Intrachromosomal Rearrangements Research Articles

Distinct Patterns of Colocalization of the CCND1 and CMYC Genes With Their Potential Translocation Partner IGH at Successive Stages of B-Cell Differentiation.

The immunoglobulin heavy chain (IGH) locus is submitted to intra-chromosomal DNA breakages and rearrangements during normal B cell differentiation that create a risk for illegitimate inter-chromosomal translocations leading to a variety of B-cell malignancies. In most Burkitt's and Mantle Cell lymphomas, specific chromosomal translocations juxtapose the IGH locus with a CMYC or Cyclin D1 (CCND1) gene, respectively. 3D-fluorescence in situ hybridization was performed on normal peripheral B lymphocytes induced to mature in vitro from a naive state to the stage where they undergo somatic hypermutation (SHM) and class switch recombination (CSR). The CCND1 genes were found very close to the IGH locus in naive B cells and further away after maturation. In contrast, the CMYC alleles became localized closer to an IGH locus at the stage of SHM/CSR. The colocalization observed between the two oncogenes and the IGH locus at successive stages of B-cell differentiation occurred in the immediate vicinity of the nucleolus, consistent with the known localization of the RAGs and AID enzymes whose function has been demonstrated in IGH physiological rearrangements. We propose that the chromosomal events leading to Mantle Cell lymphoma and Burkitt's lymphoma are favored by the colocalization of CCND1 and CMYC with IGH at the time the concerned B cells undergo VDJ recombination or SHM/CSR, respectively. J. Cell. Biochem. 117: 1506-1510, 2016. © 2016 Wiley Periodicals, Inc.

The Current Status of Solitary Fibrous Tumor: Diagnostic Features, Variants, and Genetics.

Solitary fibrous tumor (SFT) is a fibroblastic mesenchymal tumor originally described in the pleura but now shown at almost every anatomic site. Histopathologically, SFT is characteristically a circumscribed neoplasm composed of variably cellular and patternless distributions of bland spindle and ovoid cells within prominent collagenous stroma and shows diffuse expression of CD34, but it has a broad spectrum of both morphology and of biologic behavior. Many different names (particularly hemangiopericytoma) were previously used in the course of our understanding of this neoplasm but are now subsumed under the term "SFT," and the putative cell of origin was debated. However, it is now recognized that SFT is a translocation-associated neoplasm, consistently associated with NAB2-STAT6 gene fusions arising from recurrent intrachromosomal rearrangements on chromosome 12q, and this translocation is a likely major contributor to its pathogenesis. While most SFT with classical morphologic features behave in an indolent manner and those with overtly malignant histologic features tend to be aggressive neoplasms that behave as high-grade sarcomas, the behavior of SFT is unpredictable, and it is important to be aware of the propensity for aggressive behavior in a minority of histologically classical SFT and to ensure adequate clinical follow-up. Surgical excision remains the treatment gold standard; while radiotherapy and conventional chemotherapeutic agents have only shown limited efficacy, further understanding of the molecular events underlying tumorigenesis may allow the development of novel targeted treatments. We review SFT, discussing the morphologic spectrum and variants, including malignant and dedifferentiated subtypes, clinicopathological aspects, recent molecular genetic findings, and the differential diagnosis.

The 14/15 association as a paradigmatic example of tracing karyotype evolution in New World monkeys.

Fluorescence in situ hybridization (FISH), especially chromosome painting, has been extensively exploited in the phylogenetic reconstruction of primate evolution. Although chromosome painting is a key method to map translocations, it is not effective in detecting chromosome inversions, which may be up to four times more frequent than other chromosomal rearrangements. BAC-FISH instead can economically delineate marker order and reveal intrachromosomal rearrangements. However, up to now, BAC-FISH was rarely used to study the chromosomes of New World monkeys partly due to technical difficulties. In this paper, we used BAC-FISH to disentangle the complex evolutionary history of the ancestral 14/15 association in NWMs, beginning from the squirrel monkey (Saimiri boliviensis). To improve the hybridization efficiency of BAC-FISH in NWMs, we "translated" the human BACs into Callithrix jacchus (CJA) BACs, which yielded much higher hybridization efficiencies on other NWM species than human BACs. Our results disclosed 14 synteny blocks in squirrel monkeys, 7 more than with chromosome painting. We then applied a subset of CJA BACs on six other NWM species. The comparison of the hybridization pattern of these species contained phylogenetic information to discriminate evolutionary relationships. Notably Aotus was found to share an inversion with Callithrix, thus definitely assigning the genus Aotus to Cebidae. The present study can be seen as a paradigmatic approach to investigate the phylogenetics of NWMs by molecular cytogenetics.

Comparative study of the structure of chromosome 1R derived from Secale montanum and Secale cereale

AbstractWe produced 15 dissection lines of common wheat carrying segments of chromosome 1R of wild rye (Secale montanum) (1Rm) by the gametocidal system. Using the 1Rm dissection lines and previously established 24 dissection lines of chromosome 1R from cultivated rye (Secale cereale cv. ‘Imperial’) (1Ri), we conducted cytological mapping of 97 markers that were amplified in the 1Ri addition line. Sixty‐eight of the 97 markers were amplified in the 1Rm addition line. To reveal what structural differentiation occurred in chromosome 1R during domestication, we compared the cytological map of chromosome 1Ri with that of chromosome 1Rm, and also with the previously published cytological map of chromosome 1R from wheat cultivar ‘Burgas 2’ (1RB). There was one discrepancy in marker order in the satellite region between chromosomes 1Ri and 1RB, while there were four discrepancies in marker order between chromosomes 1Ri and 1Rm. These results suggested that during the domestication of rye, some intrachromosomal rearrangements had occurred in chromosome 1R, although this chromosome is regarded as the most stable chromosome in the rye genome.

Integrative Analyses of Lung Squamous Cell Carcinoma in Ten Chinese Patients with Transcriptome Sequencing

Few effective therapies have been developed for the treatment of lung squamous cell carcinoma (SQCC), in part due to a lack of understanding regarding the mechanisms underlying the initiation and development of this disease. Whole transcriptome sequencing not only provides insight into the expression of all transcribed genes, but offers an efficient approach for identifying genetic variations, including gene fusions, mutations and alternative splicing. In this study, we performed whole transcriptome sequencing of 10 patients with stage IIIA lung SQCC, and discovered a large number of single nucleotide variants (SNVs; mean of 12.2 SNVs/Mb), with C>T/G>A and A>G/T>C transitions being the most frequently observed. Additionally, a total of 132 gene fusions were identified based upon TopHat alignments, 70.5% (93/132) of which occurred as a result of intra-chromosomal rearrangements. Based on the number of supporting reads for each fusion, we further validated 20 of the 26 top gene fusions by RT-PCR and Sanger sequencing. Taken together, these data provide an in-depth view of transcriptional alterations in lung SQCC patients, and may be useful for identification of new therapeutic targets.

Abstract 1108: SPOP mutation leads to genomic instability in prostate cancer

Abstract Background: Genomic instability is a fundamental feature of human cancer, and DNA repair defects resulting in impaired genome maintenance promote pathogenesis of many types of cancers. In prostate cancer, structural genomic rearrangements, including translocations and copy number aberrations, are a key mechanism driving tumorigenesis. Recently, whole genome sequencing revealed a striking abundance, complexity, and heterogeneity of genomic rearrangements, potentially suggesting distinct mechanisms of instability in different molecular classes of prostate cancer. However, the somatic alterations underlying these phenomena remain largely undefined. Recurrent mutations in SPOP, the substrate-recognition component of an E3-ubiquitin ligase, represent the most common point mutations in primary prostate cancer, occurring in about 10% of tumors. SPOP mutations define a distinct molecular class of prostate cancer; they are mutually exclusive with TMPRSS2-ERG fusions, but harbor distinct patterns of copy number aberrations. Here, we report that SPOP mutant prostate cancers also harbor increased numbers of genomic rearrangements, and functional data suggest that SPOP mutation alters repair of DNA double strand breaks (DSB). Methods: We systematically investigated somatic alterations associated with genomic rearrangements, using a composite data set of 402 clinically localized prostate cancers. Functional analyses in vitro and in vivo were used to define pathways affected, and interrogate DNA repair phenotypes. Results: In human prostate cancers, SPOP mutation is an early event specifically associated with increased intrachromosomal genomic rearrangements. Using a zebrafish model, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation, implicating altered repair of DSB. In vitro data suggest that SPOP participates in repair of DSB, and SPOP mutation impairs homology-directed repair (HDR), instead promoting error-prone non-homologous end joining (NHEJ). Finally, SPOP mutation sensitizes prostate cancer cells to DNA damaging therapeutic agents such as PARP inhibitors. Conclusions: These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that SPOP mutant prostate cancer may be selectively responsive to DNA damaging therapeutics. Citation Format: Gunther Boysen, Christopher E. Barbieri, Davide Prandi, Sung-Suk Chae, Srilakshmi Nataraj, Mirjam Blattner, Clarisse Marotz, Limei Xu, Paola Lecca, Sagar Chhangawala, Pengbo Zou, Andrea Sboner, Francesca Demichelis, Yariv Houvras, Mark A. Rubin. SPOP mutation leads to genomic instability in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1108. doi:10.1158/1538-7445.AM2015-1108

Abstract NG01: SPOP mutation is associated with genomic instability in prostate cancer

Abstract Background: Genomic instability is a fundamental feature of human cancer, and DNA repair defects resulting in impaired genome maintenance promote pathogenesis of many types of cancers. In prostate cancer, structural genomic rearrangements, including translocations and copy number aberrations, are a key mechanism driving tumorigenesis. Recently, whole genome sequencing revealed a striking abundance, complexity, and heterogeneity of genomic rearrangements, potentially suggesting distinct mechanisms of instability in different molecular classes of prostate cancer. However, the somatic alterations underlying these phenomena remain largely undefined. Recurrent mutations in SPOP, the substrate-recognition component of an E3-ubiquitin ligase, represent the most common point mutations in primary prostate cancer, occurring in about 10% of tumors. SPOP mutations define a distinct molecular class of prostate cancer; they are mutually exclusive with TMPRSS2-ERG fusions, but harbor distinct patterns of copy number aberrations. Here, we report that SPOP mutant prostate cancers also harbor increased numbers of genomic rearrangements, and functional data suggest that SPOP mutation alters repair of DNA double strand breaks (DSB). Methods: We systematically investigated somatic alterations associated with genomic rearrangements, using a composite data set of 402 clinically localized prostate cancers. Functional analyses in vitro and in vivo were used to define pathways affected, and interrogate DNA repair phenotypes. Results: In human prostate cancers, SPOP mutation is an early event specifically associated with increased intrachromosomal genomic rearrangements. Using a zebrafish model, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation, implicating altered repair of DSB. In vitro data suggest that SPOP participates in repair of DSB, and SPOP mutation impairs homology-directed repair (HDR), instead promoting error-prone non-homologous end joining (NHEJ). Finally, SPOP mutation sensitizes prostate cancer cells to DNA damaging therapeutic agents such as PARP inhibitors. Conclusions: These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that SPOP mutant prostate cancer may be selectively responsive to DNA damaging therapeutics. Citation Format: Christopher E. Barbieri, Gunther Boysen, Davide Prandi, Sung-Suk Chae, Arun Dahiya, Srilakshmi Nataraj, Mirjam Blattner, Clarisse Marotz, Limei Xu, Julie Huang, Paola Lecca, Sagar Chhangawala, Pengbo Zhou, Andrea Sboner, Francesca Demichelis, Yariv Houvras, Mark A. Rubin. SPOP mutation is associated with genomic instability in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG01. doi:10.1158/1538-7445.AM2015-NG01

Intrachromosomal rearrangements in two representatives of the genus Saltator (Thraupidae, Passeriformes) and the occurrence of heteromorphic Z chromosomes.

Saltator is a genus within family Thraupidae, the second largest family of Passeriformes, with more than 370 species found exclusively in the New World. Despite this, only a few species have had their karyotypes analyzed, most of them only with conventional staining. The diploid number is close to 80, and chromosome morphology is similar to the usual avian karyotype. Recent studies using cross-species chromosome painting have shown that, although the chromosomal morphology and number are similar to many species of birds, Passeriformes exhibit a complex pattern of paracentric and pericentric inversions in the chromosome homologous to GGA1q in two different suborders, Oscines and Suboscines. Hence, considering the importance and species richness of Thraupidae, this study aims to analyze two species of genus Saltator, the golden-billed saltator (S. aurantiirostris) and the green-winged saltator (S. similis) by means of classical cytogenetics and cross-species chromosome painting using Gallus gallus and Leucopternis albicollis probes, and also 5S and 18S rDNA and telomeric sequences. The results show that the karyotypes of these species are similar to other species of Passeriformes. Interestingly, the Z chromosome appears heteromorphic in S. similis, varying in morphology from acrocentric to metacentric. 5S and 18S probes hybridize to one pair of microchromosomes each, and telomeric sequences produce signals only in the terminal regions of chromosomes. FISH results are very similar to the Passeriformes already analyzed by means of molecular cytogenetics (Turdus species and Elaenia spectabilis). However, the paracentric and pericentric inversions observed in Saltator are different from those detected in these species, an observation that helps to explain the probable sequence of rearrangements. As these rearrangements are found in both suborders of Passeriformes (Oscines and Suboscines), we propose that the fission of GGA1 and inversions in GGA1q have occurred very early after the radiation of this order.

A High-Resolution Comparative Chromosome Map of Cricetus cricetus and Peromyscus eremicus Reveals the Involvement of Constitutive Heterochromatin in Breakpoint Regions

Compared to humans and other mammals, rodent genomes, specifically Muroidea species, underwent intense chromosome reshuffling in which many complex structural rearrangements occurred. This fact makes them preferential animal models for studying the process of karyotype evolution. Here, we present the first combined chromosome comparative maps between 2 Cricetidae species, Cricetus cricetus and Peromyscus eremicus, and the index species Mus musculus and Rattus norvegicus. Comparative chromosome painting was done using mouse and rat paint probes together with in silico analysis from the Ensembl genome browser database. Hereby, evolutionary events (inter- and intrachromosomal rearrangements) that occurred in C. cricetus and P. eremicus since the putative ancestral Muroidea genome could be inferred, and evolutionary breakpoint regions could be detected. A colocalization of constitutive heterochromatin and evolutionary breakpoint regions in each genome was observed. Our results suggest the involvement of constitutive heterochromatin in karyotype restructuring of these species, despite the different levels of conservation of the C. cricetus (derivative) and P. eremicus (conserved) genomes.

A microsatellite-based linkage map for song sparrows (Melospiza melodia).

Although linkage maps are important tools in evolutionary biology, their availability for wild populations is limited. The population of song sparrows (Melospiza melodia) on Mandarte Island, Canada, is among the more intensively studied wild animal populations. Its long-term pedigree data, together with extensive genetic sampling, have allowed the study of a range of questions in evolutionary biology and ecology. However, the availability of genetic markers has been limited. We here describe 191 new microsatellite loci, including 160 high-quality polymorphic autosomal, 7 Z-linked and 1 W-linked markers. We used these markers to construct a linkage map for song sparrows with a total sex-averaged map length of 1731 cM and covering 35 linkage groups, and hence, these markers cover most of the 38-40 chromosomes. Female and male map lengths did not differ significantly. We then bioinformatically mapped these loci to the zebra finch (Taeniopygia guttata) genome and found that linkage groups were conserved between song sparrows and zebra finches. Compared to the zebra finch, marker order within small linkage groups was well conserved, whereas the larger linkage groups showed some intrachromosomal rearrangements. Finally, we show that as expected, recombination frequency between linked loci explained the majority of variation in gametic phase disequilibrium. Yet, there was substantial overlap in gametic phase disequilibrium between pairs of linked and unlinked loci. Given that the microsatellites described here lie on 35 of the 38-40 chromosomes, these markers will be useful for studies in this species, as well as for comparative genomics studies with other species.

MP66-01 SPOP MUTATION LEADS TO GENOMIC INSTABILITY IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2015MP66-01 SPOP MUTATION LEADS TO GENOMIC INSTABILITY IN PROSTATE CANCER Christopher Barbieri, Gunther Boysen, Davide Prandi, Sung-Suk Chae, Arun Dahiya, Srilakshmi Nataraj, Mirjam Blattner, Clarisse Marotz, Limei Xu, Julie Huang, Paola Lecca, Sagar Chhangawala, Pengbo Zhou, Andrea Sboner, Francesca Demichelis, Yariv Houvras, and Mark Rubin Christopher BarbieriChristopher Barbieri More articles by this author , Gunther BoysenGunther Boysen More articles by this author , Davide PrandiDavide Prandi More articles by this author , Sung-Suk ChaeSung-Suk Chae More articles by this author , Arun DahiyaArun Dahiya More articles by this author , Srilakshmi NatarajSrilakshmi Nataraj More articles by this author , Mirjam BlattnerMirjam Blattner More articles by this author , Clarisse MarotzClarisse Marotz More articles by this author , Limei XuLimei Xu More articles by this author , Julie HuangJulie Huang More articles by this author , Paola LeccaPaola Lecca More articles by this author , Sagar ChhangawalaSagar Chhangawala More articles by this author , Pengbo ZhouPengbo Zhou More articles by this author , Andrea SbonerAndrea Sboner More articles by this author , Francesca DemichelisFrancesca Demichelis More articles by this author , Yariv HouvrasYariv Houvras More articles by this author , and Mark RubinMark Rubin More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2354AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Genomic instability is a fundamental feature of human cancer, and DNA repair defects resulting in impaired genome maintenance promote pathogenesis of many types of cancers. In prostate cancer, structural genomic rearrangements, including translocations and copy number aberrations, are a key mechanism driving tumorigenesis. Recently, whole genome sequencing revealed a striking abundance, complexity, and heterogeneity of genomic rearrangements, potentially suggesting distinct mechanisms of instability in different molecular classes of prostate cancer. However, the somatic alterations underlying these phenomena remain largely undefined. Recurrent mutations in SPOP, the substrate-recognition component of an E3-ubiquitin ligase, represent the most common point mutations in primary prostate cancer, occurring in about 10% of tumors. SPOP mutations define a distinct molecular class of prostate cancer; they are mutually exclusive with TMPRSS2-ERG fusions, but harbor distinct patterns of copy number aberrations. Here, we report that SPOP mutant prostate cancers also harbor increased numbers of genomic rearrangements, and functional data suggest that SPOP mutation alters repair of DNA double strand breaks (DSB). METHODS We systematically investigated somatic alterations associated with genomic rearrangements, using a composite data set of 402 clinically localized prostate cancers. Functional analyses in vitro and in vivo were used to define pathways affected, and interrogate DNA repair phenotypes. RESULTS In human prostate cancers, SPOP mutation is an early event specifically associated with increased intrachromosomal genomic rearrangements. Using a zebrafish model, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation, implicating altered repair of DSB. In vitro data suggest that SPOP participates in repair of DSB, and SPOP mutation impairs homology-directed repair (HDR), instead promoting error-prone non-homologous end joining (NHEJ). Finally, SPOP mutation sensitizes prostate cancer cells to DNA damaging therapeutic agents such as PARP inhibitors. CONCLUSIONS These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that SPOP mutant prostate cancer may be selectively responsive to DNA damaging therapeutics. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e816 Peer Review Report Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christopher Barbieri More articles by this author Gunther Boysen More articles by this author Davide Prandi More articles by this author Sung-Suk Chae More articles by this author Arun Dahiya More articles by this author Srilakshmi Nataraj More articles by this author Mirjam Blattner More articles by this author Clarisse Marotz More articles by this author Limei Xu More articles by this author Julie Huang More articles by this author Paola Lecca More articles by this author Sagar Chhangawala More articles by this author Pengbo Zhou More articles by this author Andrea Sboner More articles by this author Francesca Demichelis More articles by this author Yariv Houvras More articles by this author Mark Rubin More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Disruption of NCOA2 by recurrent fusion with LACTB2 in colorectal cancer.

Whole-genome and transcriptome sequencing were used to discover novel gene fusions in a case of colon cancer. A tumor-specific LACTB2-NCOA2 fusion originating from intra-chromosomal rearrangement of chromosome 8 was identified at both DNA and RNA levels. Unlike conventional oncogenic chimeric proteins, the fusion product lacks functional domain from respective genes, indicative of an amorphic rearrangement. This chimeric LACTB2-NCOA2 transcript was detected in 6 out of 99 (6.1%) colorectal cancer (CRC) cases, where NCOA2 was significantly downregulated. Enforced expression of wild-type NCOA2 but not the LACTB2-NCOA2 fusion protein impaired the pro-tumorigenic phenotypes of CRC cells, whereas knockdown of endogenous NCOA2 in normal colonocytes had opposite effects. Mechanistically, NCOA2 inhibited Wnt/β-catenin signaling through simultaneously upregulating inhibitors and downregulating stimulators of Wnt/β-catenin pathway. Collectively, our data supports that NCOA2 is a novel negative growth regulatory gene repressing the Wnt/β-catenin pathway in CRC, where recurrent fusion with LACTB2 contributes to its disruption.

Comprehensive identification of mutations induced by heavy-ion beam irradiation in Arabidopsis thaliana.

Heavy-ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy-ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole-genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array-CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy-ion beams. Array-CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar-ion and Fe-ion irradiation, respectively, with deletion sizes ranging from 149 to 602,180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar-ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy-ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy-ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.

Whole-genome sequence of the Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod genomes

The development of efficient sequencing techniques has resulted in large numbers of genomes being available for evolutionary studies. However, only one genome is available for all amphibians, that of Xenopus tropicalis, which is distantly related from the majority of frogs. More than 96% of frogs belong to the Neobatrachia, and no genome exists for this group. This dearth of amphibian genomes greatly restricts genomic studies of amphibians and, more generally, our understanding of tetrapod genome evolution. To fill this gap, we provide the de novo genome of a Tibetan Plateau frog, Nanorana parkeri, and compare it to that of X. tropicalis and other vertebrates. This genome encodes more than 20,000 protein-coding genes, a number similar to that of Xenopus. Although the genome size of Nanorana is considerably larger than that of Xenopus (2.3 vs. 1.5 Gb), most of the difference is due to the respective number of transposable elements in the two genomes. The two frogs exhibit considerable conserved whole-genome synteny despite having diverged approximately 266 Ma, indicating a slow rate of DNA structural evolution in anurans. Multigenome synteny blocks further show that amphibians have fewer interchromosomal rearrangements than mammals but have a comparable rate of intrachromosomal rearrangements. Our analysis also identifies 11 Mb of anuran-specific highly conserved elements that will be useful for comparative genomic analyses of frogs. The Nanorana genome offers an improved understanding of evolution of tetrapod genomes and also provides a genomic reference for other evolutionary studies.

Low rate of interchromosomal rearrangements during old radiation of gekkotan lizards (Squamata: Gekkota).

Gekkotan lizards are a highly specious (∼1600 described species) clade of squamate lizards with nearly cosmopolitan distribution in warmer areas. The clade is primarily nocturnal and forms an ecologically dominant part of the world nocturnal herpetofauna. However, molecular cytogenetic methods to study the evolution of karyotypes have not been widely applied in geckos. Our aim here was to uncover the extent of chromosomal rearrangements across the whole group Gekkota and to search for putative synapomorphies supporting the newly proposed phylogenetic relationships within this clade. We applied cross-species chromosome painting with the recently derived whole-chromosomal probes from the gekkonid species Gekko japonicus to members of the major gekkotan lineages. We included members of the families Diplodactylidae, Carphodactylidae, Pygopodidae, Eublepharidae, Phyllodactylidae and Gekkonidae. Our study demonstrates relatively high chromosome conservatism across the ancient group of gekkotan lizards. We documented that many changes in chromosomal shape across geckos can be attributed to intrachromosomal rearrangements. The documented rearrangements are not totally in agreement with the recently newly erected family Phyllodactylidae. The results also pointed to homoplasy, particularly in the reuse of chromosome breakpoints, in the evolution of gecko karyotypes.

Structural Variations Identified By Whole Transcriptome Sequencing (RNA-Seq) in Childhood ALL with Different Response to Therapy

Introduction. Acute Lymphoblastic Leukemia (ALL) is the most frequent type of childhood leukemia. It is a multi-step process, characterized by the expansion of a pre-leukemic clone, accumulating cooperative genetic events required for the full transformation and clinical manifestation. Recently, the technological advances in genome-wide profiling techniques have allowed a better understanding of its molecular basis and heterogeneity. However, incidence and cure rates greatly differ among children, reflecting diverse responses to drug treatment and distinguishing risk groups. This defines the need for molecular investigations to better understand leukemia biology and improve risk prediction.Aim. We applied a whole-transcriptome sequencing approach (RNA-seq) to characterize low- (LR) versus high-risk (HR) patients, to identify new genetic lesions associated to different early response to therapy.Methods. Total RNA was extracted from primary leukemic blast samples of 10 pediatric ALL patients (4LR and 6 HR, according to minimal residual disease monitoring), included in the Italian AIEOP-BFM ALL2000 protocol. Genome-wide DNA profiling was performed by Affymetrix Cyto2.7M Arrays, RNA-seq was carried out by Illumina GAIIx platform, and validations were performed using independent approaches, such as RT-PCR and FISH. Fusion events were detected using FusionMap software, followed by a custom computational pipeline for the reduction of false positives and the identification of the most likely fusion candidates. Potential interest for leukemia was explored by testing the occurrence of these candidate fusions and con-joined genes in other RNA-seq datasets from different tumors and normal blood samples (i.e.: 15 melanomas, 2 melanocytes, 45 CEU individuals from 1K Genomes Project, plus 25 AML and 12 ALL).Results. We sequenced the transcriptome of 10 childhood ALL cases, not carrying other clinical or genetic risk factors. We performed a comprehensive whole-transcriptome analysis, comprising identification of fusion transcripts, alternative splicing and SNPs. Priority was given to fusion transcripts, which could originate from intra- or inter-chromosomal rearrangements, since they might represent potential prognostic markers or therapeutic targets for personalized treatments. We identified 127 fusion candidates. Strikingly, 123 out of 127 events were identified as intra-chromosomal, 119 of which were involving two contiguous genes or with overlapping loci (the so-called “con-joined genes”). Among the four intra-chromosomal events, the NUP214-ABL1 fusion, previously found in T-ALL and responsive to kinase inhibitors, was here identified and validated in one HR B-ALL patient, thus opening new perspectives for targeted treatment options. Finally, among the four inter-chromosomal events, the novel PAX5-POM121C fusion was identified and validated in one LR patient. Both intra- and inter-chromosomal fusions resulted private or low-frequent events, not recurrent in other tumor types, nor in normal blood samples. Among the con-joined genes, we identified a subset of 22 events not present in melanoma nor in normal blood samples, but common to the external AML and ALL datasets.Conclusion. RNA-seq represents one of the most comprehensive approaches to identify genetic alterations carried by leukemia clones. Our analyses identified novel fusion genes, originated by either inter- or intra-chromosomal rearrangements, as well as a considerable number of con-joined genes. Further evaluations will address SNPs, mutations, gene expression changes and splice variants that could be related to a different risk of relapse, and the feasibility of the screening of these candidates on a larger population of consecutive clinical cases. DisclosuresNo relevant conflicts of interest to declare.

Five Int22h Homologous Copies in Association with Intron22 Inversion Type 3

IntroductionF8 intron22 inversion is the causative gene defect in up to 45% of severe hemophilia A (HA) patients mediated by recombination between three highly homologous copies located in intron 22 (int22h-1) and two other extragenic copies (int22h-2 and int22h-3) positioned more telomerically outside the gene. Intrachromosomal rearrangement between int22h-1 and int22h-3 provide the F8 type 1 inversion while the F8 type 2 inversion could be explained by the participation of the int22-h2 sequence in the homologous recombination. However, a third type, known since 1993*, was explained by the existence of a duplicated copy of int22h-3 or int22h-2 in case of intron22 inversion type 3A or 3B respectively.MethodsThree unrelated HA patients with intron22 type 3A/3B were identified by southern blotting. To appreciate the length of the int22h extragenic duplicated, genomic hybridisation was performed using Affimetrix CytoScan High-Density array.ResultsBreakpoint analyses by CGH-array in all patients show same duplication of approximately 180kb delimited between intron 1 of CLIC2 gene and distal repeat int22h-3reflecting the presence of five genomic int22 copies. A fourth non-hemophiliac case was added since analysis by CGH technique identified duplication delineated by same boundaries.ConclusionThis study suggests the existence of genotypes harboring five int22h copies mediated by 180 kb duplication at Xq28 locus probably not associated with HA. We propose that this duplication has happened by tandem inversed duplication. Such genotype is to be considered as polymorphism and could be associated with the two kinds of F8 intron22 inversion type 3 when intrachromosomal recombination has occurred between homologous copies.*Antonarakis SE and the international consortium study. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995; 86: 2206-2212 DisclosuresNo relevant conflicts of interest to declare.

GE-03 * GENOMIC CHARACTERIZATION OF SURVIVAL OUTLIERS IN GLIOBLASTOMA MULTIFORME

Despite the general poor prognosis for patients with GBM, a proportion survives well beyond the median survival of 12-14 months following diagnosis. To elucidate molecular features associated with disproportionately protracted survival, we conducted deep genomic comparative analysis of a cohort of patients receiving standard therapy (surgery plus concurrent radiation and temozolomide) wherein outliers were identified: patients who responded (long-term survivor, LTS) versus those who failed rapidly (short-term survivor, STS). The datasets enabled interrogation for signatures indicative of tumor vulnerability. Whole genomic, epigenetic and transcriptomic analyses of 18 patients, including 8 LTS with an average 30 months overall survival (OS) and 10 STS with an average of 7 months OS were performed to capture single nucleotide variants (SNVs), indels, translocations, intra-chromosomal rearrangements, copy number variants, along with DNA methylation and mRNA expression. LTS and STS cases showed equal proportion of 7p11.2 (EGFR) amplification and 9p21.3 (CDKN2A) deletion. However, LTS GBM showed frequent chromosomal gains in 4q12 (PDGFRA and KIT) and 12q14.1 (CDK4) and deletion in 19q13.33 (BAX, BCAT2 and CD33), whereas, STS GBM showed frequent deletion in 9p11.2 (FOXD4L2 and AQP7P3) and 22q11.21 (HIC2). In addition, LTS GBM showed a 2-fold increased chromosomal instability as compared to STS GBM. By gene expression analysis, supervised clustering using the CIN70 chromosomal instability signature showed a decreased expression of these markers in LTS GBM, corroborating the genomic analysis. Finally, integrating DNA methylation data with gene expression revealed in STS GBM hypermethylation and downregulation of EPHA3, MEG, and PPP1R9A, linked to defective cell migration and adhesion. In contrast, LTS GBM showed hypomethylation and an increased gene expression of KIT. We posit that genomic instability predicts vulnerability of GBM to standard therapy and coupled with genetic and epigenetic signatures may identify patients where front-line entry into alternative, targeted regimens would be a preferred, more-efficacious management.

GE-19 * GENOMICS GUIDED THERAPEUTIC APPROACH FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME (GBM) USING NEXT GENERATION SEQUENCING (NGS) TECHNOLOGIES

BACKGROUND: The genomic heterogeneity of glioblastoma likely underlies the low response rates (8-24%) for targeted agents among unselected populations. We tested whether NGS would be useful in identifying therapeutically-actionable genetic alterations; we sought to test if this translated into improved tumor control in a patient-derived GBM model. METHODS: Tumor specimens from each patient (n = 11) that were used for analyte extraction contained between 70-80% viable tumor cellularity. Genome sequence coverage was more than 30X for both tumor and germline genomes; tumor RNA sequencing included over 100 million reads. NGS of paired tumor and germline DNA enabled detection of single nucleotide variations (SNVs), indels, translocations, intra-chromosomal rearrangements, and copy number alterations. A custom drug-matching workflow utilizing publically available databases and curated literature on reported drug effects, pharmacokinetics, and blood-brain barrier (BBB) penetration was used to map individual gene alterations in tumors with associated drug-response relationships. One specimen was tested in vitro and in vivo using a matched, patient-derived xenograft model. RESULTS: NGS for the 11 tumor panel found ~30% of cases with EGFR amplification, p16, and/or PTEN deletion. Novel potentially actionable targets included a TRIM54-FGFR3 fusion, STAG2 mutation, KIF11 and KIF15 mutation, and BRAF amplification. One recurrent, MGMT-unmethylated GBM line with a TRIM54-FGFR3 fusion underwent chemovulnerability testing. Cyquant proliferation assays demonstrated sensitivity to the pan-FGFR1-3 inhibitor AZD4547 when compared to a line without FGFR alteration (IC50 3uM vs >10uM). Furthermore, a flank PDX study demonstrated significant activity by AZD4547 when compared with either placebo or temozolomide, resulting in near complete tumor stasis at 82+ days of dosing with AZD4547. CONCLUSIONS: These results support the notion that therapeutic planning for treatment of GBM can be individualized and predicted by genome- and transcription-based analysis on the primary tumor, with the ultimate goal to design individualized GBM therapy based upon genetic markers for treatment sensitivity.

Abstract IA9: Studying therapy response and resistance in mouse models of human breast cancer

Abstract Mouse models of human cancer not only permit us to gain a detailed insight into the specific genetic changes that drive tumor development and metastasis but also provide powerful tools to study the mechanisms underlying drug response and acquired resistance. Once these processes are understood in sufficient detail it may be possible to design combination therapies that not only cause complete remissions but also eliminate remnant cells that might elicit recurrent disease. We have developed genetically engineered mouse models (GEMMs) of E-cadherin mutated lobular breast cancer. These mice develop mammary tumors that closely resemble the lobular morphology and the metastatic spectrum of the cognate tumors in humans. We have used Sleeping Beauty (SB) based insertional mutagenesis (IM) screens in conditional E-cadherin mutant mice to identify cancer genes that collaborate with E-cadherin loss in mammary tumorigenesis. Approximately 50% of all tumors carry activating SB insertions in Fgfr2. These tumors are highly sensitive to FGFR inhibitors but eventually become resistant. We are currently analyzing SB insertions in FGFRi-resistant tumors to identify genes that are causal to the resistance phenotype. We have also established GEMMs and patient-derived xenograft (PDX) models for BRCA1-deficient triple-negative breast cancer. These mice develop mammary tumors that are characterized by genomic instability and hypersensitivity to DNA-damaging agents, including platinum drugs and PARP inhibitors. Nevertheless, none of these drugs are curative: tumors grow back after drug treatment and eventually become resistant. We have used DNA methylation analysis and focused or whole-exome sequencing to identify resistance mechanisms in matched pairs of therapy-sensitive and –resistant BRCA1-deficient mammary tumors from our GEMMs and PDX models. We found that resistance of BRCA1-deficient GEMM tumors to the PARP inhibitor olaparib can be induced by several mechanisms, including activation of P-glycoprotein drug efflux transporters, type of BRCA1 founder mutation and 53BP1 loss. Therapy resistance of BRCA1-mutated PDX tumors is caused by additional intragenic deletions that lead to restoration of the open reading frame. Therapy resistance of BRCA1-methylated PDX tumors is driven by loss of BRCA1 promoter methylation or by a novel resistance mechanism involving de novo BRCA1 gene fusions created by intrachromosomal genomic rearrangements. Citation Format: Jos Jonkers. Studying therapy response and resistance in mouse models of human breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA9.