Papers by Christiaan Klijn
Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, a... more Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely conducted in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUSs have been described, thus far it has not been possible to conduct a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUSs based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay, we have analyzed 74 unclassified BRCA1 missense mutants for which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains. BRCA1 VUSs are frequently found in patients with hereditary breast or ovarian cancer and present a serious problem for clinical geneticists. This article describes the generation, validation, and application of a reliable high-throughput assay for the functional classification of BRCA1 sequence variants of uncertain significance.
PLoS ONE, 2014
Lymph-node metastasis (LNM) predict high recurrence rates in breast cancer patients. Systemic tre... more Lymph-node metastasis (LNM) predict high recurrence rates in breast cancer patients. Systemic treatment aims to eliminate (micro)metastatic cells. However decisions regarding systemic treatment depend largely on clinical and molecular characteristics of primary tumours. It remains, however, unclear to what extent metastases resemble the cognate primary breast tumours, especially on a genomic level, and as such will be eradicated by the systemic therapy chosen. In this study we used high-resolution aCGH to investigate DNA copy number differences between primary breast cancers and their paired LNMs. To date, no recurrent LNM-specific genomic aberrations have been identified using array comparative genomic hybridization (aCGH) analysis. In our study we employ a high-resolution platform and we stratify on different breast cancer subtypes, both aspects that might have underpowered previously performed studies.To test the possibility that genomic instability in triple-negative breast cancers (TNBCs) might cause increased random and potentially also recurrent copy number aberrations (CNAs) in their LNMs, we studied 10 primary TNBC-LNM pairs and 10 ER-positive (ER+) pairs and verified our findings adding additionally 5 TNBC-LNM and 22 ER+-LNM pairs. We found that all LNMs clustered nearest to their matched tumour except for two cases, of which one was due to the presence of two distinct histological components in one tumour. We found no significantly altered CNAs between tumour and their LNMs in the entire group or in the subgroups. Within the TNBC subgroup, no absolute increase in CNAs was found in the LNMs compared to their primary tumours, suggesting that increased genomic instability does not lead to more CNAs in LNMs. Our findings suggest a high clonal relationship between primary breast tumours and its LNMs, at least prior to treatment, and support the use of primary tumour characteristics to guide adjuvant systemic chemotherapy in breast cancer patients. Citation: Vollebergh MA, Klijn C, Schouten PC, Wesseling J, Israeli D, et al. (2014) Lack of Genomic Heterogeneity at High-Resolution aCGH between Primary Breast Cancers and Their Paired Lymph Node Metastases. PLoS ONE 9(8): e103177.
Nature biotechnology, 2015
Tumor-derived cell lines have served as vital models to advance our understanding of oncogene fun... more Tumor-derived cell lines have served as vital models to advance our understanding of oncogene function and therapeutic responses. Although substantial effort has been made to define the genomic constitution of cancer cell line panels, the transcriptome remains understudied. Here we describe RNA sequencing and single-nucleotide polymorphism (SNP) array analysis of 675 human cancer cell lines. We report comprehensive analyses of transcriptome features including gene expression, mutations, gene fusions and expression of non-human sequences. Of the 2,200 gene fusions catalogued, 1,435 consist of genes not previously found in fusions, providing many leads for further investigation. We combine multiple genome and transcriptome features in a pathway-based approach to enhance prediction of response to targeted therapeutics. Our results provide a valuable resource for studies that use cancer cell lines.
Genome Biology, 2010
Background: Here we present the first paired-end sequencing of tumors from genetically engineered... more Background: Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1-and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer.
Proceedings of the National Academy of Sciences, 2013
Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or... more Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or to Fanconi Anemia subtype N. PALB2 performs its tumor suppressor role, at least in part, by supporting homologous recombination-type double strand break repair (HR-DSBR) through physical interactions with BRCA1, BRCA2, and RAD51. To further understand the mechanisms underlying PALB2mediated DNA repair and tumor suppression functions, we targeted Palb2 in the mouse. Palb2-deficient murine ES cells recapitulated DNA damage defects caused by PALB2 depletion in human cells, and germline deletion of Palb2 led to early embryonic lethality. Somatic deletion of Palb2 driven by K14-Cre led to mammary tumor formation with long latency. Codeletion of both Palb2 and Tumor protein 53 (Trp53) accelerated mammary tumor formation. Like BRCA1 and BRCA2 mutant breast cancers, these tumors were defective in RAD51 focus formation, reflecting a defect in Palb2 HR-DSBR function, a strongly suspected contributor to Brca1, Brca2, and Palb2 mammary tumor development. However, unlike the case of Brca1mutant cells, Trp53bp1 deletion failed to rescue the genomic instability of Palb2-or Brca2-mutant primary lymphocytes. Therefore, Palb2-driven DNA damage control is, in part, distinct from that executed by Brca1 and more similar to that of Brca2. The mechanisms underlying Palb2 mammary tumor suppression functions can now be explored genetically in vivo. mouse model | familial breast cancer
PLoS ONE, 2013
Cancer develops through a multistep process in which normal cells progress to malignant tumors vi... more Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV) as an insertional mutagen to find both the driver genes and the networks in which they function. Using deep insertion site sequencing we identified around 31000 retroviral integration sites in 604 MMTV-induced mammary tumors from mice with mammary gland-specific deletion of Trp53, Pten heterozygous knockout mice, or wildtype strains. We identified 18 known common integration sites (CISs) and 12 previously unknown CISs marking new candidate cancer genes. Members of the Wnt, Fgf, Fgfr, Rspo and Pdgfr gene families were commonly mutated in a mutually exclusive fashion. The sequence data we generated yielded also information on the clonality of insertions in individual tumors, allowing us to develop a data-driven model of MMTV-induced tumor development. Insertional mutations near Wnt and Fgf genes mark the earliest ''initiating'' events in MMTV induced tumorigenesis, whereas Fgfr genes are targeted later during tumor progression. Our data shows that insertional mutagenesis can be used to discover the mutational networks, the timing of mutations, and the genes that initiate and drive tumor evolution.
PLoS ONE, 2007
Background. The availability of large collections of microarray datasets (compendia), or knowledg... more Background. The availability of large collections of microarray datasets (compendia), or knowledge about grouping of genes into pathways (gene sets), is typically not exploited when training predictors of disease outcome. These can be useful since a compendium increases the number of samples, while gene sets reduce the size of the feature space. This should be favorable from a machine learning perspective and result in more robust predictors. Methodology. We extracted modules of regulated genes from gene sets, and compendia. Through supervised analysis, we constructed predictors which employ modules predictive of breast cancer outcome. To validate these predictors we applied them to independent data, from the same institution (intra-dataset), and other institutions (inter-dataset). Conclusions. We show that modules derived from single breast cancer datasets achieve better performance on the validation data compared to gene-based predictors. We also show that there is a trend in compendium specificity and predictive performance: modules derived from a single breast cancer dataset, and a breast cancer specific compendium perform better compared to those derived from a human cancer compendium. Additionally, the module-based predictor provides a much richer insight into the underlying biology. Frequently selected gene sets are associated with processes such as cell cycle, E2F regulation, DNA damage response, proteasome and glycolysis. We analyzed two modules related to cell cycle, and the OCT1 transcription factor, respectively. On an individual basis, these modules provide a significant separation in survival subgroups on the training and independent validation data. Citation: van Vliet MH, Klijn CN, Wessels LFA, Reinders MJT (2007) Module-Based Outcome Prediction Using Breast Cancer Compendia. PLoS ONE 2(10): e1047.
PLoS Computational Biology, 2010
Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors follo... more Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs) are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly cooccurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T-and B-cell malignancies, respectively, showing that we can recover truly cooccurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes.
Nucleic Acids Research, 2007
Tumor formation is in part driven by DNA copy number alterations (CNAs), which can be measured us... more Tumor formation is in part driven by DNA copy number alterations (CNAs), which can be measured using microarray-based Comparative Genomic Hybridization (aCGH). Multiexperiment analysis of aCGH data from tumors allows discovery of recurrent CNAs that are potentially causal to cancer development. Until now, multiexperiment aCGH data analysis has been dependent on discretization of measurement data to a gain, loss or no-change state. Valuable biological information is lost when a heterogeneous system such as a solid tumor is reduced to these states. We have developed a new approach which inputs nondiscretized aCGH data to identify regions that are significantly aberrant across an entire tumor set. Our method is based on kernel regression and accounts for the strength of a probe's signal, its local genomic environment and the signal distribution across multiple tumors. In an analysis of 89 human breast tumors, our method showed enrichment for known cancer genes in the detected regions and identified aberrations that are strongly associated with breast cancer subtypes and clinical parameters. Furthermore, we identified 18 recurrent aberrant regions in a new dataset of 19 p53-deficient mouse mammary tumors. These regions, combined with gene expression microarray data, point to known cancer genes and novel candidate cancer genes.
Nature Communications, 2014
Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying geno... more Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.
Genome Research, 2012
Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and res... more Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.
Genome Research, 2011
Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer g... more Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative highthroughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV-and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral-and transposon-based insertional mutagenesis data.
Clinical Cancer Research, 2010
Background: Genomic instability with frequent DNA copy number alterations is one of the key hallm... more Background: Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level.
Cancer Cell, 2011
Hereditary breast cancers are frequently caused by germline BRCA1 mutations. The BRCA1(C61G) muta... more Hereditary breast cancers are frequently caused by germline BRCA1 mutations. The BRCA1(C61G) mutation in the BRCA1 RING domain is a common pathogenic missense variant, which reduces BRCA1/BARD1 heterodimerization and abrogates its ubiquitin ligase activity. To investigate the role of BRCA1 RING function in tumor suppression and therapy response, we introduced the Brca1(C61G) mutation in a conditional mouse model for BRCA1-associated breast cancer. In contrast to BRCA1-deficient mammary carcinomas, tumors carrying the Brca1(C61G) mutation responded poorly to platinum drugs and PARP inhibition and rapidly developed resistance while retaining the Brca1(C61G) mutation. These findings point to hypomorphic activity of the BRCA1-C61G protein that, although unable to prevent tumor development, affects response to therapy.
BMC Research Notes, 2010
Background: Most approaches used to find recurrent or differential DNA Copy Number Alterations (C... more Background: Most approaches used to find recurrent or differential DNA Copy Number Alterations (CNA) in array Comparative Genomic Hybridization (aCGH) data from groups of tumour samples depend on the discretization of the aCGH data to gain, loss or no-change states. This causes loss of valuable biological information in tumour samples, which are frequently heterogeneous. We have previously developed an algorithm, KC-SMART, that bases its estimate of the magnitude of the CNA at a given genomic location on kernel convolution . This accounts for the intensity of the probe signal, its local genomic environment and the signal distribution across multiple samples.
BMC Cancer, 2010
Background: Genomic gains and losses are a result of genomic instability in many types of cancers... more Background: Genomic gains and losses are a result of genomic instability in many types of cancers. BRCA1-and BRCA2-mutated breast cancers are associated with increased amounts of chromosomal aberrations, presumably due their functions in genome repair. Some of these genomic aberrations may harbor genes whose absence or overexpression may give rise to cellular growth advantage. So far, it has not been easy to identify the driver genes underlying gains and losses. A powerful approach to identify these driver genes could be a cross-species comparison of array comparative genomic hybridization (aCGH) data from cognate mouse and human tumors. Orthologous regions of mouse and human tumors that are commonly gained or lost might represent essential genomic regions selected for gain or loss during tumor development.
BMC Cancer, 2010
Background: Accurate staging of colorectal cancer (CRC) with clinicopathological parameters is im... more Background: Accurate staging of colorectal cancer (CRC) with clinicopathological parameters is important for predicting prognosis and guiding treatment but provides no information about organ site of metastases. Patterns of genomic aberrations in primary colorectal tumors may reveal a chromosomal signature for organ specific metastases.
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Papers by Christiaan Klijn