Mate-pair Sequencing Research Articles

YAP-associated chromosomal instability and cholangiocarcinoma in mice.

Deregulated Hippo pathway signaling is associated with aberrant activation of the downstream effector yes-associated protein (YAP), an emerging key oncogenic mediator in cholangiocarcinoma (CCA). In our prior work, we have demonstrated that biliary transduction of YAP along with Akt as a permissive factor induces CCA in mice. To further delineate the mechanisms associated with YAP-associated biliary oncogenesis, we have established seven malignant murine cell lines from our YAP-driven murine CCA model. These cells express the CCA markers SRY (Sex Determining Region Y)-Box 9 (SOX9), cytokeratin (CK)-7 and 19 but lack hepatocyte nuclear factor 4 alpha and alpha-smooth muscle actin, markers of hepatocellular carcinoma and cancer-associated fibroblasts, respectively. Notably, the murine CCA cells can be readily implanted into mouse livers with resultant orthotopic tumor formation. In this unique syngeneic orthotopic murine model, tumors exhibit histopathologic features resembling human CCA. We analyzed transcriptome data from YAP-associated parent CCA tumor nodules and identified a gene expression pattern associated with chromosomal instability, known as CIN25. Similarly, mate-pair sequencing of the murine CCA cells revealed chromosomal missegregation with gains and losses of several whole chromosomes demonstrating aneuploidy. Of the CIN25 genes, forkhead box M1 (Foxm1), a key cell cycle regulator, was the most significantly upregulated CIN25 gene product. Accordingly, small interfering RNA (siRNA)-mediated silencing of YAP as well as FOXM1 inhibition with thiostrepton induced CCA cell death. These preclinical data imply a role for YAP-mediated chromosomal instability in cholangiocarcinoma, and suggest FOXM1 inhibition as a therapeutic target for CCA.

SVAtools for junction detection of genome-wide chromosomal rearrangements by mate-pair sequencing (MPseq)

Mate-pair sequencing (MPseq), using long-insert, paired-end genomic libraries, is a powerful next-generation sequencing-based approach for the detection of genomic structural variants. SVAtools is a set of algorithms to detect both chromosomal rearrangements and large (>10 kb) copy number variants (CNVs) in genome-wide MPseq data. SVAtools can also predict gene disruptions and gene fusions, and characterize the genomic structure of complex rearrangements. To illustrate the power of SVAtools' junction detection methods to provide comprehensive molecular karyotypes, MPseq data were compared against a set of samples previously characterized by traditional cytogenetic methods. Karyotype, FISH and chromosomal microarray (CMA), performed for 29 patients in a clinical laboratory setting, collectively revealed 285 breakpoints in 87 rearrangements. The junction detection methods of SVAtools detected 87% of these breakpoints compared to 48%, 42% and 57% for karyotype, FISH and CMA respectively. Breakpoint resolution was also reported to 1 kb or less and additional genomic rearrangement complexities not appreciable by standard cytogenetic techniques were revealed. For example, 63% of CNVs detected by CMA were shown by SVAtools' junction detection to occur secondary to a rearrangement other than a simple deletion or tandem duplication. SVAtools with MPseq provides comprehensive and accurate whole-genome junction detection with improved breakpoint resolution, compared to karyotype, FISH, and CMA combined. This approach to molecular karyotyping offers considerable diagnostic potential for the simultaneous detection of both novel and recurrent genomic rearrangements in hereditary and neoplastic disorders.

Breakpoint mapping and haplotype analysis of translocation t(1;12)(q43;q21.1) in two apparently independent families with vascular phenotypes.

BackgroundThe risk of serious congenital anomaly for de novo balanced translocations is estimated to be at least 6%. We identified two apparently independent families with a balanced t(1;12)(q43;q21.1) as an outcome of a “Systematic Survey of Balanced Chromosomal Rearrangements in Finns.” In the first family, carriers (n = 6) manifest with learning problems in childhood, and later with unexplained neurological symptoms (chronic headache, balance problems, tremor, fatigue) and cerebral infarctions in their 50s. In the second family, two carriers suffer from tetralogy of Fallot, one from transient ischemic attack and one from migraine. The translocation cosegregates with these vascular phenotypes and neurological symptoms.Methods and ResultsWe narrowed down the breakpoint regions using mate pair sequencing. We observed conserved haplotypes around the breakpoints, pointing out that this translocation has arisen only once. The chromosome 1 breakpoint truncates a CHRM3 processed transcript, and is flanked by the 5′ end of CHRM3 and the 3′ end of RYR2. TRHDE,KCNC2, and ATXN7L3B flank the chromosome 12 breakpoint.ConclusionsThis study demonstrates a balanced t(1;12)(q43;q21.1) with conserved haplotypes on the derived chromosomes. The translocation seems to result in vascular phenotype, with or without neurological symptoms, in at least two families. We suggest that the translocation influences the positional expression of CHRM3,RYR2,TRHDE,KCNC2, and/or ATXN7L3B.

Retention of Interstitial Genes between TMPRSS2 and ERG Is Associated with Low-Risk Prostate Cancer.

TMPRSS2-ERG gene fusions occur in over 50% of prostate cancers, but their impact on clinical outcomes is not well understood. Retention of interstitial genes between TMPRSS2 and ERG has been reported to influence tumor progression in an animal model. In this study, we analyzed the status of TMPRSS2-ERG fusion genes and interstitial genes in tumors from a large cohort of men treated surgically for prostate cancer, associating alterations with biochemical progression. Through whole-genome mate pair sequencing, we mapped and classified rearrangements driving ETS family gene fusions in 133 cases of very low-, low-, intermediate-, and high-risk prostate cancer from radical prostatectomy specimens. TMPRSS2-ERG gene fusions were observed in 44% of cases, and over 90% of these fusions occurred in ERG exons 3 or 4. ERG fusions retaining interstitial sequences occurred more frequently in very low-risk tumors. These tumors also frequently displayed ERG gene fusions involving alternative 5'-partners to TMPRSS2, specifically SLC45A3 and NDRG1 and other ETS family genes, which retained interstitial TMPRSS2/ERG sequences. Lastly, tumors displaying TMPRSS2-ERG fusions that retained interstitial genes were less likely to be associated with biochemical recurrence (P = 0.028). Our results point to more favorable clinical outcomes in patients with ETS family fusion-positive prostate cancers, which retain potential tumor-suppressor genes in the interstitial regions between TMPRSS2 and ERG Identifying these patients at biopsy might improve patient management, particularly with regard to active surveillance. Cancer Res; 77(22); 6157-67. ©2017 AACR.

Identification of a t(3;4)(p1.3;q1.5) translocation breakpoint in pigs using somatic cell hybrid mapping and high-resolution mate-pair sequencing.

Reciprocal translocations are the most frequently occurring constitutional structural rearrangements in mammalian genomes. In phenotypically normal pigs, an incidence of 1/200 is estimated for such rearrangements. Even if constitutional translocations do not necessarily induce defects and diseases, they are responsible for significant economic losses in domestic animals due to reproduction failures. Over the last 30 years, advances in molecular and cytogenetic technologies have led to major improvements in the resolution of the characterization of translocation events. Characterization of translocation breakpoints helps to decipher the mechanisms that lead to such rearrangements and the functions of the genes that are involved in the translocation. Here, we describe the fine characterization of a reciprocal translocation t(3;4) (p1.3;q1.5) detected in a pig line. The breakpoint was identified at the base-pair level using a positional cloning and chromosome walking strategy in somatic cell hybrids that were generated from an animal that carries this translocation. We show that this translocation occurs within the ADAMTSL4 gene and results in a loss of expression in homozygous carriers. In addition, by taking this translocation as a model, we used a whole-genome next-generation mate-pair sequencing approach on pooled individuals to evaluate this strategy for high-throughput screening of structural rearrangements.

SVachra: a tool to identify genomic structural variation in mate pair sequencing data containing inward and outward facing reads

BackgroundCharacterization of genomic structural variation (SV) is essential to expanding the research and clinical applications of genome sequencing. Reliance upon short DNA fragment paired end sequencing has yielded a wealth of single nucleotide variants and internal sequencing read insertions-deletions, at the cost of limited SV detection. Multi-kilobase DNA fragment mate pair sequencing has supplemented the void in SV detection, but introduced new analytic challenges requiring SV detection tools specifically designed for mate pair sequencing data. Here, we introduce SVachra – Structural Variation Assessment of CHRomosomal Aberrations, a breakpoint calling program that identifies large insertions-deletions, inversions, inter- and intra-chromosomal translocations utilizing both inward and outward facing read types generated by mate pair sequencing.ResultsWe demonstrate SVachra’s utility by executing the program on large-insert (Illumina Nextera) mate pair sequencing data from the personal genome of a single subject (HS1011). An additional data set of long-read (Pacific BioSciences RSII) was also generated to validate SV calls from SVachra and other comparison SV calling programs. SVachra exhibited the highest validation rate and reported the widest distribution of SV types and size ranges when compared to other SV callers.ConclusionsSVachra is a highly specific breakpoint calling program that exhibits a more unbiased SV detection methodology than other callers.

Preferential selection and transfer of euploid noncarrier embryos in preimplantation genetic diagnosis cycles for reciprocal translocations

To develop and validate a new strategy to distinguish between balanced/euploid carrier and noncarrier embryos in preimplantation genetic diagnosis (PGD) cycles for reciprocal translocations and to successfully achieve a live birth after selective transfer of a noncarrier embryo. Retrospective and prospective study. Invitro fertilization (IVF) units. Eleven patients undergoing mate pair sequencing for identification of translocation breakpoints, followed by clinical PGD cycles. Embryo biopsy with 24-chromosome testing to determine carrier status of balanced/euploid embryos. Definition of translocation breakpoints and polymerase chain reaction (PCR) diagnostic primers, correct diagnosis of euploid embryos for carrier status, and a live birth with a normal karyotype after transfer of a noncarrier embryo. In 9 of 11 patients (82%), translocation breakpoints were successfully identified. In four patients with a term PGD pregnancy established with a balanced/euploid embryo of unknown carrier status, the correct carrier status was retrospectively determined, matching with the cytogenetic karyotype of the resulting newborns. In a prospective PGD cycle undertaken by a patient with a 46,XY,t(7;14)(q22;q24.3) translocation, the four balanced/euploid embryos identified comprised three carriers and one noncarrier. Transfer of the noncarrier embryo resulted in birth of a healthy girl who was subsequently confirmed with a normal 46,XX karyotype. The combination of mate pair sequencing and PCR breakpoint analysis of balanced reciprocal translocation derivatives is a novel, reliable, and accurate strategy for distinguishing between carrier and noncarrier balanced/euploid embryos. The method has potential application in clinical PGD cycles for patients with reciprocal translocations or other structural rearrangements.

Abstract 4814: Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts

Abstract Purpose: The treatment of ovarian cancer poses a challenge since 70% of patients will relapse with incurable platinum resistant disease. Although ovarian cancers lack frequently-occurring driver mutations or amplifications, the study presented herein examines the concept of individualized therapy based on mate pair sequencing and protein expression in ovarian cancer patient-derived xenografts. Experimental Procedure: Macrodissection of patient tumor was followed by genomic DNA isolation and next-generation sequencing using an Illumina Mate Pair protocol to identify structural genomic changes and copy number variations. Secondary analyses were carried out to select potential therapeutic targets among those. The tumors were also propagated intraperitoneally in immunocompromised mice and treated with standard chemotherapy or/and targeted therapy chosen based on genomic analyses. Results: Mate pair analysis revealed that, in general, copy number changes (amplifications, gains and losses) were common in ovarian tumors and included genes potentially targetable. Tumors from early generation patient- derived xenografts showed a landscape of genomic aberrations identical to that of original patient tumor. One PDX model in which high expression of HER2 and copy number gains at RICTOR and AKT genetic loci were revealed, was treated with either chemotherapy and Pertuzumab/trastuzumab (HER2 inhibitors), MK-8669 (mTOR inhibitor) or MK-2206 (AKT inhibitor) or chemotherapy alone. Tumor size was measured by serial abdominal ultrasound and after 28 days, the final tumor size ratio compared to baseline was 0.23, 0.36, 0.55 and 0.56, respectively, indicating that the best response was seen in the combined chemotherapy and Pertuzumab/trastuzumab cohort and that was better than in the chemotherapy only cohort. Conclusions: Patient-derived xenografts models in conjunction with genomic and protein analyses of patient tumors are valuable for testing of combination therapies including specific targeting drugs. This approach also provides actionable information for more tailored treatment of ovarian cancer patients. Citation Format: Konstantinos Leventakos, Faye R. Harris, Lin Yang, Xiaonan Hou, Saravut Weroha, Irina V. Kovtun. Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4814. doi:10.1158/1538-7445.AM2017-4814

Abstract 490: Identification of oncogenic gene fusions in primary colon cancers

Abstract Introduction: Fusion genes (such as ALK-EML) are potentially attractive targets for anti-cancer treatment. In the era of personalized medicine exploring the identification, incidence, and functionality of fusion genes may contribute to effective treatment approaches. Methods: We performed a comprehensive and unbiased screening for gene fusions in a clinically well-defined prospectively collected cohort of 278 primary stage I to III colon cancers. Illumina RNA sequencing was performed using RNA from the fresh frozen samples. The STAR fusion gene detection pipeline and GATK RNA-seq variant calling were used to identify fusion genes and detect somatic genetic variations. Gene fusions were considered relevant when recurrent, when resulting in divergent expression (outlier analysis) or when functional relevance was predicted (i.e. kinase fusions). Results: 2.5% of all samples contained a relevant gene fusion. Kinase fusions were most prevalent with a frequency of 1.8%. Three BRAF-fusions were identified, two known (TRIM24-BRAF & AGAP3-BRAF) and one novel fusion (DLG1-BRAF). Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The outlier analysis revealed four unique fusions (EML4-NTRK3, RRBP1-RET, USPX9-ERAS & EIF3E-RSPO2). The EML4-NTRK3 fusion led to increased expression of the tyrosine kinase encoding domain exons, which is retained in the fusion transcript. Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The tumor with the RRBP1-RET fusion was pan negative for known driver mutations, as reported in literature. Mate-pair sequencing of the USPX9-ERAS fusion gene region revealed that the fusion gene was caused at the genomic level by a highly local chromothripsis event on chromosome X spanning solely the region covered by USP9X and ERAS, leading to high ERAS expression. ERAS is a constitutionally active RAS protein and normally only expressed in embryonic stem cells. Analysis of phosphorylated AKT after expression of the USP9X-ERAS fusion gene in NIH-3T3 A14 cells showed activation of AKT signaling. The occurrence of one R-spondin fusion in our set (0.4%) is low compared to the previously described 9.5%, which may be explained by difference in incidence of KRAS- or BRAF-mutations in the two cohorts, since all R-spondin fusions occurred in a tumor with a KRAS- or BRAF-mutation. All fusion genes were expressed in cell lines resulting in the activation of AKT signaling. Finally, oncogenic fusions were only found in lymph node negative tumors and were not observed in tumors with classical driver mutations in BRAF and KRAS except for the R-spondin fusion. Conclusions: We found oncogenic gene fusions including novel fusions with a frequency of 2.5%, including an USP9X-ERAS fusion with strong oncogenic activity in vitro. Although recurrent fusion genes are rare events in colorectal cancer they may represent functional drivers and provide potential novel leads for personalized therapeutic strategies. Citation Format: Robert R. Coebergh van den Braak, Wigard P. Kloosterman, Mark Pieterse, Markus van Roosmalen, Anieta M. Sieuwerts, Christina Stangl, Ronne Brunekreef, Zarina S. Lalmahomed, Salo Ooft, Anne van Galen, Marcel Smid, Armel Lefebvre, Fried J. Zwartkruis, John W. Martens, John A. Foekens, Katharina Biermann, Marco J. Koudijs, Jan N. IJzermans, Emile E. Voest. Identification of oncogenic gene fusions in primary colon cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2017-490

Genomic rearrangements in sporadic lymphangioleiomyomatosis: an evolving genetic story.

Sporadic lymphangioleiomyomatosis is a progressive pulmonary cystic disease resulting from the infiltration of smooth muscle-like lymphangioleiomyomatosis cells into the lung. The migratory/metastasizing properties of the lymphangioleiomyomatosis cell together with the presence of somatic mutations, primarily in the tuberous sclerosis complex gene (TSC2), lead many to consider this a low-grade malignancy. As malignant tumors characteristically accumulate somatic structural variations, which have not been well studied in sporadic lymphangioleiomyomatosis, we utilized mate pair sequencing to define structural variations within laser capture microdissected enriched lymphangioleiomyomatosis cell populations from five sporadic lymphangioleiomyomatosis patients. Lymphangioleiomyomatosis cells were confirmed in each tissue by hematoxylin eosin stain review and by HMB-45 immunohistochemistry in four cases. A mutation panel demonstrated characteristic TSC2 driver mutations in three cases. Genomic profiles demonstrated normal diploid coverage across all chromosomes, with no aneuploidy or detectable gains/losses of whole chromosomal arms typical of neoplastic diseases. However, somatic rearrangements and smaller deletions were validated in the two cases which lacked TSC2 driver mutations. Most significantly, one of these sporadic lymphangioleiomyomatosis cases contained two different size deletions encompassing the entire TSC1 locus. The detection of a homozygous deletion of TSC1 driving a predicted case of sporadic lymphangioleiomyomatosis, consistent with the common two-hit TSC2 mutation model, has never been reported for sporadic lymphangioleiomyomatosis. However, while no evidence of the hereditary tuberous sclerosis complex disease was reported for this patient, the potential for mosaicism and sub-clinical phenotype cannot be ruled out. Nevertheless, this study demonstrates that somatic structural rearrangements are present in lymphangioleiomyomatosis disease and provides a novel method of genomic characterization of sporadic lymphangioleiomyomatosis cells, aiding in defining cases with no detected mutations by conventional methodologies. These structural rearrangements could represent additional pathogenic mechanisms in sporadic lymphangioleiomyomatosis disease, potentially affecting response to therapy and adding to the complex genetic story of this rare disease.

57 - Development of a Mate Pair Lineage Assay to Ascertain Relation of Tumors

The presence of multiple lesions typically indicates a primary tumor with associated metastases but it is not uncommon for patients to present with multiple synchronous independent primary lesions. Given significant clinical implications, it is important to accurately make this distinction. Recently it was shown that mate pair genomic sequencing can accurately determine relatedness between a patient's multiple synchronous lung carcinomas. Thus, we are designing a clinical mate pair assay to differentiate related from unrelated synchronous tumors, irrespective of type. A variety of tumor types and sets will be collected: 1) multiple separate tissue samples from the same primary tumor; 2) tissue sample from primary tumor and from confirmed metastasis in the same patient; 3) tissue sample from two confirmed independent primary tumors from the same patient. Normal tissue will be sequenced for each patient to exclude germline similarities. Following mate pair sequencing, in-house algorithms will be used for mapping, variant calling, and relatedness. Data analysis will initially follow the method established by Murphy et al. (J Clin Oncol 32:4050–4058) in which a single somatic shared event was diagnostic of lineage. Further discussions of this test will include refinements of the algorithm and false positive rate. The pathologist's assessment of tumor relatedness based on morphology and immunohistochemistry will be the gold standard to determine the accuracy of this assay. The assay will be considered successful if it correctly identifies the lineage of related and unrelated tumors at a high frequency. Additionally, progress to date, challenges, and future steps will be discussed.

Evolution of the Banana Genome (Musa acuminata) Is Impacted by Large Chromosomal Translocations.

Most banana cultivars are triploid seedless parthenocarpic clones derived from hybridization between Musa acuminata subspecies and sometimes M. balbisiana. M. acuminata subspecies were suggested to differ by a few large chromosomal rearrangements based on chromosome pairing configurations in intersubspecies hybrids. We searched for large chromosomal rearrangements in a seedy M. acuminata ssp. malaccensis banana accession through mate-pair sequencing, BAC-FISH, targeted PCR and marker (DArTseq) segregation in its progeny. We identified a heterozygous reciprocal translocation involving two distal 3 and 10 Mb segments from chromosomes 01 and 04, respectively, and showed that it generated high segregation distortion, reduced recombination and linkage between chromosomes 01 and 04 in its progeny. The two chromosome structures were found to be mutually exclusive in gametes and the rearranged structure was preferentially transmitted to the progeny. The rearranged chromosome structure was frequently found in triploid cultivars but present only in wild malaccensis ssp. accessions, thus suggesting that this rearrangement occurred in M. acuminata ssp. malaccensis. We propose a mechanism for the spread of this rearrangement in Musa diversity and suggest that this rearrangement could have played a role in the emergence of triploid cultivars.

Toward the Integration of Mate Pair and RNA Sequencing to Identify Gene Fusions in Cancer Research: A Mini Review

Toward the Integration of Mate Pair and RNA Sequencing to Identify Gene Fusions in Cancer Research: A Mini Review

Novel t(5;11)(q32;q13.4) with NUMA1-PDGFRB fusion in a myeloid neoplasm with eosinophilia with response to imatinib mesylate

We report a NUMA1-PDGFRB fusion in a myeloproliferative neoplasm with eosinophilia in a 61-year old man, with response to imatinib mesylate therapy. A t(5;11) chromosome translocation involving bands 5q32 and 11q13.4 was identified by metaphase chromosome analysis, and rearrangement of the platelet-derived growth factor receptor beta (PDGFRB) gene on 5q32 was demonstrated by FISH using a PDGFRB break-apart probe set. Bacterial artificial chromosome (BAC) FISH mapping of the PDGFRB fusion partner gene narrowed the breakpoint at 11q13.4 to a 150 kb genomic region containing three genes, including NUMA1. Mate pair sequencing analysis demonstrated NUMA1-PDGFRB fusion. The fusion protein includes coiled-coil domains of nuclear mitotic apparatus protein 1 (NuMA1, involved in protein homodimerization and heteroassociation) and tyrosine kinase domains of PDGFRB. Diverse rearrangements involving the PDGFRB gene have been identified in myeloid and lymphoid neoplasms with eosinophilia, but rearrangement of the nuclear mitotic apparatus protein 1 (NUMA1) gene has previously been reported in a human malignancy in only one instance, a NUMA1-RARA fusion caused by a t(11;17) translocation in a patient with acute promyelocytic leukemia. The NUMA1-PDGFRB fusion is the second instance of rearrangement of NUMA1, encoding an element of the mitotic apparatus, in human cancer.

TFEB-VEGFA (6p21.1) co-amplified renal cell carcinoma: a distinct entity with potential implications for clinical management.

A subset of renal cell carcinomas shows TFEB overexpression secondary to MALAT1-TFEB gene fusion. As alternate mechanisms of TFEB overexpression are likely to have the same effect, we sought to determine the frequency of amplification of TFEB and the adjacent VEGFA gene at 6p21.1. As patients with metastatic renal cell carcinomas are managed with anti-VEGF therapies, we retrospectively assessed therapeutic response in patients with amplified tumors. Amplification status was analyzed for 875 renal cell carcinomas from our institution, a consultative case and 794 cases from The Cancer Genome Atlas. Cases were classified as having low level (5-10 copies), and high-level amplification (>10 copies), and were further analyzed for adjacent oncogene copy number status (n=6; 3 single-nucleotide polymorphism genomic microarray, 3 The Cancer Genome Atlas) and structural rearrangements (n=1; mate-pair sequencing). These were then reviewed for histopathology, immunophenotype, and response to VEGF-targeted therapy on follow-up. In all, 10/875 (1.1%) institutional cases, 1 consultative case, and 3/794 (0.4%) of The Cancer Genome Atlas cases showed TFEB high-level amplification, while 14/875 (1.6%) cases showed TFEB low-level amplification. All cases had associated VEGFA amplification. This was confirmed with evaluation for copy number changes (n=6). The 6p21.1 high and low-level amplified tumors occurred in adults (mean age: 66), with over half being ≥pT3 (13/25, 52%), and most showed oncocytic, tubulopapillary features and high grade (≥grade 3: 20/22, 91%). These were aggressive tumors with metastasis and death from renal cell carcinoma in 11 (of 24, 46%) cases. Four patients received targeted therapy and had a mean survival of 31 months (range: 17-50) post nephrectomy. In summary, a group of aggressive renal cell carcinomas show genomic amplification of the 6p21.1 region including TFEB and VEGFA genes and share morphologic features. Additional studies are warranted to determine whether these patients respond to anti-VEGF therapy.

A novel cytogenetic and molecular characterization of renal metanephric adenoma: Identification of partner genes involved in translocation t(9;15)(p24;q24)

Renal metanephric adenoma (MA) is a rare benign tumor frequently misclassified when microscopic features alone are applied. The correct classification of a renal tumor is critical for diagnostic, prognostic, and therapeutic purposes. Despite the advancements in cancer genomics, up until recently relatively few genetic alterations critical to MA development have been recognized. Recent data suggest that 90% of MA have BRAFV600E mutations; the genetics of the remaining 10% are unclear. To date, only one case of a chromosomal translocation, t(9;15)(p24;q24) associated with MA has been reported. However, the potential role of the KANK1 gene, which lies near the breakpoint of the short arm of chromosome 9p24, in the etiology of MA was not examined. We identified the same cytogenetic aberration utilizing molecular cytogenetic techniques in a 22-year-old female patient, and further investigated the genes involved in the translocation that might have contributed to tumorigenesis. A series of fluorescence in situ hybridization (FISH) probes identified the rearranged genes to be KANK1 on chromosome 9 (9p24.3) and NTRK3 on chromosome 15 (15q25.3). Mate-Pair genome sequencing validated the balanced translocation between 9p24.3 and 15q25.3, involving genes KANK1 and NTRK3, respectively. BRAFV600E mutational analysis was normal. Our findings indicate that gene fusions may be one mechanism by which functionally relevant genes are altered in the development of MA. Molecular and cytogenetic analyses have elucidated a novel genetic aberration, which helps to provide a better understanding of this genomic change and assist in diagnosis and classification of new subgroups/entities in metanephric adenomas.

Digit-a tool for detection and identification of genomic interchromosomal translocations.

Structural variations (SVs) in genomic DNA can have profound effects on the evolution of living organisms, on phenotypic variations and on disease processes. A critical step in discovering the full extent of structural variations is the development of tools to characterize these variations accurately in next generation sequencing data. Toward this goal, we developed a software pipeline named digit that implements a novel measure of mapping ambiguity to discover interchromosomal SVs from mate-pair and pair-end sequencing data. The workflow robustly handles the high numbers of artifacts present in mate-pair sequencing and reduces the false positive rate while maintaining sensitivity. In the simulated data set, our workflow recovered 96% of simulated SVs. It generates a self-updating library of common translocations and allows for the investigation of patient- or group-specific events, making it suitable for discovering and cataloging chromosomal translocations associated with specific groups, traits, diseases or population structures.

Accurate Breakpoint Mapping in Apparently Balanced Translocation Families with Discordant Phenotypes Using Whole Genome Mate-Pair Sequencing.

Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on de novo translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. PTCD1, ATP5J2-PTCD1, CADPS2, and STPG1 were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike de novo translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.

Abstract A34: Targeting genes which are amplified in ovarian cancer

Abstract Over the recent years significant knowledge has been accumulated on which drugs can be therapeutically beneficial for a given patient based on mutational profiling of their tumors using selected gene panels. Much less, however, is known under which conditions targeting of genes which are amplified could be beneficial. Apart from successfully targeting HER2-positive cancers, many of which frequently carry amplified ERBB2 gene, no other targeted therapy based on DNA amplifications or gains of the genes is offered to the patients due to lack of clinical response data. Using mate pair sequencing we have profiled 60 high grade serous ovarian tumors to compare genomic rearrangements and copy number changes. The analyses revealed that these tumors carry highly rearranged genome. Copy number changes amplifications, gains and losses were very common and included genes potentially therapeutically targetable. In contrast, only a few of targetable fusion genes were identified in a set of 60 cases. The tumors were propagated in immunocompromised mice (mouse avatars), sequenced and showed identical to original patient tumor landscape of rearrangements. Using these mouse avatar models we tested whether a therapy inhibiting selected based on genomic amplification targets provided a benefit compared to standard chemotherapy. Mice which harbored tumors showing amplifications of CDKs (CDK2, 4 and 9) and ERBB3 were treated with flavopiridol and pertuzumab respectively, and response was compared to that of standard for ovarian cancer chemotherapy (carboplatin and paclitaxel combination). We observed that application of either agent, significantly inhibited tumor growth in treated mice as compared to untreated. However, neither drug alone caused tumor regression. The treatment regimen then was modified to administer chemotherapy and targeted therapy together. A mouse avatars in which genomic analyses revealed amplification at loci involving AKT3 and RICTOR genes was treated with chemotherapy alone and in combination with MK2206 (AKT inhibitor) or MK8669 (mTOR inhibitor). The high level of both target proteins was confirmed by immunoblotting. Significant regression of tumor was observed after 6 weeks of treatment. Addition of either targeted drug provided an extra benefit in killing tumor cells compared to chemotherapy alone. The mice are followed to study tumor recurrence. These data indicated that genomic information obtained using next generation sequencing can aid therapy decisions by tailoring treatments to individual tumor makeup. Avatar models in conjunction with genomic analyses of the original tumors are invaluable for testing specific targeted therapies selected based on amplification/gains of putative genes-targets and for studying mechanisms of tumor recurrence and therapy resistance. Citation Format: Faye R. Harris, Lin Yang, Xiaonan Hou, Konstantinos Leventakos, Saravut J. Weroha, George Vasmatzis, Irina V. Kovtun. Targeting genes which are amplified in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A34.

IKZF1 Regulates BRD4 Retention and Recruitement at Enhancer in Myeloma Cells: Role of BRD4 in IMiDs Resistance

Background: MYC rearrangement is a recurrent somatic event in MM and results in super-enhancers repositioning within 3Kb (centromeric or telomeric) of the MYC locus. BRD4, a member of the bromodomain family, binds H3K27Ac and regulates enhancers activity, RNA polymerase II transcription initiation and elongation and in particular it regulates MYC transcription in MM cells. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKZF1/3 and the transcriptional repression of MYC and IRF4. However the exact mechanisms through which IMiDs downregulate MYC are not well understood.Methods and Results: Using a extended panel of 12 MM cell lines we have first confirmed our previous observartion that IMIDs universally degraded IKZF1/3, however unlike in IMiDs-sensitive cell lines it failed to downregulate MYC in resistant cells. Therefore, this finding suggests that MYC transcriptional regulation rather than IKF1/3 degradation is the "bottleneck" in IMiDs anti-myeloma activity or resistance. In addition, RNA-seq of paired and serially collected primary myeloma cells with acquired resistance to IMIDsconfirmed the enrichment of genes signatures associated with MYC at the time of acquired resistance. Of note, in a library of t(4;14) MM cell lines lenalidomide significantly downregulates FGFR3 while the expression of WHSC1 was not affected. As in t(4;14) cells FGFR3 is driven by the 3' IGH enhancer while WHSC1 is under the control of the intronic Em enhancer, and in view of the known role of IKZF1 in CSR, we posit that IKZF1 regulates plasma cells enhancer activity and hence MYC expression. To validate this hypothesis we first established the genome-wide distribution of IKZF1 in MM1S, RPMI8226 and OPM2 cells by ChIP-Seq. This analysis revealed the distribution of IKZF1 across the genome, predominantly to intronic an intragenic loci with only ~ 8% or reads mapping within +/- 5 kb of genes TSS. Importantly IKZF1 was also enriched at known myeloma cells enhancers or super-enhancers such as IGLL5, TXNDC5, BCL2L1, and DUSP22. We also mapped BRD4 binding to the genome in 8226 and MM1S cell lines and confirmed previously described mapping to known B-cell enhancers. Of note IKZF1 nearly universally overlapped with all BRD4 enhancers and superenhancers loci. These findings lead us to propose that IKZF1 depletion in response to IMiDs treatment will result in enhancer destabilization and displacement of BRD4 from enhancer loci. Indeed, Chip-Seq analysis of IKZF1 and BRD4 genomic distribution pre- and post- treatment with lenalidomide (10 uM for 4 or 8h) in IMiDs sensitive cell lines significantly depleted IKZF1 and BRD4 at superenhancer loci. In contrast, in IMiDs resistant cell lines (RPMI8226) and despite IKZF1 depletion, BRD4 was largely retained at these loci. These findings suggest that in IMiDs sensitive cells, IKZF1 is the major transcription factor mediating BRD4 recruitment to enhancer and superenhancer loci. In contrast, in IMiDs resistant cells and similar to BET-bromodomain inhibitor (JQ1) resistance, transcriptional plasticity or hyper-phosphorylation of BRD4 attributable to the phosphatase PP2A inactivation may also be mediating IMiDs resistance. Indeed the levels of p-BRD4 determined by western blotting were significantly higher in IMiDs resistant INA6 and RPMI8226 cells compared to MM1S cells. Furthermore, treatment of INA6 cells with the PP2A activator phenothiazine significantly sensitized INA6 cells to lenalidomide.Lastly, in order to investigate whether IMiDs resistance was associated with acquired new MYC rearrangements with an "IKZF1_resisant enhancer," we performed mate-pair sequencing of IMiDs resistance patients (18 post acquisition of IMIDs resistance and 7 pairs of pre- and post- acquisition of IMIDs resistance). MYC structural rearrangements (indels, translocations, inversions) were detected using DELLY and MANTA. Of note several MYC rearrangement were identified including MYC duplication in 1 patient, MYC structural rearrangement (±3Kb of TSS) in 9 patients (enhancer partner: TXNDC5, IGHa2, NSMCE2, IGLL5) and a newly acquired IRF4-IGH a2 rearrangement in 1 patient with pre-existing MYC-IGLL5 rearrangement.Conclusion:We have demonstrated that IMIDs alter enhancer activity in myeloma cells by depleting IKZF1 / IKZF3 and disrupting BRD4 binding to enhancers and super-enhancers, stalling of RNA polymerase II and MYC transcription. DisclosuresNeri:Celgene and Jannsen: Consultancy, Honoraria. Bergsagel:Amgen, BMS, Novartis, Incyte: Consultancy; Novartis: Research Funding. Bahlis:Onyx: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; BMS: Honoraria; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau.