Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome.In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.     

Ribosomal DNA Is a Site of Chromosome Breakage in Aneuploid Strains of Neurospora

In wild-type strains of Neurospora crassa, the rDNA is located at a single site in the genome called the nucleolus organizer region (NOR), which forms a terminal segment on linkage group (LG) V. In the quasiterminal translocation strain T(I;V)AR190, most of the right arm of LG I moved to the distal tip of the NOR, and one or a few rDNA repeat units are moved to the truncated right arm of LG I. I report here that, in partial diploid strains derived from T(I;V)AR190, large terminal deletions result from chromosome breakage in the NOR. In most of these partial diploids, chromosome breakage is apparently frequent and the breakpoints occur in many parts of the NOR. The rDNA ends resulting from chromosome breakage are "healed" by the addition of new telomeres. Significantly, the presence of ectopic rDNA creates a new site of chromosome breakage in the genome of partial diploids. These results raise the possibility that, under certain conditions, rDNA is a region of fragility in eukaryotic chromosomes.     

A New Eurypterid (Chelicerata: Eurypterida) from the Upper Devonian Gogo Formation of Western Australia, With A Review of the Rhenopteridae

A new eurypterid, Rhenopterus waterstoni sp. nov., is described from the Gogo Formation (Frasnian, Upper Devonian) of Western Australia. This species is distinguished from related forms by the tuberculation of the anteriormost tergite and crenulated posterior margins of the carapace and opisthosomal segments. It is the only eurypterid specimen known from the Gogo Formation, the most complete eurypterid from Australia, and also the youngest representative of Rhenopterus in the fossil record. Structures retrieved from between the prosomal-opisthosomal juncture comprise polygonal tubes 30–40 μm in diameter, which are interpreted as sarcomeral sheaths of muscular tissue. Rhenopterus is reviewed: R. latus Størmer, 1936 is synonymized with R. diensti Størmer, 1936 as it is here recognized as a female sexual dimorph; R. maccarthyi (Kjellesvig-Waering, 1934) is an orthocone nautiloid.     

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.     

Compraison of mycoplasmas associated with human tumors, leukemia, and tissue cultures

Leach, R. H. (Wellcome Research Laboratories, Beckenham, Kent, England), and M. Butler. Comparison of mycoplasmas associated with human tumors, leukemia, and tissue cultures. J. Bacteriol. 91:934-941. 1966.-Mycoplasmas originally isolated by various workers from tissue cultures prepared from or inoculated with tumor or leukemic cells fell into four groups; each related to existing species or serotypes. These were Mycoplasma pulmonis, M. fermentans, M. hominis, and the GDL serotype, the last two being well known as contaminants of uninoculated cell lines. All the test strains were able to grow well in certain tissue cultures, and some caused cytopathic effects and acidity. These observations are discussed in terms of the relationship of these strains to the malignant tissues with which they were originally associated. The variable results obtained in certain biological tests on these strains emphasized the need for standardization of the conditions under which such tests may be employed to assist in identification of Mycoplasma species.     

Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins.

Scrapie and Creutzfeldt-Jakob disease are transmissible, degenerative neurological diseases caused by prions. Considerable evidence argues that prions contain protease-resistant sialoglycoproteins, designated PrPSc, encoded by a cellular gene. The prion protein (PrP) gene also encodes a normal cellular protein designated PrPC. We established clonal cell lines which support the replication of mouse scrapie or Creutzfeldt-Jakob disease prions. Mouse neuroblastoma N2a cells were exposed to mouse scrapie prions and subsequently cloned. After limited proteinase K digestion, three PrP-immunoreactive proteins with apparent molecular masses ranging between 20 and 30 kilodaltons were detected in extracts of scrapie-infected N2a cells by Western (immuno-) blotting. The authenticity of these PrPSc molecules was established by using monospecific antiserum raised against a synthetic peptide corresponding to a portion of the prion protein. Those clones synthesizing PrPSc molecules possessed scrapie prion infectivity as measured by bioassay; clones without PrPSc failed to demonstrate infectivity. Detection of PrPSc molecules in scrapie-infected N2a cells supports the contention that PrPSc is a component of the infectious scrapie particle and opens new approaches to the study of prion diseases.     

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.     

Synchronization of the desmidStaurastrum gracile

Summary Cultures ofStaurastrum gracile maintained in a liquid medium in a 168 hours light: dark regime were asynchronous. Alteration of the regime to 1410 or 186 failed to induce synchrony, as did variation of the light and dark temperatures. The cultures were synchronized by eliminating a light period to give a total dark period of 32 hours. One such cycle gave 54% synchrony, the cells entering division at the onset of the second light period after treatment. The omission of a second light period 72 hours after the first produced no increase in the level of synchrony. The level was improved by omitting a further light period 48 hours after the first to give a 321632 induction treatment which resulted in 64% synchrony. In no instance did the cells divide between the dark treatment and the synchronized wave of cytokinesis. The mechanism of this synchrony may be explained by the existence of an essential, dark-labile division precursor.This work forms part of a thesis submitted byRoy Rowley to the University of Manchester for the degree of M.Sc.     

Immunohistochemical demonstration of a 44-KD phosphoprotein in developing rat bones

Polyclonal antibodies against a 44-KD phosphoprotein (44K BPP) from rat bone were raised in rabbits, affinity-purified, and used as probes to study the protein's distribution in various types of developing bones from newborn rats. Three immunostaining procedures were applied utilizing indirect immunofluorescence, avidin-biotin-peroxidase complex, and avidin-gold complex with silver enhancement. All methods gave essentially identical and/or complementary results. Antigenicity for anti-44K BPP was detected in endochondral and membranous bone. In the latter, it was also demonstrated in the osteoid. In the woven bone of lower jaw, immunoreactivity for anti-44K BPP antibodies was found in fibroblast-shaped cells (pre-osteoblasts) that were between the bone trabeculae but not in direct contact with bony extracellular material. In addition to these presumed osteoprogenitor cells, osteoblasts as well as osteocytes were strongly stained; the cytoplasmic staining was associated with the Golgi apparatus. Occasionally immunoreactivity was detected in osteoclasts, but in these cells immunostaining was either diffusely spread in the cytoplasm or present only at sites of bone erosion. These findings support the hypothesis that the 44K BPP is a protein made by osteoblasts and is localized predominantly in bone. Furthermore, the protein appears to be expressed early in histogenesis of the bone-forming cells.     

Accuracies of genomic breeding values in American Angus beef cattle using K-means clustering for cross-validation

Background

Genomic selection is a recently developed technology that is beginning to revolutionize animal breeding. The objective of this study was to estimate marker effects to derive prediction equations for direct genomic values for 16 routinely recorded traits of American Angus beef cattle and quantify corresponding accuracies of prediction.

Methods

Deregressed estimated breeding values were used as observations in a weighted analysis to derive direct genomic values for 3570 sires genotyped using the Illumina BovineSNP50 BeadChip. These bulls were clustered into five groups using K-means clustering on pedigree estimates of additive genetic relationships between animals, with the aim of increasing within-group and decreasing between-group relationships. All five combinations of four groups were used for model training, with cross-validation performed in the group not used in training. Bivariate animal models were used for each trait to estimate the genetic correlation between deregressed estimated breeding values and direct genomic values.

Results

Accuracies of direct genomic values ranged from 0.22 to 0.69 for the studied traits, with an average of 0.44. Predictions were more accurate when animals within the validation group were more closely related to animals in the training set. When training and validation sets were formed by random allocation, the accuracies of direct genomic values ranged from 0.38 to 0.85, with an average of 0.65, reflecting the greater relationship between animals in training and validation. The accuracies of direct genomic values obtained from training on older animals and validating in younger animals were intermediate to the accuracies obtained from K-means clustering and random clustering for most traits. The genetic correlation between deregressed estimated breeding values and direct genomic values ranged from 0.15 to 0.80 for the traits studied.

Conclusions

These results suggest that genomic estimates of genetic merit can be produced in beef cattle at a young age but the recurrent inclusion of genotyped sires in retraining analyses will be necessary to routinely produce for the industry the direct genomic values with the highest accuracy.