Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which ...cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal ‘surfaceome’ in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling.
Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography–mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis.
Proteomic analysis of the exosome ‘surfaceome’ revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation.
Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.
The ability to perform comprehensive profiling of cancers at high resolution is essential for precision medicine. Liquid biopsies using shed exosomes provide high-quality nucleic acids to obtain ...molecular characterization, which may be especially useful for visceral cancers that are not amenable to routine biopsies.
We isolated shed exosomes in biofluids from three patients with pancreaticobiliary cancers (two pancreatic, one ampullary). We performed comprehensive profiling of exoDNA and exoRNA by whole genome, exome and transcriptome sequencing using the Illumina HiSeq 2500 sequencer. We assessed the feasibility of calling copy number events, detecting mutational signatures and identifying potentially actionable mutations in exoDNA sequencing data, as well as expressed point mutations and gene fusions in exoRNA sequencing data.
Whole-exome sequencing resulted in 95%–99% of the target regions covered at a mean depth of 133–490×. Genome-wide copy number profiles, and high estimates of tumor fractions (ranging from 56% to 82%), suggest robust representation of the tumor DNA within the shed exosomal compartment. Multiple actionable mutations, including alterations in NOTCH1 and BRCA2, were found in patient exoDNA samples. Further, RNA sequencing of shed exosomes identified the presence of expressed fusion genes, representing an avenue for elucidation of tumor neoantigens.
We have demonstrated high-resolution profiling of the genomic and transcriptomic landscapes of visceral cancers. A wide range of cancer-derived biomarkers could be detected within the nucleic acid cargo of shed exosomes, including copy number profiles, point mutations, insertions, deletions, gene fusions and mutational signatures. Liquid biopsies using shed exosomes has the potential to be used as a clinical tool for cancer diagnosis, therapeutic stratification and treatment monitoring, precluding the need for direct tumor sampling.
Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid ...biopsies of patients with pancreatic ductal adenocarcinoma (PDAC).
Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival.
KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls.
Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.
We report for the first time a highly divergent lineage in the Caribbean Sea for the bull shark (Carcharhinus leucas) based on the analysis of 51 mitochondrial DNA genomes of individuals collected in ...the western North Atlantic. When comparing the mtDNA control region obtained from the mitogenomes to sequences reported previously for Brazil, the Caribbean lineage remained highly divergent. These results support the existence of a discrete population in Central America due to a phylogeographic break separating the Caribbean Sea from the western North Atlantic, Gulf of Mexico and South America.
Up to half of patients with congenital autosomal recessive nonsyndromic deafness have mutations in the gene encoding the gap-junction protein connexin 26 (
GJB2
); the rest have had no identifiable ...mutations. In this study, patients with this form of deafness who had one mutant
GJB2
allele were found to have a novel 342-kb deletion that truncates the gene encoding another gap-junction protein, connexin 30 (
GJB6
). Twenty-two of the 33 subjects studied were heterozygous for both mutations and 2 were homozygous for the
GJB6
mutation.
Homozygous mutations in either
GJB2
or
GJB6
or heterozygous mutations in both genes can result in prelingual deafness.
Hearing impairment affects about 1 in 1000 newborns.
1
Cases that are present before the development of speech (prelingual onset) hamper speech acquisition and, therefore, normal communication and social integration. Early detection is essential for the application of palliative treatments and special education. Since about 50 percent of the cases of hearing impairment have genetic causes,
1
molecular diagnosis and genetic counseling are needed. However, the main obstacle to molecular diagnosis is the extreme genetic heterogeneity of nonsyndromic hearing impairment. Most cases of genetic deafness are autosomal recessive. So far, 28 loci for autosomal recessive nonsyndromic hearing impairment have been identified, which . . .
Nanoparticle tracers with small sizes and large magnetization are critical for biomedical imaging and especially for magnetic particle imaging (MPI). Small size is important for accessing future ...intracellular and neurological in vivo applications Here, we show <15 nm nanoparticles made of zero valent iron cores, iron oxide shells and coated with a strongly binding brush co-polymer are effective MPI tracers. The small nanoparticle cores create a hydrodynamic diameter that is half of the state-of-the-art iron oxide tracers while the strongly magnetic zero valent iron maintains similar MPI signal magnitude and resolution.
A high frequency of venous thromboembolism (VTE) has been observed after lung, kidney, and liver transplantation. However, data about the incidence of this complication among heart transplant (HT) ...recipients are lacking.
We analyzed the incidence, recurrence, and predisposing factors of VTE in a single-center cohort of 635 patients who underwent HT from April 1991 to April 2013. Deep venous thrombosis (DVT) and pulmonary embolism (PE) were considered as VTE episodes.
During a median post-transplant follow-up of 8.4 years, 62 VTE episodes occurred in 54 patients (8.5%). Incidence rates of VTE, DVT, and PE were, respectively, 12.7 (95% confidence interval CI, 9.7-16.3), 8.4 (95% CI, 6.0-11.4), and 7.0 (95% CI 4.8-9.7) episodes per 1,000 patient-years. Incidence rates of VTE during the first post-transplant year and beyond were, respectively, 45.1 (95% CI, 28.9-67.1) and 8.7 (95% CI 6.2-11.2) episodes per 1,000 patient-years. The incidence rate of VTE recurrence after a first VTE episode was 30.5 (95% CI, 13.2-60.2) episodes per 1,000 patient-years. By means of multivariable Cox regression, chronic renal dysfunction, older age, obesity, and the use of mammalian target of rapamycin inhibitors were identified as independent risk factors for VTE among HT recipients.
VTE is a frequent complication after HT, mainly during the first post-operative year. In view of a high recurrence rate, long-term anti-coagulation should be considered in HT recipients who experience a first VTE episode.
Abstract Objectives The enlargement of the left atrium has been identified as a marker of chronically increased left ventricular filling pressure and left ventricular diastolic dysfunction. This ...study aims to evaluate the association of indexed left atrial diameter with stroke, cardiovascular mortality, the combined event, and all-cause mortality in patients who underwent aortic valve surgery. Methods Indexed left atrial diameter was measured in 2011 adult patients (mean age, 70.9 ± 10.8 years; 58.7% were men) who underwent aortic valve surgery between January 2008 and March 2016. Results On the basis of the criteria of the American Society of Echocardiography, indexed left atrial diameter was normal in 64% of patients, mildly enlarged in 12.4% of patients, moderately enlarged in 9.2% of patients, and severely enlarged in 14.3% of patients. Over a mean follow-up period of 3.2 ± 2.1 years, there were 334 deaths and 97 strokes. Cardiovascular mortality survival at 5 years among patients with normal, mild, moderate, and severe left atrial enlargement was 91.6%, 86.8%, 77.9%, and 77.4%, respectively ( P < .001). After covariable adjustment, Cox regression analysis showed indexed left atrial diameter as an independent predictor of all-cause mortality (hazard ratio per 1-cm/m2 increment, 1.545; 95% confidence interval, 1.252-1.906, P < .001), cardiovascular death (hazard ratio per 1-cm/m2 increment, 1.971; 95% confidence interval, 1.541-2.520; P < .001), and the combined event (hazard ratio per 1-cm/m2 increment, 1.673; 95% confidence interval, 1.321-2.119; P < .001). Conclusions Indexed left atrial diameter is a strong predictor of long-term outcomes in patients with aortic valve diseases who undergo surgery.
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