Publications by authors named "Harmen J G van de Werken"

49 Publications

Generating human prostate cancer organoids from leukapheresis enriched circulating tumour cells.

Eur J Cancer 2021 Apr 27;150:179-189. Epub 2021 Apr 27.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands. Electronic address:

Background: Circulating tumour cell (CTC)-derived organoids have the potential to provide a powerful tool for personalised cancer therapy but are restrained by low CTC numbers provided by blood samples. Here, we used diagnostic leukapheresis (DLA) to enrich CTCs from patients with metastatic prostate cancer (mPCa) and explored whether organoids provide a platform for ex vivo treatment modelling.

Methods: We prospectively screened 102 patients with mPCa and performed DLA in 40 patients with ≥5 CTCs/7.5 mL blood. We enriched CTCs from DLA using white blood cell (WBC) depletion alone or combined with EpCAM selection. The enriched CTC samples were cultured in 3D to obtain organoids and used for downstream analyses.

Results: The DLA procedure resulted in a median yield of 5312 CTCs as compared with 22 CTCs in 7.5 mL of blood. Using WBC depletion, we recovered 46% of the CTCs, which reduced to 12% with subsequent EpCAM selection. From the isolated and enriched CTC samples, organoid expansion succeeded in 35%. Successful organoid cultures contained significantly higher CTC numbers at initiation. Moreover, we performed treatment modelling in one organoid cell line and identified substantial tumour heterogeneity in CTCs using single cell DNA sequencing.

Conclusions: DLA is an efficient method to enrich CTCs, although the modest success rate of culturing CTCs precludes large scale clinical application. Our data do suggest that DLA and subsequent processing provides a rich source of viable tumour cells. Therefore, DLA offers a promising alternative to biopsy procedures to obtain sufficient number of tumour cells to study sequential samples in patients with mPCa.

Trial Registration Number: NL6019.
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http://dx.doi.org/10.1016/j.ejca.2021.03.023DOI Listing
April 2021

Evaluation of AXIN1 and AXIN2 as targets of tankyrase inhibition in hepatocellular carcinoma cell lines.

Sci Rep 2021 Apr 2;11(1):7470. Epub 2021 Apr 2.

Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands.

AXIN1 mutations are observed in 8-10% of hepatocellular carcinomas (HCCs) and originally were considered to support tumor growth by aberrantly enhancing β-catenin signaling. This view has however been challenged by reports showing neither a clear nuclear β-catenin accumulation nor clearly enhanced expression of β-catenin target genes. Here, using nine HCC lines, we show that AXIN1 mutation or siRNA mediated knockdown contributes to enhanced β-catenin signaling in all AXIN1-mutant and non-mutant lines, also confirmed by reduced signaling in AXIN1-repaired SNU449 cells. Both AXIN1 and AXIN2 work synergistically to control β-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels. The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of β-catenin-dependent cancers. At concentrations that inhibit tankyrase activity, some lines (e.g. HepG2, SNU398) were clearly affected in colony formation, but in most cases apparently independent from effects on β-catenin signaling. Overall, our analyses show that AXIN1 inactivation leads to enhanced β-catenin signaling in HCC cell lines, questioning the strong statements that have been made in this regard. Enhancing AXIN activity by tankyrase monotherapy provides however no effective treatment to affect their growth exclusively through reducing β-catenin signaling.
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http://dx.doi.org/10.1038/s41598-021-87091-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018973PMC
April 2021

The miR-200c/141-ZEB2-TGFβ axis is aberrant in human T-cell prolymphocytic leukemia.

Haematologica 2021 Feb 18. Epub 2021 Feb 18.

Department of Immunology, Erasmus University Medical Center, Rotterdam.

T cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small to medium sized pro-lymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide-range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, whereas no clear T cell receptor alpha (TRA) or beta (TRB) CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of miRNAs is aberrant and often heterogeneous in T-PLL. We identified 35 miRNAs that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR-200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFβR3 and aberrant TGF-β1-induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFβ pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNAs in T-PLL and pave the way for new diagnostic targets in this disease.
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http://dx.doi.org/10.3324/haematol.2020.263756DOI Listing
February 2021

Blood-based kinase activity profiling: a potential predictor of response to immune checkpoint inhibition in metastatic cancer.

J Immunother Cancer 2020 12;8(2)

Department of Medical Oncology, Oncode Institute, Leiden University Medical Center, Leiden, The Netherlands

Background: Many cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may predict clinical outcome.

Methods: Here a total of 160 patients with advanced melanoma or non-small-cell lung cancer (NSCLC), treated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed cell death 1 (anti-PD-1), were divided into five discovery and cross-validation cohorts. The kinase activity profile was generated by analyzing phosphorylation of peripheral blood mononuclear cell lysates in a microarray comprising of 144 peptides derived from sites that are substrates for protein tyrosine kinases. Binary grouping into patients with or without clinical benefit was based on Response Evaluation Criteria in Solid Tumors V.1.1. Predictive models were trained using partial least square discriminant analysis (PLS-DA), performance of the models was evaluated by estimating the correct classification rate (CCR) using cross-validation.

Results: The kinase phosphorylation signatures segregated responders from non-responders by differences in canonical pathways governing T-cell migration, infiltration and co-stimulation. PLS-DA resulted in a CCR of 100% and 93% in the anti-CTLA-4 and anti-PD1 melanoma discovery cohorts, respectively. Cross-validation cohorts to estimate the accuracy of the predictive models showed CCRs of 83% for anti-CTLA-4 and 78% or 68% for anti-PD-1 in melanoma or NSCLC, respectively.

Conclusion: Blood-based kinase activity profiling for response prediction to immune checkpoint inhibitors in melanoma and NSCLC revealed increased kinase activity in pathways associated with T-cell function and led to a classification model with a highly accurate classification rate in cross-validation groups. The predictive value of kinase activity profiling is prospectively verified in an ongoing trial.
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http://dx.doi.org/10.1136/jitc-2020-001607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757459PMC
December 2020

The clonal relation of primary upper urinary tract urothelial carcinoma and paired urothelial carcinoma of the bladder.

Int J Cancer 2021 Feb 13;148(4):981-987. Epub 2020 Oct 13.

Department of Urology, Erasmus MC Cancer Institute, University Medical Center, Rotterdam, The Netherlands.

The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor-specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities.
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http://dx.doi.org/10.1002/ijc.33327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821318PMC
February 2021

Cystic renal-epithelial derived induced pluripotent stem cells from polycystic kidney disease patients.

Stem Cells Transl Med 2020 04 12;9(4):478-490. Epub 2020 Mar 12.

Department of Developmental Biology, Erasmus Medical Center Rotterdam (EMC), Oncode Institute, Rotterdam, The Netherlands.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient-specific iPSCs to study the function of PKD1 in kidney development and cyst formation in vitro. Somatic mutations are proposed to be the initiating event of cyst formation, and therefore, iPSCs were derived from cystic renal epithelial cells rather than fibroblasts. Mutation analysis of the ADPKD iPSCs revealed germline mutations in PKD1 but no additional somatic mutations in PKD1/PKD2. Although several somatic mutations in other genes implicated in ADPKD were identified in cystic renal epithelial cells, only few of these mutations were present in iPSCs, indicating a heterogeneous mutational landscape, and possibly in vitro cell selection before and during the reprogramming process. Whole-genome DNA methylation analysis indicated that iPSCs derived from renal epithelial cells maintain a kidney-specific DNA methylation memory. In addition, comparison of PKD1+/- and control iPSCs revealed differences in DNA methylation associated with the disease history. In conclusion, we generated and characterized iPSCs derived from cystic and healthy control renal epithelial cells, which can be used for in vitro modeling of kidney development in general and cystogenesis in particular.
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http://dx.doi.org/10.1002/sctm.18-0283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103626PMC
April 2020

Synchronous and metachronous urothelial carcinoma of the upper urinary tract and the bladder: Are they clonally related? A systematic review.

Urol Oncol 2020 06 11;38(6):590-598. Epub 2020 Feb 11.

Department of Urology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Purpose: Following radical nephroureterectomy for upper urinary tract urothelial carcinoma (UTUC), intravesical recurrence (IVR) is found in 22% to 47% of patients. Patients with a primary urothelial carcinoma of the bladder (UCB) have an increased risk of a future UTUC (1%-5%). Paired UTUC and UCB might represent clonally related tumors due to intraluminal seeding of tumor cells or might be separate entities of urothelial carcinoma caused by field cancerization. We systematically reviewed all the relevant literature to address the possible clonal relation of UTUC and paired UCB.

Materials And Methods: MEDLINE, EMBASE, and COCHRANE databases were systematically searched for relevant citations published between January 2000 and July 2019. This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Of 5038 citations identified, 86 full papers were screened, and 9 studies met the inclusion criteria.

Results: The populations studied and the molecular techniques used to assess clonality of UTUC and paired UCB differed largely over time. Eight studies reported on primary UTUC and meta- or synchronous IVR without a history of UCB. A total of 118 tumors (55 UTUC and 63 IVR) from 49 patients were included, of which 94% seemed to be clonally related. Five studies reported on primary UCB and subsequent UTUC with a total of 61 tumors (30 UCB and 31 UTUC) from 14 patients; a possible clonal origin was identified for 85% of the tumors.

Conclusion: Taking into account the limitations of microsatellite technology in comparison to Next Generation Sequencing and currently accepted concepts of tumor heterogeneity and evolution, this systematic review shows that most, if not all, UTUC and paired UCB likely are clonally related.
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http://dx.doi.org/10.1016/j.urolonc.2020.01.008DOI Listing
June 2020

The genomic landscape of metastatic castration-resistant prostate cancers reveals multiple distinct genotypes with potential clinical impact.

Nat Commun 2019 11 20;10(1):5251. Epub 2019 Nov 20.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands.

Metastatic castration-resistant prostate cancer (mCRPC) has a highly complex genomic landscape. With the recent development of novel treatments, accurate stratification strategies are needed. Here we present the whole-genome sequencing (WGS) analysis of fresh-frozen metastatic biopsies from 197 mCRPC patients. Using unsupervised clustering based on genomic features, we define eight distinct genomic clusters. We observe potentially clinically relevant genotypes, including microsatellite instability (MSI), homologous recombination deficiency (HRD) enriched with genomic deletions and BRCA2 aberrations, a tandem duplication genotype associated with CDK12 and a chromothripsis-enriched subgroup. Our data suggests that stratification on WGS characteristics may improve identification of MSI, CDK12 and HRD patients. From WGS and ChIP-seq data, we show the potential relevance of recurrent alterations in non-coding regions identified with WGS and highlight the central role of AR signaling in tumor progression. These data underline the potential value of using WGS to accurately stratify mCRPC patients into clinically actionable subgroups.
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http://dx.doi.org/10.1038/s41467-019-13084-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868175PMC
November 2019

The genomic landscape of metastatic breast cancer highlights changes in mutation and signature frequencies.

Nat Genet 2019 10 30;51(10):1450-1458. Epub 2019 Sep 30.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands.

The whole-genome sequencing of prospectively collected tissue biopsies from 442 patients with metastatic breast cancer reveals that, compared to primary breast cancer, tumor mutational burden doubles, the relative contributions of mutational signatures shift and the mutation frequency of six known driver genes increases in metastatic breast cancer. Significant associations with pretreatment are also observed. The contribution of mutational signature 17 is significantly enriched in patients pretreated with fluorouracil, taxanes, platinum and/or eribulin, whereas the de novo mutational signature I identified in this study is significantly associated with pretreatment containing platinum-based chemotherapy. Clinically relevant subgroups of tumors are identified, exhibiting either homologous recombination deficiency (13%), high tumor mutational burden (11%) or specific alterations (24%) linked to sensitivity to FDA-approved drugs. This study provides insights into the biology of metastatic breast cancer and identifies clinically useful genomic features for the future improvement of patient management.
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http://dx.doi.org/10.1038/s41588-019-0507-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858873PMC
October 2019

Identification and Characterization of a Transcribed Distal Enhancer Involved in Cardiac Kcnh2 Regulation.

Cell Rep 2019 Sep;28(10):2704-2714.e5

Amsterdam UMC, University of Amsterdam, Department of Medical Biology, Amsterdam Cardiovascular Sciences, 1105AZ Amsterdam, the Netherlands. Electronic address:

The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying I, a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer ∼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers.
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http://dx.doi.org/10.1016/j.celrep.2019.08.007DOI Listing
September 2019

Multi-Modality Analysis Improves Survival Prediction in Enucleated Uveal Melanoma Patients.

Invest Ophthalmol Vis Sci 2019 08;60(10):3595-3605

Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands.

Purpose: Uveal melanoma (UM) is characterized by multiple chromosomal rearrangements and recurrent mutated genes. The aim of this study was to investigate if copy number variations (CNV) alone and in combination with other genetic and clinico-histopathological variables can be used to stratify for disease-free survival (DFS) in enucleated patients with UM.

Methods: We analyzed single nucleotide polymorphisms (SNP) array data of primary tumors and other clinical variables of 214 UM patients from the Rotterdam Ocular Melanoma Study (ROMS) cohort. Nonweighted hierarchical clustering of SNP array data was used to identify molecular subclasses with distinct CNV patterns. The subclasses associate with mutational status of BAP1, SF3B1, or EIF1AX. Cox proportional hazard models were then used to study the predictive performance of SNP array cluster-, mutation-, and clinico-histopathological data, and their combination for study endpoint risk.

Results: Five clusters with distinct CNV patterns and concomitant mutations in BAP1, SF3B1, or EIF1AX were identified. The sample's cluster allocation contributed significantly to mutational status of samples in predicting the incidence of metastasis during a median of 45.6 (interquartile range [IQR]: 24.7-81.8) months of follow-up (P < 0.05) and vice versa. Furthermore, incorporating all data sources in one model yielded a 0.797 C-score during 100 months of follow-up.

Conclusions: UM has distinct CNV patterns that correspond to different mutated driver genes. Incorporating clinico-histopathological, cluster and mutation data in the analysis results in good performance for UM-related DFS prediction.
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http://dx.doi.org/10.1167/iovs.18-24818DOI Listing
August 2019

The mouse KLF1 Nan variant impairs nuclear condensation and erythroid maturation.

PLoS One 2019 28;14(3):e0208659. Epub 2019 Mar 28.

Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands.

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208659PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438607PMC
November 2019

Molecular heterogeneity and early metastatic clone selection in testicular germ cell cancer development.

Br J Cancer 2019 02 11;120(4):444-452. Epub 2019 Feb 11.

Department of Pathology, Wytemaweg 80, 3015 CN, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, The Netherlands.

Background: Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis.

Methods: In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed.

Results: Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of BRCA1 and BRCA2 genes.

Conclusions: Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS.
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http://dx.doi.org/10.1038/s41416-019-0381-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461884PMC
February 2019

Oncogenic STRAP Supports Hepatocellular Carcinoma Growth by Enhancing Wnt/β-Catenin Signaling.

Mol Cancer Res 2019 02 26;17(2):521-531. Epub 2018 Sep 26.

Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands.

Aberrant activation of Wnt/β-catenin signaling plays a key role in the onset and development of hepatocellular carcinomas (HCC), with about half of them acquiring mutations in either or . The serine/threonine kinase receptor-associated protein (STRAP), a scaffold protein, was recently shown to facilitate the aberrant activation of Wnt/β-catenin signaling in colorectal cancers. However, the function of STRAP in HCC remains completely unknown. Here, increased levels of STRAP were observed in human and mouse HCCs. RNA sequencing of STRAP knockout clones generated by gene editing of Huh6 and Huh7 HCC cells revealed a significant reduction in expression of various metabolic and cell-cycle-related transcripts, in line with their general slower growth observed during culture. Importantly, Wnt/β-catenin signaling was impaired in all STRAP knockout/down cell lines tested, regardless of the underlying or mutation. In accordance with β-catenin's role in (cancer) stem cell maintenance, the expressions of various stem cell markers, such as and , were reduced and concomitantly differentiation-associated genes were increased. Together, these results show that the increased STRAP protein levels observed in HCC provide growth advantage among others by enhancing Wnt/β-catenin signaling. These observations also identify STRAP as a new player in regulating β-catenin signaling in hepatocellular cancers. IMPLICATIONS: Elevated STRAP levels in hepatocellular cancers provide a growth advantage by enhancing Wnt/β-catenin signaling.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0054DOI Listing
February 2019

Paneth Cells Respond to Inflammation and Contribute to Tissue Regeneration by Acquiring Stem-like Features through SCF/c-Kit Signaling.

Cell Rep 2018 08;24(9):2312-2328.e7

Department of Pathology, University Medical Center, Rotterdam, the Netherlands. Electronic address:

IBD syndromes such as Crohn's disease and ulcerative colitis result from the inflammation of specific intestinal segments. Although many studies have reported on the regenerative response of intestinal progenitor and stem cells to tissue injury, very little is known about the response of differentiated lineages to inflammatory cues. Here, we show that acute inflammation of the mouse small intestine is followed by a dramatic loss of Lgr5 stem cells. Instead, Paneth cells re-enter the cell cycle, lose their secretory expression signature, and acquire stem-like properties, thus contributing to the tissue regenerative response to inflammation. Stem cell factor secretion upon inflammation triggers signaling through the c-Kit receptor and a cascade of downstream events culminating in GSK3β inhibition and Wnt activation in Paneth cells. Hence, the plasticity of the intestinal epithelium in response to inflammation goes well beyond stem and progenitor cells and extends to the fully differentiated and post-mitotic Paneth cells.
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http://dx.doi.org/10.1016/j.celrep.2018.07.085DOI Listing
August 2018

Functional Assay Reveals Homologous Recombination Deficiency in Breast Cancer Beyond BRCA Gene Defects.

Clin Cancer Res 2018 12 23;24(24):6277-6287. Epub 2018 Aug 23.

Department of Molecular Genetics and Oncode Institute, Erasmus MC University Medical Center Rotterdam, the Netherlands.

Purpose: Tumors of germline mutated carriers show homologous recombination (HR) deficiency (HRD), resulting in impaired DNA double-strand break (DSB) repair and high sensitivity to PARP inhibitors. Although this therapy is expected to be effective beyond germline mutated carriers, a robust validated test to detect HRD tumors is lacking. In this study, we therefore evaluated a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after irradiation of fresh breast cancer tissue: the recombination REpair CAPacity (RECAP) test.

Experimental Design: Fresh samples of 170 primary breast cancer were analyzed using the RECAP test. The molecular explanation for the HRD phenotype was investigated by exploring deficiencies, mutational signatures, tumor-infiltrating lymphocytes (TIL), and microsatellite instability (MSI).

Results: RECAP was completed successfully in 148 of 170 samples (87%). Twenty-four tumors showed HRD (16%), whereas six tumors were HR intermediate (HRi; 4%). HRD was explained by deficiencies (mutations, promoter hypermethylation, deletions) in 16 cases, whereas seven HRD tumors were non-BRCA related. HRD tumors showed an increased incidence of high TIL counts ( = 0.023) compared with HR proficient (HRP) tumors and MSI was more frequently observed in the HRD group (2/20, 10%) than expected in breast cancer (1%; = 0.017).

Conclusions: RECAP is a robust functional HR assay detecting both -deficient and -proficient HRD tumors. Functional assessment of HR in a pseudo-diagnostic setting is achievable and produces robust and interpretable results.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0063DOI Listing
December 2018

Gene length corrected trimmed mean of M-values (GeTMM) processing of RNA-seq data performs similarly in intersample analyses while improving intrasample comparisons.

BMC Bioinformatics 2018 06 22;19(1):236. Epub 2018 Jun 22.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus MC University Medical Center, 3015 CE, Rotterdam, The Netherlands.

Background: Current normalization methods for RNA-sequencing data allow either for intersample comparison to identify differentially expressed (DE) genes or for intrasample comparison for the discovery and validation of gene signatures. Most studies on optimization of normalization methods typically use simulated data to validate methodologies. We describe a new method, GeTMM, which allows for both inter- and intrasample analyses with the same normalized data set. We used actual (i.e. not simulated) RNA-seq data from 263 colon cancers (no biological replicates) and used the same read count data to compare GeTMM with the most commonly used normalization methods (i.e. TMM (used by edgeR), RLE (used by DESeq2) and TPM) with respect to distributions, effect of RNA quality, subtype-classification, recurrence score, recall of DE genes and correlation to RT-qPCR data.

Results: We observed a clear benefit for GeTMM and TPM with regard to intrasample comparison while GeTMM performed similar to TMM and RLE normalized data in intersample comparisons. Regarding DE genes, recall was found comparable among the normalization methods, while GeTMM showed the lowest number of false-positive DE genes. Remarkably, we observed limited detrimental effects in samples with low RNA quality.

Conclusions: We show that GeTMM outperforms established methods with regard to intrasample comparison while performing equivalent with regard to intersample normalization using the same normalized data. These combined properties enhance the general usefulness of RNA-seq but also the comparability to the many array-based gene expression data in the public domain.
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http://dx.doi.org/10.1186/s12859-018-2246-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013957PMC
June 2018

Investigation of the spatial structure and interactions of the genome at sub-kilobase-pair resolution using T2C.

Nat Protoc 2018 03 8;13(3):459-477. Epub 2018 Feb 8.

Department of Cell Biology, Erasmus Medical Centre, Rotterdam, the Netherlands.

Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in ∼7-8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.
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http://dx.doi.org/10.1038/nprot.2017.132DOI Listing
March 2018

SNPitty: An Intuitive Web Application for Interactive B-Allele Frequency and Copy Number Visualization of Next-Generation Sequencing Data.

J Mol Diagn 2018 03 2;20(2):166-176. Epub 2018 Jan 2.

Cancer Computational Biology Center, Erasmus MC, University Medical Center, Rotterdam, the Netherlands; Department of Urology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands. Electronic address:

Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic aberrations is, however, lacking. Therefore, the authors developed the web application SNPitty that allows interactive visualization and interrogation of variant call format files by using B-allele frequencies of single-nucleotide polymorphisms and single-nucleotide variants, coverage metrics, and copy numbers analysis results. SNPitty displays variant alleles and allelic imbalances with a focus on loss of heterozygosity and copy number variation using genome-wide heterozygous markers and somatic mutations. In addition, SNPitty is capable of generating predefined reports that summarize and highlight disease-specific targets of interest. SNPitty was validated for diagnostic interpretation of somatic events by showcasing a serial dilution series of glioma tissue. Additionally, SNPitty is demonstrated in four cancer-related scenarios encountered in daily clinical practice and on whole-exome sequencing data of peripheral blood from a Down syndrome patient. SNPitty allows detection of loss of heterozygosity, chromosomal and gene amplifications, homozygous or heterozygous deletions, somatic mutations, or any combination thereof in regions or genes of interest. Furthermore, SNPitty can be used to distinguish molecular relationships between multiple tumors from a single patient. On the basis of these data, the authors demonstrate that SNPitty is robust and user friendly in a wide range of diagnostic scenarios.
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http://dx.doi.org/10.1016/j.jmoldx.2017.11.011DOI Listing
March 2018

DOC1-Dependent Recruitment of NURD Reveals Antagonism with SWI/SNF during Epithelial-Mesenchymal Transition in Oral Cancer Cells.

Cell Rep 2017 07;20(1):61-75

Department of Biochemistry, Erasmus University Medical Center, P.O. Box 1738, 3000 DR, Rotterdam, the Netherlands. Electronic address:

The Nucleosome Remodeling and Deacetylase (NURD) complex is a key regulator of cell differentiation that has also been implicated in tumorigenesis. Loss of the NURD subunit Deleted in Oral Cancer 1 (DOC1) is associated with human oral squamous cell carcinomas (OSCCs). Here, we show that restoration of DOC1 expression in OSCC cells leads to a reversal of epithelial-mesenchymal transition (EMT). This is caused by the DOC1-dependent targeting of NURD to repress key transcriptional regulators of EMT. NURD recruitment drives extensive epigenetic reprogramming, including eviction of the SWI/SNF remodeler, formation of inaccessible chromatin, H3K27 deacetylation, and binding of PRC2 and KDM1A, followed by H3K27 methylation and H3K4 demethylation. Strikingly, depletion of SWI/SNF mimics the effects of DOC1 re-expression. Our results suggest that SWI/SNF and NURD function antagonistically to control chromatin state and transcription. We propose that disturbance of this dynamic equilibrium may lead to defects in gene expression that promote oncogenesis.
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http://dx.doi.org/10.1016/j.celrep.2017.06.020DOI Listing
July 2017

Small chromosomal regions position themselves autonomously according to their chromatin class.

Genome Res 2017 06 24;27(6):922-933. Epub 2017 Mar 24.

Department of Biology II, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes.
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http://dx.doi.org/10.1101/gr.213751.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453326PMC
June 2017

A reported 20-gene expression signature to predict lymph node-positive disease at radical cystectomy for muscle-invasive bladder cancer is clinically not applicable.

PLoS One 2017 20;12(3):e0174039. Epub 2017 Mar 20.

Department of Urology, Erasmus MC Cancer Institute, Erasmus Medical Center, Rotterdam, The Netherlands.

Background: Neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) provides a small but significant survival benefit. Nevertheless, controversies on applying NAC remain because the limited benefit must be weight against chemotherapy-related toxicity and the delay of definitive local treatment. Therefore, there is a clear clinical need for tools to guide treatment decisions on NAC in MIBC. Here, we aimed to validate a previously reported 20-gene expression signature that predicted lymph node-positive disease at radical cystectomy in clinically node-negative MIBC patients, which would be a justification for upfront chemotherapy.

Methods: We studied diagnostic transurethral resection of bladder tumors (dTURBT) of 150 MIBC patients (urothelial carcinoma) who were subsequently treated by radical cystectomy and pelvic lymph node dissection. RNA was isolated and the expression level of the 20 genes was determined on a qRT-PCR platform. Normalized Ct values were used to calculate a risk score to predict the presence of node-positive disease. The Cancer Genome Atlas (TCGA) RNA expression data was analyzed to subsequently validate the results.

Results: In a univariate regression analysis, none of the 20 genes significantly correlated with node-positive disease. The area under the curve of the risk score calculated by the 20-gene expression signature was 0.54 (95% Confidence Interval: 0.44-0.65) versus 0.67 for the model published by Smith et al. Node-negative patients had a significantly lower tumor grade at TURBT (p = 0.03), a lower pT stage (p<0.01) and less frequent lymphovascular invasion (13% versus 38%, p<0.01) at radical cystectomy than node-positive patients. In addition, in the TCGA data, none of the 20 genes was differentially expressed in node-negative versus node-positive patients.

Conclusions: We conclude that a 20-gene expression signature developed for nodal staging of MIBC at radical cystectomy could not be validated on a qRT-PCR platform in a large cohort of dTURBT specimens.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174039PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358850PMC
August 2017

The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes.

Epigenetics Chromatin 2016 24;9:58. Epub 2016 Dec 24.

Cell Biology, Department Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands.

Background: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence.

Results: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above.

Conclusions: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.
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http://dx.doi.org/10.1186/s13072-016-0089-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192698PMC
December 2016

Decoding the DNA Methylome of Mantle Cell Lymphoma in the Light of the Entire B Cell Lineage.

Cancer Cell 2016 Nov;30(5):806-821

Second Medical Department, University Hospital Schleswig-Holstein, Kiel 24116, Germany.

We analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11. Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome.
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http://dx.doi.org/10.1016/j.ccell.2016.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5805090PMC
November 2016

Identification of a regulatory domain controlling the Nppa-Nppb gene cluster during heart development and stress.

Development 2016 06 5;143(12):2135-46. Epub 2016 Apr 5.

Department of Anatomy, Embryology and Physiology, Academic Medical Center, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands

The paralogous genes Nppa and Nppb are organized in an evolutionarily conserved cluster and provide a valuable model for studying co-regulation and regulatory landscape organization during heart development and disease. Here, we analyzed the chromatin conformation, epigenetic status and enhancer potential of sequences of the Nppa-Nppb cluster in vivo Our data indicate that the regulatory landscape of the cluster is present within a 60-kb domain centered around Nppb Both promoters and several potential regulatory elements interact with each other in a similar manner in different tissues and developmental stages. The distribution of H3K27ac and the association of Pol2 across the locus changed during cardiac hypertrophy, revealing their potential involvement in stress-mediated gene regulation. Functional analysis of double-reporter transgenic mice revealed that Nppa and Nppb share developmental, but not stress-response, enhancers, responsible for their co-regulation. Moreover, the Nppb promoter was required, but not sufficient, for hypertrophy-induced Nppa expression. In summary, the developmental regulation and stress response of the Nppa-Nppb cluster involve the concerted action of multiple enhancers and epigenetic changes distributed across a structurally rigid regulatory domain.
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http://dx.doi.org/10.1242/dev.132019DOI Listing
June 2016

An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation.

Blood 2016 06 10;127(24):2991-3003. Epub 2016 Mar 10.

Department of Hematology.

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.
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http://dx.doi.org/10.1182/blood-2016-01-695759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043424PMC
June 2016

The lncRNA MIR31HG regulates p16(INK4A) expression to modulate senescence.

Nat Commun 2015 Apr 24;6:6967. Epub 2015 Apr 24.

Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaloes Vej 5, Copenhagen 2200, Denmark.

Oncogene-induced senescence (OIS) can occur in response to oncogenic insults and is considered an important tumour suppressor mechanism. Here we identify the lncRNA MIR31HG as upregulated in OIS and find that knockdown of MIR31HG promotes a strong p16(INK4A)-dependent senescence phenotype. Under normal conditions, MIR31HG is found in both nucleus and cytoplasm, but following B-RAF expression MIR31HG is located mainly in the cytoplasm. We show that MIR31HG interacts with both INK4A and MIR31HG genomic regions and with Polycomb group (PcG) proteins, and that MIR31HG is required for PcG-mediated repression of the INK4A locus. We further identify a functional enhancer, located between MIR31HG and INK4A, which becomes activated during OIS and interacts with the MIR31HG promoter. Data from melanoma patients show a negative correlation between MIR31HG and p16(INK4A) expression levels, suggesting a role for this transcript in cancer. Hence, our data provide a new lncRNA-mediated regulatory mechanism for the tumour suppressor p16(INK4A).
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http://dx.doi.org/10.1038/ncomms7967DOI Listing
April 2015