Copy Number Variation Profiles Research Articles

Rapid Phenotypic and Genomic Change in Response to Therapeutic Pressure in Prostate Cancer Inferred by High Content Analysis of Single Circulating Tumor Cells

Timely characterization of a cancer's evolution is required to predict treatment efficacy and to detect resistance early. High content analysis of single Circulating Tumor Cells (CTCs) enables sequential characterization of genotypic, morphometric and protein expression alterations in real time over the course of cancer treatment. This concept was investigated in a patient with castrate-resistant prostate cancer progressing through both chemotherapy and targeted therapy. In this case study, we integrate across four timepoints 41 genome-wide copy number variation (CNV) profiles plus morphometric parameters and androgen receptor (AR) protein levels. Remarkably, little change was observed in response to standard chemotherapy, evidenced by the fact that a unique clone (A), exhibiting highly rearranged CNV profiles and AR+ phenotype was found circulating before and after treatment. However, clinical response and subsequent progression after targeted therapy was associated with the drastic depletion of clone A, followed by the sequential emergence of two distinct CTC sub-populations that differed in both AR genotype and expression phenotype. While AR- cells with flat or pseudo-diploid CNV profiles (clone B) were identified at the time of response, a new tumor lineage of AR+ cells (clone C) with CNV altered profiles was detected during relapse. We showed that clone C, despite phylogenetically related to clone A, possessed a unique set of somatic CNV alterations, including MYC amplification, an event linked to hormone escape. Interesting, we showed that both clones acquired AR gene amplification by deploying different evolutionary paths. Overall, these data demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the multi-scale analysis of fluid biopsies to quantify and monitor disease evolution in individual patients.

An integrative exploratory analysis of –omics data from the ICGC cancer genomes lung adenocarcinoma study

It is known that all agents that cause cancer (carcinogens) also cause a change in the DNA sequence. In order to identify such often subtle changes, we attempt to integrate multiple molecular profile data sets released by the International Cancer Genome Consortium (ICGC). The list of data sets includes matched gene and microRNA expression profiles, somatic copy number variation, DNA methylation, and protein expression profiles for lung adenocarcinoma patients receiving treatments. We consider both unsupervised and supervised learning techniques (clustering and penalized regression) to identify interesting molecular markers corresponding to each type of –omics profiles that can differentiate patients. Associations between important markers of 2 types have been studied. An adaptive ensemble binary regression model has been presented that uses the entirety of available –omics profiles leading to a more accurate clinical prognosis for the patients in the given sample. This integrated study provides a more com...

Polarity gene alterations in pure invasive micropapillary carcinomas of the breast.

IntroductionPure invasive micropapillary carcinoma (IMPC) is a special type of breast carcinoma characterised by clusters of cells presenting polarity abnormalities. The biological alterations underlying this pattern remain unknown.MethodsPangenomic analysis (n = 39), TP53 (n = 43) and PIK3CA (n = 41) sequencing in a series of IMPCs were performed. A subset of cases was also analysed with whole-exome sequencing (n = 4) and RNA sequencing (n = 6). Copy number variation profiles were compared with those of oestrogen receptors and grade-matched invasive ductal carcinomas (IDCs) of no special type.ResultsUnsupervised analysis of genomic data distinguished two IMPC subsets: one (Sawtooth/8/16) exhibited a significant increase in 16p gains (71%), and the other (Firestorm/Amplifier) was characterised by a high frequency of 8q (35%), 17q (20% to 46%) and 20q (23% to 30%) amplifications and 17p loss (74%). TP53 mutations (10%) were more frequently identified in the amplifier subset, and PIK3CA mutations (4%) were detected in both subsets. Compared to IDC, IMPC exhibited specific loss of the 6q16-q22 region (45%), which is associated with downregulation of FOXO3 and SEC63 gene expression. SEC63 and FOXO3 missense mutations were identified in one case each (2%). Whole-exome sequencing combined with RNA sequencing of IMPC allowed us to identify somatic mutations in genes involved in polarity, DNAH9 and FMN2 (8% and 2%, respectively) or ciliogenesis, BBS12 and BBS9 (2% each) or genes coding for endoplasmic reticulum protein, HSP90B1 and SPTLC3 (2% each) and cytoskeleton, UBR4 and PTPN21 (2% each), regardless of the genomic subset. The intracellular biological function of the mutated genes identified by gene ontology analysis suggests a driving role in the clinicopathological characteristics of IMPC.ConclusionIn our comprehensive molecular analysis of IMPC, we identified numerous genomic alterations without any recurrent fusion genes. Recurrent somatic mutations of genes participating in cellular polarity and shape suggest that they, together with other biological alterations (such as epigenetic modifications and stromal alterations), could contribute to the morphological pattern of IMPC. Though none of the individual abnormalities demonstrated specificity for IMPC, whether their combination in IMPC may have a cumulative effect that drives the abnormal polarity of IMPC needs to be examined further with in vitro experiments.

Integrated Analysis of Copy Number Variation and Genome-Wide Expression Profiling in Colorectal Cancer Tissues

Integrative analyses of multiple genomic datasets for selected samples can provide better insight into the overall data and can enhance our knowledge of cancer. The objective of this study was to elucidate the association between copy number variation (CNV) and gene expression in colorectal cancer (CRC) samples and their corresponding non-cancerous tissues. Sixty-four paired CRC samples from the same patients were subjected to CNV profiling using the Illumina HumanOmni1-Quad assay, and validation was performed using multiplex ligation probe amplification method. Genome-wide expression profiling was performed on 15 paired samples from the same group of patients using the Affymetrix Human Gene 1.0 ST array. Significant genes obtained from both array results were then overlapped. To identify molecular pathways, the data were mapped to the KEGG database. Whole genome CNV analysis that compared primary tumor and non-cancerous epithelium revealed gains in 1638 genes and losses in 36 genes. Significant gains were mostly found in chromosome 20 at position 20q12 with a frequency of 45.31% in tumor samples. Examples of genes that were associated at this cytoband were PTPRT, EMILIN3 and CHD6. The highest number of losses was detected at chromosome 8, position 8p23.2 with 17.19% occurrence in all tumor samples. Among the genes found at this cytoband were CSMD1 and DLC1. Genome-wide expression profiling showed 709 genes to be up-regulated and 699 genes to be down-regulated in CRC compared to non-cancerous samples. Integration of these two datasets identified 56 overlapping genes, which were located in chromosomes 8, 20 and 22. MLPA confirmed that the CRC samples had the highest gains in chromosome 20 compared to the reference samples. Interpretation of the CNV data in the context of the transcriptome via integrative analyses may provide more in-depth knowledge of the genomic landscape of CRC.

Abstract P1-04-03: Genome-wide analysis of copy number variations and mutation profiles of single circulating tumour cells using massively parallel paired-end sequencing

Abstract INTRODUCTION Recent advances in single cell isolation techniques and next generation sequencing (NGS) have paved the way for the genome-wide molecular analysis of individual circulating tumour cells (CTCs) in patients with metastatic carcinomas. Here we present the results of a pilot study evaluating the feasibility and reliability of NGS of single CTC from whole blood samples. MATERIALS & METHODS Single cells of the human breast cancer cell line HCC38 were harvested from spiked blood samples in a semi-automated workflow consisting of immunomagnetic enrichment using the CellSearch system and dielectrophoretic cell sorting using the DEPArray system. DNA was isolated and amplified using the Ampli1 whole genome amplification (WGA) kit and subjected to low-coverage genome-wide paired-end sequencing for copy number variation (CNV) analysis and targeted re-sequencing of 200 cancer-related genes for somatic mutation analysis. RESULTS Single-cell WGA products of four HCC38 cells were subjected to whole genome sequencing for CNV analysis. Average coverage depth was 0,68x. At a binning window of 50 kb, detection results of CNVs in single-cell samples were highly consistent (>81% copy number concordance per bin genome wide) with CNV profiles from non-amplified multi-cell samples of the same cell line. We could demonstrate that part of the discordance was due to the acquisition of novel DNA-rearrangements in the single cells. Three of the single-cell WGA products were additionally subjected to targeted re-sequencing for mutation analysis of 200 selected genes, of which the analysis is currently ongoing. DISCUSSION Our study demonstrates the feasibility of a comprehensive genome-wide CNV analysis and targeted mutation analysis using NGS of single tumour cells isolated from whole blood samples in a highly automated isolation workflow. This approach provides a robust framework for the study of intercellular heterogeneity within the CTC population in blood samples of patients with (metastatic) breast cancer. In addition, our results document the extent of WGA-induced bias of a recently commercialized PCR-based WGA kit. These authors contributed equally to the data presented in this abstract. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-03.

Molecular Characterization of Patient-Derived Human Pancreatic Tumor Xenograft Models for Preclinical and Translational Development of Cancer Therapeutics

Preclinical evaluation of novel cancer agents requires models that accurately reflect the biology and molecular characteristics of human tumors. Molecular profiles of eight pancreatic ductal adenocarcinoma patient tumors were compared to corresponding passages of xenografts obtained by grafting tumor fragments into immunocompromised mice. Molecular characterization was performed by copy number analysis, gene expression and microRNA microarrays, mutation analysis, short tandem repeat (STR) profiling, and immunohistochemistry. Xenografts were found to be highly representative of their respective tumors, with a high degree of genetic stability observed by STR profiling and mutation analysis. Copy number variation (CNV) profiles of early and late xenograft passages were similar, with recurrent losses on chromosomes 1p, 3p, 4q, 6, 8p, 9, 10, 11q, 12p, 15q, 17, 18, 20p, and 21 and gains on 1q, 5p, 8q, 11q, 12q, 13q, 19q, and 20q. Pearson correlations of gene expression profiles of tumors and xenograft passages were above 0.88 for all models. Gene expression patterns between early and late passage xenografts were highly stable for each individual model. Changes observed in xenograft passages largely corresponded to human stromal compartment genes and inflammatory processes. While some differences exist between the primary tumors and corresponding xenografts, the molecular profiles remain stable after extensive passaging. Evidence for stability in molecular characteristics after several rounds of passaging lends confidence to clinical relevance and allows for expansion of models to generate the requisite number of animals required for cohorts used in drug screening and development studies.

MiR‐23a in amplified 19p13.13 loci targets metallothionein 2A and promotes growth in gastric cancer cells

Copy number variation (CNV) and abnormal expression of microRNAs (miRNAs) always lead to deregulation of genes in cancer, including gastric cancer (GC). However, little is known about how CNVs affect the expression of miRNAs. By integrating CNV and miRNA profiles in the same samples, we identified eight miRNAs (miR-1274a, miR-196b, miR-4298, miR-181c, miR-181d, miR-23a, miR-27a and miR-24-2) that were located in the amplified regions and were upregulated in GC. In particular, amplification of miR-23a-27a-24-2 cluster and miR-181c-181d cluster frequently occurred at 19p13.13 and were confirmed by genomic real-time PCR in another 25 paired GC samples. Moreover, in situ hybridization (ISH) experiments represented that mature miR-23a was increased in GCs (75.5%, 40/53) compared with matched normal tissues (28.6%, 14/49, P = 0.001). Knocking down of miR-23a expression inhibited BGC823 cell growth in vitro and in vivo. In addition, the potential target genes of miR-23a were investigated by integration of mRNA profile and miRNA TargetScan predictions, we found that upregulation of miR-23a and downregulation of metallothionein 2A (MT2A) were detected simultaneously in 70% (7/10) of the miRNA and mRNA profiles. Furthermore, an inverse correlation between miR-23a and MT2A expression was detected in GCs and normal tissues. Through combining luciferase assay, we confirmed that MT2A is a potential target of miR-23a. In conclusion, these results suggest that integration of CNV-miRNA-mRNA profiling is a powerful tool for identifying molecular signatures, and that miR-23a might play a role in regulating MT2A expression in GC.

Genomic heterogeneity of circulating tumor cells in castration-resistant prostate cancer (CRPC) revealed by single-cell sequencing.

5030 Background: Circulating tumor cells (CTC) provide an opportunity to sample multiple metastatic tumor sites through a single blood draw – a ”fluid biopsy.” NextGen DNA sequencing provides the means to obtain detailed genetic information from captured cells prior to and during treatment. Here we demonstrate the use of DNA sequencing to interrogate genome-wide copy number variations (CNV) at the single-cell level in CTC isolated from pts with CRPC. Methods: Pre- and post-treatment blood samples were obtained from pts treated at MSKCC. EpCAM+ events were collected singly and in groups by cytometric flow sorting and were subjected to DNA amplification and Illumina NextGeneration sequencing. Parallel samples were assayed using the Veridex CellSearch method to ensure the presence of malignant cells. Results: Samples with up to 50 EpCAM+ events analyzed in bulk displayed CNV patterns expected from published CRPC data. Subsequent single cell analyses showed that the method could reliably detect common genomic markers in CRPC, including AR amplification, PTEN and RB1 loss, and the TMPRSS-ERG fusion. Individual genomic CNV profiles obtained from 125 single cells isolated from 15 patients were then analyzed. Using unsupervised clustering, cells from each pt showed a closely related lineage structure, consistent with an evolution from a common ancestor. The degree of genomic heterogeneity within CTC from an individual pt was highly variable, with R2 correlation coefficients ranging from >0.92 (nearly homogeneous) to <0.75 (mixed populations). Two pts harbored separate subpopulations with both amplified AR and non-amplified AR cells and another displayed mixtures of genetic markers that changed over the course of treatment. Conclusions: The observed variation in complexity of CTC populations in CRPC pts underscores the importance of being able to sample and analyze multiple cells from an individual pt on multiple occasions and with real time analytics. Doing so is essential to understand and identify mechanisms of resistance so that they can be targeted effectively. Supported by STARR Cancer Consortium, NCI SPORE in Prostate Cancer; Department of Defense; Prostate Cancer Foundation.

Abstract 3815: Genomic predictors of response to preoperative chemotherapy in patients with pediatric osteosarcoma.

Abstract Purpose: It is generally accepted that sensitivity of preoperative chemotherapy, evaluated by a histological analysis using resected tumor tissue, is the most important prognostic factor for Osteosarcoma (OS). Methotrexate, doxorubicin, cisplatin (MAP regimen), and ifosfamide (IFO) are recognized as key drugs for OS, although it is still debatable whether addition of IFO for all OS patient is beneficial or not. There have been few analyses documenting molecular markers that predict the response to each regimen used in the preoperative chemotherapy of OS patients. The aim of this study is to identify possible genomic markers for the prediction of sensitivity of preoperative chemotherapy for pediatric OS patients. Methods: Pre-therapeutic biopsy tumor samples of 22 pediatric conventional OS patients treated in Chiba Cancer Center who had homogeneous clinicopathological- and therapeutic- background were analyzed. In our study, all patients were divided into 3 groups according to the histological response; group A includes 8 patients who demonstrated a good histological response to MAP regimen, group B includes 6 patients who were resistant to MAP regimen but showed good response to IFO regimen, and group C includes 8 patients who did not respond to neither MAP nor IFO regimen. Genomic DNAs were prepared from frozen biopsy samples and used for aCGH analysis with Agilent 44k DNA microarray. Results: Survival analysis revealed that patients in group A and B exhibited significantly better outcomes than in group C concerning both overall and event-free survivals. According to the results of comparative analysis of copy number variation (CNV) patterns in each group, 2 CNV regions that separated group A from group B and C (sensitivity 100%, specificity 92.9%) and 6 CNV regions that separated group C from group A and B (sensitivity 100%, specificity 100%) were identified. CNV regions specifically identified in Group C contained several genes previously reported as OS-related genes, such as CDKN2A/B, PTEN, and CDK4. Based on these CNV profiles, we established a scoring system that clearly classify the tumors into these 3 groups Re-sequencing of known cancer-related genes in these OS tumors is also underway to integrate genomic alterations into the system. Conclusion: Our results indicated that copy number changes in OS biopsy samples could be good predictors of response to preoperative chemotherapy. These signatures may be useful for the future genomics-based personalized chemotherapy for OS patients. Citation Format: Shintaro Iwata, Miki Ohira, Tsukasa Yonemoto, Takeshi Ishii, Hiroto Kamoda, Akira Nakagawara. Genomic predictors of response to preoperative chemotherapy in patients with pediatric osteosarcoma. [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 3815. doi:10.1158/1538-7445.AM2013-3815

Abstract 4035: Concurrent genes signatures for breast cancers with Han Chinese origin.

Abstract We performed genome-wide characterization of Han Chinese breast cancer at molecular level by integrating two microarray technologies: comparative genomic hybridization (CGH) for DNA copy number variations (CNV) and expression arrays. Concurrent gains and losses from the same subject across genomic and transcriptional levels may be a better approach to explore potential biomarkers for breast cancer and to identify possible candidates for target therapy. Oncogenesis of breast cancer could originate from chromosome level manifesting as CNV and persist through transcription in gene expression profiles. Genes with coherent patterns at chromosomal and transcriptional level are more likely to serve as potential biomarkers for Han Chinese breast cancer and deserve meticulous evaluation. A total of 23 array CGH and 81 gene expression microarrays (21 samples with data from both platforms) were performed. Potential targets of cancer therapy were revealed by Genomic Identification of Significant Targets in Cancer (GISTIC) from 23 array CGH. Genes with coherent patterns between CNV and gene expression profiles were identified from 21 samples with both platforms assayed. We used these concurrent genes as well as genes with significant GISTIC scores to derive signatures associated with ER, HER2 and event-free survival. Distributions of signature genes were strongly associated with chromosomal locations: chromosome 16 for ER and 17 for HER2. 37 and 13 genes were differentially expressed between distinct ER and HER2 status and were used for classifier development in our 81 samples. Predictive accuracy as high as 90% for ER and 80% for HER2 was reported during leave-one-out cross-validation while much compromised performance was observed in an independent test dataset (GSE5460). Consensus genes between our dataset and 125 Chinese breast cancers (GSE5460) were identified, and a 28 genes and a 9 genes signature for ER and HER2 was developed in combined dataset of 206 microarrays with the best predictive accuracy of 92% and 94% for ER and HER2 respectively. Breast cancer risk predictive model was built based on supervised principle components from two genes (RWDD3 and ZBTB44) and distinct survival patterns were observed between high- and low-risk groups from a combined dataset of 408 microarrays (combination of our 81 samples with GSE20685). The risk score was significantly higher in breast cancers with recurrence, metastasis or mortality than those remained event-free (0.438 versus 0.17, p<0.001). In summary, we built a breast cancer risk predictive model and distinct survival patterns were observed in Han Chinese breast cancers. Citation Format: Chi-Cheng Huang, Shih-Hsin Tu, Eric Y. Chuang. Concurrent genes signatures for breast cancers with Han Chinese origin. [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 4035. doi:10.1158/1538-7445.AM2013-4035

Abstract 4599: Sequential monitoring of single-cell copy number variation in metastatic prostate cancer.

Abstract The high-definition circulating tumor cell (HD-CTC) assay provides for an enrichment-free approach to CTC identification. Here, we utilized the HD-CTC approach to study androgen receptor (AR) expression combined with single-nucleus sequencing for genome-wide analysis of copy number variation (CNV) in sequential CTCs samples obtained from a patient with metastatic prostate cancer treated with abiraterone acetate (an androgen synthesis inhibitor). At baseline, before initiation of abiraterone treatment, we observed a balanced proportion of AR-negative and AR-positive CTCs. During a brief period of clinical response (marked with a decreased serum PSA and decreased pain) the proportion of AR-positive CTCs declined followed by a rapid increase associated with clinical progression (increased PSA and pain). CNV analysis of single CTCs revealed multiple genomic rearrangements, such as AR amplification along with the chromosomal gains and losses typical of prostate cancer, in multiple cells at baseline. During treatment response, the frequency of CNV alterations significantly declined, followed by a reemergence to a pattern of multiple, complex alterations associated with clinical progression. Detailed analysis of the CNV profiles revealed that many abnormalities were commonly shared between the CTC populations, but a number were unique to the AR+ resistant/hormone refractory CTC population including increased MYC amplification alteration and the AR amplicon that include additional adjacent genes. Remarkably, the reconstruction of tumor lineage history based on the CTC genomic profiles enables us to trace and identify the precise treatment time point where the putative therapy-resistant CTC clone emerges, under therapeutic pressure, until it eventually expanded to become the AR+ resistant CTC population at the point of therapeutic relapse. Overall, our results demonstrated that the integration of the HD-CTC enumeration technology with protein expression and single cell genomic analyses could successfully be applied to real time monitoring of ADT therapy emergent change in a prostate cancer patient, and may provide a direct roadmap for personalized cancer medicine in the near future. Citation Format: Peter Kuhn, Angel E. Dago, Asya Stepansky, Anders Carlsson, Natalie Felch, Madelyn Luttgen, Anand Kolatkar, James Hicks, Mitchell E. Gross. Sequential monitoring of single-cell copy number variation in metastatic prostate cancer. [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 4599. doi:10.1158/1538-7445.AM2013-4599

Abstract 1918: Genomic CNV testing on chromosome 17q genes reveals clinically relevant subtypes in breast cancer.

Abstract Fluorescent In Situ Hybridization (FISH) and genomic Copy Number Variation (CNV) testing may not yield identical CN status for the same gene, due to methodological differences in calling gene copies. The aim of the present study was to evaluate the clinical relevance of genomic CNV profiles on 17q (17q11.2-q12 to 17q21-q22) in the context of high-risk operable breast cancer. Methods: The CN status of NOS2, HER2, THRA, RARA and TOP2A was tested with 5’- and 3’-end-specific, exon-intron spanning qPCR CNV assays (two assays per gene). Software obtained CN values against normal DNA were evaluated from 439 informative paraffin breast cancer tissue samples with >50% tumor cells and were assessed (a) upon logarithmic transformation as continuous variables, and (b) as nominal variables with following CN categories: loss (>0-0.5 copies); no-gain or marginal accounting for DNA replication (1-4); low-gain (5-6) and high-gain (>6). For each gene, 5’- and 3’-ends were evaluated separately for gene stability and in combination for obtaining the entire gene CN status. All patients had received adjuvant anthracyclines in the frame of two phase III trials. Results: Concordance between 5’- and 3’-end CN status was observed in 94.53% of tumors for TOP2A; 91.12% for THRA; 86.79% for HER2; 69.70% for RARA; and, 53.76% for NOS2. Strong associations were observed for classic HER2 FISH status with NOS2 (p=.0007), HER2 and THRA (both p<.0001), and for classic TOP2A FISH status with HER2 (p=.0004) and THRA (p<.0001) gene CN status. Hierarchical cluster analysis based on the log-transformed CN values by using Euclidean distance and Ward's minimum variance yielded an unsupervised 7-cluster classification, whereby tumors with one or multiple low CN genes were associated with worse outcome (log-rank p=.0195 for disease free survival [DFS]). Based on this observation we used the above described CN categories and applied a deterministic profiling distinguishing tumors with no aberrations (A, n=218 [49.7%]); CN losses or complex losses/gains (B, n=29 [6.6%]); any gain in 1 gene (C, n=92 [20.9%]); and, multiple gains (D, n=100 [22.8%]). Again, patients with B-tumors had the worst outcome (p=.0123 for DFS). Upon adjustment for clinical parameters, FISH and IHC HER2 and TOP2A status, ER/PgR IHC, and breast carcinoma subtypes, B-tumors independently predicted for increased risk of relapse and death in comparison to A- and C-tumors (e.g., for DFS, B vs. A, HR: 2.3, 95%CI: 1.3-4.2, Wald-p=.0036; B vs. C, HR: 2.6, 95% CI: 1.4-5.0, Wald-p=.0031). Conclusions: Breast tumors with CN losses or complex losses/gains for the genes examined on 17q seem to carry the worst prognosis among those treated in the adjuvant setting with anthracyclines and may be worthy indentifying for different treatment selection. TOP2A FISH and CNV status are not concordant, while NOS2 and RARA seem to suffer internal CNV, which needs further validation for its biologic implications. Citation Format: Vassiliki Kotoula, Mattheos Bobos, George Kouvatseas, Christos Papadimitriou, Kyriaki Papadopoulou, Elpida Charalambous, Eleftheria Tsolaki, George Fountzilas. Genomic CNV testing on chromosome 17q genes reveals clinically relevant subtypes in breast cancer. [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 1918. doi:10.1158/1538-7445.AM2013-1918

Evaluation of calling algorithms for array-CGH

Copy number variation (CNV) detection has become an integral part many of genetic studies and new technologies promise to revolutionize our ability to detect and link them to disease. However, recent studies highlight discrepancies in the genome wide CNV profile when measured by different technologies and even by the same technology. Furthermore, the change point algorithms used to call CNVs can have substantial disagreement on the same data set. We focus this article on comparative genomic hybridization (CGH) arrays because this platform lends itself well to accurate statistical modeling. We describe some newer methodological developments in local statistics that are well suited for CNV detection and calling on CGH arrays. Then we use both simulation studies and public data to compare these new local methods with the global methods that currently dominate literature. These results offer suggestions for choosing a particular method and provide insight to the lack of reproducibility that has been seen in the field so far.

Massive screening of copy number population-scale variation in Bos taurus genome

BackgroundCopy number variations (CNVs) represent a significant source of genomic structural variation. Their length ranges from approximately one hundred to millions of base pair. Genome-wide screenings have clarified that CNVs are a ubiquitous phenomenon affecting essentially the whole genome. Although Bos taurus is one of the most important domestic animal species worldwide and one of the most studied ruminant models for metabolism, reproduction, and disease, relatively few studies have investigated CNVs in cattle and little is known about how CNVs contribute to normal phenotypic variation and to disease susceptibility in this species, compared to humans and other model organisms.ResultsHere we characterize and compare CNV profiles in 2654 animals from five dairy and beef Bos taurus breeds, using the Illumina BovineSNP50 genotyping array (54001 SNP probes). In this study we applied the two most commonly used algorithms for CNV discovery (QuantiSNP and PennCNV) and identified 4830 unique candidate CNVs belonging to 326 regions. These regions overlap with 5789 known genes, 76.7% of which are significantly co-localized with segmental duplications (SD).ConclusionsThis large scale screening significantly contributes to the enrichment of the Bos taurus CNV map, demonstrates the ubiquity, great diversity and complexity of this type of genomic variation and sets the basis for testing the influence of CNVs on Bos taurus complex functional and production traits.

A pilot study on collective effects of 22q13.31 deletions on gray matter concentration in schizophrenia.

The association of copy number variation (CNV) with schizophrenia has been reported with evidence of increased frequency of both rare and large CNVs. Yet, little is known about the impact of CNVs in brain structure. In this pilot study, we explored collective effects of all CNVs in each cytogenetic band on the risk of schizophrenia and gray matter variation measured in structural magnetic resonance imaging. With 324 participants’ CNV profiles (151 schizophrenia patients and 173 healthy controls), we first extracted specific CNV features that differ between patients and controls using a two sample t-test, and then tested their associations with gray matter concentration using a linear regression model in a subset of 301 participants. Our data first provided evidence of population structure in CNV features where elevated rare CNV burden in schizophrenia patients was confounded by the levels associated with African American subjects. We considered this ethnic group difference in the following cytoband analyses. Deletions in one cytoband 22q13.31 were observed significantly (p<0.05) more in patients than controls from all samples after controlling ethnicity, and the deletion load was also significantly (p = 1.44×10−4) associated with reduced gray matter concentration of a brain network mainly comprised of the cingulate gyrus and insula. Since 80% deletion carriers were patients, patients with deletions also showed reduced gray matter concentration compared with patients without deletions (p = 3.36×10−4). Our findings indicate that regional CNVs at 22q13.31, no matter the size, may influence the risk of schizophrenia with a remarkably increased mutation rate and with reduced gray matter concentration in the peri-limbic cortex. This proof-of-concept study suggests that the CNVs occurring at some ‘hotspots’ may in fact cause biological downstream effects and larger studies are important for confirming our initial results.

Number of rare germline CNVs and TP53 mutation types

BackgroundThe Li-Fraumeni syndrome (LFS), an inherited rare cancer predisposition syndrome characterized by a variety of early-onset tumors, is caused by different highly penetrant germline mutations in the TP53 gene; each separate mutation has dissimilar functional and phenotypic effects, which partially clarifies the reported heterogeneity between LFS families. Increases in copy number variation (CNV) have been reported in TP53 mutated individuals, and are also postulated to contribute to LFS phenotypic variability. The Brazilian p.R337H TP53 mutation has particular functional and regulatory properties that differ from most other common LFS TP53 mutations, by conferring a strikingly milder phenotype.MethodsWe compared the CNV profiles of controls, and LFS individuals carrying either p.R337H or DNA binding domain (DBD) TP53 mutations by high resolution array-CGH.ResultsAlthough we did not find any significant difference in the frequency of CNVs between LFS patients and controls, our data indicated an increased proportion of rare CNVs per genome in patients carrying DBD mutations compared to both controls (p=0.0002***) and p.R337H (0.0156*) mutants.ConclusionsThe larger accumulation of rare CNVs in DBD mutants may contribute to the reported anticipation and severity of the syndrome; likewise the fact that p.R337H individuals do not present the same magnitude of rare CNV accumulation may also explain the maintenance of this mutation at relatively high frequency in some populations.

Evaluation of chemokine-ligand pathways in pretreatment tumor biopsies as predictive biomarker of response to adoptive therapy in metastatic melanoma patients.

8576 Background: Adoptive therapy with tumor infiltrating lymphocytes (TILs) induces objective responses (OR) in approximately 50% of patients with metastatic melanoma. The recruitment of TILs through CXCR3/CCR5-ligand chemokines is believed critical for immune-mediated rejection. Here, we investigated the predictive role of a gene-signature based on CXCR3/CCR5-ligand chemokine transcripts in pre-treatment melanoma biopsies, its biological role and its relation with CCR5-Δ32 polymorphism, which encodes a protein not expressed on cell surface. Methods: Expression of CXCR3/CXCR3-ligand transcripts (i.e CXCL9, 10, 11) and CCR5/CCR5-ligand transcripts (i.e. CCL3, 4, 5) were assessed in 113 pre-treatment tumor biopsies from patients enrolled in adoptive therapy trials: 24 patients achieved a complete remission (CR), 34 a partial remission (PR), and 55 did not respond (NR). Copy number variation and gene expression profile of these target genes were assessed in 15 biopsy-derived cell lines. CCR5-Δ32 was assessed by sequencing germinal DNA. Results: CXCL9, 10 and 11 and CCL5 clustered together and were selected for hierarchical clustering analysis based on the mean-centered gene expression values. A signature characterized by the over-expression of these genes was associated with the likelihood to achieve a clinical response (OR rate: PR+CR: 65% vs 38%, High vs Low, respectively, P=0.015). Neither correlation between the copy number variation and the gene-expression of the corresponding genes, nor correlation between the transcripts of the investigated genes between tumor biopsies and the matched cell lines was detected. Transcript expression of the target genes did not differ between CCR5-Δ32 (n =20) and wild type patients (n=93). Conclusions: Coordinate over-expression of CXCR3/CCR5 ligands in pre-treatment tumor samples was associated with responsiveness to treatment. However, the lack of correlation between in vivo and ex vivo data suggest the inflammatory status characterized by the up-regulation of these inflammatory chemokine genes is an in vivo multifactorial phenomenon.

Abstract B62: Multiple genomic aberrations in BCR-ABL inhibitor-resistant cells lead to sensitivity towards MEK inhibition

Abstract Purpose: Chronic myeloid leukemia (CML) can be effectively treated with BCR-ABL inhibitors such as imatinib, dasatinib, and nilotinib; however, resistance to these inhibitors develops over time causing patients to relapse. Recently, MEK inhibition was shown to synergize with BCR-ABL inhibitors to inhibit resistant CML cells harboring the T315I mutation (1). In this study we characterized mechanisms of resistance to BCR-ABL inhibitor in acquired resistant cells without BCR-ABL mutation by genomic and phosphoprotein profiling. We further evaluated the sensitivity of these resistant clones to MEK inhibitor, GDC0973, PI3K inhibitor, GDC0941, alone or in combination. Methods: The CML cell K562 was grown in an increasing concentration of imatinib or/and dasatinib to induce acquired resistance. Clones were isolated from the resistant pool and the GI50 of each clone was measured using the Cell-Titer Glo® viability assay. Genomic DNA from selected clones was tested using Oncoscan® (Affymetrix), which surveys whole genome for copy number variation (CNV) and 400 oncogenic mutations. Cell lysates were subjected to reverse phase protein array (RPPA) analysis of phosphorylation levels of ∼100 proteins. Genomic mutation and CNV profiles, as well as protein phosphorylation patterns of the clones were compared to the parental cells to identify potential pathways involved in resistance. Results: Four imatinib-resistant clones with varying GI50s (4–30 times higher than parental cells) were fully characterized with both Oncoscan and RPPA. None contained the T315I mutation. A genomic mutation scan showed that all the clones had multiple mutations affecting various signaling pathways including TGFβ (Smad-2), Wnt (APC), PI3K (PIK3CA) and EGFR. Likewise, whole genome copy number scan showed that CNV occurred in multiple genes including copy number gains in ADAM29, BMP2, Rspondin4 (Wnt) and PKA in multiple clones, and copy number losses in ADAMTS14, LIM-kinase and FzD9 (Wnt) in multiple clones. The protein phosphorylation patterns also showed multiple changes in the resistant clones. Three of the resistant clones (K14, 15 and 25) had 6-10-fold increased pMEK and pERK, relative to the parental cells. MEk inhibition resulted in a cytostatic response in the parental cells (GI50: 1900 nM), whereas resistant clones 14, 15, and 25 were highly sensitive to GDC0973-mediated apoptosis, with GI50s ranging from 60–75 nM. Clone 6 had lower pMEK and pERK levels than the others, but was still sensitive to GDC0973 (GI50: 600 nM). Phosphorylation of proteins in the PI3K pathway such as pS6, p70S6K and p-mTOR were elevated in all of the clones as compared to the parental cells (7–11 fold); however, none of these clones were sensitive to GDC0941 alone. Our data suggest that either GDC0973 or GDC0973 alone or in combination with GDC0941 could be effective at inducing cell death in resistant clones despite their various genetic backgrounds. Conclusion: The MEK inhibitor GDC0973 caused robust cellular apoptosis in all of the resistant clones in contrast to the cytostatic effect in parental cells. All of the clones had elevated PI3K signaling as shown by enhanced PI3K substrate phosphorylation. Despite the many genetic aberrations that occurred as cells became resistant; most noticeably changes in Wnt, PKA, ADAMs and BMP2 in all of the clones, resistant cells appear to channel escape/survival signals through the MAPK and/or PI3K pathway rendering them sensitive to GDC0973 alone or in combination with GDC0941.

Abstract PR14: Germline submicroscopic chromosome imbalances in pediatric cancer

Abstract The development of cancer is a multi-step process and, therefore, the probability of cancer occurrence increases with age. Pediatric cancer then either involves a succession of infrequent events occurring in a short period of time, which supposedly only happens rarely, or alternatively, some of the steps required for cancer development are already present in the individuals' germlines. Support for the presence of genomic alterations in the germline of these patients is given by the association of pediatric cancer with congenital abnormalities, observed both as the frequent presence of congenital abnormalities among these patients and as an increase of cancer among children with congenital abnormalities. We investigated DNA copy number variations (CNVs) in a cohort of 55 pediatric cancer patients attending the AC Camargo Hospital, São Paulo, Brazil. In particular, we focused on those alterations not commonly found in the general population (rare CNVs). No selection criteria were used other than age at diagnosis &amp;lt; 20 years and the families agreeing to participate in the study. In addition to CNV profiling by array-CGH (180K – Agilent Technologies), the patients were also physically examined by a clinical geneticist. Eighteen copy number alterations were detected in 16 patients; these rare CNVs included complex rearrangements and triplications, not often observed among common variants. The size of the alterations ranged from 58 to 2,600 Kb, with an average of 438 Kb, which is over twice the average length of rare copy number variations identified with the same array-CGH platform in a sample of 100 reference individuals studied in our laboratory. Many of the sixty-four genes mapping within the chromosome imbalances detected in patients represent good candidates for cancer manifestation and/or congenital abnormalities. Our results suggest that constitutive submicroscopic chromosome imbalances are a contributor to the etiology of pediatric cancer, that the combined presence of pediatric cancer and congenital abnormalities is strongly suggestive of constitutive chromosome imbalances being present, and that further CNV studies will lead finally to identification of genes involved in predisposition to pediatric cancer. Funding: FAPESP; CNPq This abstract is also presented as Poster B7. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR14.

Social responsiveness scale-aided analysis of the clinical impact of copy number variations in autism

Recent array-based studies have detected a wealth of copy number variations (CNVs) in patients with autism spectrum disorders (ASD). Since CNVs also occur in healthy individuals, their contributions to the patient’s phenotype remain largely unclear. In a cohort of children with symptoms of ASD, diagnosis of the index patient using ADOS-G and ADI-R was performed, and the Social Responsiveness Scale (SRS) was administered to the index patients, both parents, and all available siblings. CNVs were identified using SNP arrays and confirmed by FISH or array CGH. To evaluate the clinical significance of CNVs, we analyzed three families with multiple affected children (multiplex) and six families with a single affected child (simplex) in which at least one child carried a CNV with a brain-transcribed gene. CNVs containing genes that participate in pathways previously implicated in ASD, such as the phosphoinositol signaling pathway (PIK3CA, GIRDIN), contactin-based networks of cell communication (CNTN6), and microcephalin (MCPH1) were found not to co-segregate with ASD phenotypes. In one family, a loss of CNTN5 co-segregated with disease. This indicates that most CNVs may by themselves not be sufficient to cause ASD, but still may contribute to the phenotype by additive or epistatic interactions with inherited (transmitted) mutations or non-genetic factors. Our study extends the scope of genome-wide CNV profiling beyond de novo CNVs in sporadic patients and may aid in uncovering missing heritability in genome-wide screening studies of complex psychiatric disorders.