Genomic alterations in plasma DNA from patients with metastasized prostate cancer receiving abiraterone or enzalutamide.

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.

Patient monitoring through liquid biopsies using circulating tumor DNA.

Tumors release components such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and tumor-derived extracellular vesicles into the circulation. Multiple studies have demonstrated that molecular information about tumors and metastases can be extracted from these factors, which are therefore frequently referred to as "liquid biopsies." Liquid biopsies allow the longitudinal monitoring of tumor genomes non-invasively and may hence ensure that patients receive appropriate treatments that target the molecular features of their disease. Accordingly, the number of studies employing liquid biopsy based assays has been skyrocketing in the last few years. Here, we focus on three important issues, which are of high relevance for monitoring tumor genomes. First, we analyze the relation between the allele frequency of somatic tumor-specific mutations and the tumor fraction within plasma DNA. Second, we ask how well current tumor evolution models correlate with findings in longitudinal liquid biopsy studies. And, finally, as sensitivity is one of the key challenges of mutation detection, we address the challenge of detecting mutations occurring at very low allele frequencies in plasma DNA.

Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing.

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

Systemic spread is an early step in breast cancer.

It is widely accepted that metastasis is a late event in cancer progression. Here, however, we show that tumor cells can disseminate systemically from earliest epithelial alterations in HER-2 and PyMT transgenic mice and from ductal carcinoma in situ in women. Wild-type mice transplanted with single premalignant HER-2 transgenic glands displayed disseminated tumor cells and micrometastasis in bone marrow and lungs. The number of disseminated cancer cells and their karyotypic abnormalities were similar for small and large tumors in patients and mouse models. When activated by bone marrow transplantation into wild-type recipients, 80 early-disseminated cancer cells sufficed to induce lethal carcinosis. Therefore, release from dormancy of early-disseminated cancer cells may frequently account for metachronous metastasis.

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.

Circulating tumor DNA as a liquid biopsy for cancer.

BACKGROUND: 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. CONTENT: 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. SUMMARY: 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.

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.

The dynamic range of circulating tumor DNA in metastatic breast cancer.

INTRODUCTION: The management of metastatic breast cancer needs improvement. As clinical evaluation is not very accurate in determining the progression of disease, the analysis of circulating tumor DNA (ctDNA) has evolved to a promising noninvasive marker of disease evolution. Indeed, ctDNA was reported to represent a highly sensitive biomarker of metastatic cancer disease directly reflecting tumor burden and dynamics. However, at present little is known about the dynamic range of ctDNA in patients with metastatic breast cancer. METHODS: In this study, 74 plasma DNA samples from 58 patients with metastasized breast cancer were analyzed with a microfluidic device to determine the plasma DNA size distribution and copy number changes in the plasma were identified by whole-genome sequencing (plasma-Seq). Furthermore, in an index patient we conducted whole-genome, exome, or targeted deep sequencing of the primary tumor, metastases, and circulating tumor cells (CTCs). Deep sequencing was done to accurately determine the allele fraction (AFs) of mutated DNA fragments. RESULTS: Although all patients had metastatic disease, plasma analyses demonstrated highly variable AFs of mutant fragments. We analyzed an index patient with more than 100,000 CTCs in detail. We first conducted whole-genome, exome, or targeted deep sequencing of four different regions from the primary tumor and three metastatic lymph node regions, which enabled us to establish the phylogenetic relationships of these lesions, which were consistent with a genetically homogeneous cancer. Subsequent analyses of 551 CTCs confirmed the genetically homogeneous cancer in three serial blood analyses. However, the AFs of ctDNA were only 2% to 3% in each analysis, neither reflecting the tumor burden nor the dynamics of this progressive disease. These results together with high-resolution plasma DNA fragment sizing suggested that differences in phagocytosis and DNA degradation mechanisms likely explain the variable occurrence of mutated DNA fragments in the blood of patients with cancer. CONCLUSIONS: The dynamic range of ctDNA varies substantially in patients with metastatic breast cancer. This has important implications for the use of ctDNA as a predictive and prognostic biomarker.

Establishment of tumor-specific copy number alterations from plasma DNA of patients with cancer.

With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor-specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation-detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome-wide tumor-specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring.

Multiplex genetic cancer testing identifies pathogenic mutations in TP53 and CDH1 in a patient with bilateral breast and endometrial adenocarcinoma.

BACKGROUND: Germline genetic testing for familial cancer syndromes is usually performed serially for the most likely genetic causes. In recent years the way genetic testing carried out has changed, as next generation sequencing now allows the simultaneous testing of multiple susceptibility genes at low costs. CASE PRESENTATION: Here, we present a female with bilateral breast cancer and endometrial adenocarcinoma. After simultaneous sequencing of 150 genes (890 kb) associated with hereditary cancer we identified pathogenic mutations in two high-penetrance genes, i.e. TP53 and CDH1 that would most likely not have been elucidated by serial screening of candidate genes. CONCLUSION: As the two mutated genes are located on different chromosomes and cause different cancer syndromes these findings had a tremendous impact not only on genetic counseling of the index patient and her family but also on subsequent surveillance strategies.

Genetic and epigenetic analysis of putative breast cancer stem cell models.

BACKGROUND: Cancer stem cell model hypothesizes existence of a small proportion of tumor cells capable of sustaining tumor formation, self-renewal and differentiation. In breast cancer, these cells were found to be associated with CD44⁺CD24-low and ALDH⁺ phenotype. Our study was performed to evaluate the suitability of current approaches for breast cancer stem cell analyses to evaluate heterogeneity of breast cancer cells through their extensive genetic and epigenetic characterization. METHODS: Breast cancer cell lines MCF7 and SUM159 were cultured in adherent conditions and as mammospheres. Flow cytometry sorting for CD44, CD24 and ALDH was performed. Sorted and unsorted populations, mammospheres and adherent cell cultures were subjected to DNA profiling by array CGH and methylation profiling by Epitect Methyl qPCR array. Methylation status of selected genes was further evaluated by pyrosequencing. Functional impact of methylation was evaluated by mRNA analysis for selected genes. RESULTS: Array CGH did not reveal any genomic differences. In contrast, putative breast cancer stem cells showed altered methylation levels of several genes compared to parental tumor cells. CONCLUSIONS: Our results underpin the hypothesis that epigenetic mechanisms seem to play a major role in the regulation of CSCs. However, it is also clear that more efficient methods for CSC enrichment are needed. This work underscores requirement of additional approaches to reveal heterogeneity within breast cancer.

High-resolution analyses of copy number changes in disseminated tumor cells of patients with breast cancer.

The presence of disseminated tumor cells (DTCs) in bone marrow (BM) identifies breast cancer patients with less favorable outcome. Furthermore, molecular characterization is required to investigate the malignant potential of these cells. This study presents a single-cell array comparative genomic hybridization (SCaCGH) method providing molecular analysis of immunomorphologically detected DTCs. The resolution limit of the method was estimated using the cancer cell line SK-BR-3 on 44 and 244k arrays. The technique was further tested on 28 circulating tumor cells and four hematopoietic cells (HCs) from peripheral blood (n = 8 patients). The SCaCGH method was finally applied to 24 DTCs, three immunopositive cells morphologically classified as probable HCs from breast cancer patients and five HC controls from BM (n = 7 patients plus n = 1 healthy donor). The frequency of copy number changes of the DTCs revealed similarities with primary breast tumor samples. Three of the patients had available profiles for DTCs and the corresponding tumor tissue from primary surgery. More than two-third of the analyzed DTCs disclosed equivalent changes, both to each other and to the corresponding primary disease, whereas the rest of the cells showed balanced profiles. The probable HCs revealed either balanced profiles (n = 2) or changes comparable to the tumor tissue and DTCs (n = 1), indicating morphological overlap between HCs and DTCs. Similar aberration patterns were visible in DTCs collected at diagnosis and at 3 years relapse-free follow-up. SCaCGH may be a powerful tool for the molecular characterization of DTCs.

Evolution of genomic instability in diethylnitrosamine-induced hepatocarcinogenesis in mice.

UNLABELLED: Diethylnitrosamine (DEN) is a hepatic procarcinogen which is frequently used as an inducer of hepatocellular carcinoma (HCC) in mice. Although mice after DEN exposure are among the most widely used models for liver tumorigenesis, a detailed, mechanistic characterization of the longitudinal changes in the respective tumor genomes has never been performed. Here we established the chronological order of genetic alterations during DEN carcinogenesis by examining mice at different points in time. Tumor samples were isolated by laser microdissection and subjected to array-comparative genomic hybridization (array-CGH) and sequencing analysis. Chromosomal gains and losses were observed in tumors by week 32 and increased significantly by week 56. Loss of distal chromosome 4q, including the tumor suppressors Runx3 and Nr0b2/Shp, was a frequent early event and persisted during all tumor stages. Surprisingly, sequencing revealed that ß-catenin mutations occurred late and were clearly preceded by chromosomal instability. Thus, contrary to common belief, ß-catenin mutations and activation of the Wnt/ß-catenin pathway are not involved in tumor initiation in this model of chemical hepatocarcinogenesis. CONCLUSION: Our study suggests that the majority of the current knowledge about genomic changes in HCC is based on advanced tumor lesions and that systematic analyses of the chronologic order including early lesions may reveal new, unexpected findings.

Defining 'chromosomal instability'.

Most scientists agree that the majority of human solid malignant tumors are characterized by chromosomal instability (CIN) involving gain or loss of whole chromosomes or fractions of chromosomes. CIN is thought to be an early event during tumorigenesis and might therefore be involved in tumor initiation. Despite its frequent occurrence in tumors and its potential importance in tumor evolution, CIN is poorly defined and is used inconsistently and imprecisely. Here, we provide criteria to define CIN and argue that few experimental approaches are capable of assessing the presence of CIN. Accurate assessment of CIN is crucial to elucidate whether CIN is a driving force for tumorigenesis and whether a chromosomally unstable genome is necessary for tumor progression.

Combined molecular genetic and cytogenetic analysis from single cells after isothermal whole-genome amplification.

BACKGROUND: Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS: We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS: DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS: This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays.

Clinical DNA is often available in limited quantities requiring whole-genome amplification for subsequent genome-wide assessment of copy-number variation (CNV) by array-CGH. In pre-implantation diagnosis and analysis of micrometastases, even merely single cells are available for analysis. However, procedures allowing high-resolution analyses of CNVs from single cells well below resolution limits of conventional cytogenetics are lacking. Here, we applied amplification products of single cells and of cell pools (5 or 10 cells) from patients with developmental delay, cancer cell lines and polar bodies to various oligo tiling array platforms with a median probe spacing as high as 65 bp. Our high-resolution analyses reveal that the low amounts of template DNA do not result in a completely unbiased whole genome amplification but that stochastic amplification artifacts, which become more obvious on array platforms with tiling path resolution, cause significant noise. We implemented a new evaluation algorithm specifically for the identification of small gains and losses in such very noisy ratio profiles. Our data suggest that when assessed with sufficiently sensitive methods high-resolution oligo-arrays allow a reliable identification of CNVs as small as 500 kb in cell pools (5 or 10 cells), and of 2.6-3.0 Mb in single cells.

Effect of genome-wide association studies, direct-to-consumer genetic testing, and high-speed sequencing technologies on predictive genetic counselling for cancer risk.

Genetic counselling is offered to patients with various hereditary cancers. At-risk family members can be identified by predictive testing and included in specifically designed screening and prevention programmes. Since genetic testing has far-reaching ethical and medical outcomes, it is usually done according to well-defined guidelines developed by medical societies, entailing extensive interaction between family members and medical health providers. This established procedure is now changing after three new developments. First, data from genome-wide association studies have identified many new loci in the human genome, which could moderately change cancer risk. Second, although inclusion of low-risk cancer genes in patients' risk assessments represents an unresolved challenge, several companies already offer such tests in a direct-to-consumer format over the internet. These tests shift control of genetic testing from medical professionals to individuals. Third, development of high-speed sequencing technologies multiplies available genomic data and enables rapid sequencing of entire human genomes at low costs. This innovation will pave the way for personalised genetics and new options for predictive testing. Here, we use the example of genetic counselling in colon cancer to describe how these developments will change genetic testing in families at risk of cancer and in the general population.