Genomische analyses van hoog-risico prostaatkanker

PCa is the second most frequently diagnosed cancer in males worldwide. A wide range of genomic alterations, including point mutations, copy number changes and rearrangements, can lead to the development of cancer. Due to the heterogeneity of PCa, it still remains a clinical challenge to differentiate indolent from very aggressive tumors. A better molecular profiling of the primary tumors should enable a better classification of the disease, ultimately providing information that could direct a more personalized treatment. One approach is to study the contribution of somatic base pair substitutions to the oncogenic process.Cancer cell lines are commonly used as laboratory resources to study basic molecular and cellular biology. For prostate cancer (PCa), LNCaPs are the most commonly used cells. However, information on protein-changing mutations, genetic heterogeneity and genetic (in)stability is largely lacking for these cells. In a first part, we used exome-sequencing to focus on missense and nonsense single nucleotide variants and short insertions and deletions. We detected 1802 non-synonymous point mutations and 218 small insertions and deletions. While most detected mutations were undescribed so far, we confirmed the known mutations in the androgen receptor and the PTEN gene. Surprisingly, we confirmed 38 out of 42 mutations in DNA and RNA from different monoclonal and polyclonal LNCaP derivatives. From this, we deduced that LNCaP cells are heterozygous for a large number of variants and that both the variant and the wild type allele can be simultaneously expressed as mRNA. The fact that mutations in the E-cadherin, CDK4, Notch1 and PlexinB1 genes are absent in some subclones, strongly indicates a degree of genetic instability. Finally, to help identify the mutations that are most likely drivers of the oncogenic process, we developed an in silico protocol, which can be adopted for other exome analyses. We provided an extensive database of genetic variations in the exome of LNCaP cells, and these should be taken into consideration when using LNCaPs as a model for PCa. The progression of PCa from androgen-dependent to androgen-independent poses an important clinical question, as the mechanisms leading to metastatic PCa are not well understood. C4-2B cells are derivatives of LNCaP cells, as they were derived from a bone metastasis that grew in nude mice after inoculation with the LNCaP-derived, castration-resistant C4-2 cells. The combination of LNCaP and C4-2B cells thus forms an excellent preclinical model to study the development of metastatic castration-resistant PCa. Because of the importance of this progression model, a second part of this study characterized both cell lines more thoroughly using exome and transcriptome sequencing to obtain point mutations and differential expression patterns. Exome sequencing detected 2188 and 3840 mutations in LNCaP and C4-2B cells respectively, of which 1784 were found in both cell lines. The use of more recent algorithms resulted in a higher sensitivity to detect point mutations, increasing the number of mutations detected in LNCaP from 1802 to 2188. Surprisingly, the parental LNCaP cells contained more than 400 mutations that were not found in the C4-2B exome. Moreover, more than half of the mutations found in the exomes of both cell lines were confirmed by analyzing the transcriptome sequencing data. The transcriptome data also revealed that 457 genes show increased expression and 246 genes show decreased expression in C4-2B cells as compared to LNCaP cells. Based on the list of C4-2B-specific point mutations and the list of differentially expressed genes, we detected changes in the focal adhesion and ECM-receptor interaction pathways which converged on the myosin light chain kinase gene. Whether this contributes to the metastatic potential of C4-2B cells remains to be investigated. To conclude, we provide lists of mutated genes and differentially expressed genes in the LNCaP and C4-2B PCa cell lines to all researchers interested in using these cells as preclinical models.A final component of this project used tissue from 27 patients with high-risk primary PCa. We performed exome sequencing and copy number profiling of 27 primary prostate tumors and their normal tissue pairs. Tumors having a PSA > 20 ng/ml, or Gleason score ≥ 8 or clinical stage ≥ T2c are known to have a high risk on disease recurrence after treatment. In addition to amplifications and deletions that were described before, we identified a novel recurrent amplification on 7p22.3. Exome sequencing revealed one hypermutated sample containing 451 mutations, compared to an average of 19 mutations in the other samples, indicating that DNA repair is compromised in this sample. This hypermutated tumor indeed harbored a mutation in the DNA-repair gene Replication Factor C. This mutation is predicted to affect the interaction with PCNA and hence the recruitment of DNA polymerase to PCNA. In a second tumor, we detected a novel point mutation in the TET1 gene. This methylcytosine dioxygenase converts 5-methylcytosine to 5-hydroxymethylcytosine, which leads to demethylation of cytosines and might lead to changes in gene expression or chromatin organization. Immunoprecipitation of methylated and hydroxymethylated DNA followed by deep-sequencing performed on the tumor sample containing the A1908S TET1 mutation demonstrated an overall hypo-hydroxymethylation and hypermethylation at specific genomic loci, when compared to two other tumor samples without mutation in TET1. This effect was corroborated by the in vitro effect of the mutation on the dioxygenase activity as assessed by dot blot assays. We further showed that there is an overlap between groups of androgen-regulated genes and the TET1-regulated genes. Moreover, in cotransfection experiments, TET1 seems to act as a coactivator of the androgen receptor. From the above data, we conclude that the A1908S TET1 mutation as detected in a primary PCa leads to partial loss of TET1 tumor suppressor activity.nrpages: 145status: publishe

Oncothesis: Genomic analysis of high-risk prostate cancer

Whole exome sequencing was performed on 38 high-risk prostate cancer (PCa) samples. We confirmed recurrent mutations in PCa-specific genes, but also identified genes not reported to be mutated, like TET1. This DNA hydroxymethylase converts methylcytosines to hydroxymethylcytosines as a first step in DNA demethylation. By immunohistochemistry, we detected decreased TET1 protein levels in tumor compared to surrounding non-tumor tissue. DNA hydroxymethylation followed the same course. Furthermore, TET1 mRNA expression levels are an independent predictor of metastasis-free survival in a larger retrospective cohort, indicating an important role for TET1 and hydroxymethylation in PCa. The LNCaP and C4-2B cell lines form an excellent preclinical model to study the development of metastatic castration-resistant PCa. Both exome and transcriptome sequencing was performed: more than half of the mutations found in the exomes were confirmed in the RNA-seq data. Combining C4-2B-specific mutations with differentially expressed genes allowed the detection of changes in focal adhesion and ECM-receptor interactions, which might contribute to the metastatic potential of C4-2B cells.status: accepte

Novel insight in mechanisms of Enzalutamide resistance in prostate cancer

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Androgen regulation of the TMPRSS2 gene and the effect of a SNP in an androgen response element

More than 50% of prostate cancers have undergone a genomic reorganization that juxtaposes the androgen-regulated promoter of TMPRSS2 and the protein coding parts of several ETS oncogenes. These gene fusions lead to prostate-specific and androgen-induced ETS expression and are associated with aggressive lesions, poor prognosis, and early-onset prostate cancer. In this study, we showed that an enhancer at 13 kb upstream of the TMPRSS2 transcription start site is crucial for the androgen regulation of the TMPRSS2 gene when tested in bacterial artificial chromosomal vectors. Within this enhancer, we identified the exact androgen receptor binding sequence. This newly identified androgen response element is situated next to two binding sites for the pioneer factor GATA2, which were identified by DNase I footprinting. Both the androgen response element and the GATA-2 binding sites are involved in the enhancer activity. Importantly, a single nucleotide polymorphism (rs8134378) within this androgen response element reduces binding and transactivation by the androgen receptor. The presence of this SNP might have implications on the expression and/or formation levels of TMPRSS2 fusions, because both have been shown to be influenced by androgens

Describing the Reportable Range Is Important for Reliable Treatment Decisions A Multiple Laboratory Study for Molecular Tumor Profiling Using Next-Generation Sequencing

Because interpretation of next-generation sequencing (NGS) data remains challenging, optimization of the NGS process is needed to obtain correct sequencing results. Therefore, extensive validation and continuous monitoring of the quality is essential. NGS performance was compared with traditional detection methods and technical quality of nine NGS technologies was assessed. First, nine formalin-fixed, paraffin-embedded patient samples were analyzed by 114 laboratories by using different detection methods. No significant differences in performance were observed between analyses with NGS and traditional techniques. Second, two DNA control samples were analyzed for a selected number of variants by 26 participants with the use of nine different NGS technologies. Quality control metrics were analyzed from raw data files and a survey about routine procedures. Results showed large differences in coverages, but observed variant allele frequencies in raw data files were in line with predefined variant allele frequencies. Many false negative results were found because of low-quality regions, which were not reported as such. It is recommended to disclose the reportable range, the fraction of targeted genomic regions for which calls of acceptable quality can be generated, to avoid any errors in therapy decisions. NGS can be a reliable technique, only if essential quality control during analysis is applied and reported.status: publishe

Establishment, Characterization, and Imaging of a First Platinum-resistant Penile Cancer Patient-derived Xenograft in Nude Mice: A eUROGEN Project

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Recurrent MALAT1-GLI1 oncogenic fusion and GLI1 upregulation define a subset of plexiform fibromyxoma

Plexiform fibromyxomas are rare neoplasms, being officially recognized as a distinct entity among benign mesenchymal gastric tumours in the 2010 WHO Classification of Tumors of the Digestive System. Characteristically, these tumours have a multinodular/plexiform growth pattern, and histologically contain variably cellular areas of bland myofibroblastic-type spindle cells embedded in an abundant myxoid matrix, rich in capillary-type vessels. As of yet, the molecular and/or genetic features of these tumours are unknown. Here, we describe a recurrent translocation t(11;12)(q11;q13) involving the long noncoding gene MALAT1 (metastasis associated lung adenocarcinoma transcript 1) and the gene GLI1 (glioma-associated oncogene homologue 1) in a subgroup of these tumours. The presence of the fusion transcript in our index case was confirmed using polymerase chain reaction on genomic DNA followed by Sanger sequencing. We showed that the truncated GLI1 protein is overexpressed and retains its capacity to transcriptionally activate its target genes. A specific FISH assay was developed to detect the novel MALAT1-GLI1 translocation in formalin-fixed paraffin-embedded material. This resulted in the identification of two additional cases with this fusion, and two cases with polysomy of the GLI1 gene. Finally, immunohistochemistry revealed that the GLI1 protein is exclusively overexpressed in those cases that harbour GLI1/12q13 genomic alterations. In conclusion, overexpression of GLI1 through a recurrent MALAT1-GLI1 translocation or GLI1 upregulation delineates a pathogenically distinct subgroup of plexiform fibromyxomas with activation of the Sonic Hedgehog signalling pathway.status: publishe