Genome-wide DNA Copy Number Research Articles

PO-390 Image-guided stereotactic molecular profiling of samples taken in- and outside conventional tumour boundaries highlight extensive infiltration of tumour cells in diffuse gliomas

IntroductionDiffuse gliomas are highly infiltrative tumours of the central nervous system that lack effective treatment options and suffer from poor survival. We and others have used DNA methylation and gene expression profiling to stratify these tumours into clinically relevant subtypes. Nevertheless, diffuse gliomas are notoriously heterogeneous and a single biopsy is unlikely to reflect all tumour cell populations. Advanced sampling schemes guided by imaging are needed for accurate delineation of heterogeneity.Material and methodsWe obtained 74 stereotactic image-guided biopsies in eight patients with glioma prior to craniotomy in addition to 102 multi-sector glioma samples from 25 patients from outside sources. Patients underwent extensive imaging including standard magnetic resonance imaging and various advanced modalities. For each patient, multiple biopsies were taken from the tumour core, tumour margin and outside of visible imaging abnormalities and each sample was histologically assessed by two board-certified pathologists. We performed methylation profiling on all samples. We used supervised machine learning to determine methylation subtype composition and predict transcriptional subtypes in each sample. Genome wide DNA copy number was inferred.Results and discussionsPrincipal component analysis of DNA methylation separated samples based on IDH status and tumour cell content. Tumour signal could be detected in samples far outside tumour margins on standard MRI, despite demonstrating lower tumour purity and inconclusive tumour subtypes. Samples taken from inside imaging abnormalities demonstrated concordant methylation subtypes, suggesting that this classifier is reliable and not subject to heterogeneity due to microenvironment cell populations as often the case with gene expression subtyping. Despite uniformity in classification, IDH mutant tumours demonstrated a higher degree of epigenetic heterogeneity compared to wild-type tumours, reflecting hypermethylation established by IDH oncometabolites. Although still imperfect, advanced imaging substantially improved tumour delineation. While samples from inside imaging abnormalities are optimal for diagnostic use, tumour cells infiltrate well beyond these boundaries and treatment of these regions should be considered when safe and feasible.ConclusionThese data show methylation profiling is stable across multiple biopsies and could serve as a powerful diagnostic biomarker. Moreover, advanced imaging can more accurately delineate tumour margins and could help direct treatment.

Genes co-amplified with ERBB2 or MET as novel potential cancer-promoting genes in gastric cancer.

Gastric cancer (GC), one of the most common cancers worldwide, has a high mortality rate due to limited treatment options. Identifying novel and promising molecular targets is a major challenge that must be overcome if treatment of advanced GC is to be successful. Here, we used comparative genomic hybridization and gene expression microarrays to examine genome-wide DNA copy number alterations (CNAs) and global gene expression in 38 GC samples from old and young patients. We identified frequent CNAs, which included copy number gains on chromosomes 3q, 7p, 8q, 20p, and 20q and copy number losses on chromosomes 19p and 21p. The most frequently gained region was 7p21.1 (55%), whereas the most frequently deleted region was 21p11.1 (50%). Recurrent highly amplified regions 17q12 and 7q31.1-7q31.31 harbored two well-known oncogenes: ERBB2 and MET. Correlation analysis of CNAs and gene expression levels identified CAPZA2 (co-amplified with MET) and genes GRB7, MIEN1, PGAP3, and STARD3 (co-amplified with ERBB2) as potential candidate cancer-promoting genes (CPGs). Public dataset analysis confirmed co-amplification of these genes with MET or ERBB2 in GC tissue samples, and revealed that high expression (except for PGAP3) was significantly associated with shorter overall survival. Knockdown of these genes using small interfering RNA led to significant suppression of GC cell proliferation and migration. Reduced GC cell proliferation mediated by CAPZA2 knockdown was attributable to attenuated cell cycle progression and increased apoptosis. This study identified novel candidate CPGs co-amplified with MET or ERBB2, and suggests that they play a functional role in GC.

Sweepstake evolution revealed by population-genetic analysis of copy-number alterations in single genomes of breast cancer.

Single-cell sequencing is a promising technology that can address cancer cell evolution by identifying genetic alterations in individual cells. In a recent study, genome-wide DNA copy numbers of single cells were accurately quantified by single-cell sequencing in breast cancers. Phylogenetic-tree analysis revealed genetically distinct populations, each consisting of homogeneous cells. Bioinformatics methods based on population genetics should be further developed to quantitatively analyse the single-cell sequencing data. We developed a bioinformatics framework that was combined with molecular-evolution theories to analyse copy-number losses. This analysis revealed that most deletions in the breast cancers at the single-cell level were generated by simple stochastic processes. A non-standard type of coalescent theory, the multiple-merger coalescent model, aided by approximate Bayesian computation fit well with the data, allowing us to estimate the population-genetic parameters in addition to false-positive and false-negative rates. The estimated parameters suggest that the cancer cells underwent sweepstake evolution, where only one or very few parental cells produced a descendent cell population. We conclude that breast cancer cells successively substitute in a tumour mass, and the high reproduction of only a portion of cancer cells may confer high adaptability to this cancer.

Abstract 5403: Reproducible elevation of RNA versus DNA mutation signal in low purity breast tumors

Abstract Background: Accurate detection of somatic mutations is critical for informing targeted therapy options. Prevalent non-cancer cell admixture complicates this detection in breast cancer. Conventional mutation detection relies on DNA sequencing; however in prior work, we demonstrated that combining RNA and DNA sequencing increases mutation signal strength, or mutant allele fraction (MAF). The ratio of RNA MAF versus DNA MAF (RNA:DNA MAF) was greatest in low purity breast tumors. We hypothesized that this elevation is biologically driven and would be conserved in a second, distinct tissue specimen of the same tumors. Here, we compare mutation characteristics between two tissue blocks in a cohort of breast tumors (n = 8) to evaluate possible preservation of RNA versus DNA mutation signal throughout the tumor. Methods: We selected four high purity and four low purity breast tumors (“Block1”) from The Cancer Genome Atlas (TCGA) cohort and associated ABSOLUTE purity analysis. For these tumors, we acquired a second tissue block (“Block2”) not analyzed by TCGA, cut sections, analyzed sections by H&E stains, and extracted nucleic acids. Whole genome DNA sequencing and mRNA sequencing was performed for Block2 specimens using Illumina X and NextSeq 500 sequencers, respectively. Somatic mutations in Block2 were detected using UNCeqR and compared to published UNCeqR somatic mutations from TCGA. We then evaluate MAF characteristics in the entire TCGA breast tumor cohort (n = 695). Results: Tumor purity estimates, determined by histology and by sequencing, were reduced in Block2 of the low purity tumor set versus the high purity tumor set, consistent with Block1 analysis. Molecular properties of genome-wide gene expression and somatic DNA copy number were highly similar between block-mated specimens (p < 0.01). We then identified expressed mutations present in Block1 and Block2 of the same tumor and compared the MAFs on these common mutations. DNA MAF and RNA MAF were each significantly correlated between Block1 and Block2 (p < 1e-12 in both cases). The average RNA:DNA MAF was 2.5 for the cohort, indicating that RNA mutation signal is greater than DNA in general. In Block2 specimens, the RNA:DNA MAFs were significantly greater in the low purity tumor set than the high purity tumor set (mean 2.7 versus 2.1, p < 6e-5), reflecting the same trend observed in Block1 specimens. Analyzing the entire TCGA cohort, RNA:DNA MAF was positively correlated with proliferation pathway gene expression (p < 3e-16 ) and was greatest in the Basal subtype versus other subtypes (p < 2e-9). Conclusion: Mutant allele fraction both of DNA and of RNA was conserved across breast tumor subsections. Low purity and basal subtype breast tumors had elevated RNA:DNA MAF supporting a relationship to underlying biology and identifying classes of tumors with pronounced benefit for DNA and RNA integrated mutation analysis. Citation Format: Jerez Te, Coralie Viollet, Xijun Zhang, Jatinder Singh, Jeffrey A. Hooke, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Clifton L. Dalgard, Matthew D. Wilkerson. Reproducible elevation of RNA versus DNA mutation signal in low purity breast tumors [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 5403. doi:10.1158/1538-7445.AM2017-5403

Clinical and molecular features associated with long-term survival of elderly patients with glioblastoma.

2013 Background: Glioblastomas in elderly patients are associated with particularly poor outcome, with only few patients demonstrating long-term survival (LTS). Methods: To better characterize clinical and molecular correlates of LTS in elderly glioblastoma patients, we searched the German Glioma Network (GGN) database for patients aged 71 years or more with histological confirmation of glioblastoma and survival of at least two years after diagnosis. Results: Of 2071 glioblastoma patients enrolled in the GGN from 2004-2012, 425 patients were aged 71 years or more; of these, 27 patients (6.4%) survived for 2 years or more (median survival: 37.1 months, 95% confidence interval: 30.0-44.2 months). A comparison of these 27 patients with the 398 patients who survived shorter than 2 years (median survival: 6.2, 95% confidence interval: 5.2-7.2 months) revealed more intensive up-front treatment and a trend towards higher initial Karnofsky performance score as distinguishing clinical factors. Molecular analyses additionally showed more frequent O6-methylguanine-DNA methyltransferase ( MGMT) promoter methylation in the LTS patients. Isocitrate dehydrogenase (IDH) mutation was restricted to single patients and the frequency of telomerase reverse transcriptase ( TERT) promoter mutation did not differ between groups. Genome-wide DNA copy number and methylation profiling using 450k microarray analysis performed for 16 LTS patients and 40 control patients revealed limited differential DNA methylation and no specific copy number profiles linked to LTS. Conclusions: Collectively, our findings confirm that LTS is rare in elderly patients with glioblastoma and that clinical and tumor-associated molecular factors linked to LTS resemble those in standard age patients, except for less common IDH mutation.

Histone 3.3 hotspot mutations in conventional osteosarcomas: a comprehensive clinical and molecular characterization of six H3F3A mutated cases

BackgroundHistone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone (GCTBs; 96%), chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, histopathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown.MethodsIn this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes H3F3A and H3F3B. H3.3 mutant osteosarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and H3F3A G34W/L mutant GCTBs.ResultsSix osteosarcomas (6/106) carried H3F3A hotspot mutations. No mutations were found in H3F3B. All patients with H3F3A mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberrations that are commonly encountered in high-grade osteosarcomas also occurred in H3F3A mutant osteosarcomas. Unlike a single osteosarcoma with a H3F3A K27M mutation, the DNA methylation profiles of H3F3A G34W/R mutant osteosarcomas were clearly different from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most differentially methylated promoters between H3F3A G34W/R mutant and H3.3 wild-type osteosarcomas were in KLLN/PTEN (p < 0.00005) and HIST1H2BB (p < 0.0005).ConclusionsH3.3 mutations in osteosarcomas may occur in H3F3A at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying H3F3A G34W/R mutations are associated with epigenetic dysregulation of KLLN/PTEN and HIST1H2BB.

Malignant canine mammary tumours: Preliminary genomic insights using oligonucleotide array comparative genomic hybridisation analysis

Neoplastic mammary disease in female dogs represents a major health concern for dog owners and veterinarians, but the genomic basis of the disease is poorly understood. In this study, we performed high resolution oligonucleotide array comparative genomic hybridisation (oaCGH) to assess genome wide DNA copy number changes in 10 malignant canine mammary tumours from seven female dogs, including multiple tumours collected at one time from each of three female dogs. In all but two tumours, genomic imbalances were detected, with losses being more common than gains. Canine chromosomes 9, 22, 26, 27, 34 and X were most frequently affected. Dissimilar oaCGH ratio profiles were observed in multiple tumours from the same dogs, providing preliminary evidence for probable independent pathogenesis. Analysis of adjacent samples of one tumour revealed regional differences in the number of genomic imbalances, suggesting heterogeneity within tumours.

Initiation of aberrant DNA methylation patterns and heterogeneity in precancerous lesions of human hepatocellular cancer

ABSTRACTWhile intratumor heterogeneity contributes to disease progression, metastasis, and resistance to chemotherapy, it also provides a route to understanding the evolution and drivers of disease. Defects in epigenetic landscapes are intimately linked to pathogenesis of a variety of human diseases, with epigenetic deregulation promoting tumorigenesis. Understanding epigenetic heterogeneity is crucial in hepatocellular carcinoma (HCC), where epigenetic alterations are frequent, early, and pathogenic events. We determined genome-wide DNA methylation and copy number variation leveraging the Infinium 450K in a series of regenerative nodules from within single patient livers. Bioinformatics strategies were used to ascertain within-patient heterogeneity, link epigenetic changes to clinical features, and determine their relevance to disease pathogenesis. Our data demonstrate that DNA methylation and copy number alterations evolve during the pre-neoplastic phase of HCC and independently segregate regenerative nodules into distinct clusters. Regenerative nodules with a high frequency of epigenetic changes have significantly lower copy number variation, suggesting that individual nodules have differential enrichment of epigenetic and genetic components, with both contributing to disease progression. Regenerative nodules were scored based on ‘epigenetic progression’ with higher scores associated with increased proliferation measured by Ki67 staining. Early events observed in epigenetically ‘aggressive’ nodules are enriched for genes involved in liver cancer. Our study demonstrates that marked epigenetic and genetic heterogeneity exists in early pre-neoplastic liver tissue within individual patients, emphasizing the potential contributions of each mechanism to driving liver disease progression, and it unveils strategies for identifying epigenetic drivers of hepatocellular carcinoma.

Genome-wide DNA copy number analysis in clonally expanded human ovarian cancer cells with distinct invasive/migratory capacities.

Ovarian cancer has the worst prognosis of any gynecological malignancy, and generally presents with metastasis at advanced stages. Copy number variation (CNV) frequently contributes to the alteration of oncogenic drivers. In this study, we sought to identify genetic targets in heterogeneous clones from human ovarian cancers cells. We used array-based technology to systematically assess all the genes with CNVs in cell models clonally expanded from A2780 and SKOV3 ovarian cancer cell lines with distinct highly and minimally invasive/migratory capacities. We found that copy number alterations differed between matched highly and minimally invasive/migratory subclones, differentially affecting specific functional processes including immune response processes, DNA damage repair, cell cycle and cell proliferation. We also identified seven genes as strong candidates, including DDB1, ERCC1, ERCC2, PRPF19, BCAT1, CDKN1B and MARK4, by integrating the above data with gene expression and clinical outcome data. Thus, by determining the molecular signatures of heterogeneous invasive/migratory ovarian cancer cells, we identified genes that could be specifically targeted for the treatment and prognosis of advanced ovarian cancers.

Genomic profiling of canine mast cell tumors identifies DNA copy number aberrations associated with KIT mutations and high histological grade.

Mast cell tumor (MCT) is the most common skin malignancy of domestic dogs and presents with a widely variable clinical behavior. Although activating KIT mutations are present in approximately 20% of canine MCTs, molecular etiology is largely unknown for the majority of this cancer. Characterization of genomic alterations in canine MCTs may identify genomic regions and/or genes responsible for their development and progression, facilitating the discovery of new therapeutic targets and improved clinical management of this heterogeneous cancer. We performed genome-wide DNA copy number analysis of 109 primary MCTs derived from three popular canine breeds (the Boxer, Labrador Retriever, and Pug) as well as nontarget breeds using oligonucleotide array comparative genomic hybridization (oaCGH). We demonstrated a stepwise accumulation of numerical DNA copy number aberrations (CNAs) as tumor grade increases. DNA sequencing analysis revealed that KIT mutations were found less frequently in the Pug tumors and were strongly associated with high histological grade. Tumors with KIT mutations showed genome-wide aberrant copy number profiles, with frequent CNAs involving genes in the p53 and RB pathways, whereas CNAs were very limited in tumors with wild-type KIT. We evaluated the presence of four CNAs to predict aggressive tumor phenotypes. This approach predicted aggressive tumors with a sensitivity of 78-94% and specificity of 88-93%, when using oaCGH and droplet digital PCR platforms. Further investigation of genome regions identified in this study may lead to the development of a molecular tool for classification and prognosis, as well as identification of therapeutic target molecules.

Comprehensive genomic characterization of five canine lymphoid tumor cell lines.

BackgroundLeukemia/lymphoma cell lines have been critical in the investigation of the pathogenesis and therapy of hematological malignancies. While human LL cell lines have generally been found to recapitulate the primary tumors from which they were derived, appropriate characterization including cytogenetic and transcriptional assessment is crucial for assessing their clinical predictive value.ResultsIn the following study, five canine LL cell lines, CLBL-1, Ema, TL-1 (Nody-1), UL-1, and 3132, were characterized using extensive immunophenotyping, karyotypic analysis, oligonucleotide array comparative genomic hybridization (oaCGH), and gene expression profiling. Genome-wide DNA copy number data from the cell lines were also directly compared with 299 primary canine round cell tumors to determine whether the cell lines represent primary tumors, and, if so, what subtype each most closely resembled.ConclusionsBased on integrated analyses, CLBL-1 was classified as B-cell lymphoma, Ema and TL-1 as T-cell lymphoma, and UL-1 as T-cell acute lymphoblastic leukemia. 3132, originally classified as a B-cell lymphoma, was reclassified as a histiocytic sarcoma based on characteristic cytogenomic properties. In combination, these data begin to elucidate the clinical predictive value of these cell lines which will enhance the appropriate selection of in vitro models for future studies of canine hematological malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0836-z) contains supplementary material, which is available to authorized users.

Abstract 136: Refining the molecular profile of colorectal tumors to expand prevention and treatment opportunities

Abstract The completion of The Cancer Genome Atlas (TCGA) project for colorectal cancer (CRC) is ushering in a new phase of identifying treatment strategies tailored to the molecular profile of each person's tumor. Precision medicine approaches to cancer treatment rely on the identification of molecular profiles that can be used to identify effective therapies and can be used in a targeted sequencing setting to make treatment decisions. The initial TCGA colorectal effort included 276 samples and focused on integrating data from exome sequencing with genome-wide DNA copy number alterations (CNAs), DNA methylation, and mRNA and microRNA expression. Since then a total of 626 samples have been completed with the potential to refine CRC subtypes, identify novel mutated pathways, and further functional understanding. Such a large data set presents opportunities to identify new recurrent drug targets and to stratify patients into groups that are predictive of treatment response. However, large data sets also present substantial challenges, since hand-curation becomes intractable, while computational tools can be overwhelmed by hypermutation and copy number changes. Here we present a comprehensive molecular analysis of all 626 TCGA colorectal cancer samples, including exome sequencing, CNAs, DNA methylation, and mRNA expression. For each data type, we identified recurrently altered genes. Using MutSigCV on 525 samples yielded 27 and 87 significantly mutated genes in non-hypermutated and hypermutated samples, respectively, a substantial increase over the 15 and 17 somatically recurrently mutated genes identified using MutSig in non-hypermutated and hypermutated samples, respectively, in the previously published TCGA colorectal study. For example, PTEN, a known tumor suppressor, was not reported as significantly recurrently mutated in the initial TCGA non-hypermutated set; however, it was in the larger non-hypermutated set, demonstrating the power of a larger data set for assessing the significance and relative frequency of mutations in the context of known subtypes. In addition, we integrated the somatic mutation data, copy number data, LOH data, and hyper-methylation data to identify genes, like MLH1, that are recurrently disrupted by different mechanisms. We also considered somatic mutations that are likely gain-of-function mutations based on nonrandom clustering; and we used recurrent indels to identify loss-of-function drivers in samples positive for microsatellite instability (MSI). We further classified each sample using the previously identified subtypes – BRAF+, KRAS+, APC+, CTNNB1+ (beta-catenin+), TGFBR2/SMAD4+, PTEN+ and PIK3CA+, and R-spondin fusion positive, as well as CpG Island Methylator Phenotype (CIMP) and MSI - in order to refine the relevant molecular signatures driving CRC etiology and thereby prevention and treatment paradigms. Citation Format: Catherine S. Grasso, Eve Shinbrot, Ming Yu, Max Liesersen, Mark Chaisson, Andrew Chan, Charles Connolly, James Dai, Margaret Du, Charles Fuchs, Levi Garraway, Marios Giannakis, Tabitha Harrison, Li Hsu, Jeroen Huyghe, Jasmine Mu, Shuji Ogino, Colin Pritchard, Stephen Salipante, Wei Sun, Syed H. Zaidi, Ni Zhao, William Grady, Ben Raphael, Thomas Hudson, David Wheeler, Ulrike Peters. Refining the molecular profile of colorectal tumors to expand prevention and treatment opportunities. [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 136.

The genetic landscape of dural marginal zone lymphomas.

The dura is a rare site of involvement by marginal zone lymphoma (MZL) and the biology of dural MZL is not well understood. We performed genome-wide DNA copy number and targeted mutational analysis of 14 dural MZL to determine the genetic landscape of this entity. Monoallelic and biallelic inactivation of TNFAIP3 by mutation (n=5) or loss (n=1) was observed in 6/9 (67%) dural MZL exhibiting plasmacytic differentiation, including 3 IgG4+ cases. In contrast, activating NOTCH2 mutations were detected in 4/5 (80%) dural MZL displaying variable monocytoid morphology. Inactivating TBL1XR1 mutations were identified in all NOTCH2 mutated cases. Recurrent mutations in KLHL6 (n=2) and MLL2 (n=2) were also detected. Gains at 6p25.3 (n=2) and losses at 1p36.32 (n=3) were common chromosomal imbalances, with loss of heterozygosity (LOH) of these loci observed in a subset of cases. Translocations involving the IGH or MALT1 genes were not identified. Our results indicate genetic similarities between dural MZL and other MZL subtypes. However, recurrent and mutually exclusive genetic alterations of TNFAIP3 and NOTCH2 appear to be associated with distinct disease phenotypes in dural MZL.

Genome-Wide DNA Copy Number Analysis of Acute Lymphoblastic Leukemia Identifies New Genetic Markers Associated with Clinical Outcome.

Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL) is still a challenge. Aims: To characterize the presence of additional DNA copy number alterations (CNAs) in children and adults with ALL by whole-genome oligonucleotide array (aCGH) analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults). The NimbleGen CGH 12x135K array (Roche) was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q). CNAs were associated with age, phenotype, genetic subtype and overall survival (OS). In the whole cohort of children, the losses on 14q32.33 (p = 0.019) and 15q13.2 (p = 0.04) were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001) and Xp21.1 (p = 0.029), and the loss of 17p (p = 0.014) were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL.

Longitudinal copy number, whole exome and targeted deep sequencing of 'good risk' IGHV-mutated CLL patients with progressive disease.

The biological features of IGHV-M chronic lymphocytic leukemia responsible for disease progression are still poorly understood. We undertook a longitudinal study close to diagnosis, pre-treatment and post relapse in 13 patients presenting with cMBL or Stage A disease and good-risk biomarkers (IGHV-M genes, no del(17p) or del(11q) and low CD38 expression) who nevertheless developed progressive disease, of whom 10 have required therapy. Using cytogenetics, fluorescence in situ hybridisation, genome-wide DNA methylation and copy number analysis together with whole exome, targeted deep- and Sanger sequencing at diagnosis, we identified mutations in established chronic lymphocytic leukemia driver genes in nine patients (69%), non-coding mutations (PAX5 enhancer region) in three patients and genomic complexity in two patients. Branching evolutionary trajectories predominated (n=9/13), revealing intra-tumoural epi- and genetic heterogeneity and sub-clonal competition before therapy. Of the patients subsequently requiring treatment, two had sub-clonal TP53 mutations that would not be detected by standard methodologies, three qualified for the very-low-risk category defined by integrated mutational and cytogenetic analysis and yet had established or putative driver mutations and one patient developed progressive, therapy-refractory disease associated with the emergence of an IGHV-U clone. These data suggest that extended genomic and immunogenetic screening may have clinical utility in patients with apparent good-risk disease.

Abstract 52: QDNAseq: A bioinformatics pipeline for DNA copy number analysis from shallow whole genome sequencing with noise levels near the probabilistic lower limit imposed by read counting

Abstract The emerging field of precision medicine considers the patient's individual genetic make up, environment, and lifestyle to arrive at individualized treatment. For cancer patients, analysis of the tumour genome can reveal actionable alterations, including mutations (e.g., BRAF mutation in melanoma), translocations (e.g., t(9;22)(q34;q11) in CML) and copy number changes (e.g., HER2 amplification in breast cancer). Formalin-fixed paraffin embedded (FFPE) biopsy or surgical pathology samples are often the only materials available for analysis, highlighting the need for genomic analysis techniques that perform reliably with such materials. We present a complete pipeline for DNA copy number profiling by sequencing called quantitative DNA sequencing (QDNAseq), which offers a cost effective procedure for genome-wide DNA copy number analysis, facilitates correct biological interpretations from samples with a varying range of quality and is suitable for routine pathology diagnostics from fresh, frozen and FFPE materials. QDNAseq is implemented in R and made available through BioConductor and Galaxy. The pipeline requires as little as ∼0.1× genome coverage (∼6 million single-end 50nt reads per sample) and utilizes a depth of coverage (DOC) approach. A two-dimensional LOESS correction is applied for GC content and mappability and data from the 1000 genomes project are used to compile a blacklist of problematic genome regions. Downstream analyses have been included in QDNAseq by adapting algorithms previously developed for segmentation (DNAcopy) and the calling of gains and losses (CGHcall). QDNAseq is able to produce profiles with noise levels near the probabilistic lower limit imposed by read counting and yields a higher resolution than is available from microarrays. Some samples, however, have variances clearly above the probabilistic lower limit and are characterized by a wave pattern. This pattern originates in the sample, as it is reproducible, both in magnitude and in shape along the genome. Much of this wave bias can be reduced by regression with profiles from matched normal FFPE DNA samples using the algorithm implemented in NoWaves. The QDNAseq procedure is highly cost effective and robust. We have processed over 1500 clinical samples obtained from the FFPE archives of more than 25 institutions from Europe and North America. Citation Format: Daoud Sie, Ilari Scheinin, Stef Lieshout, van, Martijn Cordes, Daniel Pinkel, Donna G. Albertson, Mark A. Wiel, van de, Bauke Ylstra. QDNAseq: A bioinformatics pipeline for DNA copy number analysis from shallow whole genome sequencing with noise levels near the probabilistic lower limit imposed by read counting. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 52.

The molecular landscape of extraskeletal osteosarcoma: A clinicopathological and molecular biomarker study.

Extraskeletal osteosarcoma (ESOSA) is a rare soft tissue neoplasm representing <5% of osteosarcomas and <1% of all soft‐tissue sarcomas. Herein, we investigate the clinicopathological and molecular features of ESOSA and explore potential parameters that may affect outcome. Thirty‐two cases were retrieved and histomorphology was reviewed. Clinical history and follow‐up were obtained through electronic record review. DNA from formalin‐fixed paraffin‐embedded (FFPE) tissue was extracted and processed from 27 cases. Genome‐wide DNA copy number (CN) alterations and allelic imbalances were analyzed by single nucleotide polymorphism array using Affymetrix OncoScan FFPE Assay. Massive high‐throughput deep parallel sequencing was performed using a customized panel targeting 410 cancer genes. Log rank, Fisher's exact test and Cox proportional hazards were used for statistical analysis. In this series of 32 patients (male n = 12, female n = 20), the average age was 66 years (19–93) and median follow up was 24 months (range 6–120 months). Frequent genomic alterations included CN losses in tumour suppressor genes including CDKN2A (70%), TP53 (56%) and RB1 (49%). Mutations affecting methylation/demethylation, chromatin remodeling and WNT/SHH pathways were identified in 40%, 27%, and 27%, respectively. PIK3CA and TERT promoter variant mutations were identified in 11% of the cases. Cases harbouring simultaneous TP53 and RB1 biallelic CN losses were associated with worse overall survival and local recurrence (p = 0.04, p = 0.02, respectively). CDKN2A losses and positive margins were also associated with worse overall survival (p = 0.002; p = 0.03, respectively). Our findings suggest that age above 60, positive margin status, simultaneous biallelic TP53 and RB1 losses and CDKN2A loss are associated with a worse outcome in ESOSA. Comparison between conventional paediatric osteosarcoma and ESOSA shows that, while both share genetic similarities, there are notable dissimilarities and mechanistic differences in the molecular pathways involved in ESOSA.

A cultured approach to canine urothelial carcinoma: molecular characterization of five cell lines.

BackgroundUrothelial carcinoma (UC), also known as transitional cell carcinoma (TCC), of the bladder is the most common neoplasm affecting the canine urogenital system. To facilitate study of the disease in vitro, cell line models have been established from primary tumor biopsies. Their resemblance to the primary disease, however, has not been well defined. In the present study, we evaluated five canine UC cell lines via oligonucleotide array comparative genomic hybridization (oaCGH), fluorescence in situ hybridization (FISH), and gene expression analysis.ResultsComparison of genome wide DNA copy number profiles of the cell lines with primary biopsy specimens revealed redundancies in genomic aberrations, indicating that the cell lines retain the gross genomic architecture of primary tumors. As in the primary tumors, gain of canine chromosomes 13 and 36 and loss of chromosome 19 were among the most frequent aberrations evident in the cell lines. FISH analysis revealed chromosome structural aberrations, including tandem duplications, bi-armed chromosomes, and chromosome fusions, suggesting genome instability during neoplastic transformation. Gene expression profiling highlighted numerous differentially expressed genes, including many previously shown as dysregulated in primary canine UC and human bladder cancer. Pathway enrichment analysis emphasized pathways suspected to be at the crux of UC pathogenesis, including xenobiotic and lipid compound metabolism.ConclusionsThese data support valid use of the canine UC cell lines evaluated by confirming they provide an accurate and practical means to interrogate the UC at a molecular level. Moreover, the cell lines may provide a valuable model for furthering our understanding of aberrant metabolic pathways in UC development.Electronic supplementary materialThe online version of this article (doi:10.1186/s40575-015-0028-3) contains supplementary material, which is available to authorized users.

Molecular dissection of ependymomas.

Molecular dissection of ependymomas.

Genome wide DNA copy number analysis in cholangiocarcinoma using high resolution molecular inversion probe single nucleotide polymorphism assay

In order to study molecular similarities and differences of intrahepatic (IH-CCA) and extrahepatic (EH-CCA) cholangiocarcinoma, 24 FFPE tumor samples (13 IH-CCA, 11 EH-CCA) were analyzed for whole genome copy number variations (CNVs) using a new high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay.Common in both tumor subtypes the most frequent losses were detected on chromosome 1p, 3p, 6q and 9 while gains were mostly seen in 1q, 8q as well as complete chromosome 17 and 20. Applying the statistical GISTIC (Genomic Identification of Significant Targets in Cancer) tool we identified potential novel candidate tumor suppressor- (DBC1, FHIT, PPP2R2A) and oncogenes (LYN, FGF19, GRB7, PTPN1) within these regions of chromosomal instability.Next to common aberrations in IH-CCA and EH-CCA, we additionally found significant differences in copy number variations on chromosome 3 and 14. Moreover, due to the fact that mutations in the Isocitrate dehydrogenase (IDH-1 and IDH-2) genes are more frequent in our IH-CCA than in our EH-CCA samples, we suggest that the tumor subtypes have a different molecular profile.In conclusion, new possible target genes within regions of high significant copy number aberrations were detected using a high-density Molecular Inversion Probe Single Nucleotide Polymorphism (MIP SNP) assay, which opens a future perspective of fast routine copy number and marker gene identification for gene targeted therapy.