Sensitive and robust liquid biopsy-based detection of PIK3CA mutations in hormone-receptor-positive metastatic breast cancer patients.

BACKGROUND: The benefit of alpelisib in hormone-receptor-positive (HR+) metastatic breast cancer patients provided clinical evidence for the increasing importance of PIK3CA testing. We performed a comparison of liquid biopsy and tissue-based detection of PIK3CA mutations. MATERIALS AND METHODS: PIK3CA hotspot mutation analysis using a high-resolution SiMSen-Seq assay was performed in plasma from 93/99 eligible patients with HR+/HER2- breast cancer. Additionally, mFAST-SeqS was used to estimate the tumour fractions in plasma samples. In 72/93 patients, matched tissue was available and analysed using a customised Ion Torrent panel. RESULTS: PIK3CA mutations were detected in 48.6% of tissue samples and 47.3% of plasma samples, with identical PIK3CA mutation detected in 24/72 (33.3%) patients both in tissue and plasma. In 10 (13.9%) patients, mutations were only found in plasma, and in 6 (8.3%) patients, PIK3CA mutations found in tissue were not detectable in ctDNA. In 49/93 plasma samples without detectable PIK3CA mutations, 22 (44.9%) samples had elevated tumour fractions, implying true negative results. CONCLUSION: SiMSen-Seq-based detection of PIK3CA mutations in plasma shows advantageous concordance with the tissue analyses. A combination with an untargeted approach for detecting ctDNA fractions may confirm a negative PIK3CA result and enhance the performance of the SiMSen-Seq test.

An hourglass mechanism controls torpor bout length in hibernating garden dormice.

Hibernating mammals drastically lower their rate of oxygen consumption and body temperature (Tb) for several weeks, but regularly rewarm and stay euthermic for brief periods (<30 h). It has been hypothesized that these periodic arousals are driven by the development of a metabolic imbalance during torpor; that is, the accumulation or the depletion of metabolites or the accrual of cellular damage that can be eliminated only in the euthermic state. We obtained oxygen consumption (as a proxy of metabolic rate) and Tb at 7 min intervals over entire torpor-arousal cycles in the garden dormouse (Eliomys quercinus). Torpor bout duration was highly dependent on mean oxygen consumption during the torpor bout. Oxygen consumption during torpor, in turn, was elevated by Tb, which fluctuated only slightly in dormice kept at ∼3-8°C. This corresponds to a well-known effect of higher Tb on shortening torpor bout lengths in hibernators. Arousal duration was independent from prior torpor length, but arousal mean oxygen consumption increased with prior torpor Tb. These results, particularly the effect of torpor oxygen consumption on torpor bout length, point to an hourglass mechanism of torpor control, i.e. the correction of a metabolic imbalance during arousal. This conclusion is in line with previous comparative studies providing evidence for significant interspecific inverse relationships between the duration of torpor bouts and metabolism in torpor. Thus, a simple hourglass mechanism is sufficient to explain torpor/arousal cycles, without the need to involve non-temperature-compensated circadian rhythms.

Circulating Tumor DNA as a Marker for Treatment Response in Metastatic Melanoma Patients Using Next-Generation Sequencing-A Prospective Feasibility Study.

We prospectively performed a longitudinal analysis of circulating tumor DNA (ctDNA) from 149 plasma samples and CT scans in Stage III and IV metastatic melanoma patients (n = 20) treated with targeted agents or immunotherapy using two custom next-generation sequencing (NGS) Ion AmpliSeq™ HD panels including 60 and 81 amplicons in 18 genes, respectively. Concordance of matching cancer-associated mutations in tissue and plasma was 73.3%. Mutant allele frequency (MAF) levels showed a range from 0.04% to 28.7%, well detectable with NGS technologies utilizing single molecule tagging like the AmpliSeq™ HD workflow. Median followup time of the tissue and/or plasma positive cohort (n = 15) was 24.6 months and median progression-free survival (PFS) was 7.8 months. Higher MAF ≥ 1% at baseline was not significantly associated with a risk of progression (Odds Ratio = 0.15; p = 0.155). Although a trend could be seen, MAF levels did not differ significantly over time between patients with and without a PFS event (p = 0.745). Depending on the cell-free DNA amount, NGS achieved a sensitivity down to 0.1% MAF and allowed for parallel analysis of multiple mutations and previously unknown mutations. Our study indicates that NGS gene panels could be useful for monitoring disease burden during therapy with ctDNA in melanoma patients.

Investigation of cardiorespiratory effects of the selective 5-HT4 agonist BIMU-8 in etorphine-immobilised goats (Capra aegagrus hircus) in a randomized, blinded and controlled trial.

BACKGROUND: Opioid-induced respiratory compromise remains a significant challenge in etorphine-immobilised wildlife. Serotonergic agonists offer a potential avenue for preventing or treating opioid-induced respiratory compromise. We therefore aimed to determine whether the selective 5-hydroxytryptamine receptor 4 (5-HT4) agonist, BIMU-8, reverses opioid-induced respiratory compromise in etorphine-immobilised goats. METHODS: Seven healthy adult goats were immobilised with etorphine, then treated with BIMU-8 or sterile water 5 minutes later in a randomised, prospective cross-over study. Cardiorespiratory variables were measured at 1-minute intervals from 4 minutes before etorphine to 15 minutes after its administration. Arterial blood gas analyses were also performed before and after etorphine administration and the respective treatments. RESULTS: Intravenous injection of BIMU-8 attenuated etorphine-induced respiratory compromise, as indicated by improvements, compared to baseline and between treatments, in respiratory rate (fR ), peripheral arterial blood oxygen saturation (SpO2 ), partial pressure of arterial oxygen (PaO2 ) and the alveolar-arterial oxygen partial pressure gradient (P(A-a)O2 ). BIMU-8 caused an increase in heart rate and a temporary decrease in arterial blood pressure. Mild movements and slight muscle spasm occurred but BIMU-8 did not reverse immobilisation. CONCLUSION: Our results indicate that BIMU-8 may be a potential drug candidate for the treatment, or prevention, of etorphine-induced respiratory compromise in immobilised ungulates.

Dynamic Function and Composition Shift in Circulating Innate Immune Cells in Hibernating Garden Dormice.

Hibernation is characterized by successive torpor bouts during which metabolic rate is down-regulated to 2-4% of euthermic levels along with core body temperatures (T b ) ranging between 0 and 10°C. One characteristic of the torpid state, which is periodically interrupted by a few hours of euthermic phases or arousals during hibernation, resides in an overall impairment of the immune system. The most striking change during torpor is the reduction of circulating white blood cells up to 90%, while their numbers rise to near summer euthermic level upon rewarming. However, potential changes in responsiveness and function of neutrophil granulocytes, accounting for the primary cellular innate immune defense, are unknown. Here we present the first data on shifts in oxidative burst capacity, i.e., the ability to produce reactive oxygen species (ROS), of neutrophils during hibernation. Using a chemiluminescence assay, we measured real-time ROS production in whole blood of hibernating garden dormice (Eliomys quercinus) in early or late torpor, and upon arousals. Accounting for changes in neutrophil numbers along the torpor-arousal cycle, we found significant differences, between torpid and euthermic states, in the neutrophil oxidative burst capacity (NOC), with shallow cell responses during torpor and a highly significant increase by up to 30-fold during arousals. Further, we observed a significant reduction of NOC from aroused animals with euthermic T b of 36.95 ± 0.37°C, when tested at 6°C, whereas no change occurred in NOC from torpid individuals reaching constant T b of 4.67 ± 0.42°C, when measured at 35°C. This dynamic indicates that the reduction in NOC during torpor may be temperature-compensated. These results linked to the understanding of immune function during the torpor-arousal cycle might have clinical relevance in the context of therapeutic hypothermia and reperfusion injury.

Real-World Evidence Data on Metastatic Renal-Cell Carcinoma Treatment in Austria: The RELACS Study.

BACKGROUND: Treatment decisions in routine clinical practice are based on reports of clinical trials, which represent highly selected populations. Limited studies reported real-world evidences representing routine clinical practices in patients with renal-cell carcinoma (RCC) in Europe. The aim of this retrospective, noninterventional chart review was to collect data on the treatment landscape for patients with advanced/metastatic RCC in routine clinical practice in a broader patient population in Austria. PATIENTS AND METHODS: Patients with advanced/metastatic RCC receiving systemic treatment between June 2010 and June 2016 across 12 centers in Austria were included. Parameters were entered into an electronic case report form from the participating sites via the application Hermesoft electronic data capture system. Progression-free survival (PFS) and overall survival (OS) were the 2 primary end points. RESULTS: The median PFS and OS were 12 months and 44 months, respectively (first-line PFS was 14 months for pazopanib and 13 months for sunitinib; first-line OS was 44 months for pazopanib and 48 months for sunitinib). Factors influencing the OS were sex, with female patients at a significantly higher risk than male patients (hazard ratio = 1.719), Eastern Cooperative Oncology Group performance status > 0 increased the risk twice (hazard ratio = 2.048), and number of metastases > 3 before the first line doubled the risk compared to metastases (hazard ratio = 2.064). CONCLUSION: OS in this retrospective chart review was considerably longer than the previous reports in real-world patients, underlining the benefit of current RCC treatment options in routine clinical practice.

Dietary Lipids Affect the Onset of Hibernation in the Garden Dormouse (Eliomys quercinus): Implications for Cardiac Function.

Dietary lipids strongly influence patterns of hibernation in heterotherms. Increased dietary uptake of n-6 polyunsaturated fatty acids (PUFAs), particularly of linoleic acid (LA, C18:2 n-6), enables animals to reach lower body temperatures (Tb), lengthens torpor bout duration, and results in lower energy expenditure during hibernation. Conversely, dietary n-3 PUFA impacts negatively on hibernation performance. PUFA in surrounding phospholipids (PLs) presumably modulate the temperature-dependent activity of the sarcoplasmic reticulum (SR) Ca2+ ATPase 2 (SERCA2) and thus determine the threshold Tb still allowing proper heart function during torpor. We tested the effect of diets enriched with 10% of either corn oil ("CO," high n-6 PUFA, e.g., LA) or menhaden oil ["MO," long-chain n-3 PUFA, e.g., docosahexaenoic acid (DHA)] on hibernation performance and SERCA2 activity levels during torpor in garden dormice, an insectivorous, fat-storing hibernator. Prior to hibernation, individuals fed the MO diet showed an almost nine-times higher DHA levels and 30% lower LA proportions in white adipose tissue (WAT), reflecting the fatty acid composition of SR membranes, compared to CO-diet fed animals. When fed the MO diet, dormice significantly delayed their mean onset of hibernation by almost 4 days (range: 0-12 days), compared with CO-diet fed animals. Hibernation onset correlated positively with WAT-DHA levels and negatively with WAT-LA proportions prior to hibernation. Subsequently, hibernating patterns were similar between the two dietary groups, despite a significant difference in WAT-LA but not in WAT-DHA levels in mid-hibernation. SR-PL fatty acid composition and SERCA2 activity were identical in torpid individuals from the two dietary groups in mid-hibernation. In line with our previous findings on Syrian hamsters, a granivorous, food-storing hibernator, SERCA2 activity correlated positively with LA and negatively with DHA levels of SR-PL in torpid dormice, although SERCA2 activity was about three-times higher in garden dormice than in Syrian hamsters at similar PL-DHA proportions. Similarly, minimal Tb during torpor decreased as SERCA2 activity increased. We conclude that: (1) fatty acid composition of SR membranes modulates cardiac SERCA2 activity, hence determining the minimum Tb tolerated by hibernators, and (2) high DHA levels prevent hibernators from entering into torpor, but the critical levels differ substantially between species.

The insensitive dormouse: reproduction skipping is not caused by chronic stress in Glis glis.

Entire populations of edible dormice (Glis glis) can skip reproduction in years without mast seeding of deciduous trees (particularly beech or oak seed), because juveniles require high-calorie seeds for growth and fattening prior to hibernation. We hypothesized that, in mast failure years, female dormice may be forced to spend larger amounts of time foraging for low-quality food, which would increase their exposure to predators, mainly owls. This may lead to chronic stress, i.e. long-term increased secretion of glucocorticoids (GC), which can have inhibitory effects on reproductive function in both female and male mammals. We monitored reproduction in free-living female dormice over 3 years with varying levels of food availability, and performed a supplemental feeding experiment. To measure stress hormone levels, we determined fecal GC metabolite (GCM) concentrations collected during the day, which reflect hormone secretion rates in the previous nocturnal activity phase. We found that year-to-year differences in beech mast significantly affected fecal GCM levels and reproduction. However, contrary to our hypothesis, GCM levels were lowest in a non-mast year without reproduction, and significantly elevated in full-mast and intermediate years, as well as under supplemental feeding. Variation in owl density in our study area had no influence on GCM levels. Consequently, we conclude that down-regulation of gonads and reproduction skipping in mast failure years in this species is not caused by chronic stress. Thus, in edible dormice, delayed reproduction apparently is profitable in response to the absence of energy-rich food in non-mast years, but not in response to chronic stress.

Implications of being born late in the active season for growth, fattening, torpor use, winter survival and fecundity.

For hibernators, being born late in the active season may have important effects on growth and fattening, hence on winter survival and reproduction. This study investigated differences in growth, fattening, energetic responses, winter survival and fecundity between early-born ('EB') and late-born ('LB') juvenile garden dormice (Eliomys quercinus). LB juveniles grew and gained mass twice as fast as EB individuals. Torpor use was low during intensive growth, that are, first weeks of body mass gain, but increased during pre-hibernation fattening. LB juveniles showed higher torpor use, reached similar body sizes but lower fat content than EB individuals before hibernation. Finally, LB individuals showed similar patterns of hibernation, but higher proportion of breeders during the following year than EB dormice. These results suggest that torpor is incompatible with growth but promotes fattening and consolidates pre-hibernation fat depots. In garden dormice, being born late in the reproductive season is associated with a fast life history.

RADIOGRAPHY IN THE FIELD: ASSESSING A LIGHTWEIGHT, HANDHELD, BATTERY-POWERED DENTISTRY UNIT FOR FIELD DIAGNOSTIC APPLICATIONS.

Radiography units are not used commonly in wildlife medicine field settings, primarily because of their weight and requirement for a power supply. In this study, a portable, battery-powered, and lightweight radiography unit, originally developed for dentistry, was assessed for its potential field applications. Radiographs of various animal species (ranging in weight from 14 g to 1,000 kg) were imaged using varying source image distance (SID) and exposure time. The quality of these images was evaluated for their resolution, image noise, and motion blur. When required, image resolutions were further enhanced using computed radiography postprocessing. Other parameters evaluated were the freehand use of the device, its battery durability, the maximum obtainable image size, and multiple use of a single computed radiography cassette. Using an SID of 60 cm, radiographs delivered adequate image quality. The quality, however, was found deteriorated in images of larger animals (>50 kg) or thicker tissues (>15 cm). The use of a tripod proved unnecessary in most cases, and its exclusion greatly facilitated equipment handling. Under field conditions, the battery was depleted after a total running time of 1.6 hr or 36 radiographs. The maximum size of a radiographic image reached a diameter of 40 cm, and radiation shielding allowed the multiple use of a single computed radiography cassette. Taken together, the radiography unit evaluated in this study presented a balanced compromise between portability and radiograph quality for field use. However, the unit image resolution cannot replace those of the fixed standard radiography units commonly used in veterinary medicine.

Arterial pH and Blood Lactate Levels of Anesthetized Mongolian Khulan ( Equus hemionus hemionus) in the Mongolian Gobi Correlate with Induction Time.

Research and conservation of wide-ranging wild equids in most cases necessitate capture and handling of individuals. For free-roaming Mongolian khulan ( Equus hemionus hemionus), also known as the khulan, capture involves a strenuous, high-speed chase, and physiologic responses have yet to be elucidated. We analyzed sequential arterial blood gas (ABG) samples as a proxy for respiratory and metabolic status of khulan during capture-related anesthesia. We recorded precise chase and induction times and monitored vital parameters and ABG from free-ranging khulan during anesthesia performed for GPS collaring. At the initiation of anesthesia, animals had ABG values similar to those recorded for thoroughbred horses ( Equus caballus ) after maximal exercise. Longer induction times resulted in higher arterial pH (P<0.001) and lower blood lactate (P<0.002). This trend of improvement continued over the course of anesthesia. The most important factor explaining pH and lactate was the time that elapsed between cessation of the chase and obtaining the first ABG sample, which, under field conditions, is tightly linked to induction time. All animals recovered uneventfully. Our data show that khulan recover and shift their metabolic status back toward expected normal values during opioid-based field anesthesia.

Basking hamsters reduce resting metabolism, body temperature and energy costs during rewarming from torpor.

Basking can substantially reduce thermoregulatory energy expenditure of mammals. We tested the hypothesis that the largely white winter fur of hamsters (Phodopus sungorus), originating from Asian steppes, may be related to camouflage to permit sun basking on or near snow. Winter-acclimated hamsters in our study were largely white and had a high proclivity to bask when resting and torpid. Resting hamsters reduced metabolic rate (MR) significantly (>30%) when basking at ambient temperatures (Ta) of ∼15 and 0°C. Interestingly, body temperature (Tb) also was significantly reduced from 34.7±0.6°C (Ta 15°C, not basking) to 30.4±2.0°C (Ta 0°C, basking), which resulted in an extremely low (<50% of predicted) apparent thermal conductance. Induced torpor (food withheld) during respirometry at Ta 15°C occurred on 83.3±36.0% of days and the minimum torpor MR was 36% of basal MR at an average Tb of 22.0±2.6°C; movement to the basking lamp occurred at Tb<20.0°C. Energy expenditure for rewarming was significantly reduced (by >50%) during radiant heat-assisted rewarming; however, radiant heat per se without an endogenous contribution by animals did not strongly affect metabolism and Tb during torpor. Our data show that basking substantially modifies thermal energetics in hamsters, with a drop of resting Tb and MR not previously observed and a reduction of rewarming costs. The energy savings afforded by basking in hamsters suggest that this behaviour is of energetic significance not only for mammals living in deserts, where basking is common, but also for P. sungorus and probably other cold-climate mammals.

How to spend the summer? Free-living dormice (Glis glis) can hibernate for 11 months in non-reproductive years.

Edible dormice are arboreal rodents adapted to yearly fluctuations in seed production of European beech, a major food source for this species. In years of low beech seed abundance, dormice skip reproduction and non-reproductive dormice fed ad libitum in captivity can display summer dormancy in addition to winter hibernation. To test whether summer dormancy, that is, a very early onset of hibernation, actually occurs in free-living dormice, we monitored core body temperature (Tb) over ~12 months in 17 animals during a year of beech seeding failure in the Vienna Woods. We found that 8 out of 17 dormice indeed re-entered hibernation as early as in June/July, with five of them having extreme hibernation durations of 11 months or more (total range: 7.8-11.4 months). Thus, we show for the first time that a free-living mammal relying on natural food resources can continuously hibernate for >11 months. Early onset of hibernation was associated with high body mass in the spring, but the distribution of hibernation onset was bimodal with prolonged hibernation starting either early (prior to July 28) or late (after August 30). This could not be explained by differences in body mass alone. Animals with a late hibernation onset continued to maintain high nocturnal Tb's throughout summer but used short, shallow torpor bouts (mean duration 7.44 ± 0.9 h), as well as occasional multiday torpor for up to 161 h.

Comparison of S(+)-ketamine and ketamine, with medetomidine, for field anaesthesia in the European brown hare (Lepus europaeus).

OBJECTIVE: To compare anaesthesia and recovery parameters of racemic ketamine or S(+)-ketamine in combination with medetomidine for intramuscular (IM) field anaesthesia in the European brown hare (EBH) (Lepus europaeus). STUDY DESIGN: Randomized, prospective, blinded clinical trial. ANIMALS: 20 adult EBH (eight male, 12 female), mean ± SD weight 3360 341). METHODS: Medetomidine (0.2 mg kg(-1) ) and ketamine (30 mg kg(-1) ) (K-M group) or S(+)-ketamine (15 mg kg(-1) ) (S-M group) were administered by IM injection. Time until first effect and loss of righting reflex were recorded. During sedation and anaesthesia heart rate, saturation of arterial haemoglobin, respiratory rate, side stream end tidal CO(2) (Pe'CO(2) ), non invasive blood pressure, body temperature, cardiorespiratory parameters, palpebral reflex, jaw tone and nociception were recorded every 5 minutes. Medetomidine was antagonized with IM atipamezole (1 mg kg(-1) ) 45 minutes after treatment injection. Time until first head lift, standing and total recovery time (T-Recov) were recorded. Incidences of falling and involuntary movements during recovery were counted. Recovery quality was scored by visual analogue scale. Descriptive statistics were used to visualize maintenance data. All other data were included in multiple linear regression models. RESULTS: Surgical anesthesia was not produced reliably with either protocol. Hypoxaemia occurred in both groups (SpO(2) < 90%). During recovery, falling was noted significantly less often (p < 0.001) in the S-M group (13 ± 7) versus the K-M group (27 ± 13). T-Recov was long, lasting for more than 3 hours in individuals with no significant differences between groups. CONCLUSION AND CLINICAL RELEVANCE: S(+)-ketamine showed only minor advantages over racemic ketamine. Surgical anaesthesia was not achieved reliably with either protocol. Oxygen supplementation should be considered to prevent hypoxaemia. Further research is needed to develop an injectable field protocol adequate for surgical procedures, but with a rapid smooth recovery.