Jelena Belic

Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer

Genomic alterations in metastatic prostate cancer remain incompletely characterized. Here we analyse 493 prostate cancer cases from the TCGA database and perform whole-genome plasma sequencing on 95 plasma samples derived from 43 patients with metastatic prostate cancer. From these samples, we identify established driver aberrations in a cancer-related gene in nearly all cases (97.7%), including driver gene fusions (TMPRSS2:ERG), driver focal deletions (PTEN, RYBP and SHQ1) and driver amplifications (AR and MYC). In serial plasma analyses, we observe changes in focal amplifications in 40% of cases. The mean time interval between new amplifications was 26.4 weeks (range: 5–52 weeks), suggesting that they represent rapid adaptations to selection pressure. An increase in neuron-specific enolase is accompanied by clonal pattern changes in the tumour genome, most consistent with subclonal diversification of the tumour. Our findings suggest a high plasticity of prostate cancer genomes with newly occurring focal amplifications as a driving force in progression.

Rapid Identification of Plasma DNA Samples with Increased ctDNA Levels by a Modified FAST-SeqS Approach

BACKGROUND Recent progress in the analysis of cell-free DNA fragments [cell-free circulating tumor DNA (ctDNA)] now allows monitoring of tumor genomes by noninvasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly variable allele frequencies of ctDNA. The comprehensive analysis of tumor genomes is greatly facilitated when plasma DNA has increased amounts of ctDNA. Therefore, a fast and cost-effective prescreening method to identify such plasma samples without previous knowledge about alterations in the respective tumor genome could assist in the selection of samples suitable for further extensive qualitative analysis. METHODS We adapted the recently described Fast Aneuploidy Screening Test-Sequencing System (FAST-SeqS) method, which was originally established as a simple, effective, noninvasive screening method for fetal aneuploidy from maternal blood. RESULTS We show that our modified FAST-SeqS method (mFAST-SeqS) can be used as a prescreening tool for an estimation of ctDNA percentage. With a combined evaluation of genome-wide and chromosome arm-specific z-scores from dilution series with cell line DNA and by comparisons of plasma-Seq profiles with data from mFAST-SeqS, we established a detection limit of ≥10% mutant alleles. Plasma samples with an mFAST-SeqS z-score >5 showed results that were highly concordant with those of copy number profiles obtained from our previously described plasma-Seq approach. CONCLUSIONS Advantages of this approach include the speed and cost-effectiveness of the assay and that no prior knowledge about the genetic composition of tumor samples is necessary to identify plasma DNA samples with >10% ctDNA content.

Genomic alterations in plasma DNA from patients with metastasized prostate cancer receiving abiraterone or enzalutamide: Prostate cancer and plasma DNA analyses

In patients with metastatic castrate‐resistant prostate cancer (mCRPC), circulating tumor DNA (ctDNA) analysis offers novel opportunities for the development of non‐invasive biomarkers informative of treatment response with novel agents targeting the androgen‐receptor (AR) pathway, such as abiraterone or enzalutamide. However, the relationship between ctDNA abundance, detectable somatic genomic alterations and clinical progression of mCRPC remains unexplored. Our study aimed to investigate changes in plasma DNA during disease progression and their associations with clinical variables in mCRPC patients. We analyzed ctDNA in two cohorts including 94 plasma samples from 25 treatment courses (23 patients) and 334 plasma samples from 125 patients, respectively. We conducted whole‐genome sequencing (plasma‐Seq) for genome‐wide profiling of somatic copy number alterations and targeted sequencing of 31 prostate cancer‐associated genes. The combination of plasma‐Seq with targeted AR analyses identified prostate cancer‐related genomic alterations in 16 of 25 (64%) treatment courses in the first cohort, in which we demonstrated that AR amplification does not always correlate with poor abiraterone and enzalutamide therapy outcome. As we observed a wide variability of ctDNA levels, we evaluated ctDNA levels and their association with clinical parameters and included the second, larger cohort for these analyses. Employing altogether 428 longitudinal plasma samples from 148 patients, we identified the presence of bone metastases, increased lactate dehydrogenase and prostate‐specific antigen (PSA) as having the strongest association with high ctDNA levels. In summary, ctDNA alterations are observable in the majority of patients with mCRPC and may eventually be useful to guide clinical decision‐making in this setting.

mFast-SeqS as a Monitoring and Pre-screening Tool for Tumor-Specific Aneuploidy in Plasma DNA

Recent progress in the analysis of cell-free DNA fragments (cell-free circulating tumor DNA, ctDNA) now allows monitoring of tumor genomes by non-invasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly variable allele frequencies of ctDNA. Comprehensive genome-wide analysis of tumor genomes is greatly facilitated when plasma DNA has increased amounts of ctDNA. In order to develop a fast and cost-effective pre-screening method for the identification of plasma samples suitable for further extensive qualitative analysis, we adapted the recently described FAST-SeqS method. We show that our modified FAST-SeqS method (mFAST-SeqS) can be used as a pre-screening tool for an estimation of the ctDNA percentage. Moreover, since the genome-wide mFAST-SeqS z-scores correlate with the actual tumor content in plasma samples, changes in ctDNA levels associated with response to treatment can be easily monitored without prior knowledge of the genetic composition of tumor samples.

Potentials, challenges and limitations of a molecular characterization of circulating tumor DNA for the management of cancer patients

Abstract: A liquid profiling, i.e. the analysis of cell-free circulating tumor DNA (ctDNA), enables a continuous non-invasive monitoring of tumor-specific changes during the entire course of the disease with respect to early detection, identification of minimal residual disease, assessment of treatment response and monitoring tumor evolution. Technological improvements, advances in understanding the nature of ctDNA, the implementation of ctDNA analyses in clinical trials as well as efforts for the establishment of benchmarks, will bring an actual widespread clinic use within reach in the near future. However, despite this progress there are still hurdles that have to be overcome, which are discussed in this review. Moreover, present knowledge and new findings about the biology of ctDNA as well as selected potential clinical applications for metastatic cancer patients are pointed out.

Abstract PR05: Analysis of ctDNA using the mFastSeqS and plasma–Seq methods for screening and therapy monitoring in prostate cancer patients

Background: Prostate cancer is the most common malignancy in males. Prostate cancer progression can be inhibited by androgen-deprivation therapy, but nearly all patients eventually develop castration-resistance prostate cancer (CRPC). In the management of prostate cancer treatment, there are still unsolved questions as to how patients can best be matched to targeted therapies in accordance with the characteristics of their tumor genome. Recent progress in the analysis of cell-free circulating tumor DNA (ctDNA) now allows for the monitoring of tumor genomes by non-invasive means. Therefore, there is an unmet need to develop methods which will allow cost-effective pre-screening and therapy monitoring. Methods: We analyzed a total of 71 metastatic prostate cancer patients with our recently published plasma-Seq method in order to establish genome-wide copy number aberrations (CNAs). Furthermore, we used a targeted resequencing approach to screen for mutations in a set of 58 cancer-associated genes and for the presence of TMPRSS-ERG fusions. In order to assess the amount of ctDNA in plasma, we used a modified FAST-Seq (mFAST-Seq) approach in a subset of samples. Follow-up samples were available from a total of 30 patients, which enabled us to monitor the constantly changing tumor genomes. Results: Using plasma-Seq, we observed a variety of copy number changes characteristic of prostate cancer, i.e. 8q gains as well as losses at 3p, 8p, 10q, 13q, 17p and 21q in 67% of patients. AR gene amplifications were found in 70% of metastatic castration-resistant patients. Furthermore, when analyzing follow-up samples, we observed the occurrence of novel copy number aberrations and clonal shifts in one-third of the patients. In one extreme case we observed the loss of an AR focal amplification, which was accompanied by shifts in mutant allele frequencies for TP53 and EP300 mutations from 76.9% to 94.8% and from 14.4% to undetectable levels, respectively, after switching to cytotoxic chemotherapy. In a subset of samples (n=61), we performed mFAST-SeqS in order to estimate the ctDNA content and to identify samples with a ctDNA content of 10% or more. For samples that did not show any CNAs after plasma-Seq, a low amount of ctDNA ( 10%), we observed highly consistent results (r=0.667) for plasma-Seq and mFastSeqS copy number profiles. Conclusion: Plasma-Seq serves as a non-invasive method for monitoring a patient9s response to therapy and may identify the occurrence of novel changes associated with resistance to a given therapy. The mFastSeqS approach has the advantage that no prior knowledge about the genetic composition of tumor samples is needed for the identification of plasma DNA samples with more than 10% of ctDNA content. Plasma DNA analyses may evolve to become a novel tool for the monitoring of patients with cancer and for the development of personalized medicine. This abstract is also presented as Poster 28. Citation Format: Jelena Belic, Ellen Heitzer, Peter Ulz, Martina Auer, Katja Fischereder, Thomas Bauernhofer, Jochen B. Geigl, Michael R. Speicher. Analysis of ctDNA using the mFastSeqS and plasma–Seq methods for screening and therapy monitoring in prostate cancer patients. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr PR05.

Abstract PR02: Somatic CRISPR/Cas9-mediated tumor suppressor disruption enables versatile brain tumor modeling

Modeling cancer in mice through engineering of candidate genes in the germline has long been the gold standard for the validation of putative oncogenes or tumor suppressor genes (TSGs). For TSGs, whereby loss-of-function (LOF) mutations act as a driver for malignant transformation, this has traditionally been accomplished using constitutive or cell type-specific knockout strategies mediated by homologous recombination in embryonic stem cells. While this allows evaluation of cell type-specific susceptibility to malignant transformation, generation of genetically engineered mouse models (GEMMs) is a time consuming process. For in vivo investigation of a large number of molecular alterations, such as the many new candidates currently emerging from large-scale tumor genome sequencing efforts, a faster and more flexible method is required. We therefore sought to adapt the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-guided endonuclease technique for the somatic disruption of candidate TSGs and thereby complement aforementioned already existing models like GEMM. To provide a flexible and effective method for investigating somatic LOF alterations and their influence on tumorigenesis in vivo, we established in vivo somatic gene transfer of CRISPR/Cas9-encoding vectors using polyethylenimine or in utero electroporation, respectively, allowing for in vivo targeting of TSGs in the developing murine brain. We demonstrate the utility of this approach by deleting the tumor suppressor Ptch1, which resulted in the development of cerebellar tumors resembling sonic hedgehog (SHH) subgroup medulloblastoma both at the histopathological as well as the molecular level. Furthermore, we show that multiple genes can be disrupted with this approach, using in utero electroporation of guide RNAs (gRNAs) targeting Trp53, Pten and Nf1 into the forebrain of mice. This resulted in induction of glioblastoma with 100% penetrance. Using whole genome sequencing (WGS) we characterized the MB-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumors. Taken together, these approaches provide a fast and convenient way for validating the emerging wealth of novel candidate TSGs and the generation of faithful animal models of human cancer. Citation Format: Marc Zuckermann, Volker Hovestadt, Christiane B. Knobbe-Thomsen, Marc Zapatka, Paul A. Northcott, Kathrin Schramm, Jelena Belic, David T.W. Jones, Barbara Tschida, Branden Moriarity, David Largaespada, Martine F. Roussel, Andrey Korshunov, Guido Reifenberger, Stefan M. Pfister, Peter Lichter, Daisuke Kawauchi, Jan Gronych. Somatic CRISPR/Cas9-mediated tumor suppressor disruption enables versatile brain tumor modeling. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr PR02.