EGAGenomicsEllen Heitzerhttps://ega-archive.org/dacs/EGAC00001000072EGAEGA DAC EGAC00001000072EGAellen.heitzer@medunigraz.atThe analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine. cfDNA consists predominantly of nucleosome-protected DNA shed into the bloodstream by cells undergoing apoptosis. We performed whole-genome sequencing of plasma DNA and identified two discrete regions at transcription start sites (TSSs) where nucleosome occupancy results in different read depth coverage patterns for expressed and silent genes. By employing machine learning for gene classification, we found that the plasma DNA read depth patterns from healthy donors reflected the expression signature of hematopoietic cells. In patients with cancer having metastatic disease, we were able to classify expressed cancer driver genes in regions with somatic copy number gains with high accuracy. We were able to determine the expressed isoform of genes with several TSSs, as confirmed by RNA-seq analysis of the matching primary tumor. Our analyses provide functional information about cells releasing their DNA into the circulation.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.<h4>Background</h4>Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively.<h4>Methods</h4>We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC).<h4>Results</h4>The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone.<h4>Conclusions</h4>The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'.<h4>Introduction</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4A(V78M) demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing.Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing.Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X.The dynamic range of circulating tumor DNA in metastatic breast cancer.Inferring expressed genes by whole-genome sequencing of plasma DNA.Mohan Sumitra S, Heitzer Ellen E, Ulz Peter P, Lafer Ingrid I, Lax Sigurd S, Auer Martina M, Pichler Martin M, Gerger Armin A, Eisner Florian F, Hoefler Gerald G, Bauernhofer Thomas T, Geigl Jochen B JB, Speicher Michael R MRHeitzer Ellen E, Ulz Peter P, Belic Jelena J, Gutschi Stefan S, Quehenberger Franz F, Fischereder Katja K, Benezeder Theresa T, Auer Martina M, Pischler Carina C, Mannweiler Sebastian S, Pichler Martin M, Eisner Florian F, Haeusler Martin M, Riethdorf Sabine S, Pantel Klaus K, Samonigg Hellmut H, Hoefler Gerald G, Augustin Herbert H, Geigl Jochen B JB, Speicher Michael R MRUlz Peter P, Thallinger Gerhard G GG, Auer Martina M, Graf Ricarda R, Kashofer Karl K, Jahn Stephan W SW, Abete Luca L, Pristauz Gunda G, Petru Edgar E, Geigl Jochen B JB, Heitzer Ellen E, Speicher Michael R MRHeidary Maryam M, Auer Martina M, Ulz Peter P, Heitzer Ellen E, Petru Edgar E, Gasch Christin C, Riethdorf Sabine S, Mauermann Oliver O, Lafer Ingrid I, Pristauz Gunda G, Lax Sigurd S, Pantel Klaus K, Geigl Jochen B JB, Speicher Michael R MRSchulz Eduard E, Klampfl Petra P, Holzapfel Stefanie S, Janecke Andreas R AR, Ulz Peter P, Renner Wilfried W, Kashofer Karl K, Nojima Satoshi S, Leitner Anita A, Zebisch Armin A, Wölfler Albert A, Hofer Sybille S, Gerger Armin A, Lax Sigurd S, Beham-Schmid Christine C, Steinke Verena V, Heitzer Ellen E, Geigl Jochen B JB, Windpassinger Christian C, Hoefler Gerald G, Speicher Michael R MR, Boland C Richard CR, Kumanogoh Atsushi A, Sill Heinz HfalseDAC CTC - Plasma-DNAData Access Committee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