The 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.

To our knowledge, L1CAM has been shown to be the best-ever published prognostic factor in FIGO stage I, type I endometrial cancers and shows clear superiority over the standardly used multifactor risk score. L1CAM expression in type I cancers indicates the need for adjuvant treatment. This adhesion molecule might serve as a treatment target for the fully humanized anti-L1CAM antibody currently under development for clinical use.

Mechanical interaction between cells – specifically distortion of tensional homeostasis-emerged as an important aspect of breast cancer genesis and progression. We investigated the biophysical characteristics of mechanosensitive ion channels (MSCs) in the malignant MCF-7 breast cancer cell line. MSCs turned out to be the most abundant ion channel species and could be activated by negative pressure at the outer side of the cell membrane in a saturable manner. Assessing single channel conductance (GΛ) for different monovalent cations revealed an increase in the succession: Li+ < Na+ < K+ ≈Rb+ ≈ Cs+. Divalent cations permeated also with the order: Ca2+ < Ba2+. Comparison of biophysical properties enabled us to identify MSCs in MCF-7 as ion channels formed by the Piezo1 protein. Using patch clamp technique no functional MSCs were observed in the benign MCF-10A mammary epithelial cell line. Blocking of MSCs by GsMTx-4 resulted in decreased motility of MCF-7, but not of MCF-10A cells, underscoring a possible role of Piezo1 in invasion and metastatic propagation. The role of Piezo1 in biology and progression of breast cancer is further substantiated by markedly reduced overall survival in patients with increased Piezo1 mRNA levels in the primary tumor.

Digital pathology is on the verge of becoming a mainstream option for routine diagnostics. Faster whole slide image scanning has paved the way for this development, but implementation on a large scale is challenging on technical, logistical, and financial levels. Comparative studies have published reassuring data on safety and feasibility, but implementation experiences highlight the need for training and the knowledge of pitfalls. Up to half of the pathologists are reluctant to sign out reports on only digital slides and are concerned about reporting without the tool that has represented their profession since its beginning. Guidelines by international pathology organizations aim to safeguard histology in the digital realm, from image acquisition over the setup of work-stations to long-term image archiving, but must be considered a starting point only. Cost-efficiency analyses and occupational health issues need to be addressed comprehensively. Image analysis is blended into the traditional work-flow, and the approval of artificial intelligence for routine diagnostics starts to challenge human evaluation as the gold standard. Here we discuss experiences from past digital pathology implementations, future possibilities through the addition of artificial intelligence, technical and occupational health challenges, and possible changes to the pathologist’s profession.

Cell-free DNA (cfDNA) from plasma is an intensively investigated biomarker. In the field of oncology, numerous publications have demonstrated that analyses of cancer cell-derived DNA in the circulation, referred to as circulating tumor DNA (ctDNA), can be used to track tumor dynamics in real time [1][2][3][4][5] . cfDNA fragments have been associated with the release of DNA from apoptotic cells after enzymatic processing since the distribution of their lengths has a mode near 166 bp in most analyses, a size which corresponds approximately to the DNA wrapped around a nucleosome (~147 bp) plus a linker fragment (~20 bp) [6][7][8] . Indeed, evidence that cfDNA reflects nucleosome footprints was recently reported 9 .Importantly, micrococcal nuclease (MNase) assays, in which MNase digestion is used to produce mononucleosome-bound DNA fragments to define nucleosome positions in genomes, have revealed specific nucleosome patterns at promoters, which profoundly influence gene regulation [10][11][12][13] . In actively transcribed genes, the promoter region, i.e. the region of about 150 bp upstream . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/049478 doi: bioRxiv preprint first posted online Apr. 20, 2016; 3 of the transcriptional start site (TSS), is a nucleosome-depleted region (NDR) that facilitates access to the bulky transcriptional machinery, which is flanked by arrays of well-positioned nucleosomes [11][12][13] . Furthermore, a reduction in nucleosome occupancy was found which extended up to 1 kb into the gene body, resulting in reduced frequencies of mapping reads 11,13 . Inactive promoters, by contrast, exhibited neither a pronounced depletion nor strong positioning and phasing of nucleosomes 13 .Our investigation was hence threefold: to determine whether plasma DNA is able to reflect such expression-specific nucleosome occupancy at promoters, to assess if plasma DNA possesses the sensitivity and accuracy to predict whether genes are expressed or not, and furthermore, to determine if blood samples from patients with cancer are informative for expressed cancer driver genes. To this end, we conducted WGS of plasma DNA from 50 male and 54 female donors, 179paired-end sequenced plasma samples and 2 patients with metastasized breast cancer and we generated altogether over ~414 Gbp of raw sequencing data and ~2.6 billion mapped plasma sequence reads. We then analyzed 426 additional plasma samples from cancer patients for their suitability for nucleosome promoter analysis.Analyses of 179 paired-end sequenced plasma DNA samples confirmed the expected unimodal size distribution of plasma nuclear DNA fragments with a narrow range and mode at 166 bp, which differed from plasma mitochondrial DNA, in which higher-order nucleosome packaging is absent, thus leaving it more exposed to enzymatic cleavage 14 (Fig. 1a)...