In-house Developed Pipeline Research Articles

Candidate lethal haplotypes and causal mutations in Angus cattle

BackgroundIf unmanaged, high rates of inbreeding in livestock populations adversely impact their reproductive fitness. In beef cattle, historical selection strategies have increased the frequency of several segregating fatal autosomal recessive polymorphisms. Selective breeding has also decreased the extent of haplotypic diversity genome-wide. By identifying haplotypes for which homozygotes are not observed but would be expected based on their frequency, candidates for developmentally lethal recessive loci can be localized. This analysis comes without the need for observation of the loss-associated phenotype (e.g., failure to implant, first trimester abortion, deformity at birth). In this study, haplotypes were estimated for 3961 registered Angus individuals using 52,545 SNP loci using findhap v2, which exploited the complex pedigree among the individuals in this population.ResultsSeven loci were detected to possess haplotypes that were not observed in homozygous form despite a sufficiently high frequency and pedigree-based expectation of homozygote occurrence. These haplotypes were identified as candidates for harboring autosomal recessive lethal alleles. Of the genotyped individuals, 109 were resequenced to an average 27X depth of coverage to identify putative loss-of-function alleles genome-wide and had variants called using a custom in-house developed pipeline. For the candidate lethal-harboring haplotypes present in these bulls, sequence-called genotypes were used to identify concordant variants. In addition, whole-genome sequence imputation of variants was performed into the set of 3961 genotyped animals using the 109 resequenced animals to identify candidate lethal recessive variants at the seven loci. Following the imputation, no variants were identified that were fully concordant with the marker-based diplotypes.ConclusionsSelective breeding programs could utilize the predicted lethal haplotypes associated with SNP genotypes. Sequencing and other methods for identifying the causal variants underlying these haplotypes can allow for more efficient methods of management such as gene editing. These two methods in total will reduce the negative impacts of inbreeding on fertility and maximize overall genetic gains.

Abstract P1-07-09: A multi-OMIC analysis to explore the impact of “actionable” genomic alterations on protein pathway activation: Clinical implication for precision medicine in metastatic breast cancer

Abstract Background: While genomic alterations are central players in tumor progression, proteins are the targets for precision therapy. The degree by which “actionable” genomic alterations translate into activated/altered proteins and pathway is still under investigation. Using a multi-OMIC approach from the SideOut 2 metastatic breast cancer (MBC) trial, this study explored the concordance between selected “actionable” genomic alterations and protein expression/activation. Methods: Snap frozen biopsies from 29 MBC patients enrolled in a prospective phase II trial were used for this analysis. Exome WES and RNASeq data was processed using an in-house developed pipeline and identified amplification of CCND1 (6/29), FGFR1 (4/29), and FGF 3, 4, 5, and 19 (4/29) as some of most frequent “actionable” genomic alterations in our MBC cohort. Signaling analysis of the 29 cases was performed using Reverse Phase Protein Microarray coupled with Laser Capture Microdissection. Protein expression/phosphorylation was measured in a continuous scale and classified based on quartile distribution. Concordance between CCND1 amplification and Cyclin D1 expression, along with the activation of FOXM1 T600 and Rb S780, was explored. Amplification of the FGFR1 locus or its ligands was correlated with the level of activation/phosphorylation of FGFR1 Y653/654. Results: While Cyclin D1 protein expression was greater than the population mean for 4/6 (67%) patients with CCND1 amplification, only 2/6 (33%) patients with CCND1 amplification had Cyclin D1 level within the top quartile of the population (n=29). FOXM1 T600 activation was independent from CCND1 amplification, with high levels of FOXM1 T600 predominantly in the CCND1 wild-type population. Only 1/6 (17%) patients with CCND1 amplification had FOXM1 T600 level similar to the top quartile of the population while a second patient was above the population median. Activation of Rb S780 was above the population median, but below the top quartile, in 2/6 (33%) CCND1 amplified patients. Similarly, none of the patients with activation of FGFR Y653/654 equal to the top quartile harbored an FGFR1 amplification. Only 1/4 (25%) patients carrying an FGFR1 amplification had an activation of FGFR Y653/654 above the population median. Similarly, 1/4 (25%) patients with FGF ligand amplification showed FGFR Y653/654 level within the top quartile while three patients had FGFR Y653/654 activation below the population median. No significant results were found between proteomic (below/above the median) and genomic characteristics by Fisher test (p>0.05). Conclusion: Molecular genotyping of “actionable” cancer targets alone may be insufficient in predicting whether the actual drug target protein is expressed and/or activated in any given patient's tumor. Although these results need further validation, the combination of genomic and proteomic data may represent a more informative approach for identifying real molecular drivers of individual lesions as well as “actionable” protein/phosphoprotein targets in the absence of genomic events. Multi-OMIC approaches may lead to more effective stratification in precision medicine trials. Citation Format: Pierobon M, Wong S, Reeded A, Anthony S, Robert N, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Carpten J, Petricoin EF. A multi-OMIC analysis to explore the impact of “actionable” genomic alterations on protein pathway activation: Clinical implication for precision medicine in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-09.

A Novel Homozygous CTC1 Germline Mutation Associated with Bone Marrow Failure

Iron deficiency in young adult males is rare and necessitates prompt investigations. Here we describe a previously healthy 22-year-old male suffering from gastrointestinal bleeding leading to iron deficiency anemia and explained by a constitutional disease. His diagnosis was obtained after detecting a homozygous CTC1 mutation in his germline DNA. Conserved telomere maintenance component 1 (CTC1) is a part of the conserved telomere-associated complex (CST) participating in telomere replication. CST seems to have a role in resolving replication stress also in non-telomeric regions, but little is known about the exact mechanisms. Biallelic, typically compound heterozygous mutations in CTC1 are known to cause cerebroretinal microangiopathy with calcifications and cysts(CRMCC), which is considered as a telomere disease. It is a highly pleiotropic multi-organ disease characterized by abnormalities e.g. in the brain, retina, intestinal vasculature, as well as bone marrow failure (BMF). Some CRMCC patients have dyskeratosis congenita (DC) -like skin, nail, mucosal, and hair changes. Most patients are diagnosed in early childhood and die before teens. To date 37 patients with CRMCC and CTC1 mutations have been reported.The patient was referred to hematology due to iron deficiency anemia with coinciding thrombocytopenia. He also had a notable lymphopenia but no tendency for viral infections or other signs of immunodeficiency. Imaging revealed a patent umbilical vein, widened portal vein, and hepatosplenomegaly without parenchymal changes or lymphadenopathy. Gaucher's disease was excluded by glucocerebrosidase and genetic tests. Histology of a liver biopsy was normal. Based on these findings an idiopathic portal hypertension was diagnosed. Endoscopy revealed gastric antral vascular ectasia. The bone marrow was hypoplastic and pancytopenia persisted after supplementation with iron. He had no known family history of BMF. The patient's telomere length analyzed by a semi-quantitative PCR-test was within the normal range compared to healthy controls of the same age group. We then sequenced the patient's germline exome. Roche MedExome kit was used to target the coding regions of the genome and sequencing conducted using an Illumina HiSeq1500 sequencer. The quality of the data was assessed and the sequence data analyzed using an in-house developed analysis pipeline. The mutation analysis was targeted to genes previously reported to cause immune deficiencies, myelodysplastic syndrome and BMF. The average sequence coverage was 59x, translating into 95% of the exome being covered with a minimum of 20x.Common polymorphisms were excluded from the analysis. The patient was identified to carry a homozygous variation in CTC1 exon 12; c.1994T>G, p.V665G (rs199473676). The variant has previously been reported pathogenic, but all previously reported cases of p.V665G are compound heterozygous (in the majority of cases V665G and a frameshift mutation). The minor allele frequency for this variant in Finns is 0.0015 (10/6612 alleles) and 0.0001 (13/120608) in total (ExAC database) suggesting this mutation is a Finnish founder mutation. We validated the finding by capillary sequencing. The variant was detected heterozygous in both parents ruling out a larger deletion and verifying the mode of inheritance. A healthy sister did not carry the mutation. Identification of CTC1 p.V665G homozygosity raised a high suspicion of CRMCC and further investigations were indicated. Retinal examination as well as brain MRI/MRA revealed multiple lesions characteristic for CRMCC.This is the first report on homozygous CTC1 p.V665G and the second describing CTC1-homozygosity associated with CRMCC, strengthening the disease-causality of biallelic missense mutations in CTC1. The identification of a homozygous CTC1 p.V665G may also open new prospects for studying the effects of the mutation. Our case shows the importance and capacity of new genomic techniques in clinical management, as well: The patient's primary phenotype with iron deficiency anemia, thrombopenia and hepatosplenomegaly could have been explained by only an idiopathic portal hypertension. However, with exome sequencing we were able to reveal a more complex disease entity with major impact on patient's future care and genetic counseling, highlighting the value of germline sequencing in modern diagnostics. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3171: Powerful target capture/NGS approach reveals extensive genetic changes including SNVs, CNVs and gross chromosomal rearrangements in colorectal cancers

Abstract Introduction: Accurate and comprehensive molecular analyses of genetic changes in cancer tissues are critical for cancer management, treatment, and genetic counseling. Study of the complete molecular profiles in patient's blood, cacinoma tissue and surrounding pathological normal tissues will help us understand the etiology of colorectal cancers (CRC) as either hereditary or sporadic. In addition, this study will also reveal somatic changes and possible targets for therapy. Multigene target capture followed by deep next generation sequencing (NGS) provides a powerful approach to simultaneously detect both single nucleotide variants (SNVs) and exonic copy number variants (CNVs) in a time and cost effective fashion. Method: DNA samples were prepared from blood, freshly frozen normal surrounding and carcinoma or polyp tissues from 20 patients with CRC and 10 patients with colorectal polyps. A custom probe library containing 183 cancer related genes was used to capture the target sequences followed by NGS with deep coverage (average depth per base is ∼1000X). Both SNVs and CNVs were analyzed based on in-house developed analytical algorithm and bioinformatics pipeline. Results: This study revealed extensive genetic changes in colon carcinoma tissues. At least 40% of the colon carcinoma samples harbor loss-of-function pathogenic germline mutations in cancer related genes. However, loss of function germline mutations were not found in the polyps. Germline missense variants of unknown significance were identified in 60% of the polyps. Multiple deleterious somatic mutations are identified in all carcinoma tissues but only 1-2 somatic mutations per sample are found in the polyps. Almost every carcinoma tissue harbor gross chromosomal rearrangement, which is only found in about 50% of the polyp tissues.. NGS based CNV analysis revealed 2 germline events (FANCD2 E15-16 del; whole ALK gene dup). Somatic CNV events are more frequently observed in carcinoma tissues. Gross changes at chromosomal levels were also detected by using the NGS CNV analytical algorithm. Recurrent somatic pathogenic variants in genes associated with CRC, such as APC, TP53, and KRAS were identified while novel likely pathogenic variants in novel genes possibly linked to CRC were also discovered for further investigation. Heterogeneity of carcinoma tissues within the same individual was suggested by comparing allele frequency of different somatic mutations. Numerous loss of heterozygosity was observed in both carcinoma and polyp tissues. Conclusion: Target capture/NGS approach allows simultaneous analyses of SNVs and CNVs with quantitative data to distinguish between germline and somatic mutations. These analyses reveal abundant and valuable molecular information for target therapy, patient management, genetic counseling, and new insights of possible pathogenic mechanism Citation Format: Hongzheng Dai, Xia Tian, Stella Chen, Yue Wang, Tzu-Wei Yang, Chun-Che Lin, Ming-Chang Tsai, Chih-Hong Wang, Chi-Chou Huang, Chin-Ying Shih, Guang-Yuh Chiou, Yuh-Jyh Jong, Lee-Jun Wong. Powerful target capture/NGS approach reveals extensive genetic changes including SNVs, CNVs and gross chromosomal rearrangements in colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3171.

Abstract P2-05-21: The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer

Abstract Background: The identification of organ-specific targetable signatures may help design more effective treatment for patients with metastatic breast cancer (MBC). We took a multi-OMIC approach to assess whether the AKT-mTOR pathway is globally activated during metastatic progression or whether it represents an organ-specific target. Methods: Snap frozen biopsies from 25 MBC patients enrolled in a prospective phase II trial were used. Sites of metastasis were classified as liver (n=8) and others (n=17), the latter including cutaneous, lung, lymph nodes, and intra-abdominal lesions. Signaling analysis of the 25 cases was performed using Reverse Phase Protein Microarray (RPPA) coupled with Laser Capture Microdissection. Activation of the AKT-mTOR pathway was quantified as phosphorylation of AKT (S473) and the mTOR target p70S6 (T389). Matched exome (WES) and RNASeq data were available for 17 of 25 patients, five with liver metastases. Sequencing data was processed using an in-house developed pipeline to identify somatic events including coding mutations, copy number alterations, gene fusions, and differential expression. Activation of the AKT-mTOR pathway and sequencing data were compared between hepatic and non-hepatic lesions using an integrated RPPA and genomic approach. Results: Among liver metastases, AKT was activated in 4 of the 8 (50.0%) patients, while 6 of the 8 cases (75.0%) showed activation of p70S6. Sequencing data revealed mutation of PIK3CA in 4 of the 5 liver metastases (80.0%). Three of the PIK3CA mutated specimens with catalytic domain mutations (codons 1023 and 147) demonstrated co-activation of AKT and p70S6, while the fourth case, containing a helical domain mutation (E542K), had activation of p70S6 only. The PIK3CA wild-type liver metastasis demonstrated low activation of AKT and p70S6. For non-hepatic metastases AKT was activated in 2 of the 17 cases (11.8%) and p70S6 in 5 of the 17 patients (29.4%). Discussion: Although these results need further validation, activation of the AKT-mTOR pathway appears to be a hepatic specific signature in MBC and could be used for the selection of targeted agents for hepatic lesions. Citation Format: Pierobon M, Wong S, Reeder A, Anthony SP, Robert NJ, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Petricoin EF, Carpten J. The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-21.

Abstract A2-59: Resistance mechanisms to erlotinib in the non-small cell lung cancer cell line, HCC827 examined by RNA-seq

Abstract Background: Erlotinib, an EGFR selective reversible inhibitor, has dramatically changed the treatment of non-small cell lung cancer (NSCLC) as approximately 70% of patients show significant tumor regression upon treatment. However, all patients eventually relapse due to development of acquired resistance, which in 43-50% of cases is caused by a secondary mutation (T790M) in EGFR, and in 5-15% of cases is caused by MET amplification. However, a majority of resistance cases are still unexplained. Consequently, our aim was to identify novel resistance mechanisms – and potentially new drug targets - in erlotinib-resistant subclones of the NSCLC cell line HCC827. Materials and methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively), and prepared cDNA libraries of purified RNA from biological duplicates using TruSeq® Stranded Total RNA Ribo-Zero™ Gold (Illumina) prior to sequencing on an Illumina HiSeq platform (100bp paired end). The resistant subclones were examined both in presence and absence of erlotinib. The data was analyzed by an in-house developed pipeline including quality control by Trim Galore v0.3.3, mapping of reads to HG19 by TopHat2 v.2.0.10 and differential expression analysis by CuffDiff from the Cufflinks package v.2.2.1. Results: Significant differences in viability between the resistant clones and parental HCC827 were observed when incubated with erlotinib. Importantly, the resistant clones did not acquire the T790M mutation or other EGFR/KRAS mutations, indicating that other mechanisms are responsible for the resistance in these cells. We identified 303-581 transcripts being >2-fold upregulated (p < 0.001), and 460-679 transcripts being >2-fold downregulated (p < 0.001) in the three resistant clones treated with erlotinib compared to the parental cell line, HCC827. Among others, we found networks such as growth and proliferation, migration and oxidative stress to be upregulated by Ingenuity Pathway Analysis (IPA), whereas adherens junction signaling was downregulated. More specifically, transcripts encoding potential resistance drivers such as AXL kinase and Fibroblast Growth Factor Receptor 1 (FGFR1) were found consistently upregulated together with a number of central metabolic enzymes, which may also be important for resistance. Vimentin and CD44 encoding transcripts involved with epithelial-to-mesenchymal transition were consistently upregulated and CDH1 encoding E-cadherin was downregulated, indicating that EMT may be correlated with resistance to erlotinib. EGFR was either not regulated or downregulated in the three clones. MET was not regulated, whereas p53 expression was consistently downregulated. Conclusions: We established 3 erlotinib-resistant NSCLC subclones, which did not harbor any of the common resistance mutations, thus allowing for the identification of novel resistance mechanisms in these subclones. Cancer-related networks such as proliferation and migration were upregulated in the resistant clones, supporting the validity of the model. While EGFR was either not regulated or down-regulated, other potential resistance drivers such as AXL kinase and FGFR1, among others, were found consistently upregulated in the three resistant clones, which may indicate a cooperative bypass signaling mechanism to obtain resistance to erlotinib. Citation Format: Kirstine Jacobsen, Nicolas Alcaraz, Rikke Raaen Lund, Henrik Jørn Ditzel. Resistance mechanisms to erlotinib in the non-small cell lung cancer cell line, HCC827 examined by RNA-seq. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-59.

OC-030 Investigation of faecal volatile organic compounds as biomarkers for the diagnosis of necrotising entercolitis

Introduction Necrotising enterocolitis (NEC) is the most common serious gastrointestinal complication of preterm birth. Clinical signs and symptoms are often non-specific and may only become apparent once disease is established. A biomarker to indicate early disease would allow earlier treatment and may improve outcome. We have reported a pilot study showing changes in faecal volatile organic compounds (VOCs) profile in the prodromal period of NEC. Here, we report a much larger series with more comprehensive clinical and laboratory data. Method Daily faecal samples were collected prospectively from 1375 apparently healthy preterm babies, born between 23 and 34 weeks gestation, in 8 NICUs over a 2-year period. Over 13,000 samples were catalogued. Samples from 70 healthy preterm babies and 34 preterm babies who developed NEC (Bell’s stage II and III) were analysed. In short, 50–100 mg of faeces underwent headspace gas extraction using solid phase micro-extraction followed by gas chromatography–mass spectrometry using our optimised method. Each NEC patient was matched with 1 to 3 healthy control patients. Samples from NEC patients between 1 to 6 days prior to diagnosis and matched samples from controls were selected. Compounds were identified using an in-house developed pipeline involving the AMDIS software, the NIST database and the R packages Metab and XCMS. Univariate and multivariate analysis were performed on the clinical and VOC data. The features best describing the differences between NEC and control samples were used to build a classifier for the diagnosis of NEC. Results Six VOCs and birthweight were significant in multivariable analysis. Birthweight was the strongest predictor. Of the VOCs, 3 were positively associated with NEC and 3 negatively associated. A logistic regression model was developed to diagnose NEC overall, then for each of the 6 days that preceded the clinical diagnosis. Overall the model had an area under the ROC (AUROC) curve of 0.83. When estimated for each day, the AUROC ranged from 0.78 to 0.9. Conclusion To the best of our knowledge, this is the first large scale metabolomics study of faecal VOCs aiming at classifying NEC patients. The data indicate the VOCs may be used to predict NEC up to 6 days before the condition is diagnosed using current methods. This may have an important impact on reducing the mortality of this devastating condition. Further investigation of VOCs may also elucidate the pathogenesis of NEC. Disclosure of interest None Declared.

Diagnosis of autosomal dominant polycystic kidney disease using efficient PKD1 and PKD2 targeted next-generation sequencing.

Molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) relies on mutation screening of PKD1 and PKD2, which is complicated by extensive allelic heterogeneity and the presence of six highly homologous sequences of PKD1. To date, specific sequencing of PKD1 requires laborious long-range amplifications. The high cost and long turnaround time of PKD1 and PKD2 mutation analysis using conventional techniques limits its widespread application in clinical settings. We performed targeted next-generation sequencing (NGS) of PKD1 and PKD2. Pooled barcoded DNA patient libraries were enriched by in-solution hybridization with PKD1 and PKD2 capture probes. Bioinformatics analysis was performed using an in-house developed pipeline. We validated the assay in a cohort of 36 patients with previously known PKD1 and PKD2 mutations and five control individuals. Then, we used the same assay and bioinformatics analysis in a discovery cohort of 12 uncharacterized patients. We detected 35 out of 36 known definitely, highly likely, and likely pathogenic mutations in the validation cohort, including two large deletions. In the discovery cohort, we detected 11 different pathogenic mutations in 10 out of 12 patients. This study demonstrates that laborious long-range PCRs of the repeated PKD1 region can be avoided by in-solution enrichment of PKD1 and PKD2 and NGS. This strategy significantly reduces the cost and time for simultaneous PKD1 and PKD2 sequence analysis, facilitating routine genetic diagnostics of ADPKD.

Abstract P2-07-03: High-throughput germline mutation screening for hereditary breast cancer in southern Chinese patients by massively parallel DNA sequencing

Abstract Introduction: Breast cancer is the most common malignancy and 3rd leading cause of deaths among the female population in Hong Kong. Since the establishment of The Hong Kong Hereditary Breast Cancer Family Registry in 2007, 1344 patients with breast and/or ovarian cancer who met the selection criteria were recruited for genetic testing in Hong Kong. Since 2011 we started to employ next-generation DNA sequencing (NGS) to expedite the analysis workflow and expand the panel of genes for sequencing. Aim: To evaluate the workflow of NGS in mutation screening of BRCA1, BRCA2, TP53 and PTEN genes, and compared with the sequence data obtained by Sanger sequencing. Methods: We sequenced BRCA1, BRCA2, TP53 and PTEN genes in peripheral blood samples of 410 patients, 53 positive controls and 107 healthy local individuals using 454 GS Junior System. Generation of barcoded amplicon libraries was streamlined by microfluidic PCR using Fluidigm Access Array System. Sequencing data were analyzed by an in-house developed fully automatic bioinformatics pipeline, which mainly consists of GS Amplicon Variant Analyzer, SAMtools and Ensembl Variant Effect Predictor. All putative mutations identified were validated by Sanger sequencing. Furthermore, the frequency of BRCA1, BRCA2 and PTEN missense variants of unknown significance (VUS) identified in the cohort were compared among 107 healthy local individuals and 1000 Genomes project samples. The VUS were also subjected to a panel of in silico prediction methods including PolyPhen and SIFT. Results: Among 410 patients, there were 7 in BRCA1, 6 in BRCA2 and 1 in TP53 mutations found, including 1 novel recurrent BRCA2 (c.7007G>T) and 1 novel founder BRCA2 (c.5164_5165delAG) mutations. Based on multiple criteria, 12 in BRCA1, 12 in BRCA2 and 1 in PTEN VUS could be prioritized for further investigation. The bioinformatics pipeline was extensively evaluated with Sanger-validated controls. The evaluation determined minimum sequencing coverage needed in this sequencing platform for accurate analysis. The pipeline accuracy was demonstrated by successful detecting mutations from 53 positive controls, including single nucleotide variants, insertions and deletions in different sequence context. Conclusion: BRCA1, BRCA2, TP53 and PTEN mutation screening of 410 patients were expedited by high-throughput DNA sequencing. This method could detect 14 positive cases, including recurrent mutations, in a shorter period of time when compared with Sanger full gene sequencing. High-risk patients who are negative for the gene panel may need further investigation other than screening for BRCA1/2. The in-house developed bioinformatics pipeline was validated to detect various types of mutations and potentially become a conventional platform for genetic screening. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-03.

Abstract 4021: Transcriptomic landscape of Ewing sarcoma based on RNAseq.

Abstract Ewing sarcoma is the second most common primary bone tumor in children, constituting 3% of all pediatric tumors. The 5 year survival rate is 59% to 76% for children younger than 15 years and from 20% to 49% for those aged 15 to 19 years. 85% of Ewing sarcoma are characterized by a chromosomal translocation between the Ewing sarcoma gene (EWS) breaking point and the ETS family of transcription factor FLI1, which creates a new type of fusion transcription factor with novel functions. While EWS-FLI1 is the predominating translocation event, other types of translocations between EWS and other ETS transcription factors have been reported. Mesenchymal stem cells (hMSC) are thought to be the cell of origin for Ewing sarcoma. The molecular mechanisms of transformation of hMSCs with ectopic EWS-FLI1 expression has been extensively studied, however, a complete transformation of hMSCs into Ewing sarcoma tumor cells is not yet achieved suggesting there are other elements required for the transformation of hMSCs. RNAseq has proved to be a powerful new method for capturing multiple arrays of information simultaneously such as gene expression, nucleotide variation, alternative splicing patterns and fusion transcripts. RNAseq has been used only once in the literature in identification of a novel fusion transcript in Ewing sarcoma tumor cells. Here we obtained RNAseq data from Ewing sarcoma patients and cell lines to gather novel insight into the pathogenesis of Ewing sarcoma. We obtained sequencing data from two Ewing sarcoma cell lines (A673 and SK-PN-DW); human brain vascular pericytes (HBVP), 8 patient tumors and 3 matched normal control tissues. Initial data analysis was performed through an in-house developed pipeline that uses publicly available software and further data analysis was performed using R with various packages. We describe the gene expression changes in patients compared to normal tissues as well as HBVP. We also discovered a group of novel fusion transcripts that we subsequently confirmed with RT-PCR. Since EWS is also an RNA splicing protein, changes in alternative splicing are also described. Citation Format: Ahmet Zehir, Srikanth R. Ambati, Malcolm AS Moore. Transcriptomic landscape of Ewing sarcoma based on RNAseq. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4021. doi:10.1158/1538-7445.AM2013-4021