Matched Germline Research Articles

Subclonal Driver Mutations Predict Shorter Progression Free Survival in Chronic Lymphocytic Leukemia Following First-Line Chemo(immuno)Therapy: Results from the CLL8 Trial

Intra-tumoral genetic heterogeneity and clonal evolution play a central role in disease relapse, and constitute one of the foremost obstacles to cancer cure. To measure intra-tumoral genetic heterogeneity, we recently developed an analytic framework which utilizes whole-exome sequencing (WES) to calculate the proportion of cancer cells within a sample harboring each mutation. Applying this approach to a clinically heterogeneous cohort of chronic lymphocytic leukemia (CLL), we previously discovered that the intra-tumoral genetic heterogeneity in primary patient samples prior to treatment initiation anticipates clonal evolution and is linked to adverse clinical outcome with therapy (Landau et al., Cell 2013).To definitively evaluate the value of intra-tumoral heterogeneity in predicting clinical outcome in CLL, we performed an analogous analysis of pre-treatment samples collected from study subjects enrolled on the CLL8 trial of the GCLLSG (Hallek et al., Lancet 2010). In this multicenter phase III study, 817 patients were randomized to receive as first-line therapy fludarabine and cyclophosphamide either alone or in combination with rituximab. Paired pre-treatment tumor DNA from CD19+ selected peripheral blood mononuclear cells (PBMC), and non-tumor DNA from CD19- PBMC were available from 309 subjects (61% IGHV unmutated, 26% del(11q), 4.5% del(17p)). From these 309 sample pairs, we performed SNP array analysis and WES. Somatic mutations were identified as single nucleotide variants (SNVs), indels and copy number alterations (CNAs), detected in the tumor DNA but not in matched germline DNA. Subsequently, we utilized the algorithm ABSOLUTE to calculate the purity and ploidy of each sample, and classify every mutation as either clonal (i.e., present in all leukemic cells in the sample) vs. subclonal (i.e., present in a subset of leukemic cells). Progression free survival (PFS) was defined as the primary outcome measure.Preliminary sequencing results for 275 of 309 CLL samples are currently available. Across these samples, WES identified 7290 somatic SNVs and indels. Overall mutation rate for these samples was 0.8/Mb, similar to prior large-scale reports of DNA sequencing of CLL. By applying the algorithm MutSig, which identifies putative driver genes recurrently mutated more than expected by chance, we detected 16 of 24 previously described CLL drivers (e.g., TP53, ATM, SF3B1 and NOTCH1) to significantly affect this cohort. In addition, due to the large sample size we have identified 9 novel putative drivers in CLL including MGA, IKZF3 and ARID1A. CNA analysis identified the previously reported recurrent CNAs including (del(13q), del(11q), del(17p), trisomy 12 and del(8p)).At this time, subclonal analysis by ABSOLUTE is available for 216 of 309 CLLs. Across the 216 samples, we identified 2041 clonal and 2106 subclonal mutations. A putative driver gene mutation was identified as clonal in 102 (47.2%) patients, and as subclonal in 81 (37.5%) patients. The presence of a clonal driver was not significantly associated with differences inPFS (logrank P= 0.236). In contrast, the presence of a subclonal driver was associated with a significantly shortened PFS (logrank P= 0.006, Figure 1A-B). A Cox regression model that included the presence of a subclonal driver as well as the treatment arm assignment (FC vs. FCR), retained the presence of a subclonal driver as statistically significant in association with shorter PFS (HR 1.56 [95%CI 1.12-2.19], P=0.009). The presence of a subclonal driver was furthermore associated with shorter overall survival (logrank P=0.02, Fig 1C-D).This preliminary analysis demonstrates that pre-treatment characterization of intra-tumoral genetic heterogeneity can identify patients at risk for inferior outcome with first-line fludarabine-based therapy in CLL. With completion of this analysis for the entire cohort of 309 patients, we plan to perform a multivariable analysis to evaluate the clinical impact of clonal and subclonal driver mutations in CLL. Further ongoing studies include a longitudinal genetic evaluation of the subset of patients with subsequent disease relapse in order to unravel the evolutionary dynamics underlying disease progression. [Display omitted] DisclosuresFischer:Roche: Travel grants Other. Hallek:Janssen, Pharmacyclics: Consultancy, Research Funding.

Development of a DNA-Based Targeted Assay Suitable for Accurate Quantification of Chromosomal Deletions in Myelodysplastic Syndromes with Deletion (5q) and Other Clonal Diseases

IntroductionThe acquisition of large-scale chromosomal lesions is a frequent event in malignant disorders. One example is the recurrent deletion of chromosome 5q (del(5q)) in myelodysplastic syndromes (MDS). The detection and monitoring of such deletions are important elements in routine clinical diagnostics and cancer genomic studies. Currently established methods for their assessment are metaphase cytogenetics (MC), fluorescence in situ hybridization (FISH) and micro-array based techniques. However, each of these methods harbours specific disadvantages as they depend on (viable) cells, are expensive, labour-intensive or only semi-quantitative. One possible approach to interrogate chromosomal deletions constitutes the assessment of allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Therefore, we aimed to establish a robust, quick and inexpensive PCR assay that measures allelic imbalance at such STR loci in order to reliably estimate frequencies of cells carrying del(5q) from only minute amounts of DNA.MethodsGenomic DNA (gDNA) was isolated from bone marrow (BM) or blood cells of MDS and acute myeloid leukemia (AML) patients with cytogenetically confirmed del(5q). Based on NCBI UniSTS database, we designed 12 fluorochrome-labelled PCR amplicons with size ranges of 100-400 bp that surround STR loci between chromosomal bands 5q21 and 5q31. Using only 10 ng gDNA, all 12 PCR amplicons were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis on an ABI 3130 Genetic Analyzer. Allele size quantification of informative heterozygous loci was performed using ABI Genemapper software. Furthermore, size calculations of individual alleles were corrected for PCR-stutter, which was estimated from corresponding loci in homozygous samples. Finally, using mesenchymal stromal cells as a germline control, the degree of skewing in the allelic ratios of all informative STR markers in the tumor sample relative to the corresponding allelic ratios in the control was averaged and subsequently translated into fractions of cells carrying the del(5q) lesion.ResultsApplication of our novel assay for quantification of del(5q) burden in n=559 samples from n=67 patients revealed a high frequency of informative markers with an average of 7 heterozygous STR loci per patient. The data shows a strong inter-marker concordance with a standard deviation (SD) of 2.3% for del(5q) cell frequencies. Moreover, duplicate analysis of 328 samples revealed an average SD of 0.86%. Most importantly, paired analysis of the proportion of del(5q) cells estimated using interphase-FISH and our PCR assay was carried out for n=9 samples and resulted in strong correlation with r²=0.93. A serial dilution series with deleted and non-deleted gDNA also revealed highly concordant results with r²=0.96. When comparing matched germline and tumor STR profiles, no case of microsatellite instability was detectable in our MDS/AML cohort thus highlighting the suitability of STR based lesion quantification in this disease entity. Furthermore, we could use our large dataset to calculate amplification efficiencies for each locus in order to predict “surrogate” germline profiles eventually allowing us to calculate del(5q) frequencies in tumor samples that lack germline controls. Finally, our assay reliably confirmed molecular remission in BM from del(5q) MDS patients after Lenalidomide treatment, in agreement with concomitant MC analyses.ConclusionUsing a multiplexed PCR assay for measurement of STRs in deleted chromosomal regions we present a highly adaptable tool for precise quantification of large scale lesions. We show a very good correlation with established methods exemplarily for del(5q) lesions. Even without available germline control our assay provides robust results. Requiring only minute amounts of gDNA, this assay is ideally suitable for copy number quantification in samples for which only residual archival gDNA and no cells for FISH analysis are available. Due to the small amplicon sizes it should also be useful for investigation of fragmented gDNA from formalin-fixed archival specimen. In summary, our newly developed assay offers a mean to obtain quantitative data for basically any large scale chromosomal deletion which contains STRs and should be easily applicable to other clonal diseases. DisclosuresNolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract A22: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. It is treated with a combination of surgery, chemotherapy, and radiation, and while most children are cured, survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and CTNNB1) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors. We also sequenced matched germline DNA by whole-exome sequencing (15 cases) or targeted Sanger sequencing (29 cases) to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1, which were mutually exclusive with known Wilms tumor mutations in WT1 and CTNNB1. The ribonucleases DROSHA and DICER1 perform two key steps in the biogenesis of microRNAs (miRNAs), which are ∼22-nt non-coding RNAs that regulate the stability and translation of target mRNAs. DROSHA and DICER1 mutations occurred at or near conserved metal-binding residues in their ribonuclease (RNase) IIIB domains. In vitro processing assays modeling these mutations showed that they partially or completely disrupted the ribonuclease activity of DROSHA and DICER1. In addition, next-generation sequencing of small RNAs in these tumors revealed that the DICER1 mutations preferentially impaired processing of miRNAs derived from the 5′ arm of pre-miRNA hairpins, including the let-7 tumor suppressor miRNA family. Processing of 5′-derived miRNAs is specifically dependent on DICER1 RNase IIIB activity. Although DROSHA-mutant tumors did not exhibit skewed expression of 5′ vs. 3′-derived miRNAs, expression of let-7 and other putative tumor suppressor miRNAs was reduced in these tumors as well. While germline and somatic mutations in DICER1 have been reported in some cancers, this is the first report of recurrent DROSHA mutations in any human tumor type. Moreover, these results suggest that impairment of miRNA processing by somatic mutation of DROSHA and DICER1 defines a novel subclass of Wilms tumor. Citation Format: Dinesh Rakheja, Kenneth S. Chen, Yangjian Liu, Abhay A. Shukla, Sara Hildebrand, Vanessa Schmid, Xiaoyong Sun, Xin Feng, Tsung-Cheng Chang, Shama Khokhar, Nitin J. Karandikar, James S. Malter, Joshua T. Mendell, James F. Amatruda. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A22.

Abstract LB-204: Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. It is treated with a combination of surgery, chemotherapy, and radiation, and, while most children are cured, survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and CTNNB1) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors. We also sequenced matched germline DNA by whole-exome sequencing (15 cases) or targeted Sanger sequencing of candidate mutations (29 cases) to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1 which were mutually exclusive with known Wilms tumor mutations in WT1 and CTNNB1. The ribonucleases DROSHA and DICER1 perform two key steps in the biogenesis of microRNAs (miRNAs), which are ∼22-nt non-coding RNAs that regulate the stability and translation of target mRNAs. DROSHA and DICER1 mutations occurred at or near conserved metal-binding residues in their ribonuclease (RNase) IIIB domains. In vitro processing assays modeling these mutations showed that they partially or completely disrupt the ribonuclease activity of DROSHA and DICER1. In addition, next-generation sequencing of small RNAs in these tumors revealed that the DICER1 mutations preferentially impaired processing of miRNAs derived from the 5′ arm of pre-miRNA hairpins, including the let-7 tumor suppressor miRNA family. Although DROSHA-mutant tumors did not exhibit skewed expression of 5′ vs. 3′-derived miRNAs, expression of let-7 and other putative tumor suppressor miRNAs was reduced in these tumors as well. While germline and somatic mutations in DICER1 have been reported in some cancers, this is the first report of recurrent DROSHA mutations in any human tumor type. Moreover, these results suggest that impairment of miRNA processing by somatic mutation of DROSHA and DICER1 defines a novel subclass of Wilms tumor. Citation Format: Dinesh Rakheja, Kenneth S. Chen, Yangjian Liu, Tsung-Cheng Chang, Abhay A. Shukla, James Malter, Joshua T. Mendell, James F. Amatruda. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis in Wilms tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-204. doi:10.1158/1538-7445.AM2014-LB-204

Molecular, immunohistochemical and morphologic analysis of urothelial carcinoma with ERBB2/ERBB3 mutations

<h3>Background</h3> Aberrations in ERBB2 (both amplification and mutations) and ERBB3 represent a novel therapeutic avenue for HER2/HER3-targeted therapy. A minority of urothelial carcinoma (UC) is known to have ERBB2 amplification but little is known about ERBB2 and ERBB3 mutations in this disease. We identified a cohort of UC with ERBB2/ERBB3 mutations using a next generation sequencing platform and performed detailed histopathologic evaluation and immunohistochemistry for HER2. <h3>Methods</h3> A total of 138 UC cases were studied. Frozen tumor tissue and matching germline DNA from peripheral blood were analyzed using a targeted, deep-sequencing assay designed to identify point mutations, indels, and copy number alterations in 300 cancer-associated genes, including ERBB2 and ERBB3. HER2 IHC was performed on the confirmed mutant UCs and on 14 randomly selected cases from the cohort that were without ERBB2/ERBB3 mutations or amplification. <h3>Results</h3> ERBB2 mutations were identified in 15 cases (11%). There were 14 missense mutations and one frameshift mutation. Two HER2 mutant UCs also exhibited ERBB2 amplification. ERBB3 mutations were identified in 17 cases (12%), all of which were missense mutations. Three cases had concurrent ERBB2 and ERBB3 mutations (2%).The majority of the ERBB2/ERBB3 mutant tumors did not exhibit HER2 overexpression as determined by intense and diffuse membranous staining by IHC. Only 4 mutant cases had 3+ staining, 2 of which also had HER2 amplification. Many tumors with wild-type ERBB2 and ERBB3 expressed HER2 at variable intensity but none as 3+. <h3>Conclusions</h3> Mutations in ERBB2 and ERBB3 are present in a significant minority of urothelial carcinoma and may represent targets for anti-HER2 and anti-HER3 therapy. HER2 immunohistochemistry is not a sensitive method for identifying tumors with ERBB2/ERBB3 mutations.

IgBLAST: an immunoglobulin variable domain sequence analysis tool

The variable domain of an immunoglobulin (IG) sequence is encoded by multiple genes, including the variable (V) gene, the diversity (D) gene and the joining (J) gene. Analysis of IG sequences typically requires identification of each gene, as well as a comparison of sequence variations in the context of defined regions. General purpose tools, such as the BLAST program, have only limited use for such tasks, as the rearranged nature of an IG sequence and the variable length of each gene requires multiple rounds of BLAST searches for a single IG sequence. Additionally, manual assembly of different genes is difficult and error-prone. To address these issues and to facilitate other common tasks in analysing IG sequences, we have developed the sequence analysis tool IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/). With this tool, users can view the matches to the germline V, D and J genes, details at rearrangement junctions, the delineation of IG V domain framework regions and complementarity determining regions. IgBLAST has the capability to analyse nucleotide and protein sequences and can process sequences in batches. Furthermore, IgBLAST allows searches against the germline gene databases and other sequence databases simultaneously to minimize the chance of missing possibly the best matching germline V gene.

Abstract 4594: Recurrent FGFR1 hotspot mutations represent a novel therapeutic target in childhood astrocytoma.

Abstract Introduction: Pilocytic astrocytoma (PA) is the most common childhood brain tumor. This tumor can occur throughout the central nervous system, with roughly 50% of cases arising outside of the cerebellum. Tumors in these non-cerebellar locations are often difficult to treat surgically, leading to multiple tumor recurrences. PA can therefore become a chronic disease, with patients experiencing substantial morbidities. Alterations in the MAPK pathway, particularly BRAF, have previously been identified in ∼80% of PAs. Interestingly, however, the majority of those cases without a recognized change are non-cerebellar. Methods: To investigate the full range of genetic alterations occurring in PA, we used Illumina HiSeq technologies to perform whole-genome sequencing of matched tumor and germline DNA from 47 patients, with corresponding RNA sequencing data for 35 tumors. Results: The average somatic mutation rate in PA was extremely low, at 0.065/Mb, with an average of only 1.8 non-synonymous coding single nucleotide variants (SNVs) per tumor - almost ten times lower than we have recently reported for medulloblastoma. We found several novel alterations in known PA-related genes, including two new oncogenic BRAF fusions. Most strikingly, however, we identified mutations at two hotspots in the FGFR1 receptor tyrosine kinase in 4/6 centrally located PAs lacking any other MAPK pathway change. Two of these cases also carried a mutation in a downstream adaptor protein, PTPN11 (Shp2). Interestingly, germline mutations of PTPN11 are associated with Noonan syndrome (NS), and there are case reports of NS patients developing PAs. Screening of additional non-cerebellar PAs revealed four further cases with an FGFR1 mutation. All PAs, regardless of MAPK pathway alteration, displayed highly elevated expression of FGF2, indicating a general role for ligand-mediated activation of the FGFR1/MAPK pathway in PA tumorigenesis. Notably, the same FGFR1 mutations were also identified in four midline pediatric glioblastomas (GBM), a highly malignant brain tumor, suggesting a possible common origin for a subset of these two entities, despite their dramatically different clinical course. Conclusion: Altogether, MAPK alterations were identified in 96% of PAs, with very few other changes, confirming the concept of PA as a single-pathway disease. Our results also suggest that a subset of centrally located, FGFR1-driven pediatric PAs and GBMs may share common origins. Most importantly, they reveal a novel therapeutic target in clinically relevant subsets of childhood glioma. Citation Format: David TW Jones, Barbara Hutter, Natalie Jäger, Andrey Korshunov, Marcel Kool, Sally R. Lambert, Dong Anh Khuong Quang, Adam M. Fontebasso, Marina Ryzhova, Hans-Jörg Warnatz, Thomas Zichner, Jan O. Korbel, Stephan Wolf, Marie-Laure Yaspo, Keith L. Ligon, Mark W. Kieran, Benedikt Brors, Jörg Felsberg, Guido Reifenberger, V. Peter Collins, Nada Jabado, Roland Eils, Peter Lichter, Stefan M. Pfister, ICGC PedBrain Tumor Project. Recurrent FGFR1 hotspot mutations represent a novel therapeutic target in childhood astrocytoma. [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 4594. doi:10.1158/1538-7445.AM2013-4594

The Pediatric Cancer Genome Project.

Subject terms: Cancer genomics• Paediatric cancer• Sequencing At a glance Figures View all figures Figure 1: Frequency of cancer diagnoses and leukemia subtypes in children and adults. (a) The frequency of cancer types in children (left) and adults (right) on the basis of 2012 Surveillance, Epidemiology and End Results (SEER) data. Each chart is organized with cancers listed from the most common to the least common in a clockwise fashion. (b) The frequency of T-cell lineage (blue text) and B-cell lineage (black text) subtypes of acute lymphoblastic leukemia (ALL) in children (left) and adults (right). Each chart is organized with ALL subtypes listed from the most common to the least common in a clockwise fashion. iAMP21, intrachromosomal amplification of chromosome 21. Full size image View in article Figure 2: Genetic landscape of 15 different types of pediatric cancers determined from whole-genome sequencing of 260 tumors and matching germline samples. The number of somatic mutations in each sample, including single-nucleotide variations (SNVs), insertion and/or deletion events (indels) and structural variations, is shown as the height in the three-dimensional graph. Only high-quality variations or validated somatic mutations are included in the summary. CDS, protein-coding regions; tier 1, mutations in annotated genes; tier 2, mutations in non-coding conserved or regulatory regions; tier 3, mutations in non-repetitive, non-coding and non-conserved regions; tier 4, mutations in repetitive regions. Tier 2 and tier 3/tier 4 mutations were rescaled to 1/10 and 1/100 of the original counts to maintain a consistent scale with the results for other somatic lesions. INF, infant ALL; CBF, core-binding-factor acute myeloid leukemia; TALL, T-cell ALL; AMLM7, acute megakaryoblastic leukemia; HYPO, hypodiploid ALL; PHALL, Philadelphia chromosome–positive BCR-ABL1 ALL; RB, retinoblastoma; RHB, rhabdomyosarcoma; NBL, neuroblastoma; OS, osteosarcoma; ACT, adrenocortical carcinoma; HGG, high-grade glioblastoma; LGG, low-grade glioma; EPD, ependymoma; MB, medulloblastoma. Full size image View in article

Abstract 4873: Comprehensive analysis of 160 whole-genome sequences reveals striking telomere alteration patterns in 9 pediatric cancers

Abstract Although telomere shortening has long been recognized as a feature of cancer genomes, a comprehensive characterization of telomere alteration in pediatric cancer has not yet been performed. As part of the St. Jude Children's Research Hospital-Washington University Pediatric Cancer Genome Project (PCGP), we are performing whole genome sequence analysis on 600 pediatric cancers and matched germ line samples. This database will allow us to carry out a systematic investigation of telomere alteration across a broad range of pediatric cancers. To characterize the relative level of telomere length within a tumor, we have developed an approach that utilizes the WGS data and identifies all sequencing reads containing the telomeric repeat (TTAGGG)4. This read count is then normalized for genome-wide coverage and compared to the result from the matched germline sample to define the relative level of telomere amplification or loss in the tumor. To date we have analyzed 160 tumor/normal pairs collected from 9 distinct pediatric cancers types (Early T Progenitor Acute Lymphoblastic Leukemia (ALL), Infant ALL, Hypo-diploid ALL, Medulloblastoma, Retinoblastoma, Neuroblastoma, Osteosarcoma, Rhabdomyosarcoma, and Adrenocortical Carcinoma). Our analysis revealed normal telomere length or minimal loss in Early T Progenitor Acute Lymphoblastic Leukemia (ALL), Hypo-Diploid ALL, Infant-ALL, Medulloblastoma, Retinoblastoma, and Rhabdomyosarcoma. By contrast a subset of three distinct tumor types (Neurobalstoma, Oteosarcoma, and Adrenocortical Carcinoma) were characterized by marked increases in the number of telomere reads in the tumor consistent with telomere lengthening. These latter findings were validated in neuroblastoma by quantitative polymerase chain reaction (13/14) and fluorescence in situ hybridization (7/7). The presence of long telomeres was statistically associated with an increase in the number of somatic structural variations in tumor, with this association most evident in osteosarcoma. These data suggest that some pediatric cancers are associated with marked abnormalities in telomere maintenance leading to expansion in the telomere repeats. Defining the specific mutational processes that are responsible for these telomere defects and expanding the dataset to all 600 cancers in the project should help to define the mechanisms through which these alterations contribute to the transformed phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4873. doi:1538-7445.AM2012-4873

Abstract SY25-01: Analysis of next-generation sequencing data for cancer genomes: challenges and pitfalls

Abstract The characterization of the landscape of genetic lesions that underlie cancer has been significantly advanced with the recent application of next-generation sequencing (NGS) technology. This methodology can be used to sequence selected subsets of genes, the whole exome, the whole genome, or the expressed transcriptome within a cancer cell. By comparing the acquired sequences from both a cancer and matched normal tissue sample from the same patients, one should be able to identify almost all somatic lesions within the cancer. As part of the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project (PCGP), we have undertaken the approach of performing whole genome sequencing (WGS) on 600 pediatric cancers and matched control tissue (1200 total genomes). Although the acquisition of the primary sequence is a formidable challenge, the analysis of these data is where the real work begins. Unfortunately, the majority of published NGS analysis methods were developed to identify germ line variation and therefore perform sub-optimally when applied to the task of identifying somatic mutations in cancer genomes. This is in part a result of the distinct difference in logic that must be used to accurately identify all somatic lesions within a cancer. A cancer genome typically exists within a heterogenous DNA sample that is composed of normal cells admixed with an oligoclonal tumor sample. Moreover, the range of somatic lesions seen in cancer is broader than what is seen as part of germ line genetic variation, with some cancers having exceedingly complex genomes containing focal insertions, deletions, inversions, intra-chromosomal and inter-chromosomal rearrangements and large copy number abnormalities. The accurate identification of these lesions requires not only the presence of the lesions within the cancer DNA, but also their absence from the matched germ line sample. To approach these problems, we, as well as others, have recently developed new analytical approaches to enhance our ability to identify the somatic mutations in cancer. The starting point for these analyses is ≥75 bp paired-end sequencing reads from patient matched tumor and normal DNA samples. Our goal is to identify all somatic single-nucleotide variation (SNV), small insertion/deletion (indel), copy number alteration (CNA) and structural variation (SV) that occur within the cancer DNA sample. Paired tumor-normal NGS data were analyzed together to ensure sensitivity for detecting DNA alterations in tumor and for confirming their absence in the matched normal sample. Somatic lesions initially identified by mapped NGS reads were further analyzed using more accurate algorithms to correct errors cause by suboptimal NGS mapping. The sensitivity of the methods we have developed depends on the read depth, but with WGS at 30X haploid coverage we are able to detect mono-allelic mutation present in as low as ∼25% of the analyzed cellular populations. This sensitivity can be significantly enhanced with greater read densities. Key among the methods we have developed are two new algorithms focused on identifying gross DNA alterations: CREST (Clipping REveals STructure) for SV analysis and CONSERTING (COpy Number SEgmentation by Regression Tree) for CNA analysis. CREST uses sequencing reads with partial alignments to the reference human genome (so-called soft-clipped reads) to directly map the breakpoints of somatic SVs. CONSERTING integrates read depth analysis with SV detection and adjust for sequencing artifacts, coverage bias and germ line CNVs. Together, these methods identify somatic lesions with a high validation rate (92-98% of SNV and Indels, 80% for SVs). In this talk, I will highlight the NGS analytic pipeline we have developed and the recent discoveries that have emerged through its application to pediatric cancer genomes. In addition, I will point out some of the significant challenges that remain to be tacked in order for us to identify the full landscape and functional consequences of the somatic mutations in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY25-01. doi:1538-7445.AM2012-SY25-01

Abstract 5075: Genome-wide analysis of DNA copy number variations in osteosarcoma

Abstract Objectives: Copy number variations (CNVs) such as deletions, amplifications and duplications across the whole genome may contribute to Osteosarcoma (OS) tumorigenesis. Detection of genomic regions containing these changes is extremely important for both the basic understanding of OS and its diagnosis. In this study we performed Whole Genome Genotyping (WGG) using high-density SNP arrays to detect CNVs in OS tumors and blood. A major advantage of this technology is the simultaneous detection of both allelic frequencies and CNVs. Methods: WGG analysis was performed using 35 high-grade intramedullary OS and 8 parosteal sarcoma tumor DNAs. 10 matching germline DNAs from blood were also included into the analysis. 250 nanograms of DNA were hybridized onto the Illumina 610 Quad BeadChips using the Infinium II WGG assay. The WGG data was analysed using Bead Studio and Partek software. Results: SNP array based Whole Genome Genotyping revealed a large number of chromosomal aberrations in OS. Preliminary analysis of 35 OS tumor DNAs showed high levels of recurrent amplification of 1p36, 3p12.1, 6q14, 12q13-15, 17p11.2, 20p12; and deletion of 3q26.31, 6p21, 6q16.3, 7q21, 7q35, 8q24.13, 9p21 and 17p13 regions. Parosteal tumor DNAs displayed a high level of amplification of the 12q13-15 amplicon. Amplification status of genes from 17p11.2 (PMP22, NCOR1, COPS3 and TOM1L2) and 12q14 (CDK4 and MDM2) was validated by Real-Time PCR. Some tumors exhibited whole chromosome arm deletions and/or duplications. Conclusions: OS tumors exhibit chromosomal alterations that can be detected using WGG. Comparison of normal and tumour DNAs from the same subjects can be used to identify CNV associated with OS development and progression. Current analyses include comparisons of CNV data and gene expression profiles of the same OS tumours to discover genes that may be biologically relevant targets in OS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5075. doi:1538-7445.AM2012-5075

Mitotic recombination of chromosome arm 17q as a cause of loss of heterozygosity of NF1 in neurofibromatosis type 1-associated glomus tumors.

Neurofibromatosis type 1 (NF1) is a common, autosomal dominant, tumor-predisposition syndrome that arises secondary to mutations in NF1. Glomus tumors are painful benign tumors that originate from the glomus body in the fingers and toes due to biallelic inactivation of NF1. We karyotyped cultures from four previously reported and one new glomus tumor and hybridized tumor (and matching germline) DNA on Illumina HumanOmni1-Quad SNP arrays (≈ 1 × 10(6) SNPs). Two tumors displayed evidence of copy-neutral loss of heterozygosity of chromosome arm 17q not observed in the germline sample, consistent with a mitotic recombination event. One of these two tumors, NF1-G12, featured extreme polyploidy (near-tetraploidy, near-hexaploidy, or near-septaploidy) across all chromosomes. In the remaining four tumors, there were few cytogenetic abnormalities observed, and copy-number analysis was consistent with diploidy in all chromosomes. This is the first study of glomus tumors cytogenetics, to our knowledge, and the first to report biallelic inactivation of NF1 secondary to mitotic recombination of chromosome arm 17q in multiple NF1-associated glomus tumors. We have observed mitotic recombination in 22% of molecularly characterized NF1-associated glomus tumors, suggesting that it is a not uncommon mechanism in the reduction to homozygosity of the NF1 germline mutation in these tumors. In tumor NF1-G12, we hypothesize that mitotic recombination also "unmasked" (reduced to homozygosity) a hypomorphic germline allele in a gene on chromosome arm 17q associated with chromosomal instability, resulting in the extreme polyploidy.

Distinct Expression Patterns in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) Characterized by Uniparental Disomy.

Genome-wide single nucleotide polymorphism (SNP) analyses have revealed uniparental disomy (UPD) to be a common event in cytogenetically normal acute myeloid leukemia (CN-AML) occurring in approximately 20% of cases. Acquired UPD results in copy number neutral loss of heterozygosity (LOH). Comparing matched tumor and germline DNA samples recurrent acquired UPDs affecting chromosomes 11p and 13q were identified. As DNA microarray-based gene expression profiling (GEP) has recently been shown to powerfully capture the molecular heterogeneity of leukemia, we sought to identify gene expression patterns associated with recurrent UPD in CN-AML.We profiled a set of clinically annotated CN-AML specimens (n=66) entered on a multicenter trial for patients <60 years (AMLSG 07-04) which had been characterized by either 50k or 500k Affymetrix SNP microarrays. All cases were analyzed using Affymetrix microarrays (Human Genome U133 Plus 2.0 Arrays). In this data set we investigated 12 UPDs (affecting chromosomes 1p, 2p, 6p, 11p, 13q and 19q) and applied supervised analyses to define gene-expression patterns associated with UPDs on chromosome 11p and 13q.For the case with an acquired UPD on 19q a gene dosage effect could be demonstrated. Genes located in the 36 Mb large UPD region showed a significantly lower average expression (p<0.001; t-test). Similarly, we observed a gene dosage effect in one of the UPDs observed on chromosome 1 (p=0.0097; t-test), whereas for the other UPDs no significant association between LOH and gene expression levels could be identified. Despite small sample numbers supervised analyses revealed a biologically meaningful gene expression signatures associated with acquired UPD 11p and 13q. In accordance with the association of UPD 13q with FLT3-ITD, the UPD13q gene expression signature was enriched for genes associated with FLT3-ITD. The UPD11p expression pattern was characterized by genes found to be down-regulated in CEBPAmut CN-AML cases, such as down-regulation of homeobox genes HOXA9, HOXA10, HOXB2, and MEIS1. Notably, the UPD11p signature was also characterized by the expression of e.g. UGT2B28, P2RX5, PGDS, CAPN1, NDFIP1, and TRIB2, an expression profile that has been shown to be associated with CEBPAmut CN-AML as well as AML cases with epigenetic CEBPA silencing.Thus, our findings represent a starting point to further dissect CN-AML characterized by recurrent UPD, and ongoing analyses will provide additional insights into leukemia biology.

Systematic genomic screen for tyrosine kinase mutations in CLL

Systematic genomic screen for tyrosine kinase mutations in CLL

Detection of microsatellite instability in endometrial cancer: advantages of a panel of five mononucleotide repeats over the National Cancer Institute panel of markers

The aim of this study was to find the optimal set of microsatellite markers for diagnosis of the microsatellite instability (MSI) phenotype in endometrial cancers. We compared the sensitivity, specificity and ease of use of a reference panel of five markers originally recommended by the National Cancer Institute (NCI) for colorectal cancer and a panel of five quasi-monomorphic mononucleotide repeat markers (pentaplex PCR system). We used these panels for establishing the MSI status of a series of 80 sporadic endometrial adenocarcinomas by comparing the allelic profiles of the markers between tumor and matching germline DNA. Both panels detected the same subset of 21 out of 80 (26%) endometrial MSI carcinomas. However, in the MSI cases, the mean instability of the five mononucleotide repeats was 96.1% as compared with a mean instability of 69.8% for the three dinucleotide repeats of the NCI panel, indicating a superiority of mononucleotide repeats over dinucleotide repeats in detecting MSI. The fact that the two panels of markers detect the same set of MSI tumors is due to the presence of two mononucleotide repeats within the NCI panel. As demonstrated previously in gastric and colon MSI cases, the pentaplex PCR reaction using mononucleotide repeats is thus an easier and more sensitive method than the NCI panel, for the screening of MSI status in endometrial tumors.