Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA.

BACKGROUND: The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. PATIENTS AND METHODS: We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. RESULTS: Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. CONCLUSION: We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available.

Dyspnea Postpulmonary Embolism From Physiological Dead Space Proportion and Stroke Volume Defects During Exercise.

BACKGROUND: Many patients with pulmonary embolism (PE) report dyspnea on exertion following long-term treatment. Increased physiological dead space proportion (VD/VT) and decreased cardiac stroke volume reserve may distinguish persistent effects of PE itself from symptoms reflecting comorbid conditions or deconditioning. METHODS: This retrospective study analyzed a consecutive series of incremental symptom-limited cardiopulmonary exercise tests that had been ordered to evaluate persistent dyspnea on exertion following long-term treatment for acute PE. Physiological VD/VT was determined at anaerobic threshold from exhaled CO2 and transcutaneous Pco2 (validated against Paco2 measurements). Cardiac stroke volume reserve was estimated at rest and at anaerobic threshold by using oxygen consumption/pulse and previously validated estimates of the arteriovenous oxygen content difference. RESULTS: Cardiopulmonary exercise tests were performed on 40 patients with post-PE dyspnea. In 65.0% (95% CI, 50.2-79.8), VD/VT at anaerobic threshold was abnormally elevated, stroke volume reserve was decreased, or both defects occurred. VD/VT at anaerobic threshold was abnormally elevated (≥ 0.27) in 35.0% (95% CI, 20.2-49.8). VD/VT at anaerobic threshold significantly correlated with the extent of unmatched perfusion defects on subsequent ventilation-perfusion scans (P = .0085). In 55.0% (95% CI, 39.6-70.4), stroke volume reserve at anaerobic threshold was abnormally decreased (≤ 128% of the resting value). Both defects were present in 25.0% (95% CI, 11.6-38.4). CONCLUSIONS: Increased VD/VT at anaerobic threshold and decreased stroke volume reserve during exercise are common among patients with dyspnea on exertion after long-term treatment of PE. The defects can be disclosed noninvasively by using cardiopulmonary exercise testing.

Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients.

INTRODUCTION: Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients. MATERIALS AND METHODS: A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients. RESULTS: Baseline cfDNA concentration correlated with pre-treatment tumour burden (ρ = 0.52, P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/µl identified two distinct prognostic groups (HR = 2.22 for high cfDNA, P = 0.004). Patients with cfDNA ≥89 pg/µl had shorter OS (10.0 versus 22.7 months, P = 0.009; HR = 2.22 for high cfDNA, P = 0.004) and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden (ρ = 0.49, P = 0.002). In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(µl*cm3), P = 0.024). CONCLUSIONS: We have demonstrated that cfDNA is a surrogate marker of tumour burden in metastatic melanoma patients, and that it is prognostic for overall survival.

A cross-sectional survey of Australian chicken farms to identify risk factors associated with seropositivity to Newcastle-disease virus.

Several outbreaks of virulent Newcastle-disease occurred in Australia in 1998-2000. We conducted a cross-sectional survey of 753 Australian chicken farms to identify risk factors associated with the seroprevalence of chicken flocks with Newcastle-disease virus (NDV). We had a 99.7% response rate to the survey and the overall prevalence of NDV seropositive farms was 39.8%. Associations were analysed for the layer, chicken-meat and breeder production sectors in sector-specific logistic-regression models using 187, 198 and 146 farms, respectively. In the layer sector, increased risk of seroprevalence was associated with increasing age of the chickens, and decreased risk when the nearest-neighbour poultry farm was >10 km distant (odds ratio (OR)=0.30). In the chicken-meat sector, increased risk of seroprevalence was associated with location in the Sydney basin (OR=13.67), eastern Victoria (OR=26.10) or western Victoria (OR=5.43), and decreased risk when the nearest-neighbour poultry farm was greater than 0.5 km distant (OR=0.34). In the breeder sector, increased risk of seroprevalence was associated with increasing age of the chickens, the presence of wild birds on the farm (OR=5.28) and location in eastern Victoria (OR=16.19). A conditional logistic-regression for 112 pairs of farms matched for age, survey region and production sector identified a distance of >1.0 km to the nearest-neighbour poultry farm (OR=0.24) and ownership by owner 2 (OR=0.02), owner 5 (OR=0.11) or owner 9 (OR=0.25) as significant in reducing the risk of NDV seroprevalence. Our survey found that high levels of biosecurity and hygiene practices had been adopted by most farms.

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice.

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 µL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a ß-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.

Serosurvey of selected zoonotic agents in polar bears (Ursus maritimus).

Between 1982 and 1999 blood samples were collected from 500 polar bears (Ursus maritimus) captured in the Beaufort and Chukchi seas, to determine the seroprevalence of Brucella species, Toxoplasma gondii, and Trichinella species infections. The bears were classified into four age groups, cubs, yearlings, subadults and adults. Brucella and Toxoplasma antibodies were detected by agglutination (a buffered acidified card antigen and rapid automated presumptive test for brucellosis and a commercial latex agglutination test for toxoplasmosis); an ELISA was used to detect Trichinella antibodies. The overall seroprevalence of Brucella species was 5 per cent, and subadults and yearlings were 2-62 times (95 per cent confidence interval 1.02 to 6.82) more likely to be seropositive for Brucella species than adults and their cubs. The antibody prevalence for Toxoplasma gondii was 6 per cent, and for Trichinella species 55.6 per cent. The prevalence of antibodies to Trichinella species increased with age (P<0.001).

Vacuum-assisted wound closure provides early fascial reapproximation in trauma patients with open abdomens.

BACKGROUND: Damage control and decompressive laparotomies salvage severely injured patients who would have previously died. Unfortunately, many of these patients develop open abdomens. A variety of management strategies exist. The end result in many cases, however, is a large ventral hernia that requires a complex repair 6 to 12 months after discharge. We instituted vacuum-assisted wound closure (VAWC) to achieve early fascial closure and eliminate the need for delayed procedures. METHODS: For 12 months ending June 2000, 14 of 698 trauma intensive care unit admissions developed open abdomens and were managed with VAWC dressing. This was changed every 48 hours in the operating room with serial fascial approximation until complete closure. RESULTS: Fascial closure was achieved in 13 patients (92%) in 9.9 +/- 1.9 days, and 2.8 +/- 0.6 VAWC dressing changes were performed. There were 2 wound infections, no eviscerations, and no enteric fistulas. CONCLUSIONS: Use of VAWC can safely achieve early fascial closure in more than 90% of trauma patients with open abdomens.

Are alpha-gliadins glycosylated?

Alpha-gliadins isolated by carboxymethylcellulose chromatography contain noncovalently bound glucose probably due to contaminating proteoglycans and to material shed from the column. Traces of carbohydrate remain strongly bound to alpha-gliadins even after harsh denaturation, but our results indicate alpha-gliadins are not glycoproteins. Suggestions that gliadins are glycoproteins are probably due to contamination with this glucose and the presence of these proteoglycans.

Ototoxicity after high-dose chemotherapy with cyclophosphamide, thiotepa and carboplatin followed by stem cell transplantation in patients with breast cancer.

Our purpose was to determine the risk of ototoxicity in breast cancer patients receiving a myeloablative regimen consisting of cyclophosphamide 6000 mg/m2, thiotepa 500 mg/m2 and carboplatin 800 mg/m2 (CTCb) followed by stem cell transplantation. Fourteen consecutive patients with breast cancer were treated with high dose chemotherapy consisting of the CTCb regimen followed by stem cell transplantation. A pretransplant complete hearing study was obtained which consisted of hearing case history, audiometry and tympanometry. In addition, DPOAE (Distortion Product Otoaccoustic Emissions) was done to evaluate measurable changes in the cochlear (outer hair cell) functioning. Pre-transplant, all patients had no clinical evidence of hearing impairment and hearing studies were normal. Eleven patients had hearing studies and a telephone interview posttransplant. One patient was lost to follow-up and two patients died. One of the 11 patients tested had an abnormal post-transplant hearing study but none of them had clinically detectable hearing impairment. In our prospective study of breast cancer patients treated with the CTCb regimen, we did not observe clinically detectable hearing impairment in any of the patients tested.

Effects of environmental heat and intake of tall fescue seed infested with Acremonium coenophialum on the acid-base status of young bulls.

Effects of high environmental temperature and dietary intake of tall fescue (Festuca arundinacea) seed containing the endophyte Acremonium coenophialum on bovine acid-base status were studied using 3 groups of bull calves (2 Simmental, 1 Angus). Experimental animals were housed in controlled-climate chambers and subjected to gradual increases in environmental temperature, first while being fed an endophyte-free diet and then while being fed a diet containing 17% endophyte-infested fescue seed. Marked acid-base disturbances were not observed in any animals. In general, Pco2, HCO3-, base excess, and arterial blood pH values were reduced in response to heat stress, both with endophyte-free and endophyte-containing diets. In most individuals anion gap increased. These results reflected metabolic compensation for mild chronic alveolar hyperventilation and retention of organic acids. These findings suggest that, under conditions similar to those found during the summer in central Missouri, normal cattle should not be at great risk of developing respiratory alkalosis or other severe acid-base disturbances as a result of heat stress and/or intake of tall fescue endophyte.

Geographical distribution of organochlorine pesticides (OCPs) in polar bears (Ursus maritimus) in the Norwegian and Russian Arctic.

Geographical variation of organochlorine pesticides (OCPs) was studied in blood samples from 90 adult female polar bear (Ursus maritimus) from Svalbard, Franz Josef Land, Kara Sea, East-Siberian Sea and Chukchi Sea. In all regions, oxychlordane was the dominant OCP. Regional differences in mean levels of HCB, oxychlordane, trans-nonachlor, alpha-HCH, beta-HCH and p,p'-DDE were found. The highest levels of oxychlordane, trans-nonachlor and DDE were found in polar bears from Franz Josef Land and Kara Sea. HCB level was lowest in polar bears from Svalbard. Polar bears from Chukchi Sea had the highest level of alpha- and beta-HCH. The lowest alpha-HCH concentration was found in bears from Kara Sea. In all the bears, summation operator HCHs was dominated by beta-HCH. The geographical variation in OCP levels and pattern may suggest regional differences in pollution sources and different feeding habits in the different regions. Polar bears from the Western Russian Arctic were exposed to higher levels of chlordanes and p,p'-DDE than polar bears from locations westwards and eastwards from this region. This may imply the presence of a significant pollution source in the Russian Arctic area. The study suggests that the western Russian Arctic is the most contaminated region of the Arctic and warrants further research.

Influence of forage level on passage rate, digestibility and performance of cattle.

Two trials (feedlot and metabolism) were conducted to evaluate the influence of level of chopped tall fescue hay (FH) in high concentrate diets on average daily gain (ADG), liquid and particulate passage rates, digestibility and in situ digestibility of corn. In the feedlot trial, 36 Hereford steers were fed diets containing 15, 30 and 50% FH in combination with 74, 59 and 39% whole shelled corn (WSC) and a soybean meal supplement. Steers offered 15, 30 and 50% FH consumed 9.0, 9.0 and 7.6 kg dry matter (DM) per d; gained 1.19, .89 and .67 kg; and had DM to gain ratios of 7.6, 10.1 and 11.5, respectively. A negative correlation was observed between fecal pH and ADG (r = -.52) and between fecal pH and fecal starch (r = -.40). In a 4 X 4 Latin-square trial, four cannulated steers were fed 4, 8, 16 or 24% FH in combination with 86, 82, 74 or 66% WSC and a soybean meal supplement. After 14 d of adaptation, steers were offered ytterbium (Yb)-labelled WSC and were ruminally pulse-dosed with chromium ethylenediaminetetraacetic acid (Cr-EDTA) on the first day of the collection period. Steers fed 4, 8, 16 or 24% FH had the following particulate passage rates: 2.3, 2.7, 2.7 and 2.9%/h from fecal analyses; 2.3, 1.7, 2.4 and 2.8%/h from ruminal analyses; 6.0, 5.3, 6.3 and 8.1%/h for liquid, respectively. With increasing FH level, liquid passage rate exhibited linear and quadratic effects (P less than .05), while particulate passage rate (rumen sampling) showed linear and cubic effects (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Thermoregulatory ability of beef heifers following intake of endophyte-infected tall fescue during controlled heat challenge.

Consumption of endophyte-infected tall fescue during summer months can result in severe hyperthermia in cattle. Six heifers (296+/-8.3 kg BW) were used to determine changes in body temperature control that occur with consumption of an endophyte-infected tall fescue diet during controlled heat challenge. All animals were exposed in five separate periods to a step increase in ambient temperature (Ta) from 21 to 31 degrees C while fed E+ (5 microg ergovaline x kg(-1) x d(-1)) or endophyte-free (E-) diets. Core body temperature (Tcore) was monitored continuously using implanted, telemetric temperature transmitters. Heat production and heat loss were also measured at selected times to identify primary effects of E+ on thermal balance. Pretreatment Tcore exhibited a diurnal rhythm at a constant Ta of 21 degrees C, with high and low values at 2300 and 1300, respectively. An increase in daily averaged Tcore (P < 0.001) occurred with an increase in Ta from 21 to 31 degrees C. Likewise, all phases of the daily cycle increased equally during this challenge. This increase at 31 degrees C was associated with higher levels of respiration rate, skin temperature, respiratory vaporization, and skin vaporization (P < 0.05) and lower blood levels of thyroxine (P < 0.05). Intake of the E+ diet further elevated Tcore in heifers during the short-term heat challenge (2 d), and the effect was most pronounced at 0000 to 0300 and declined thereafter. The increase in Tcore during E+ treatment was associated with an increase in respiration rate (P < 0.05), whereas metabolic heat production, skin temperature, skin vaporization and respiratory vaporization were unaffected. These results show that consumption of an E+ diet during continuous heat challenge results in a marked increase in core body temperature, especially during nighttime exposure to heat stress, due primarily to a reduction in cutaneous heat transfer, with no effect on heat production or other measured avenues of heat loss.

Performance, forage utilization, and ergovaline consumption by beef cows grazing endophyte fungus-infected tall fescue, endophyte fungus-free tall fescue, or orchardgrass pastures.

Two 120-d trials (May to September, 1988 and 1989) determined the effects of grazing tall fescue (two varieties) or orchardgrass on forage intake and performance by beef cows. Each summer, 48 cow-calf pairs grazed endophyte-infected Kentucky-31 tall fescue (KY-31), endophyte-free Mozark tall fescue (MOZARK), or Hallmark orchardgrass (OG) pastures (16 pairs/treatment). Forage OM intakes and digestibilities were determined during June and August each year. Cow and calf BW and milk production were determined every 28 d. During June of both years, OM intakes did not differ (P greater than .10) among treatments. During August of 1988, intakes were 18% lower (P less than .05) by KY-31 cows (1.6% of BW) than by MOZARK or OG cows (average 1.95% of BW); however, no differences (P greater than .10) were measured in August of 1989. Estimates of ergovaline consumption during June from KY-31 were between 4.2 (1988) and 6.0 mg/d (1989), whereas August estimates were between 1.1 (1988) and 2.8 mg/d (1989). Ergovaline in MOZARK estrusa was below detection limits, except in August of 1989. Cows that grazed KY-31 lost three times (P less than .01) more BW than cows that grazed MOZARK or OG (42 vs 9 and 13 kg, respectively). Milk production by KY-31 cows was 25% lower (P less than .01) than that by cows that grazed MOZARK or OG (6.0 vs average of 8.0 kg/d). Similarly, slower (P less than .01) calf gains were noted for KY-31 than for MOZARK or OG (.72 vs .89 and .88 kg/d, respectively). Cows grazing KY-31 experienced accelerated BW loss and reduced milk production and weaned lighter calves than did cows grazing MOZARK or OG. Decreased performance was not explained by consistently reduced forage intakes; hence, altered nutrient utilization was suspected.

Temperature-dependent responses of rats to ergovaline derived from endophyte-infected tall fescue.

Ergovaline, found in Acremonium coenophialum-infected tall fescue, is considered to be responsible for many symptoms associated with fescue toxicosis. Rats were tested to determine time-related thermoregulatory responses to acute treatment with ergovaline during specific thermal challenges. Isolated ergovaline was administered to rats (15 micrograms/kg body mass, i.p.) at controlled ambient temperatures (Ta) of 7 to 9 degrees C (cold) and 31 to 33 degrees C (hot). Treatment at cold Ta resulted in a decrease in rectal temperature (Tre) from 38.0 to 37.0 degrees C at 50 min after injection (P < .05) without complete return to preinjection value at 100 min. Tail temperature (Ttail) exhibited a concomitant 1C degree decrease (P < .05) after ergovaline injection at cold Ta. Also, metabolic heat production decreased from 15.7 to 11.7 W/kg at 20 min after injection of ergovaline (P < .05), with return to normal value at 40 min after injection. Injection of ergovaline at hot Ta increased Tre from 39.0 to 40.6 degrees C at 80 min after injection (P < .05), with no return to preinjection value at 100 min. This was attributed to a reduction in heat transfer across the tail as indicated by the decrease in Ttail from 37.1 to 36.4 degrees C at 40 min after injection (P < .05) and an initial increase in metabolic heat production from 8.4 to 9.4 W/kg at 3 min after injection (P < .05). Ambient temperature was found to be a major determinant of ergovaline response, which included alterations in both thermogenic and thermolytic mechanisms that control thermal balance.

Performance, body composition and carcass characteristics of finishing steers as influenced by previous forage systems.

To evaluate effects of previous forage systems on feedlot performance, yearling Hereford steers (average initial weight of 249 kg) were grazed on tall fescue (TF), smooth bromegrass-red clover (BG-RC) or orchardgrass-red clover (OG-RC) pastures before finishing. Serial slaughter was utilized during the first 2 yr of this study to determine changes in carcass characteristics throughout finishing, while steers were slaughtered at approximately 29% body fat during the third year. Steers grazing TF entered the feedlot at lighter weights and maintained lighter weights throughout finishing (P less than .05) even though dry matter intakes and feed conversions were similar (P greater than .05) among treatments. Steers that previously grazed TF had less (P less than .05) body fat, body protein, fat thickness and marbling, smaller (P less than .05) ribeye areas and lower (P less than .05) USDA yield and quality grades than than those that grazed BG-RC and OG-RC. However, linear contrasts indicated that steers grazing TF were compensating in ribeye area, marbling and quality grade as days in feedlot increased. This was confirmed in the third-year study, since carcass characteristics were similar among all steers. During the third year, linear and quadratic contrasts indicated that steers that grazed TF partially compensated in body protein. As days in feedlot increased, live and hot carcass weights, body fat, fat thickness, kidney, pelvic and heart fat, marbling, and USDA yield and quality grades increased (P less than .05), while ribeye area and body protein plateaued. Although carcass characteristics were similar among treatments, steers backgrounded on TF entered the feedlot at lighter weights, and partially compensated in weight after 134 d of finishing.

Videothermometry for assay of fescue foot in cattle.

Surface temperatures of hoofs of calves given toxic anion fractions of tall fescue were measured with an infrared sensitive camera. These changes expressed in terms of a weighted average coronary band temperature relate to clinical signs of fescue foot. The weighted average coronary band temperature values for control calves given saline solution were 27 to 31 C; those values of test calves given anion fractions of tall fescue were as low as 22 +/- 1 C. Videothermometry provides an independent, permanent, objective measure that is useful in assessing fescue-foot potential of tall fescue fractions by intraperitoneal injection. Videothermometry may also serve as a clinical means for determining progression of the total fescue foot syndrome.

Induction of fescue foot syndrome in cattle by fractionated extracts of toxic fescue hay.

Tall fescue (Festuca arundinacea Shreb) hay from a source known to cause "fescue foot" in grazing cattle was extracted with 80% ethanol. The ethanolic extract was further refined and fractionated into cation,nion, and neutral f fractions by ion-exchange chromatography. The cation fraction was partitioned with alkaline-chloroform to give chloroform-extractable cation and residual cation fractions. All fractions plus the crude ethanolic extract were assayed for toxic activity by intraperitoneal injection into 12 calves (weighting 152.4 to 241.3 kg each) over a 14-day period. Clinical signs of fescue foot were observed on the 5th day in calves given the anion and crude ethanolic extracts. Lameness, swelling, and reddening of the rear coronary bands, discoloration of the tip of the tail, and other signs of fescue foot were seen. Microscopically, coronary bands and tail tips of affected calves had blood vessels with thick walls and small lumens.

Occurrence of rabies in a wolf population in northeastern Alaska.

Nine Alaskan wolves (Canis lupus) were found dead during spring and summer 1985; five of seven animals tested for rabies virus were positive. The 1985 epizootic altered annual den use patterns by wolves in northeastern Alaska, but did not appear to affect population size. We propose that rabies in arctic wolves may be more common than previously thought.

Serological evidence of morbillivirus infection in polar bears (Ursus maritimus) from Alaska and Russia.

One-hundred-and-ninety-one samples of blood serum collected from 186 polar bears (Ursus maritimus) between 1987 and 1992 were analysed for morbillivirus antibodies. The samples were collected in the Bering, Chukchi and East Siberian seas. Sixty-eight samples (35.6 per cent) had morbillivirus antibody titres > 5; the percentage of positive samples ranged from 26.2 to 46.2 per cent from year to year. The proportions of adults, sub-adults and cubs which were seropositive were 43.9, 35.7 and 37.9 per cent respectively. Some seropositive dams had seronegative young and some that were seronegative had seropositive young. One litter of two cubs, in which the dam was seronegative, had one seropositive and one seronegative cub. Seropositive bears occurred in all the areas from which the samples were collected but there was a significantly greater incidence in the bears sampled in Russia. The high prevalence of seropositive bears over the period suggests that the bear morbillivirus is endemic in these regions of the Arctic, but its source is unknown.