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Cancer treatments have evolved from indiscriminate cytotoxic agents to selective genome- and immune-targeted drugs that have transformed outcomes for some malignancies.1 Tumor complexity and heterogeneity suggest that the “precision medicine” paradigm of cancer therapy requires treatment to be personalized to the individual patient.2–6 To date, precision oncology trials have been based upon molecular matching with predetermined monotherapies.7–14 Several of these trials have been hindered by very low matching rates, often in the 5–10% range,15 and low response rates. Low matching rates may be due to the use of limited gene panels, restrictive molecular matching algorithms, lack of drug availability or the deterioration and death of end-stage patients before therapy can be implemented. We hypothesized that personalized treatment with combination therapies would improve outcomes in patients with refractory malignancies. As a first test of this concept, we implemented a cross-institutional, prospective study (I-PREDICT, NCT02534675) that used tumor DNA sequencing and timely recommendations for individualized treatment with combination therapies. We found that administration of customized multi-drug regimens was feasible, with 49% of consented patients receiving personalized treatment. Targeting of a larger fraction of identified molecular alterations, yielding a higher “matching score,” was correlated with significantly improved disease control rates, as well as longer progression-free and overall survival rates, as compared to when fewer somatic alterations were targeted. Our findings suggest that the current clinical trial paradigm for precision oncology, which pairs one driver mutation with one drug, may be optimized by treating molecularly complex and heterogeneous cancers with combinations of customized agents.

Background Animal-attached devices can be used on cryptic species to measure their movement and behaviour, enabling unprecedented insights into fundamental aspects of animal ecology and behaviour. However, direct observations of subjects are often still necessary to translate biologging data accurately into meaningful behaviours. As many elusive species cannot easily be observed in the wild, captive or domestic surrogates are typically used to calibrate data from devices. However, the utility of this approach remains equivocal. Methods Here, we assess the validity of using captive conspecifics, and phylogenetically-similar domesticated counterparts (surrogate species) for calibrating behaviour classification. Tri-axial accelerometers and tri-axial magnetometers were used with behavioural observations to build random forest models to predict the behaviours. We applied these methods using captive Alpine ibex (Capra ibex) and a domestic counterpart, pygmy goats (Capra aegagrus hircus), to predict the behaviour including terrain slope for locomotion behaviours of captive Alpine ibex. Results Behavioural classification of captive Alpine ibex and domestic pygmy goats was highly accurate (> 98%). Model performance was reduced when using data split per individual, i.e., classifying behaviour of individuals not used to train models (mean ± sd = 56.1 ± 11%). Behavioural classifications using domestic counterparts, i.e., pygmy goat observations to predict ibex behaviour, however, were not sufficient to predict all behaviours of a phylogenetically similar species accurately (> 55%). Conclusions We demonstrate methods to refine the use of random forest models to classify behaviours of both captive and free-living animal species. We suggest there are two main reasons for reduced accuracy when using a domestic counterpart to predict the behaviour of a wild species in captivity; domestication leading to morphological differences and the terrain of the environment in which the animals were observed. We also identify limitations when behaviour is predicted in individuals that are not used to train models. Our results demonstrate that biologging device calibration needs to be conducted using: (i) with similar conspecifics, and (ii) in an area where they can perform behaviours on terrain that reflects that of species in the wild.

Background Tri-axial accelerometers are frequently deployed on terrestrial quadrupedal mammals using collars, because they are easy to fit and are thought to have minimal impact on the subject. Collar-attached devices are not fixed to the body and can move independently of the body. This may result in inaccurate measures of acceleration, reducing the accuracy of measured body movement. We determined the effect of collar size and collar weight on acceleration measured by a collar-mounted accelerometer on a quadruped mammal. The aim was to suggest best practice for sizes and weights of collars on which to deploy tri-axial accelerometers. Using pygmy goats, Capra aegagrus hircus, which were trained to walk at different speeds (0.8–3.0 km/h) on a treadmill, we measured body acceleration using a collar-mounted tri-axial accelerometer, with different collar sizes (individual neck circumference + 1 cm to + 9 cm) and collar weight (0.4% to 1.2% of individual weight). Results There was a significant effect of collar size, collar weight and walking speed on measured acceleration. Measured acceleration was less accurate and more variable when collars were looser and heavier. To measure body acceleration more accurately, we found that collar size should be within 5 cm or 16% of an individual’s neck circumference when it was heavy (up to 1.2% of animal’s body weight) or within 7 cm (33%) of neck circumference if the collar was light (up to 0.6% of animal body weight). Conclusion We suggest that not only reporting collar size and weight for welfare purposes, but it is also important to consider these aspects for scientific rigour, to ensure data are collected as accurately as possible. We provide guidelines for researchers fitting collar-attached devices to ensure a higher degree of accuracy of recorded body acceleration.

Background This retrospective study was undertaken to determine if the plasma circulating tumor DNA (ctDNA) level and tumor biological features in patients with advanced solid tumors affected the detection of genomic alterations (GAs) by a plasma ctDNA assay. Method Cell-free DNA (cfDNA) extracted from frozen plasma (N = 35) or fresh whole blood (N = 90) samples were subjected to a 62-gene hybrid capture-based next-generation sequencing assay FoundationACT. Concordance was analyzed for 51 matched FoundationACT and FoundationOne (tissue) cases. The maximum somatic allele frequency (MSAF) was used to estimate the amount of tumor fraction of cfDNA in each sample. The detection of GAs was correlated with the amount of cfDNA, MSAF, total tumor anatomic burden (dimensional sum), and total tumor metabolic burden (SUVmax sum) of the largest ten tumor lesions on PET/CT scans. Results FoundationACT detected GAs in 69 of 81 (85%) cases with MSAF > 0. Forty-two of 51 (82%) cases had ≥ 1 concordance GAs matched with FoundationOne, and 22 (52%) matched to the National Comprehensive Cancer Network (NCCN)-recommended molecular targets. FoundationACT also detected 8 unique molecular targets, which changed the therapy in 7 (88%) patients who did not have tumor rebiopsy or sufficient tumor DNA for genomic profiling assay. In all samples (N = 81), GAs were detected in plasma cfDNA from cancer patients with high MSAF quantity (P = 0.0006) or high tumor metabolic burden (P = 0.0006) regardless of cfDNA quantity (P = 0.2362). Conclusion This study supports the utility of using plasma-based genomic assays in cancer patients with high plasma MSAF level or high tumor metabolic burden.

Inflammatory breast cancer (IBC) is a distinct clinicopathologic entity that carries a worse prognosis relative to non-IBC breast cancer even when matched for standard biomarkers (ER/PR/HER2). The objective of this study was to identify opportunities for benefit from targeted therapy, which are not currently identifiable in the standard workup for advanced breast cancer. Comprehensive genomic profiling on 53 IBC formalin-fixed paraffin-embedded specimens (mean, 800× + coverage) using the hybrid capture-based FoundationOne assay. Academic and community oncology clinics. From a series of 2208 clinical cases of advanced/refractory invasive breast cancers, 53 cases with IBC were identified. The presence of clinically relevant genomic alterations (CRGA) in IBC and responses to targeted therapies. CRGA were defined as genomic alterations (GA) associated with on label targeted therapies and targeted therapies in mechanism-driven clinical trials. For the 44 IBCs with available biomarker data, 19 (39 %) were ER-/PR-/HER2- (triple-negative breast cancer, TNBC). For patients in which the clinical HER2 status was known, 11 (25 %) were HER2+ with complete (100 %) concordance with ERBB2 (HER2) amplification detected by the CGP assay. The 53 sequenced IBC cases harbored a total of 266 GA with an average of 5.0 GA/tumor (range 1-15). At least one alteration associated with an FDA approved therapy or clinical trial was identified in 51/53 (96 %) of cases with an average of 2.6 CRGA/case. The most frequently altered genes were TP53 (62 %), MYC (32 %), PIK3CA (28 %), ERBB2 (26 %), FGFR1 (17 %), BRCA2 (15 %), and PTEN (15 %). In the TNBC subset of IBC, 8/19 (42 %) showed MYC amplification (median copy number 8X, range 7-20) as compared to 9/32 (28 %) in non-TNBC IBC (median copy number 7X, range 6-21). Comprehensive genomic profiling uncovered a high frequency of GA in IBC with 96 % of cases harboring at least 1 CRGA. The clinical benefit of selected targeted therapies in individual IBC cases suggests that a further study of CGP in IBC is warranted.

SUMMARY Multiple somatic rearrangements are often found in cancer genomes. However, the underlying processes of rearrangement and their contribution to cancer development are poorly characterised. Here, we employed a paired-end sequencing strategy to identify somatic rearrangements in breast cancer genomes. There are more rearrangements in some breast cancers than previously appreciated. Rearrangements are more frequent over gene footprints and most are intrachromosomal. Multiple architectures of rearrangement are present, but tandem duplications are common in some cancers, perhaps reflecting a specific defect in DNA maintenance. Short overlapping sequences at most rearrangement junctions suggest that these have been mediated by non-homologous end-joining DNA repair, although varying sequence patterns indicate that multiple processes of this type are operative. Several expressed in-frame fusion genes were identified but none were recurrent. The study provides a new perspective on cancer genomes, highlighting the diversity of somatic rearrangements and their potential contribution to cancer development.

The Wellcome Trust Sanger Institute is one of the world’s largest genome centres, and a substantial amount of our sequencing is performed on ‘next generation’ massively parallel sequencing technologies: in June 2008 the quantity of purity filtered sequence data generated by our Genome Analyzer (Illumina) platforms reached 1 terabase, and our average weekly Illumina production output is currently 64gigabases (Gb). Here we describe a set of improvements we have made to the standard Illumina protocols to make the library preparation more reliable in a high throughput environment, to reduce bias, tighten insert size distribution, and reliably obtain high yields of data.

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

Ferredoxins that contain 2[4Fe-4S]2+/+ clusters can be divided into two classes. The "clostridial-type" ferredoxins have two CysXXCysXXCysXXXCysPro motifs. The "photosynthetic bacterial and nif-related" ferredoxins have one motif of that type and one more unusual CysXXCysX7–9CysXXXCysPro motif. In Azotobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purification of a small anionic [Fe-S] protein with yields of ∼3 mg per 500 g cell paste. NH2-terminal sequence analysis shows that this protein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now commonly used for a very different cb-type cytochrome oxidase we have renamed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of "photosynthetic bacterial and nif-related" 2[4Fe-4S]2+/1+ ferredoxins that includes Chromatium vinosum ferredoxin. Studies of the purified protein by Fe analysis, absorption, CD and EPR spectroscopies and electrochemistry confirm this characterization; the reduction potentials of the two clusters are –440 mV vs SHE. The fact that A. vinelandii synthesizes three different proteins with the same sequence motif, each of which is likely to have a different function, shows that although sequence motifs may be used reliably to classify ferredoxins by cluster type they cannot yet be used reliably for classifying ferredoxins by function.

Nature 469, 539–542 (2011) In this Letter, the accession number for the gene expression data in the Gene Expression Omnibus was wrongly published as GEO22316. The correct accession number is GSE22316. We thank P. Boutros for pointing this out. This has been corrected in the HTML and PDF versions online.

1. The motor supply to the closer muscles of the snapper and pincer claws in alpheid shrimp was examined using electrical stimulation and recording techniques. Closer muscles of both claws are innervated by two excitatory and two inhibitory axons. Details of the relative distribution of the two excitatory axons to both sets of closer muscle fibers in each claw are similar. 3. Properties of synaptic transmission at the neuromuscular junction in the pincer claw resemble those in developing or regenerating muscle, with frequent failures and fluctuating ejp amplitudes. 4. We conclude that gross reorganization of claw motor innervation does not occur during the phenomenon of pincer-snapper transformation. Moreover the developmentally primitive state of neuromuscular transmission in the pincer provides a clue to possible control mechanisms in the transformation of the closer muscle.

1. The gross anatomy of the nerves and muscles in the pincer and snapper claws ofAlpheus armillatus is described. In both claws there are three muscles: the opener muscle, the main closer muscle, Cl1, and the small closer muscle, Cl2. 2. The number of fibers in each of the two closer muscles is similar for a pair of claws removed from the same animal. However, the size of individual closer muscle fibers in the snapper is about two times greater, both in length and diameter, than in the pincer. 3. Sarcomeres in fibers of the opener muscle and the small closer muscle are similar in length for the pincer and the snapper. In contrast, sarcomeres in the main closer muscle of the pincer are shorter than those in the snapper Cl2 muscle. 4. Excitatory junctional potentials (ejp's) recorded from single pincer Clx muscle fibers at 1 Hz were always small in amplitude (≦12 mV) and facilitated poorly at 10 Hz. Snapper Cl1 ejp's ranged up to about. 30 mV in amplitude and displayed a large degree of facilitation at 10 Hz. 5. During the process of pincer transformation into a snapper claw, parallel changes occur in both the sarcomere length of Cl1 muscle fibers and the degree of ejp facilitation.

We have examined the effects of temperature changes on the stretcher muscle and its motor supply in a crab (Pachygrapsus crassipes). An increase in temperature caused a decrease in the amplitude of evoked excitatory junctional potentials (ejp's). Above a critical threshold a single action potential in the excitor (E) or specific inhibitor (SI) axon provoked multiple spikes in the appropriate axon and concomitant ejp's or inhibitory junctional potentials (ijp's) in the stretcher muscle fibers. The critical temperature for generation of peripheral spikes was dependent upon the crab's thermal history.

Neuromuscular transmission at temperatures between 8 °c and 24 °c was investigated in a leg muscle of the shore crab,Pachygrapsus crassipes, acclimated to 12 °c, to determine whether diversity of synaptic performance at different endings of the same motor axon could be maintained over a wide temperature range.

1. The present paper examines the changes that take place in the synaptic physiology and morphology of the main closer (Cl1) muscle during simultaneous regeneration of pincer and snapper claws. 2. Regenerating chelipeds appear one moult cycle after removal of the original claws. Both chelipeds are small but exhibit discrete differences in skeletal structure, Cl1 muscle morphology, and synaptic physiology. 3. After the first moult cycle excitatory and inhibitory synaptic terminals on Cl1 muscle fibers are formed at about the same time. Synaptic contacts are initially made on fibers in the proximal portion of the main closer muscle, and only later during the second moult cycle are synaptic connections made on more distally located Cl1 fibers. 4. After two moult cycles, the pincer main closer muscle is composed of fibers of different diameters and sarcomeres that exhibit a bimodal length distribution. The thicker fibers are located centrally in the muscle and have sarcomeres 2 and 3 μm long, while intermediate length sarcomeres are characteristic of thinner Cl1 fibers on the lateral and medial margins. 5. Prior to the third moult cycle, snapper Cl1 sarcomeres exhibit a unimodal distribution, whereas after 3 moults the sarcomeres show a bimodal distribution. Data from a series of snapper claws in different stages of regeneration revealed that there is a close matching between the synaptic physiology and the morphology of the main closer muscle fibers.

PERIPHERAL nerves exert strong trophic influences on the muscles they supply. Although not well understood, the crucial role of nerves in regulating development, regeneration and normal maintenance of limb muscles in vertebrate and invertebrate animals is assumed to depend on a direct anatomical relationship between the axons and their targets. The trophic nervous mechanisms which operate during limb regeneration and development may be studied profitably in crustaceans, many of which possess opposite limbs which are morphologically and functionally dissimilar. Homarid lobsters, for example, have one enlarged heavy crusher claw and a more slender, highly serrated ripper claw on the opposite side. Sexually mature male fiddler crabs have a greatly enlarged claw on one side only, and many other crustaceans show similar bilateral differences in relative claw size. The snapping shrimps (Family Alpheidae) show especially dramatic cheliped asymmetries. In these animals, one of the two claws is tremendously enlarged and also differs greatly in external morphology from its opposite number (Fig. 1). Snapping shrimps use their enlarged ‘snapper’ claw mainly in aggressive encounters with conspecific shrimp1. Exoskeletal and/or muscular arrangements permit the dactyl of the snapper claw to close against the propodite with great force2. This action generates an intense jet of water from a specialised region of the propodite and also produces an audible snap. We describe here changes not only in the size and function, but also in the morphology of the terminal limb segments in this shrimp following damage to a distant anatomically separate nervous pathway. At least one, if not several, synapses intervene between the damaged neurones and those which make functional contact with muscles in the affected limb. Thus, these findings imply that peripheral nerves need not make direct anatomical contact with the targets of their trophic influences.

MAGNETIC circular dichroism (MCD) is the differential absorption of left and right circularly polarized light induced by a magnetic field1–4. By contrast with natural circular dichroism (CD), MCD is a property of all materials. Until now no instrumentation has been available for measuring CD and MCD in the near infrared (IR) spectral region with the high sensitivity routine in the visible and near ultraviolet. Some CD studies at wavelengths greater than 1 µm have been made on iron-sulphur proteins5,6. No MCD measurements have been made in this region. The recent development of photoelastic modulators (PEM)7 has made this possible and at the University of Southern California we have built a near IR CD and MCD instrument incorporating an ‘infrasil’ quartz PEM8. The instrument has a sensitivity of close to 10−5 absorbance units over its spectral range of 800–3,000 nm and employs magnetic fields up to 60 kgauss. We believe this will permit important new studies of metalloproteins and here report initial results on haemoproteins.