Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions.

BACKGROUND: In a significant percentage of advanced non-small-cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses. We prospectively analyzed if circulating-free DNA (cfDNA) purified from blood can be used as a surrogate in this setting to select patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). PATIENTS AND METHODS: Blood samples were collected in 119 hospitals from 1138 advanced NSCLC patients at presentation (n = 1033) or at progression to EGFR-TKIs (n = 105) with no biopsy or insufficient tumor tissue. Serum and plasma were sent to a central laboratory, cfDNA purified and EGFR mutations analyzed and quantified using a real-time PCR assay. Response data from a subset of patients (n = 18) were retrospectively collected. RESULTS: Of 1033 NSCLC patients at presentation, 1026 were assessable; with a prevalence of males and former or current smokers. Sensitizing mutations were found in the cfDNA of 113 patients (11%); with a majority of females, never smokers and exon 19 deletions. Thirty-one patients were positive only in plasma and 11 in serum alone and mutation load was higher in plasma and in cases with exon 19 deletions. More than 50% of samples had <10 pg mutated genomes/µl with allelic fractions below 0.25%. Patients treated first line with TKIs based exclusively on EGFR positivity in blood had an ORR of 72% and a median PFS of 11 months. Of 105 patients screened after progression to EGFR-TKIs, sensitizing mutations were found in 56.2% and the p.T790M resistance mutation in 35.2%. CONCLUSIONS: Large-scale EGFR testing in the blood of unselected advanced NSCLC patients is feasible and can be used to select patients for targeted therapy when testing cannot be done in tissue. The characteristics and clinical outcomes to TKI treatment of the EGFR-mutated patients identified are undistinguishable from those positive in tumor.

Narrowing the agronomic yield gap with improved nitrogen use efficiency: a modeling approach.

Improving nitrogen use efficiency (NUE) in the major cereals is critical for more sustainable nitrogen use in high-input agriculture, but our understanding of the potential for NUE improvement is limited by a paucity of reliable on-farm measurements. Limited on-farm data suggest that agronomic NUE (AE(N)) is lower and more variable than data from trials conducted at research stations, on which much of our understanding of AE(N) has been built. The purpose of this study was to determine the magnitude and causes of variability in AE(N) across an agricultural region, which we refer to as the achievement distribution of AE(N). The distribution of simulated AE(N) in 80 farmers' fields in an irrigated wheat system in the Yaqui Valley, Mexico, was compared with trials at a local research center (International Wheat and Maize Improvement Center; CIMMYT). An agroecosystem simulation model WNMM was used to understand factors controlling yield, AE(N), gaseous N emissions, and nitrate leaching in the region. Simulated AE(N) in the Yaqui Valley was highly variable, and mean on-farm AE(N) was 44% lower than trials with similar fertilization rates at CIMMYT. Variability in residual N supply was the most important factor determining simulated AE(N). Better split applications of N fertilizer led to almost a doubling of AE(N), increased profit, and reduced N pollution, and even larger improvements were possible with technologies that allow for direct measurement of soil N supply and plant N demand, such as site-specific nitrogen management.

Simulating the influence of vernalization, photoperiod and optimum temperature on wheat developmental rates.

BACKGROUND AND AIMS: Accurately representing development is essential for applying crop simulations to investigate the effects of climate, genotypes or crop management. Development in wheat (Triticum aestivum, T. durum) is primarily driven by temperature, but affected by vernalization and photoperiod, and is often simulated by reducing thermal-time accumulation using vernalization or photoperiod factors or limiting accumulation when a lower optimum temperature (T(optl)) is exceeded. In this study T(optl) and methods for representing effects of vernalization and photoperiod on anthesis were examined using a range of planting dates and genotypes. METHODS: An examination was made of T(optl) values of 15, 20, 25 and 50 degrees C, and either the most limiting or the multiplicative value of the vernalization and photoperiod development rate factors for simulating anthesis. Field data were from replicated trials at Ludhiana, Punjab, India with July through to December planting dates and seven cultivars varying in vernalization response. KEY RESULTS: Simulations of anthesis were similar for T(optl) values of 20, 25 and 50 degrees C, but a T(optl) of 15 degrees C resulted in a consistent bias towards predicting anthesis late for early planting dates. Results for T(optl) above 15 degrees C may have occurred because mean temperatures rarely exceeded 20 degrees C before anthesis for many planting dates. For cultivars having a strong vernalization response, anthesis was more accurately simulated when vernalization and photoperiod factors were multiplied rather than using the most limiting of the two factors. CONCLUSIONS: Setting T(optl) to a high value (30 degrees C) and multiplying the vernalization and photoperiod factors resulted in accurately simulating anthesis for a wide range of planting dates and genotypes. However, for environments where average temperatures exceed 20 degrees C for much of the pre-anthesis period, a lower T(optl) (23 degrees C) might be appropriate. These results highlight the value of testing a model over a wide range of environments.

Multi-Temporal and Spectral Analysis of High-Resolution Hyperspectral Airborne Imagery for Precision Agriculture: Assessment of Wheat Grain Yield and Grain Protein Content.

This study evaluates the potential of high resolution hyperspectral airborne imagery to capture within-field variability of durum wheat grain yield (GY) and grain protein content (GPC) in two commercial fields in the Yaqui Valley (northwestern Mexico). Through a weekly/biweekly airborne flight campaign, we acquired 10 mosaics with a micro-hyperspectral Vis-NIR imaging sensor ranging from 400-850 nanometres (nm). Just before harvest, 114 georeferenced grain samples were obtained manually. Using spectral exploratory analysis, we calculated narrow-band physiological spectral indices-normalized difference spectral index (NDSI) and ratio spectral index (RSI)-from every single hyperspectral mosaic using complete two by two combinations of wavelengths. We applied two methods for the multi-temporal hyperspectral exploratory analysis: (a) Temporal Principal Component Analysis (tPCA) on wavelengths across all images and (b) the integration of vegetation indices over time based on area under the curve (AUC) calculations. For GY, the best R2 (0.32) were found using both the spectral (NDSI-Ri, 750 to 840 nm and Rj, ±720-736 nm) and the multi-temporal AUC exploratory analysis (EVI and OSAVI through AUC) methods. For GPC, all exploratory analysis methods tested revealed (a) a low to very low coefficient of determination (R2 ≤ 0.21), (b) a relatively low overall prediction error (RMSE: 0.45-0.49%), compared to results from other literature studies, and (c) that the spectral exploratory analysis approach is slightly better than the multi-temporal approaches, with early season NDSI of 700 with 574 nm and late season NDSI of 707 with 523 nm as the best indicators. Using residual maps from the regression analyses of NDSIs and GPC, we visualized GPC within-field variability and showed that up to 75% of the field area could be mapped with relatively good predictability (residual class: -0.25 to 0.25%), therefore showing the potential of remote sensing imagery to capture the within-field variation of GPC under conventional agricultural practices.

[Single fibre electromyography in 18 patients with amyotrophic lateral sclerosis]. / Electromiograma de fibra única en 18 pacientes con esclerosis lateral amiotrófica.

INTRODUCTION: Single fibre electromyography (SFEMG) is a method that complements conventional electromyography and studies the activity of individual muscle fibres and of the neuromuscular junction. AIMS. We attempt to demonstrate the presence of reinnervation disorders by studying Jitter, fibre density (FD) and the presence of neurogenic blocks in patients with amyotrophic lateral sclerosis (ALS). PATIENTS AND METHODS: SFEMG was carried out on 18 patients diagnosed with ALS. All the patients were submitted to a voluntary SFEMG in the extensor digitorum communis muscle of the fingers on the most affected side. RESULTS: The SFEMG in the extensor digitorum communis muscle of the fingers showed alterations in 13 out of the 18 patients studied (72%). FD was the most reliable parameter for translation of the motor neuron instability and increased in all cases. This was followed by the mean and the maximum individual (62%) Jitter and, lastly, the percentage of blocks (38%). CONCLUSIONS: In ALS, the parameters studied with SFEMG were modified in 72% of the cases studied. There is an overall correlation between the length of time the patient has had the disease and the modifications in the SFEMG (21.5 versus 13.7 months). The degree of clinical involvement is higher (6.5/13) in those who present an altered SFEMG compared to those who present a normal SFEMG (5.3/13).

Matrix metalloproteinase expression is related to hemorrhagic transformation after cardioembolic stroke.

BACKGROUND AND PURPOSE: In animal models of cerebral ischemia, matrix metalloproteinase (MMP) expression was significantly increased and related to blood-brain barrier disruption, edema formation, and hemorrhagic transformation (HT). MMP inhibitors reduce HT after embolic ischemia in tissue-type plasminogen activator-treated animals. We aimed to determine the relationship between MMPs and HT after human ischemic stroke. METHODS: Serial MMP-2 and MMP-9 determinations were performed by means of ELISA in 39 cardioembolic strokes in the middle cerebral artery territory. Hemorrhagic events were classified according to clinical and CT criteria (hemorrhagic infarction [HI] and parenchymal hematoma [PH]). HT was evaluated on CT at 48 hours (early HT) and again between day 5 and 7 (late HT). RESULTS: HT was present in 41% of the patients (43.75% early HI, 25% early PH and 31.25% late HI). MMP-2 values were within normal range and were unrelated to HT. Increased expression of MMP-9 (normal range <97 ng/mL) was found among patients with and without HT (159.3+/-82 versus 143.9+/-112.6 ng/mL; P=0.64). According to HT subtypes, the highest baseline MMP-9 levels corresponded to patients with late HI (240.4+/-111.2 versus 102.5+/-76.7 ng/mL for all other patients, P=0.002). Baseline MMP-9 was the only variable associated with late HI in the multiple logistic regression model (OR 9; CI 1.46, 55.24; P=0.010). Peak of MMP-9 at the 24-hour time point (250.6 ng/mL) was found before appearance of PH. CONCLUSIONS: MMPs are involved in some subtypes of HT after human cardioembolic stroke. Baseline MMP-9 level predicts late HI and a 24-hour peak precedes early PH.

Matrix metalloproteinase expression after human cardioembolic stroke: temporal profile and relation to neurological impairment.

BACKGROUND AND PURPOSE: Uncontrolled expression of matrix metalloproteinases (MMPs) can result in tissue injury and inflammation. In animal models of cerebral ischemia, the expression of MMP-2 and MMP-9 was significantly increased. However, their role in human stroke in vivo remains unknown. Therefore, we sought to determine the temporal profile of MMP expression in patients with acute ischemic stroke and to investigate its relationship to stroke severity, location of arterial occlusion, and total infarct volume. METHODS: Serial MMP-2 and MMP-9 determinations were made in 39 patients with cardioembolic strokes that involved the middle cerebral artery territory by means of enzyme-linked immunosorbent assay. Blood samples, transcranial Doppler recordings, and National Institutes of Health Stroke Scale (NIHSS) scores were obtained at baseline and at 12, 24, and 48 hours after stroke onset. Infarct volume was measured with CT scanning at 48 hours. RESULTS: No correlation was found between MMP-2 and NIHSS score at any time point, although a close relation appeared between mean MMP-9 and final NIHSS score (r=0.486, P=0.002). MMP-9 value was the only factor associated with the final NIHSS score in the multiple logistic regression model (OR 4.54, 95% CI 1.5 to 13.75). A cut-point of MMP-9 142.18 ng/mL had a positive predictive value of 94.4% to assess a patient's NIHSS (<8 or >/=8) by the end of the study. Final MMP-2 and MMP-9 levels were significantly lower when recanalization occurred (528+/-144.3 versus 681.4+/-239.2 ng/mL, P=0.031 for MMP-2; 110.2+/-100.9 versus 244.8+/-130 ng/mL, P=0.004 for MMP-9). A positive correlation was found between mean MMP-9 and infarct volume (r=0.385, P=0.022). CONCLUSIONS: MMPs are involved in the acute phase of human ischemic stroke. MMP-9 levels are associated with neurological deficit, middle cerebral artery occlusion, and infarct volume.

Effects of two different doses of hirudin on APTT, determined with eight different reagents.

The APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study. Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica-based reagents. In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica-containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

Patients with geleophysic dysplasia are not always geleophysic.

We report on two boys with facial anomalies, small hands and feet, joint contractures, thick skin, unusual tiptoe gait and lysosome-like inclusions in the hepatocytes, compatible with a diagnosis of geleophysic dysplasia (GD). One of them also had fibrosis and fatty degeneration of the liver. In both, the facial appearance was different and neither had short stature nor progressive cardiac valvular disease. These clinical findings, consistent with a mild form of GD, support the notion that this disorder may have a broader spectrum than initially suspected.

Heparin cofactor II: a new marker for pre-eclampsia.

The plasma levels of heparin cofactor II were determined in pregnant women who were either normotensive, or had essential hypertension, gestational hypertension or pre-eclampsia during the third trimester and 72 h after delivery. Heparin cofactor II levels in the pre-eclampsia group were depressed. The clinical relevance of this finding is the potential utility of heparin cofactor II plasma levels in the differential diagnostic between non-proteinuric hypertension and pre-eclampsia.

Evaluation of thrombin-antithrombin complexes and fibrin fragment D in carbohydrate-induced acute laminitis.

Carbohydrate-induced laminitis has been associated with decreased platelet survival, decreased blood flow to the hoof wall and with the deposition of platelets and microthrombi within venules in the dermal laminae. To evaluate further the systemic prothrombotic events occurring in the prodromal stages of laminitis, plasma samples from control and laminitis-affected ponies and horses were tested for the presence of thrombin-antithrombin (TAT) complexes and fibrin fragment D (D-dimer). No statistically significant differences between the control and laminitis-affected animals were observed for either the D-dimer or the TAT complexes. Few of the values for individual animals exceeded the reference ranges for control animals. These data indicate that the prothrombotic events observed in carbohydrate-induced laminitis may not be associated with systemic activation of the coagulation or fibrinolytic systems.

Hemorheological, coagulative and fibrinolytic changes during autologous blood donation.

BACKGROUND: Clinical data suggest that autologous blood donation may prevent postsurgical venous thrombosis. If confirmed, this is probably due to beneficial effects in rheologic and hematologic variables which may be changed in patients as a result of repeated bleeding. STUDY DESIGN AND METHODS: To ascertain this point, we studied variations in hematological, hemorheological, coagulative and fibrinolytic parameters in 30 patients undergoing autologous blood donation. RESULTS: Whole blood viscosity (WBV), plasma viscosity and blood viscosity adjusted to 40% hematocrit, progressively and substantially decreased throughout the successive bleeding at all the shear rates considered. WBV was further reduced by presurgical hemodilution with autologous plasma which decreased the platelet and leukocyte count. The hemostasis and fibrinolysis variables, however, underwent no clinically significative changes. CONCLUSION: Repeated bleedings change most hemorheological variables. By decreasing cytocrit and viscosity, reducing aggregability and increasing blood cell deformability an optimal milieu to help prevent thrombosis is artificially created.

[Correlation between the expression of proinflammatory cytokines and matrix metalloproteinases in the acute phase of an ischemic stroke]. / Correlación entre la expresión de citocinas proinflamatorias y metaloproteinasas de matriz en la fase aguda del ictus isquémico.

INTRODUCTION: Proinflammatory cytokines are the main responsible for the onset of postischemic inflammatory cascade. Recently, the deleterious effect of matrix metalloproteinases (MMPs) in the acute phase of stroke has been described. Animal models suggest a link between both families. OBJECTIVE: We aimed to investigate possible relations between the MMP overproduction and proinflammatory cytokine expression after human ischemic stroke. PATIENTS AND METHODS: From all consecutive stroke patients attended during a 10 months period, we selected and prospectively studied those presenting as a cardioembolic stroke involving the MCA territory. MMP 9, MMP 2 and IL 6 were serially measured by means of ELISA at study entry and at 12, 24 and 48 hours after symptoms onset. RESULTS: A total of 39 patients were studied. A positive correlation was found between mean expression of both MMPs and IL 6 (r= 0.33, p= 0.040 for MMP 2 y r= 0.45, p= 0.004 for MMP 9). From all measured timepoints, the best obtained correlation was that of MMP 9 with IL 6 at 24 hours (r= 0.418, p= 0.010). At 24 h a peak value of IL 6 was observed. Baseline MMP 2 and MMP 9 levels showed a trend to correlate with that peak of IL 6 (r= 0.329, p= 0.061 for MMP 2 y r= 0.325, p= 0.061 for MMP 9). CONCLUSION: MMP expression correlates with the inflammatory cascade activation after acute cardioembolic stroke.