Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue ...origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.
Abstract
The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for ...point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy.
Graphical Abstract
Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from ...fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.
The homologous recombination repair (HRR) pathway repairs DNA double-strand breaks in an error-free manner. Mutations in HRR genes can result in increased mutation rate and genomic rearrangements, ...and are associated with numerous genetic disorders and cancer. Despite intensive research, the HRR pathway is not yet fully mapped. Phylogenetic profiling analysis, which detects functional linkage between genes using coevolution, is a powerful approach to identify factors in many pathways. Nevertheless, phylogenetic profiling has limited predictive power when analyzing pathways with complex evolutionary dynamics such as the HRR. To map novel HRR genes systematically, we developed clade phylogenetic profiling (CladePP). CladePP detects local coevolution across hundreds of genomes and points to the evolutionary scale (e.g., mammals, vertebrates, animals, plants) at which coevolution occurred. We found that multiscale coevolution analysis is significantly more biologically relevant and sensitive to detect gene function. By using CladePP, we identified dozens of unrecognized genes that coevolved with the HRR pathway, either globally across all eukaryotes or locally in different clades. We validated eight genes in functional biological assays to have a role in DNA repair at both the cellular and organismal levels. These genes are expected to play a role in the HRR pathway and might lead to a better understanding of missing heredity in HRR-associated cancers (e.g., heredity breast and ovarian cancer). Our platform presents an innovative approach to predict gene function, identify novel factors related to different diseases and pathways, and characterize gene evolution.
Inequalities among the western population, combined with the introduction of new treatment options for cancer, have challenged endeavors to provide equal care to patients with cancer. Israel's highly ...developed healthcare system and mandatory National Health Insurance afforded an opportunity to study geographic variation over time in mortality following cancer diagnosis.
This historical prospective cohort study included a nationally representative cohort that was assessed by the Israeli Central Bureau of Statistics 1995 census and followed until 2011. The cancer incidence (1995-2009) was ascertained by the Israel National Cancer Registry. We analyzed the effect on patient outcome of living in a given district, according to the Israeli Central Bureau of Statistics classification. Patients were stratified by the year of diagnosis (1995-1997, 1998-2000, etc.), and associations were adjusted for age, ethnicity, and districts. We excluded patients with malignancies associated with screening program (breast, prostate, colon, and cervical cancers).
This study included 26,173 patients living in 13 residential districts. During the last years (2007-2009) of the study, the hazard ratio (HR) for risk of death was high in 8/13 districts (61.5%), compared to 4/13 (30.7%) during 2004-2006, and 0/13 (0%) during 2001-2003. Districts that were less likely to be associated with increased risk of death were located in the center of Israel and in metropolitan areas, compared to the peripheral regions. Furthermore, HRs were substantially higher in the last years of the study (2007-2009, HRs rose to 1.69, 95%CI: 1.38-2.08) compared to the earlier years (2004-2006, HRs rose to 1.35, 95%CI: 1.13-1.62).
Our findings suggested that geographic variation for mortality following cancer diagnosis have increased over time. Our results provide policy makers with vital information regarding the need for targeted interventions, mainly in peripheral regions.
Radiotherapy has an important role in the treatment of brain metastases but carries risk of short and/or long-term toxicity, termed radiation-induced brain injury (RBI). As the diagnosis of RBI is ...crucial for correct patient management, there is an unmet need for reliable biomarkers for RBI. The aim of this proof-of concept study is to determine the utility of brain-derived circulating free DNA (BncfDNA), identified by specific methylation patterns for neurons, astrocytes, and oligodendrocytes, as biomarkers brain injury induced by radiotherapy. Twenty-four patients with brain metastases were monitored clinically and radiologically before, during and after brain radiotherapy, and blood for BncfDNA analysis (98 samples) was concurrently collected. Sixteen patients were treated with whole brain radiotherapy and eight patients with stereotactic radiosurgery. During follow-up nine RBI events were detected, and all correlated with significant increase in BncfDNA levels compared to baseline. Additionally, resolution of RBI correlated with a decrease in BncfDNA. Changes in BncfDNA were independent of tumor response. Elevated BncfDNA levels reflects brain cell injury incurred by radiotherapy. further research is needed to establish BncfDNA as a novel plasma-based biomarker for brain injury induced by radiotherapy.
During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each ...pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.
The oncogenic role and clinical relevance of BRCA mutations in NSCLC remain unclear. We aim to evaluate the characteristics and clinical outcomes of patients with NSCLC harboring BRCA mutations ...treated at Hadassah Medical Center (HMC). We retrospectively assessed all patients with advanced NSCLC who underwent next-generation sequencing (NGS) and were found to have pathogenic somatic BRCA mutations (p-BRCA). We compared clinical outcomes in NSCLC patients with wild-type BRCA (wt-BRCA) matched by age, stage, gender, smoking, PDL-1 and driver mutations. Between 2015 and 2022, we evaluated 598 patients with advanced NSCLC using NGS and found 26 patients with p-BRCA, of whom 17 (65.4%) were carriers of germline BRCA variants and represented 1% of all BRCA carriers HMC. The median age of diagnosis was 67 years old (40–78), 13 patients (50%) had a history of smoking and 9 patients (34.6%) had additional driver mutations (EGFR, ALK, BRAF, MET or ERBB2). Objective response rate and median progression-free survival (PFS) for first-line platinum-based chemotherapy in the p-BRCA group compared to wt-BRCA controls were 72.2% and 16 months (CI 95%, 5–22), compared to 47.4% and 7 months (CI 95%, 5–9), respectively, and HR for PFS was 0.41 (CI 95%, 0.17–0.97). Six patients in the p-BRCA group were treated with advanced-line poly (adenosine-phosphate-ribose) polymerase inhibitors (PARPi), with a durable response observed in four patients (66%). In this cohort, patients with NSCLC harboring p-BRCA exhibit high-sensitivity PARPi and a prolonged response to platinum, suggesting some oncogenic role for BRCA mutations in NSCLC. The results support further prospective trials of the treatment of NSCLC harboring p-BRCA with PARPi.
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