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Patients with Lynch syndrome often present with multiple synchronous or metachronous colorectal cancers (CRCs). The presence of multiple CRCs with distinct genetic profiles and driver mutations could complicate treatment as each cancer may respond differently to therapy. Studies of sporadic CRCs suggested that synchronous tumors have distinct etiologies, but could not rule out differences in genetic background. The presence of multiple cancers in a patient with a predisposing mutation provides an opportunity to profile synchronous cancers in the same genetic background. Here, we describe the case of a patient with Lynch syndrome that presented with six synchronous CRCs. Microsatellite instability (MSI) and genomic profiling indicated that each lesion had a unique pattern of instability and a distinct profile of affected genes. These findings support the idea that in Lynch syndrome, synchronous CRCs can develop in parallel with distinct mutation profiles and that these differences may inform treatment decisions.

Comprehensive genetic profiling is increasingly important for the clinical workup of hematologic tumors, as specific alterations are now linked to diagnostic characterization, prognostic stratification and therapy selection. To characterize relevant genetic and genomic alterations in myeloid malignancies maximally, we utilized a comprehensive strategy spanning fluorescence in situ hybridization (FISH), classical karyotyping, Chromosomal Microarray (CMA) for detection of copy number variants (CNVs) and Next generation Sequencing (NGS) analysis. In our cohort of 569 patients spanning the myeloid spectrum, NGS and CMA testing frequently identified mutations and copy number changes in the majority of genes with important clinical associations, such as TP53, TET2, RUNX1, SRSF2, APC and ATM. Most importantly, NGS and CMA uncovered medically actionable aberrations in 75.6% of cases normal by FISH/cytogenetics testing. NGS identified mutations in 65.5% of samples normal by CMA, cytogenetics and FISH, whereas CNVs were detected in 10.1% cases that were normal by all other methodologies. Finally, FISH or cytogenetics, or both, were abnormal in 14.1% of cases where NGS or CMA failed to detect any changes. Multiple mutations and CNVs were found to coexist, with potential implications for patient stratification. Thus, high throughput genomic tumor profiling through targeted DNA sequencing and CNV analysis complements conventional methods and leads to more frequent detection of actionable alterations.

The Drosophila CNS midline glia (MG) are multifunctional cells that ensheath and provide trophic support to commissural axons, and direct embryonic development by employing a variety of signaling molecules. These glia consist of two functionally distinct populations: the anterior MG (AMG) and posterior MG (PMG). Only the AMG ensheath axon commissures, whereas the function of the non-ensheathing PMG is unknown. The Drosophila MG have proven to be an excellent system for studying glial proliferation, cell fate, apoptosis, and axon–glial interactions. However, insight into how AMG migrate and acquire their specific positions within the axon–glial scaffold has been lacking. In this paper, we use time-lapse imaging, single-cell analysis, and embryo staining to comprehensively describe the proliferation, migration, and apoptosis of the Drosophila MG. We identified 3 groups of MG that differed in the trajectories of their initial inward migration: AMG that migrate inward and to the anterior before undergoing apoptosis, AMG that migrate inward and to the posterior to ensheath commissural axons, and PMG that migrate inward and to the anterior to contact the commissural axons before undergoing apoptosis. In a second phase of their migration, the surviving AMG stereotypically migrated posteriorly to specific positions surrounding the commissures, and their final position was correlated with their location prior to migration. Most noteworthy are AMG that migrated between the commissures from a ventral to a dorsal position. Single-cell analysis indicated that individual AMG possessed wide-ranging and elaborate membrane extensions that partially ensheathed both commissures. These results provide a strong foundation for future genetic experiments to identify mutants affecting MG development, particularly in guidance cues that may direct migration. Drosophila MG are homologous in structure and function to the glial-like cells that populate the vertebrate CNS floorplate, and study of Drosophila MG will provide useful insights into floorplate development and function.

The study of how transcriptional control and cell signaling influence neurons and glia to acquire their differentiated properties is fundamental to understanding CNS development and function. The Drosophila CNS midline cells are an excellent system for studying these issues because they consist of a small population of diverse cells with well-defined gene expression profiles. In this paper, the origins and differentiation of midline neurons and glia were analyzed. Midline precursor (MP) cells each divide once giving rise to two neurons; here, we use a combination of single-cell gene expression mapping and time-lapse imaging to identify individual MPs, their locations, movements and stereotyped patterns of division. The role of Notch signaling was investigated by analyzing 37 midline-expressed genes in Notch pathway mutant and misexpression embryos. Notch signaling had opposing functions: it inhibited neurogenesis in MP1,3,4 and promoted neurogenesis in MP5,6. Notch signaling also promoted midline glial and median neuroblast cell fate. This latter result suggests that the median neuroblast resembles brain neuroblasts that require Notch signaling, rather than nerve cord neuroblasts, the formation of which is inhibited by Notch signaling. Asymmetric MP daughter cell fates also depend on Notch signaling. One member of each pair of MP3-6 daughter cells was responsive to Notch signaling. By contrast, the other daughter cell asymmetrically acquired Numb, which inhibited Notch signaling, leading to a different fate choice. In summary, this paper describes the formation and division of MPs and multiple roles for Notch signaling in midline cell development, providing a foundation for comprehensive molecular analyses.

The Drosophila embryonic Central Nervous System (CNS) develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of our knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, we combined loss of function with ectopic activation of Egfr activity. We observe that ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. Through this work, we identified the genetic criteria required for the development of the medial and intermediate column cell fates. We also show that ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. Finally, we demonstrate that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells.

The development of targeted therapies based on specific genomic alterations has altered the treatment and management of lung and colorectal cancers. Chromosomal microarray (CMA) has allowed identification of copy number variations (CNVs) in lung and colorectal cancers in great detail, and next-generation sequencing (NGS) is used extensively to analyze the genome of cancers for molecular subtyping and use of molecularly guided therapies. The main objective of this study was to evaluate the utility of combining CMA and NGS for a comprehensive genomic assessment of lung and colorectal adenocarcinomas, especially for detecting drug targets. We compared the results from NGS and CMA data from 60 lung and 51 colorectal tumors. From CMA analysis, 33% were amplified, 89% showed gains, 75% showed losses and 41% demonstrated loss of heterozygosity; pathogenic variants were identified in 81% of colon and 67% lung specimens through NGS. KRAS mutations commonly occurred with loss in TP53 and there was significant loss of BRCA1 and NF1 among male patients with lung cancer. For clinically actionable targets, 23% had targetable CNVs when no pathogenic variants were detected by NGS. The data thus indicate that combining the two approaches provides significant benefit in a routine clinical setting not available by NGS alone.

The two pairs of wings that are characteristic of ancestral pterygotes (winged insects) have often undergone evolutionary modification. In the fruitfly, Drosophila melanogaster, differences between the membranous forewings and the modified hindwings (halteres) depend on the Hox gene Ultrabithorax (Ubx). The Drosophila forewings develop without Hox input, while Ubx represses genes that are important for wing development, promoting haltere identity. However, the idea that Hox input is important to the morphologically specialized wing derivatives such as halteres, and not the more ancestral wings, requires examination in other insect orders. In beetles, such as Tribolium castaneum, it is the forewings that are modified (to form elytra), while the hindwings retain a morphologically more ancestral identity. Here we show that in this beetle Ubx 'de-specializes' the hindwings, which are transformed to elytra when the gene is knocked down. We also show evidence that elytra result from a Hox-free state, despite their diverged morphology. Ubx function in the hindwing seems necessary for a change in the expression of spalt, iroquois and achaete-scute homologues from elytron-like to more typical wing-like patterns. This counteracting effect of Ubx in beetle hindwings represents a previously unknown mode of wing diversification in insects.

The Drosophila CNS midline cells constitute a specialized set of interneurons, motorneurons, and glia. The utility of the CNS midline cells as a neurogenomic system to study CNS development derives from the ability to easily identify CNS midline-expressed genes. For this study, we used a variety of sources to identify 281 putative midline-expressed genes, including enhancer trap lines, microarray data, published accounts, and the Berkeley Drosophila Genome Project (BDGP) gene expression data. For each gene, we analyzed expression at all stages of embryonic CNS development and categorized expression patterns with regard to specific midline cell types. Of the 281 candidates, we identified 224 midline-expressed genes, which include transcription factors, signaling proteins, and transposable elements. We find that 58 genes are expressed in mesectodermal precursor cells, 138 in midline primordium cells, and 143 in mature midline cells—50 in midline glia, 106 in midline neurons. Additionally, we identified 27 genes expressed in glial and mesodermal cells associated with the midline cells. This work provides the basis for future research that will generate a complete cellular and molecular map of CNS midline development, thus allowing for detailed genetic and molecular studies of neuronal and glial development and function.

The study of achaete-scute (ac/sc) genes has recently become a paradigm to understand the evolution and development of the arthropod nervous system. We describe the identification and characterization of the ac/sc genes in the coleopteran insect species Tribolium castaneum. We have identified two Tribolium ac/sc genes -achaete-scute homolog (Tc-ASH) a proneural gene and asense (Tc-ase) a neural precursor gene that reside in a gene complex. Focusing on the embryonic central nervous system we find that Tc-ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies we show that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. Furthermore, we find that Tc-ase is expressed in all neural precursors suggesting an important and conserved role for asense genes in insect nervous system development. Our analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development.

Background The Drosophila CNS midline cells are an excellent model system to study neuronal and glial development because of their diversity of cell types and the relative ease in identifying and studying the function of midline-expressed genes. In situ hybridization experiments generated a large dataset of midline gene expression patterns. To help synthesize these data and make them available to the scientific community, we developed a web-accessible database. Description MidExDB (Drosophila CNS Midline Gene Expression Database) is comprised of images and data from our in situ hybridization experiments that examined midline gene expression. Multiple search tools are available to allow each type of data to be viewed and compared. Descriptions of each midline cell type and their development are included as background information. Conclusion MidExDB integrates large-scale gene expression data with the ability to identify individual cell types providing the foundation for detailed genetic, molecular, and biochemical studies of CNS midline cell neuronal and glial development and function. This information has general relevance for the study of nervous system development in other organisms, and also provides insight into transcriptional regulation.

Glia play crucial roles in ensheathing axons, a process that requires an intricate series of glia-neuron interactions. The membrane-anchored protein Wrapper is present in Drosophila midline glia and is required for ensheathment of commissural axons. By contrast, Neurexin IV is present on the membranes of neurons and commissural axons, and is highly concentrated at their interfaces with midline glia. Analysis of Neurexin IV and wrapper mutant embryos revealed identical defects in glial migration,ensheathment and glial subdivision of the commissures. Mutant and misexpression experiments indicated that Neurexin IV membrane localization is dependent on interactions with Wrapper. Cell culture aggregation assays and biochemical experiments demonstrated the ability of Neurexin IV to promote cell adhesion by binding to Wrapper. These results show that neuronal-expressed Neurexin IV and midline glial-expressed Wrapper act as heterophilic adhesion molecules that mediate multiple cellular events involved in glia-neuron interactions.

Background: NCCN guidelines and a continuously expanding collection of high-impact publications recommend interrogating hematologic neoplasms for biomarkers that yield diagnostic, prognostic, and/or therapeutic information. The advent of comprehensive, high-throughput genomic profiling technologies has enabled the detection of multiple genomic alterations in an efficient and cost effective manner, and provides insights into disease initiation, progression, therapy response and identification of therapeutic targets not available through conventional methods. Design: Based on NCCN guidelines and literature review, we designed a genomic profiling strategy composed of a targeted amplicon-based next generation sequencing (NGS) assay and cytogenomic microarray (CGA) to identify clinically actionable genomic alterations. In the current context, "actionable" was defined as helping establish a diagnosis through demonstration of "clonal hematopoiesis" as recommended by NCCN guidelines, informing prognosis, and/or providing a potential therapeutic target. These assays complement the routine work-up of hematologic tumors, which include flow cytometry, morphologic evaluation and FISH/cytogenetic analyses. Initial data from implementation of this testing strategy in our broad community-based practice are presented. Results: 865 patient samples were analyzed, which included the following: AML (168), ALL (24), MDS (194), MDS/MPN (34), CLL (82), MPN (156), samples with suspicion of a myeloid stem cell disorder (107) and others. Of the cases evaluated by NGS and CGA, genomic aberrations were detected in 70% and 48%, respectively. Conventional cytogenetic analyses revealed abnormalities in 38% of the cases for which conclusive results were obtained; abnormal FISH results were observed in 44.7%. In cases where conventional cytogenetics and FISH tests were negative, 70% were abnormal by either NGS or CGA (~81% in cases with evidence of a myeloid stem cell disorder or acute leukemia) (Figure 1). Importantly, 12% of 51 cases with normal FISH, cytogenetics and NGS results were abnormal by CGA, and 60% of 111 cases with normal FISH, cytogenetics and CGA results had actionable mutations detected by NGS. CGA and NGS aberrations were frequently detected in MPN or MDS/MPN cases with negative cytogenetics and FISH results. For example, among 156 MPN cases, CGA and NGS abnormalities were observed in 42% and 65% of cases respectively, while only 11% of cases had abnormal cytogenetics results and 10% of cases were FISH positive (Figure 2). In contrast, in cases without actionable mutations detected by NGS and CGA, which also had been analyzed by FISH/cytogenetics, 11% of 89 were cytogenetically abnormal, and 36% of 75 had genomic alterations detected by FISH. Conclusions: High throughput genomic tumor profiling through targeted DNA sequencing and analysis of copy number alterations complements conventional methods of tumor interrogation and leads to more frequent detection of actionable alterations. This is especially apparent in the context of morphologic and/or clinical suspicion of a myeloid stem cell disorder. These data indicate that integrating multiple strategies to identify informative biomarkers can enhance diagnosis, prognosis and/or therapy in hematologic disorders. Most importantly, we have observed changes in clinical management decisions across different disease states including AML, ALL, other myeloid malignancies, and CLL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ma: PathGroup: Employment. Gottwals:PathGroup: Employment. Ho:PathGroup: Employment. Fasig:PathGroup: Employment. Rietz:PathGroup: Employment. Hartley:PathGroup: Employment. Kravstov:PathGroup: Employment. Spence:PathGroup: Employment. Connor:PathGroup: Employment. Turnicky:PathGroup: Employment. Prescott:PathGroup: Employment. Lennon:PathGroup: Employment. Sathanoori:PathGroup: Employment. Flinn:Celgene Corporation: Research Funding. Berdeja:Celgene: Research Funding; Takeda: Research Funding; Curis: Research Funding; Abbvie: Research Funding; Onyx: Research Funding; MEI: Research Funding; Acetylon: Research Funding; Novartis: Research Funding; Array: Research Funding; Janssen: Research Funding; BMS: Research Funding. Wheeler:PathGroup: Employment. Coldren:PathGroup: Employment. Chandra:PathGroup: Employment. Mukherjee:PathGroup: Employment. Casey:PathGroup: Employment.

The achaete-scute (ac/sc) genes are a highly conserved family of transcription factors that play important roles in the development of neural cells in both vertebrates and invertebrates. As such, the study of arthropod ac/sc gene expression during neurogenesis has become a model system for investigating the evolution of neural patterning. To date, ac/sc gene expression has been investigated in insects, chelicerates, and myriapods. Here we present the identification of two ac/sc genes from the branchiopod crustacean Triops longicaudatus. Triops longicaudatus achaete-scute homologs1 and 2 (Tl-ASH1 and Tl-ASH2) exhibit dynamic and distinct expression profiles during Triops neurogenesis. Tl-ASH1 expression initiates in nearly all cells of the neurogenic region and subsequently in clusters of cells evenly spaced along the length of the developing limbs. In contrast, Tl-ASH2 initiates expression after Tl-ASH1. In the CNS, only a subset of Tl-ASH1 cells appears to express Tl-ASH2. Similarly, in the PNS individual Tl-ASH2 positive cells appear to arise from the clusters of Tl-ASH1 expressing cells. Shortly after activating Tl-ASH2 expression, these cells enlarge and divide. The expression dynamics of ac/sc genes in Triops parallel those observed in insects and contrasts with those found in chelicerates and myriapods.

Understanding the generation of neuronal and glial diversity is one of the major goals of developmental neuroscience. The Drosophila CNS midline cells constitute a simple neurogenomic system to study neurogenesis, cell fate acquisition, and neuronal function. Previously, we identified and determined the developmental expression profiles of 224 midline-expressed genes. Here, the expression of 59 transcription factors, signaling proteins, and neural function genes was analyzed using multi-label confocal imaging, and their expression patterns mapped at the single-cell level at multiple stages of CNS development. These maps uniquely identify individual cells and predict potential regulatory events and combinatorial protein interactions that may occur in each midline cell type during their development. Analysis of neural function genes, including those encoding peptide neurotransmitters, neurotransmitter biosynthetic enzymes, transporters, and neurotransmitter receptors, allows functional characterization of each neuronal cell type. This work is essential for a comprehensive genetic analysis of midline cell development that will likely have widespread significance given the high degree of evolutionary conservation of the genes analyzed.

The Drosophila columnar genes are key regulators of neural precursor formation and patterning along the dorsal–ventral axis of the developing CNS and include ventral nerve cord defective (vnd), intermediate nerve cord defective (ind), muscle segment homeodomain (msh), and Epidermal growth factor receptor (Egfr). To investigate the evolution of neural pattern formation, we identified and determined the expression patterns of Tribolium vnd, ind, and msh, and found that they are expressed in the medial, intermediate, and lateral columns of the developing CNS, respectively, in patterns similar, but not identical, to their Drosophila orthologs. The pattern of Egfr activity suggests that the genetic regulatory mechanisms that initiate Tc-vnd expression are similar in Drosophila and Tribolium, whereas those that initiate Tc-ind have diverged. RNAi analyses of gene function show that Tc-vnd and Tc-ind promote the formation of medial and intermediate column neural precursors and that vnd-mediated repression of ind establishes the boundary between the medial and intermediate columns. These data suggest that columnar gene expression and function underlie neural pattern formation in Drosophila, Tribolium, and potentially all insects, but that subtle spatiotemporal differences in expression of these genes may produce species-specific morphological differences.