Targeted therapies have markedly changed the treatment of cancer over the past 10 years. However, almost all tumors acquire resistance to systemic treatment as a result of tumor heterogeneity, clonal ...evolution, and selection. Although genotyping is the most currently used method for categorizing tumors for clinical decisions, tumor tissues provide only a snapshot, or are often difficult to obtain. To overcome these issues, methods are needed for a rapid, cost-effective, and noninvasive identification of biomarkers at various time points during the course of disease. Because cell-free circulating tumor DNA (ctDNA) is a potential surrogate for the entire tumor genome, the use of ctDNA as a liquid biopsy may help to obtain the genetic follow-up data that are urgently needed.
This review includes recent studies exploring the diagnostic, prognostic, and predictive potential of ctDNA as a liquid biopsy in cancer. In addition, it covers biological and technical aspects, including recent advances in the analytical sensitivity and accuracy of DNA analysis as well as hurdles that have to be overcome before implementation into clinical routine.
Although the analysis of ctDNA is a promising area, and despite all efforts to develop suitable tools for a comprehensive analysis of tumor genomes from plasma DNA, the liquid biopsy is not yet routinely used as a clinical application. Harmonization of preanalytical and analytical procedures is needed to provide clinical standards to validate the liquid biopsy as a clinical biomarker in well-designed and sufficiently powered multicenter studies.
Cell-free DNA (cfDNA) is evolving into a widely used prognostic and predictive biomarker, particularly in oncology. However, its versatile clinical use precedes a profound understanding of the ...underlying biology of cfDNA release. There is much evidence to suggest that cfDNA is mainly derived from dying (i.e., apoptotic) cells. However, numerous cancer studies have shown that cfDNA is informative about acquired resistance to given therapies, which is present in living, proliferating tumor subclones. To explain this contradiction, we review current insights regarding cfDNA release, in particular the interplay between apoptosis and proliferation. We describe how improved knowledge about cfDNA biology could be used for novel therapeutic strategies and how this may affect patient management.
Cells release DNA into the circulation, which is referred to as ‘cell-free DNA’ (cfDNA). In healthy individuals, cfDNA is mainly derived from hematopoietic cells.In patients with cancer, tumor cells release their DNA in addition, which is then present in different proportions as ‘circulating tumor DNA’ (ctDNA) within cfDNA.Recently an arsenal of sophisticated methods to extract a multitude of information from cfDNA and/or ctDNA has been developed, which can be used for various clinical purposes, to assess distinct physiologic conditions, or as a tool for basic research.The release of cfDNA is thought to be tightly linked to programmed cell death or apoptosis; however, at present the underlying biology of cfDNA release remains largely unknown.Leveraging the full potential of cfDNA as a biomarker will require improved knowledge of all mechanisms and factors involved in cfDNA release.
New non-invasive approaches that can complement and improve on current strategies for colorectal cancer (CRC) screening and management are urgently needed. A growing number of publications have ...documented that components of tumors, which are shed into the circulation, can be detected in the form of liquid biopsies and can be used to detect CRC at early stages, to predict response to certain therapies and to detect CRC recurrence in a minimally invasive way. The analysis of circulating tumor DNA (ctDNA), tumor-derived cells (CTC, circulating tumor cells) or circulating microRNA (miRNA) in blood and other body fluids, have a great potential to improve different aspects of CRC management. The challenge now is to find which types of components, biofluids and detection methods would be the most suitable to be applied in the different steps of CRC detection and treatment. This chapter will provide an up to date review on ctDNA, CTCs and circulating miRNAs as new biomarkers for CRC, either for clinical management or early detection, highlighting their advantages and limitations.
We constructed a panel of S. pombe strains expressing DNA polymerase ε variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES459F, and used these to compare mutation ...rates determined by canavanine resistance with other selective methods. Canavanine-resistance mutation rates are broadly similar to those seen with reversion of the ade-485 mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of ura4+ or ura5+ genes). Inactivation of several genes has been associated with canavanine resistance in S. pombe but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an R175C mutation in the any1/arn1 gene. This gene encodes an α-arrestin-like protein involved in mediating Pub1 ubiquitylation of target proteins, and the phenotypic resistance to canavanine by this single mutation is similar to that shown by the original "can1-1" strain, which also has the any1R175C mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The any1R175C strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first ...comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse.
Deregulation of transcription factors (TFs) is an important driver of tumorigenesis, but non-invasive assays for assessing transcription factor activity are lacking. Here we develop and validate a ...minimally invasive method for assessing TF activity based on cell-free DNA sequencing and nucleosome footprint analysis. We analyze whole genome sequencing data for >1,000 cell-free DNA samples from cancer patients and healthy controls using a bioinformatics pipeline developed by us that infers accessibility of TF binding sites from cell-free DNA fragmentation patterns. We observe patient-specific as well as tumor-specific patterns, including accurate prediction of tumor subtypes in prostate cancer, with important clinical implications for the management of patients. Furthermore, we show that cell-free DNA TF profiling is capable of detection of early-stage colorectal carcinomas. Our approach for mapping tumor-specific transcription factor binding in vivo based on blood samples makes a key part of the noncoding genome amenable to clinical analysis.
Precision medicine refers to the choosing of targeted therapies based on genetic data. Due to the increasing availability of data from large-scale tumor genome sequencing projects, genome-driven ...oncology may have enormous potential to change the clinical management of patients with cancer. To this end, components of tumors, which are shed into the circulation, i.e., circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or extracellular vesicles, are increasingly being used for monitoring tumor genomes. A growing number of publications have documented that these "liquid biopsies" are informative regarding response to given therapies, are capable of detecting relapse with lead time compared to standard measures, and reveal mechanisms of resistance. However, the majority of published studies relate to advanced tumor stages and the use of liquid biopsies for detection of very early malignant disease stages is less well documented. In early disease stages, strategies for analysis are in principle relatively similar to advanced stages. However, at these early stages, several factors pose particular difficulties and challenges, including the lower frequency and volume of aberrations, potentially confounding phenomena such as clonal expansions of non-tumorous tissues or the accumulation of cancer-associated mutations with age, and the incomplete insight into driver alterations. Here we discuss biology, technical complexities and clinical significance for early cancer detection and their impact on precision oncology.
The role of subclonal
mutations, defined by a variant allele frequency of <20%, has not been addressed in acute myeloid leukemia yet. We, therefore, analyzed their prognostic value in a cohort of ...1,537 patients with newly diagnosed disease, prospectively treated within three trials of the "German-Austrian Acute Myeloid Leukemia Study Group". Mutational analysis was performed by targeted deep sequencing and patients with
mutations were categorized by their variant allele frequency into groups with frequencies >40%, 20%-40% and <20%. A total of 108
mutations were found in 98 patients (6.4%). Among these, 61 patients had variant allele frequencies >40%, 19 had variant allele frequencies between 20%-40% and 18 had frequencies <20%. Compared to specimens with clonal
mutations, those with subclonal ones showed significantly fewer complex karyotypes and chromosomal losses. In either
-mutated group, patients experienced significantly fewer complete responses (
<0.001) and had worse overall and event-free survival rates (
<0.0001). In Cox regression analyses adjusting for age, white blood cell count, cytogenetic risk and type of acute myeloid leukemia, the adverse prognostic effect of
mutations remained significant for all
-mutated subgroups. These data suggest that subclonal
mutations are a novel prognostic parameter in acute myeloid leukemia and emphasize the usefulness of next-generation sequencing technologies for risk stratification in this disorder. The study was registered at ClinicalTrials.gov with number NCT00146120.
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.
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