metastatic spread. Subclonal analysis reveals evidence of monoclonal seeding in 41 of 42 patients. Pathway analysis of transcriptomic data reveals that downregulated pathways in metastases are mainly immune-related. Further deconvolution analysis reveals significantly lower infiltration of various immune cell types in metastases with the exception of CD4+ T cells and M2 macrophages. These results are in line with lower densities of immune cells and higher CD4/CD8 ratios in metastases shown by IHC. Analysis of transcriptomic data from autopsy cases and animal models confirms that immunosuppression is also present in extracranial metastases. Significantly higher somatic copy number aberration and allelic imbalance burdens are identified in metastases. Conclusions Metastasis is a molecularly late event, and immunosuppression driven by different molecular events, including somatic copy number aberration, may be a common characteristic of tumors with metastatic plasticity.

Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia characterized by leukemic blasts presenting myeloid and lymphoid markers. Here we report data from integrated genomic analysis on 31 MPAL samples and compare molecular profiling with that from acute myeloid leukemia (AML), B cell acute lymphoblastic leukemia (B-ALL), and T cell acute lymphoblastic leukemia (T-ALL). Consistent with the mixed immunophenotype, both AML-type and ALL-type mutations are detected in MPAL. Myeloid-B and myeloid-T MPAL show distinct mutation and methylation signatures that are associated with differences in lineage-commitment gene expressions. Genome-wide methylation comparison among MPAL, AML, B-ALL, and T-ALL sub-classifies MPAL into AML-type and ALL-type MPAL, which is associated with better clinical response when lineage-matched therapy is given. These results elucidate the genetic and epigenetic heterogeneity of MPAL and its genetic distinction from AML, B-ALL, and T-ALL and further provide proof of concept for a molecularly guided precision therapy approach in MPAL.

BACKGROUND: AML is a group of clinically heterogeneous diseases. We hypothesized that heterogeneous presentation of AML is a reflection of equally heterogeneous genetic process during the leukemogenesis.

We aimed to identify phenotype-defining mutations by correlating the mutation and phenotype landscape in AML.

Purpose The aim of the current study was to determine whether the degree of mutation clearance at remission predicts the risk of relapse in patients with acute myeloid leukemia (AML). Patients and Methods One hundred thirty-one previously untreated patients with AML who received intensive induction chemotherapy and attained morphologic complete remission (CR) at day 30 were studied. Pretreatment and CR bone marrow were analyzed using targeted capture DNA sequencing. We analyzed the association between mutation clearance (MC) on the basis of variant allele frequency (VAF) at CR (MC2.5: if the VAF of residual mutations was < 2.5%; MC1.0: if the VAF was < 1%; and complete MC [CMC]: if no detectable residual mutations) and event-free survival, overall survival (OS), and cumulative incidence of relapse (CIR). Results MC1.0 and CMC were associated with significantly better OS (2-year OS: 75% v 61% in MC1.0 v non-MC1.0; P = .0465; 2-year OS: 77% v 60% in CMC v non-CMC; P = .0303) and lower CIR (2-year CIR: 26% v 46% in MC1.0 v non-MC 1.0; P = .0349; 2 year-CIR: 24% v 46% in CMC v non-CMC; P = .03), whereas there was no significant difference in any of the above outcomes by MC2.5. Multivariable analysis adjusting for age, cytogenetic risk, allogeneic stem-cell transplantation, and flow cytometry-based minimal residual disease revealed that patients with CMC had significantly better event-free survival (hazard ratio [HR], 0.43; P = .0083), OS (HR, 0.47; P = .04), and CIR (HR, 0.27; P < .001) than did patients without CMC. These prognostic associations were stronger when preleukemic mutations, such as DNMT3A, TET2, and ASXL1, were removed from the analysis. Conclusion Clearance of somatic mutation at CR, particularly in nonpreleukemic genes, was associated with significantly better survival and less risk of relapse. Somatic mutations in nonpreleukemic genes may function as a molecular minimal residual disease marker in AML.

Introduction Minimal residual disease (MRD) measured by multi-color flow cytometry has been associated with an increased risk of relapse in patients with acute myeloid leukemia (AML). There is also a strong interest in using somatic mutation as an MRD marker and persistence of NPM1 mutation after 2 cycles of induction therapy has been shown to increase the risk of relapse in AML (Ivey et al, NEJM 2016). However, only one third of AML patients carry NPM1 mutation and prognostic impact of persistent mutations in the context of other co-occurring mutations and association with flow cytometry measured MRD is not well understood. Methods We studied 95 patients with previously untreated AML who received frontline induction chemotherapy and subsequently achieved complete remission (CR). Paired bone marrow samples obtained at pre-treatment and CR (median day 31) were analyzed by targeted capture exome sequencing of 295 genes (median 309x coverage). Mutect and Pindel was used to call single nucleotide variants (SNVs) and small indels, respectively. We defined two levels of mutation clearance (MC) based on the variant allele frequency (VAF) of the detected mutations at CR: 1) MC2.5, if persistent mutation with VAF Results Of 95 patients, 49 (52%) were male and 46 (48%) were female. The median age was 53 (range: 17-76). Conventional karyotyping showed one (1%) patient with favorable cytogenetics (CG), 70 (74%) with intermediate CG, and 21 (22%) with adverse CG (all defined by ELN criteria). Eighty nine (94%) patients had de novo AML and 6 (6%) had secondary AML. Thirty-nine (41%) patients underwent allogeneic stem cell transplant in first CR. In the pre-treatment samples of 95 AML patients, we detected 299 high-confidence somatic mutations (192 SNVs and 107 indels) in 60 genes in 85 (89%) patients. The most frequently mutated genes were NPM1 (31%) followed by DNMT3A (29%), FLT3 (18%), and IDH2 (18%). In the matching CR samples, 84 (28%) and 108 (36%) mutations persisted at the level of VAF ≥2.5% and ≥1%, respectively, which corresponded to 41 (48%) and 31 (36%) patients achieving MC2.5 and MC1.0, respectively. Pattern of MC was distinct between mutated genes and affected molecular pathways (Table1). Mutations associated with clonal hematopoiesis of indeterminate potential (CHIP), DNA methylation, and splicing had low rate of MC, whereas mutations in transcription factor genes or receptor tyrosine kinase (RTK) pathway had high rate of MC. With the median follow-up of 41 months (95% CI: 39-47), patients who achieved MC1.0 had significantly better RFS, EFS, OS and lower CIR than those without MC1.0, while there was no significant difference in any of the above outcomes by MC2.5 (Table 2). Flow cytometry-based MRD (flow-MRD) data were available in 92 (97%) patients at CR. Positive flow-MRD was associated with significantly worse RFS, EFS, OS and CIR (Table2). Among 61 patients with negative flow-MRD, 25 (41%) and 32 (52%) had persistent mutations with VAF ≥2.5% and ≥1%, respectively. Multivariable analysis considering mutations that were prognostic in univariable analysis (NPM1, IDH1), age, ELN-defined adverse cytogenetics, flow-MRD and MC revealed that MC1.0 was a significant prognostic factor for RFS (p = 0.03, HR 0.36, 95%CI: 0.14-0.91) and CIR (p = 0.03, HR 0.42, 95%CI: 0.19-0.92). MC1.0 in DNA methylation (2-year EFS: 85% (95% CI: 51-96) in MC1.0 vs. 40% (95% CI: 23-56) in non-MC1.0, p = 0.01; 2-year OS: 85% (95% CI: 51-96) in MC1.0 vs. 58% (95% CI: 39-72) in non-MC1.0, p = 0.03) or in chromatin-cohesin pathways (2-year EFS: 73% (95% CI: 37-90) in MC1.0 vs. 15% (95% CI: 3-39) in non-MC1.0, p Conclusion In patients with AML treated with induction chemotherapy, persistent somatic mutation at CR with VAF >1% was associated with increased risk of death and relapse. Assessment of both mutation clearance and flow-based MRD at CR may help further risk stratification of AML. Download : Download high-res image (138KB) Download : Download full-size image Disclosures Daver: Immunogen: Research Funding; Kiromic: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Incyte Corporation: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Jazz: Consultancy; Pfizer Inc.: Consultancy, Research Funding. Cortes: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Sun Pharma: Research Funding; BMS: Consultancy, Research Funding. Jabbour: Bristol-Myers Squibb: Consultancy. Kantarjian: Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Pfizer: Research Funding; Amgen: Research Funding; Novartis: Research Funding. Takahashi: Symbio Pharmaceuticals: Consultancy.

7023 Background: MPAL is a rare subgroup of acute leukemia characterized by both myeloid and lymphoid phenotypes. Genetic basis of MPAL is not well understood. Methods: We studied 31 patients (pts) with MPAL (median age 53) that met 2008 WHO criteria. Bone marrow samples were studied by targeted capture sequencing of 295 genes (median 393x), RNA sequencing, and Infinium methylation EPIC array (Illumina). Mutational landscape was compared to 194 AML, 71 B-ALL, and 6 T-ALL cases. Promoter methylation pattern was compared to the data from 194 AML (TCGA), 505 B-ALL and 101 T-ALL cases (Nordlund et al. Genome Biology. 2013). Results: Eighteen (58%) pts had myeloid-T and 13 (42%) had myeloid-B phenotype. Four pts had t(9;22), 1 had 11q23 rarrangement, and 8 had complex karyotype. MPAL had similar number of mutations with AML but had higher number of mutations than B-ALL or T-ALL. Both AML-type and ALL-type mutations were detected in MPAL, supporting the mixed phenotypic features. However, NPM1, CEBPA and GATA2 mutations were specific to AML and were not found in MPAL. Myeloid-T and myeloid-B showed distinct patterns of mutations, in which DNMT3A, IDH2, NOTCH1, IL7R, and FBXW7 mutations were enriched in myeloid-T whereas RUNX1 mutations were enriched in myeloid-B. Myeloid-T and myeloid-B also showed distinct patterns of promoter methylation. Overall, myeloid-T had more hypermethylated CpG loci than myeloid-B. Genes that have essential role in T-cell receptor (TCR) pathway ( CD3D, CD7, CD247, LCK, PRKCQ, CCR9, and TCL1A) were differentially methylated and differentially expressed between myeloid-T and myeloid-B. RNA sequencing revealed several known translocations such as, NSD1-NUP98, and KMT2A-MLLT4, in addition to the novel fusion proteins such as FOXP1-DNAJC15, RUNX1-NAP1L1, and BCL2-TM9SF3. Unbiased hierarchical clustering of MPAL, AML, B-ALL and T-ALL by promoter methylation revealed that myeloid-T had consistent similarity with T-ALL, while myeloid-B showed random similarity with either B-ALL or AML. Conclusions: MPAL is genetically heterogeneous and myeloid-T and myeloid-B shows distinct patterns of mutations, methylation and gene expressions. Therapy for MPAL may need to be tailored based on the genetic profiles.

Background: The precise molecular mechanisms underlying metastasis of nonsmall cell lung cancers (NSCLC) are largely unknown. Two recent studies comparing genomic landscapes of primary NSCLC tumors and paired brain metastases suggested branched evolution, where all metastatic and primary tumors shared a common ancestor yet continued to evolve independently. The integrated genomic and transcriptomic profiles of primary NSCLC and metastases have not been studied in any details. Methods: We performed whole exome sequencing (WES) and RNA sequencing (RNA-seq) of surgically resected primary tumors and paired distant metastases from 7 patients with NSCLC. Results: Totally, 6,945 somatic mutations, including 1,702 non-silent (stop-gain, stop-loss, frameshift, splicing site and nonsynonymous) mutations were identified by WES. Metastases trended to have larger mutation burdens compared to paired primary tumors, although the difference was not statistically different (average 595 mutations per tumor in primary tumors versus 852 mutations per tumor in metastases, respectively, p = 0.54). On average, 51% of all mutations (24% to 93%) were shared between primary tumors and metastases. We identified 14 canonical cancer gene mutations in this cohort of patients, defined as mutations that lead to amino acid changes identical to those found previously in cancer genes or disrupting mutations in tumor suppressor genes, all of which were shared between primary tumors and paired distant metastases. In addition, metastases resembled paired primary NSCLC tumors closely in regard to somatic copy number aberration profiles and mutation signatures. Pathway analysis from RNA-seq data demonstrated that 25 of the 35 signal transduction pathways that were significantly down regulated in metastases relative to primary NSCLC tumors were related to immune activation. Validation study with a larger patient cohort is in progress. Conclusions: Although branched evolution is a common phenomenon during metastasis of NSCLC, majority of canonical cancer gene mutations are probably early molecular events likely acquired before metastasis initiates. Mutation mechanism may be determined early during carcinogenesis and preserved during cancer evolution even at the metastatic sites. Immune suppression may be one characteristic feature of cancer cells of metastatic capacity. Citation Format: Jianjun Zhang, Chia-Chin Wu, Jianhua Zhang, Junya Fujimoto, Xingzhi Song, Xizeng Mao, Huadong Sun, Sahil Seth, Rebecca Thornton, Marcus Coyle, Latasha Little, Curtis Gumbs, Carmen Behrens, Chi-Wan Chow, Erik Sulman, Ganesh Rao, Stephen Swisher, Ignacio Wistuba, John Heymach, Andrew Futreal, Daniel Gomez. Integrated exome and transcriptome sequencing of primary lung cancers and paired distant metastases. [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 156.

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats.Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits.Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.