Whole-genome sequencing reveals clinically relevant insights into the aetiology of familial breast cancers.

BACKGROUND: Whole-genome sequencing (WGS) is a powerful method for revealing the diversity and complexity of the somatic mutation burden of tumours. Here, we investigated the utility of tumour and matched germline WGS for understanding aetiology and treatment opportunities for high-risk individuals with familial breast cancer. PATIENTS AND METHODS: We carried out WGS on 78 paired germline and tumour DNA samples from individuals carrying pathogenic variants in BRCA1 (n = 26) or BRCA2 (n = 22) or from non-carriers (non-BRCA1/2; n = 30). RESULTS: Matched germline/tumour WGS and somatic mutational signature analysis revealed patients with unreported, dual pathogenic germline variants in cancer risk genes (BRCA1/BRCA2; BRCA1/MUTYH). The strategy identified that 100% of tumours from BRCA1 carriers and 91% of tumours from BRCA2 carriers exhibited biallelic inactivation of the respective gene, together with somatic mutational signatures suggestive of a functional deficiency in homologous recombination. A set of non-BRCA1/2 tumours also had somatic signatures indicative of BRCA-deficiency, including tumours with BRCA1 promoter methylation, and tumours from carriers of a PALB2 pathogenic germline variant and a BRCA2 variant of uncertain significance. A subset of 13 non-BRCA1/2 tumours from early onset cases were BRCA-proficient, yet displayed complex clustered structural rearrangements associated with the amplification of oncogenes and pathogenic germline variants in TP53, ATM and CHEK2. CONCLUSIONS: Our study highlights the role that WGS of matched germline/tumour DNA and the somatic mutational signatures can play in the discovery of pathogenic germline variants and for providing supporting evidence for variant pathogenicity. WGS-derived signatures were more robust than germline status and other genomic predictors of homologous recombination deficiency, thus impacting the selection of platinum-based or PARP inhibitor therapy. In this first examination of non-BRCA1/2 tumours by WGS, we illustrate the considerable heterogeneity of these tumour genomes and highlight that complex genomic rearrangements may drive tumourigenesis in a subset of cases.

Comparison of actionable events detected in cancer genomes by whole-genome sequencing, in silico whole-exome and mutation panels.

BACKGROUND: Next-generation sequencing is used in cancer research to identify somatic and germline mutations, which can predict sensitivity or resistance to therapies, and may be a useful tool to reveal drug repurposing opportunities between tumour types. Multigene panels are used in clinical practice for detecting targetable mutations. However, the value of clinical whole-exome sequencing (WES) and whole-genome sequencing (WGS) for cancer care is less defined, specifically as the majority of variants found using these technologies are of uncertain significance. PATIENTS AND METHODS: We used the Cancer Genome Interpreter and WGS in 726 tumours spanning 10 cancer types to identify drug repurposing opportunities. We compare the ability of WGS to detect actionable variants, tumour mutation burden (TMB) and microsatellite instability (MSI) by using in silico down-sampled data to mimic WES, a comprehensive sequencing panel and a hotspot mutation panel. RESULTS: We reveal drug repurposing opportunities as numerous biomarkers are shared across many solid tumour types. Comprehensive panels identify the majority of approved actionable mutations, with WGS detecting more candidate actionable mutations for biomarkers currently in clinical trials. Moreover, estimated values for TMB and MSI vary when calculated from WGS, WES and panel data, and are dependent on whether all mutations or only non-synonymous mutations were used. Our results suggest that TMB and MSI thresholds should not only be tumour-dependent, but also be sequencing platform-dependent. CONCLUSIONS: There is a large opportunity to repurpose cancer drugs, and these data suggest that comprehensive sequencing is an invaluable source of information to guide clinical decisions by facilitating precision medicine and may provide a wealth of information for future studies. Furthermore, the sequencing and analysis approach used to estimate TMB may have clinical implications if a hard threshold is used to indicate which patients may respond to immunotherapy.

Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains.

Hepatocyte growth factor/scatter factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the beta-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (K(d)=2.4, 3.3 and 1.4 microM). The same domains also bound heparin with decreasing affinities (N>K1>>SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the beta-chain moiety (amino acid residues 308-514) of the Met beta-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy.

Bicuculline epileptogenesis in the rat.

Bicuculline has been applied electrophoretically from a fluid filled microelectrode at different depths within the primary somatosensory area of the cerebral cortex of rats anaesthetized with urethane. The delay between onset of drug application at a constant rate and onset of spontaneous focal interictal epileptiform discharges (FIEDs), detected by a nearby recording microelectrode, was least when bicuculline was applied at a depth of 0.65 mm below the pial surface. The subsequent frequency of FIEDs and their voltage excursion were also greatest at this depth. The relationship between the delay of onset of epileptiform spiking and the depth of drug application was very similar to that previously determined for penicillin. This similarity of the sensitivity profiles suggests that the epileptogenic actions of the two agents may be attributable to a common mechanism. At low concentrations, both agents specifically block GABAergic inhibitory synaptic transmission in brain tissue. This is likely to be the mechanism of their epileptogenic effects. Other synaptic and non-synaptic mechanisms cannot, however, be ruled out because of the high concentrations which are achieved locally when a chemical is applied from a point source.

Penicillin epilepsy in the rat: the responses of different layers of the cortex cerebri.

Na penicillin G has been electrophoretically applied from a fluid-filled micropipette at different rates and depths within the somatosensory area of the cerebral cortex of rats anaesthetized with urethane. With low fluxes of penicillin, the chemical has to reach a critical concentration in the deep part of layer III and produces focal interictal epileptiform discharges (FIEDs) there, irrespective of the depth of application. Larger penicillin fluxes result in FIEDs maximal at the site of the tip of the penicillin electrode. Calculations showed that diffusion together with the need for the penicillin concentration to reach a threshold throughout a critical mass of tissue accounts for the observation that application of penicillin into a relatively insensitive layer of cortex can initiate spiking confined either to that lamina or to a distant more sensitive lamina, the site of the FIED depending on the rate of penicillin application. With a large penicillin flux, FIEDs persisted for many hours so that subsequent trials had to be undertaken in the presence of background spiking. Despite this, it proved possible to determine the rank order of sensitivity of the different cortical laminae. This was, in decreasing order of sensitivity, III > IV >> II > Va >> Vb. Evidence is presented to suggest that the difference in sensitivity of different cortical depths is not due to a difference in packing density of the sensitive neural elements; there seem to be genuine laminar gradients in the sensitivity of the neuronal structures in which synchronized electrical activity is induced.

Penicillin epileptogenesis in the rat: diffusion and the differential laminar sensitivity of the cortex cerebri.

When penicillin is applied electrophoretically from a fluid-filled microelectrode into the substance of the somatosensory cortex of the rat under urethane anaesthesia, the latent period for production of interictal epileptic spikes is least when the electrode lies 0.7 mm below the cortical surface. With low electrophoretic currents of --50 to 100 nA the increase in latent period as the tip of the electrode is placed further and further away from this level can be quantitatively accounted for by the time taken for penicillin to diffuse and to reach a threshold concentration throughout a critical mass of tissue at the 0.7 mm level. With these low currents, the generators of the interictal spikes are confined to a band of cortex centred at the 0.7 mm level. This is true even when the penicillin is applied away from the sensitive layer; in this circumstance the duration of electrophoresis needed to evoke interictal spikes is greater but when they do eventually appear the spikes are generated at the 0.7 mm layer. Histologically, the sensitive layer has been identified as the deep part of layer III. So far as the generation of interictal spikes is concerned, there is no evidence that, with low electrophoretic currents, penicillin has effects other than at the deep part of layer III; all the available evidence indicates that the penicillin has to diffuse to this layer and produces its effects there.

Sci-Fri AM: YIS-04: Scatter correction of cone beam optical computed tomography for polymer gel dosimetry.

Optical computed tomography (OptCT) may become the preferred scanning method for gel dosimetry dose validations, due to its high sensitivity and relatively low cost. Cone beam computed tomography (CBCT) arrangements are advantageous because of reduced scan time. However, CBCT arrangements are more sensitive to errors associated with scatter than other CT configurations. Unfortunately in polymer gel dosimetry this problem is amplified as the primary mode of beam attenuation is through scatter. Thus, managing and reducing the effects of scatter remains an important challenge for cone beam OptCT. In this work we examine two schemes for reducing the effects of scatter in the Vista cone beam OptCT system. First, we employed a pair of anti-scatter polarizing planes to reduce the magnitude of stray light reaching the camera. Secondly, we implemented a beam stop array (BSA) sampling method -which has been successful in correcting for scatter in X-ray CBCT- to obtain scatter field measurements that are subtracted from CT projections removing veiling glare. While both implementations reduced scatter related artifacts, the BSA technique yielded greater improvement without obvious image degradation. Comparative studies between absorbing dye standards and colloidal scattering standards with the same spectrophotometric optical attenuation revealed that application of the BSA technique nullified OptCT measurement disagreements between scattering and absorbing systems. When the BSA scatter correction technique was applied to polymer gel dosimetry 3%3mm agreement rose from 79.2% to 99.82%. These findings underscore the strength of the BSA sampling technique and its utility in cone beam OptCT.

Poster - Thurs Eve-42: A revision of the γ-evaluation: Initial interpretation of dose disagreements on γ-vector fields.

Gamma evaluations are a common clinical tool used as a quantitative comparison between dose-distributions, combining both dose difference and distance to agreement criteria. Because gamma evaluations permit rapid analysis of agreement between complex dose distributions, they are often a preferred comparison method for assessing delivery of conformal radiotherapy distributions. Although the comparison provides a useful measure of agreement between distributions when the index is less than one, the scalar gamma value provides little information into the clinical significance or source of disagreements of failing gamma values (i.e., when γ>1). Previously, Stock et al., have presented the gamma angle as an indicator of the relative influence of the distance to agreement versus the dose difference on gamma. We present a modification to the gamma evaluation such that the complete 3D gamma vector information is considered. The predictive nature of each vector component was investigated by simulating various dose disagreements in test distributions. Misalignment tests revealed that the mean gamma vector components indicate the offset direction and relative magnitude for all test distributions. The mean dose component of the gamma vector was prognostic of double Gaussian overdoses and underdoses in a virtual conformal delivery. The response of the vector field depends on properties distinctive to each distribution, such as the local dose gradient. Understanding how these unique properties affect the vector field may permit better diagnosis of dose disagreement sources. Other vector field properties, such as curl and divergence, may yet provide more information for interpreting the cause and significance of γ>1.

The intracortical neuronal connectivity subserving focal epileptiform activity in rat neocortex.

In the anaesthetized rat, regions of the somatosensory cortex have been subpially isolated, leaving intact the cortical blood supply and the connectivity via the white matter. Application of penicillin or strychnine into layer IV of intact cortex resulted in enhancement of amplitude and prolongation of evoked potentials together with the appearance of spontaneous epileptiform discharges. Within a partially isolated region of cortex, spontaneous and evoked potentials occurred as in normal cortex, but application of convulsant drug resulted in no changes in evoked potentials and in no spontaneous spiking. With incisions for which the surface profile measured 0.9 x 0.9 mm, full-depth isolation resulted in interruption of the propensity for epilepsy, whereas half-depth incisions left epileptic manifestations unimpaired. With the surface profile measuring 0.5 x 0.5 mm, half-depth isolation was sufficient to prevent epileptic activity. Results from isolated regions of various geometries and sizes indicated that the ability of cortical neurones to generate epileptic activity depends on the amount of connectivity with surrounding cortex. The propensity of cortex to become epileptic is thus a mass action effect and the 'epileptic neuronal aggregate' is operationally different from anatomically based modular organizations such as thalamo-cortical or cortico-cortical columns. In the small barrel field of the somatosensory cortex, partial isolations that prevented the appearance of spontaneous epileptiform spiking contained many barrels, indicating that a single thalamo-cortical module contains insufficient inherent lateral connectivity to support epileptiform activity. Theoretical considerations indicated that the excitability of a neurone depends both on its monosynaptic connections with other neurones and on the connectivity of these latter with neurones further afield. The interruption of epileptiform activity by partial isolation could be mimicked by a computer model in which connectivity was mediated via short synaptic paths. The model exhibited self-sustaining synchronized neural activity that could be prevented by interruption solely of polysynaptic paths.

Analysis of the activity of one type of spontaneously discharging unit in the cortex cerebri of the anaesthetized rat.

1. Extracellular records from one type of spontaneously active element in the cortex of rats anaesthetized with urethane have been analysed statistically.2. Intensity functions reveal that the frequency of discharge of a unit tends to be modulated periodically, the period being 1(1/2)-2 sec under fairly deep anaesthesia. When anaesthesia becomes lighter, long intervals occur rarely; the unitary discharge becomes less regular and the intensity function becomes flat. Variations in depth of anaesthesia are accompanied by changes in amplitude of the periodic modulation; the period of the modulation is not significantly altered.3. Summed interval histograms indicate that the long intervals are separated by groups of short intervals in a significantly regular fashion. The number of short intervals in a group varies from one to about fifteen, according to the mean frequency of discharge of the unit.4. Serial correlation coefficients reflect the observations of 2 and 3 above but indicate no additional serial correlation. The existence of long intervals obscures any serial patterning of short intervals during bursts.5. Simultaneous records from two units show that the periods of activity in the two units are highly synchronized.6. It is probable that all these features are due to a process which causes periodic synchronous excitability changes of all the spontaneously active elements of the type studied.

Units in the cerebral cortex of the anaesthetized rat and the correlations between their discharges.

1. Extracellular records have been made of spontaneous unitary activity in the cortex of the anaesthetized rat. If the animal is anaesthetized with sufficient urethane almost to abolish reflex contractions of leg muscles when a hind paw is stimulated, two types of spontaneously active unit are found. One type of unit is found in all areas of the cortex which have been studied, and gives high voltage discharges. The other has only been foun in restricted areas of cortex and gives low voltage discharges. 2. The activity of the former type of unit has been analysed. In the unstimulated animal, groups of relatively frequent discharges are separated by periods of quiescence. Apart from this, the discharge pattern appears irregular on naked-eye examination. Synchronous with these periods of frequent discharge, the electrocorticogram exhibits rapid oscillations. Peripheral stimulation of the animal can modify the pattern of discharge of this type of unit. Details of these observations are described. 3. The results are described of experiments in which simultaneous records have been taken from pairs of units in the cerebral cortex. The intensity of correlation between the two units has been measured as a function of temporal displacement (lag) between the two records. 4. With fairly deep urethane anaesthesia, the highest correlation between the two unitary discharges occurs at zero lag if the two units are close together or if they are far apart but in the same coronal line, e.g. if the recording electrodes lie in homologous areas of the two cerebral hemispheres. If one unit is in the anterior pole of a hemisphere and the other in the posterior pole, the discharges are usually synchronous. However, it has sometimes been found that the anterior of the pair of units discharges impulses which precede those of the posterior unit by up to 100 msec. The highest intensity of correlation is then appropriately displaced to 100 msec. 5. The correlation fluctuates rhythmically with a period of 1-8-2 sec, slowly damps out and is symmetrical around the point of highest correlation. 6. Increasing the sensory bombardment of the cortex reduces or abolishes the correlation between the activity of pairs of units. 7. Under Nembutal anaesthesia sufficient to abolish reflex contractions of leg muscles when a hind paw is pinched, no spontaneous cortical unitary activity has been found. When anaesthesia is ligher, spontaneous activity does occur and consists of bursts of up to six impulses at a frequency of 300-500/sec. When simultaneous records from two units have been made, no correlation has been found between their activity.

Phases in the development of a penicillin epileptiform focus in rat neocortex.

Somatosensory evoked potentials and potentials evoked by direct cortical stimulation were recorded from layer IV of the somatosensory area of the cerebral cortex in urethane anaesthetised rats. Penicillin was expelled electrophoretically from the tip of a drug-filled micropipette at constant rates into layer IV. Small fluxes of penicillin (with electrophoretic currents of -50 to -90 nA) resulted in the appearance, after a delay of 1-2 min, of an enhancement of amplitude in the voltage of both types of evoked potential, unaccompanied by any prolongation of the waveform or spontaneous focal epileptiform discharges. The amplitude of the enhanced evoked potential exhibited a strength-response curve which was a scaled-up version of the curve before penicillin, the scaling factor reflecting the enhancement of amplitude. As the interval between a pair of stimuli was increased, the magnitude of the response to the second stimulus recovered, following a time course similar to that before penicillin. With larger fluxes of penicillin (with electrophoretic currents of -250 to -1000 nA) the amplitude of evoked potentials rose more rapidly and to a higher level; as the concentration of penicillin rose, this enhancement of amplitude led into a second phase, in which there was additionally an increase in the duration of the evoked potentials and the appearance of spontaneous epileptiform discharges. The evoked potentials in this situation showed physiological properties different from those before penicillin application. The strength-response curve exhibited a discontinuity, indicating the evoked potential to be the sum of a physiological response and an epileptiform discharge, the former being graded with stimulus strength and the latter being all or none.(ABSTRACT TRUNCATED AT 250 WORDS)

Cobalt applied to the sensorimotor area of the cortex cerebri of the rat.

1. In anaesthetized rats, the area of the cerebral cortex where the electrical response to stimulation of the fore or hind paw has a minimum latency was found to bear a fairly constant relation to bony landmarks of the skull. Reasons are given for accepting the site of minimum latency as being the area most closely associated with the periphery.2. In animals under anaesthesia, the cortical areas responding with minimum latency to fore- and hind paw stimulation are spatially distinct.3. Cobalt powder has been applied to one of the two cortical areas where the electrical response to contralateral fore- or hind paw stimulation is a minimum. The animals have been allowed to recover and the time course of the resulting muscular jerks has been followed.4. Jerks occur most frequently and earliest in the contralateral forelimb after the application of cobalt to the forelimb area of the cortex. When the cobalt is placed on the hind limb area, these jerks appear first and occur most frequently in the hind limb. This implies that the cortical areas most closely connected with fore and hind limbs are physiologically distinct in the absence of anaesthesia.5. The limb jerks start to appear after 24 hr, occur with increasing frequency until the 10th day and thereafter decline. The decline in jerking is attributed to the formation around the cobalt of a connective tissue capsule.

Interaction of cortical evoked potentials in the rat.

1. Unitary and mass potentials were recorded with glass micropipettes at different depths in and around the primary somatosensory area of the cortex in rats anaesthetized with urethane; in addition, surface mass potentials were recorded with chlorided silver ball electrodes. Potentials were evoked by stimuli to the contralateral forepaw and the contralateral cortex. Observations were confined to potentials evoked within 20 msec of stimulation.2. If forepaw stimuli were applied at times when the cortex was not showing spontaneous activity, one component of the evoked response was a widespread depth negativity. This was accompanied by a smaller surface positivity which had the same time course. It could be recorded over the same area of cortex. Unitary activity was found with the same latent period and spatial distribution as the depth negativity. These components of the evoked mass and unitary responses to forepaw stimulation were absent if times of spontaneous cortical activity were chosen for delivering the stimuli.3. Contralateral cortical stimuli evoked mass activity similar to the component of the mass response to forepaw stimulation described in 2. Stimuli given simultaneously to the two sites elicited less unitary activity than the sum of the unitary activity evoked by the two stimuli separately. The mass activity exhibited the interactions which would be expected on the hypothesis that it is generated by the unitary activity. The time course of the interaction is described.4. The interaction was confined to the components of the evoked potentials which were only present if stimuli were applied when the cortex was quiescent. Stimuli applied when the cortex was active evoked mass and unitary potentials which showed no interaction.

Comparison of penicillin epileptogenesis in rat somatosensory and motor cortex.

The relative sensitivities of somatosensory and motor areas of the cerebral cortex in penicillin epileptogenesis were compared in urethane-anaesthetized rats. Penicillin was applied electrophoretically from a fluid-filled micro-electrode. Spontaneous focal interictal epileptiform discharges were detected by a nearby recording electrode. In motor cortex, every cortical layer was less sensitive in penicillin epileptogenesis than the corresponding layer in somatosensory cortex; epileptic spikes occurred later, were of lower amplitude and were less frequent. In motor cortex, the sensitive depth extended from the deep part of layer III to the upper part of layer V. It seemed possible that penicillin applied to motor cortex might be producing its effects by diffusing back to the sensitive somatosensory area. This was excluded by applying penicillin to motor cortex whilst recording from both somatosensory and motor areas and demonstrating that the spikes were found in motor but not in somatosensory cortex.

Nonlinear pharmacokinetics of ethanol: the disproportionate AUC-dose relationship.

Whole venous blood concentrations of ethyl alcohol were measured following the constant rate intravenous infusion of ethanol to four Beagle dogs. Five different doses (0.1-0.8 g ethanol/kg body weight) were administered at scheduled intervals. The area under the blood ethanol concentration-time curves (AUC) was found to demonstrate a markedly nonlinear relationship with the administered dose. Simulations of one and two compartment open models with Michaelis-Menten elimination kinetics and zero-order input are presented with their theoretical AUC-dose relationships.