Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

Improved Aflatoxins and Fumonisins Forecasting Models for Maize (PREMA and PREFUM), Using Combined Mechanistic and Bayesian Network Modeling-Serbia as a Case Study.

Contamination of maize with aflatoxins and fumonisins is one of the major food safety concerns worldwide. Knowing the contamination in advance can help to reduce food safety risks and related health issues and economic losses. The current study aimed to develop forecasting models for the contamination of maize grown in Serbia with aflatoxins and fumonisins. An integrated modeling approach was used, linking mechanistic modeling with artificial intelligence, in particular Bayesian network (BN) modeling. Two of such combined models, i.e., the prediction model for aflatoxins (PREMA) and for fumonisins (PREFUM) in maize, were developed. Data used for developing PREMA were from 867 maize samples, collected in Serbia during the period from 2012 to 2018, of which 190 were also used for developing PREFUM. Both datasets were split randomly in a model training set and a model validation set. With corresponding geographical and meteorological data, the so-called risk indices for total aflatoxins and total fumonisins were calculated using existing mechanistic models. Subsequently, these risk indices were used as input variables for developing the BN models, together with the longitudes and latitudes of the sites at which the samples were collected and related weather data. PREMA and PREFUM were internally and externally validated, resulting in a prediction accuracy of PREMA of, respectively, 83 and 70%, and of PREFUM of 76% and 80%. The capability of PREMA and PREFUM for predicting aflatoxins and fumonisins contamination using data from the early maize growth stages only was explored as well, and promising results were obtained. The integrated approach combining two different modeling techniques, as developed in the current study, was able to overcome the obstacles of unbalanced data and deficiency of the datasets, which are often seen in historical observational data from the food safety domain. The models provide predictions for mycotoxin contamination at the field level; this information can assist stakeholders of the maize supply chain, including farmers, buyers/collectors, and food safety authorities, to take timely decisions for improved mycotoxin control. The developed models can be further validated by applying them into practice, and they can be extended to other European maize growing areas.

Drug-induced torsade de pointes arrhythmias in the chronic AV block dog are perpetuated by focal activity.

BACKGROUND: The electrically remodeled canine heart after chronic AV block (CAVB) has a high susceptibility for drug-induced torsade de pointes (TdP) arrhythmias. Although focal mechanisms have been considered for initiation, there is still controversy about whether reentry is the dominant mechanism for perpetuation of TdP. In this animal model with known nonuniform prolongation of repolarization, the mechanism of perpetuation of TdP arrhythmia was explored. METHODS AND RESULTS: Seventeen TdP-sensitive CAVB and 10 sinus rhythm (SR) dogs were studied. In 6 animals, 66 needle electrodes were evenly distributed transmurally to record 240 unipolar local electrograms simultaneously. Activation times and activation recovery intervals were determined before and during ibutilide-induced TdP. In 12 CAVB and 9 SR dogs, left ventricular (LV) and right ventricular (RV) epicardial electrograms were recorded with a 208-point multiterminal grid electrode allowing conduction velocity (CV) and ventricular effective refractory period (VERP) measurements. Biopsy specimens were processed for connexin43 (Cx43) expression and collagen content. Ventricular myocytes were isolated to determine sodium current (I(Na)) density and cell dimensions. Computer simulations were used to assess the effects of changes therein. In CAVB, VERP and ARI were increased, whereas CV was unaltered in LV. Transversal but not longitudinal CV was increased in RV. I(Na) was reduced by 37% in LV but unaltered in RV. LV and RV cell size were increased, but collagen and Cx43 content remained unchanged. Simulations showed increase in CV of RV as a consequence of increased cell size at normal I(Na). Ibutilide increased ARI, ERP, and maximal transmural dispersion of ERP (45 ± 25 to 120 ± 65 ms; P < 0.05). Twenty-eight of 47 episodes of self-terminating TdP (43 ± 72 beats) were analyzed. The majority (> 90%) of beats were focal; reentry was observed only occasionally. CONCLUSIONS: Focal activity is the dominant mechanism involved in perpetuation of ibutilide-induced TdP in CAVB dogs based on detailed 3D mapping. This conclusion is in line with unaltered conduction and documented increase in VERP.

Submicron plasticity: yield stress, dislocation avalanches, and velocity distribution.

The existence of a well-defined yield stress, where a macroscopic crystal begins to plastically flow, has been a basic observation in materials science. In contrast with macroscopic samples, in microcrystals the strain accumulates in random bursts, which makes controlled plastic formation difficult. Here we study by 2D and 3D simulations the plastic deformation of submicron objects under increasing stress. We show that, while the stress-strain relation of individual samples exhibits jumps, its average and mean deviation still specify a well-defined critical stress. The statistical background of this phenomenon is analyzed through the velocity distribution of dislocations, revealing a universal cubic decay and the appearance of a shoulder due to dislocation avalanches.

Dislocation avalanches, strain bursts, and the problem of plastic forming at the micrometer scale.

Under stress, many crystalline materials exhibit irreversible plastic deformation caused by the motion of lattice dislocations. In plastically deformed microcrystals, internal dislocation avalanches lead to jumps in the stress-strain curves (strain bursts), whereas in macroscopic samples plasticity appears as a smooth process. By combining three-dimensional simulations of the dynamics of interacting dislocations with statistical analysis of the corresponding deformation behavior, we determined the distribution of strain changes during dislocation avalanches and established its dependence on microcrystal size. Our results suggest that for sample dimensions on the micrometer and submicrometer scale, large strain fluctuations may make it difficult to control the resulting shape in a plastic-forming process.

Cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted mice.

BACKGROUND: Our department worked out a modified surgical form of spleen autotransplantation earlier, named "spleen apron method" introduced already into the clinical practice. Recently we tested the immunological changes in a group of patients autotransplanted with about 10-15% of their spleen, what was the at least always implantable amount after the severe splenic injuries. In the current work we aimed at measuring some cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted inbred mice two and eight months after the operations in order to get more unambiguous results than that we could obtain in our patients with this technique. MATERIALS AND METHODS: We divided 96 two months old Balb/c female mice into eight groups (n = 12/group). The group of controls, sham operated, splenectomized and autotransplanted animals with two and eight months of survival time after the operations. During the autotransplantation we inserted the same amount of spleen, five slices, "chips," about 10-15% of total mass of spleen, into the greater omentum similarly as it was used in the patients. The concentration of serum proteins were measured by laser nephelometry. The lymphocyte subsets were analyzed by flow cytometry. RESULTS: We found that two months after the operations the number of CD 19+ B-cells increased in the splenectomized but decreased in the autotransplanted animals. Eight months after the operations the number of both CD3+ T and CD19+ B lymphocytes decreased both in the splenectomized and autotransplanted animals compared to the controls and sham operated mice. However, the numbers of T and B cells were slightly but not significantly higher in the autotransplanted than in the splenectomized mice. The serum level of IgM was also decreased in the splenectomized and autotransplanted mice at both time points, however, eight months after the operations the concentration of IgM was significantly higher in the autotransplanted group than in the splenectomized animals. CONCLUSION: The effects of autotransplanted "chips" were different at the various ages of the animals. Additionally, they showed some immunological benefit being quantitatively in accordance to the amount of the transplanted spleen. The elevated level of serum IgM what we found in the autotransplanted mice even with this amount of transplanted spleen eight months after the operations, however, might have the potentially greatest importance compared to splenectomy. These experiments can prove that the attempts for autotransplantation may have real perspectives but their efficacy depends on the amount of the successfully transplanted (saved) mass of spleen.

Distribution of peripheral blood cells in mice after splenectomy or autotransplantation.

Our aim was to compare the distribution changes of peripheral leukocytes and erythrocytes in splenectomized and spleen-autotransplanted BALB/c female mice (n = 96), 2 and 8 months after surgery. In total, there were eight groups of animals: splenectomy, autotransplantation, sham, and untreated controls at both time points. We used the spleen-apron method of Furka et al. (Khirurgiia (Mosk) 1989;9:125-127), inserting five spleen chips into the greater omentum, for autotransplantation. Quantitative and qualitative blood cell counts and the phagocytic activity of cells (stimulated with zymosan) were determined. In splenectomized animals, the number of neutrophils significantly increased 8 months after surgery. The greatest phagocytic activity of neutrophils, however, was observed in autotransplanted animals of the same age. In splenectomized animals, erythrocyte volumes were significantly higher in the second postoperative month, but normalized by the eighth month. In conclusion, spleen autotransplantation has some beneficial effects, including clearing erythrocytes and preserving the phagocytic activity of neutrophils in peripheral blood.

Phosphorus-doped and undoped glassy carbon indicator electrodes in controlled-current potentiometric titrations of bromide- or chloride-containing active ingredients in some pharmaceutical preparations.

Phosphorus-doped glassy carbon (as a novel material) and glassy carbon (Sigri commercial sample) were applied as potentiometric indicator electrodes in the titrimetric determination of active components with bromide or chloride in their molecules in different pharmaceutical preparations (Buscopan, Prostigmine, Isoptin, Bedoxin, Akineton and Trodon). After the necessary pre-treatment of the electrode surfaces and sample dissolution, the halide was titrated with a standard solution of silver nitrate (indirect determination). Amounts of 10-20 micromol of the investigated active ingredients per titration were determined with a relative standard deviation that, depending on the nature of indicator electrode, determined molecules and filler components, was in the range of 0.3-2.7%. The results obtained were compared with those of the official methods and with those obtained by potentiometric titrations using silver electrode. The titrimetric procedures developed are relatively fast, easy, economical and can be used to analyse of a large number of pharmaceutical products.

Multiorgan transplantation with a new organ-chip technique in mice: preliminary histological data.

A simple model was developed for multiorgan liver-kidney-spleen-intestine transplantation on 108 inbred mice. Donor operations included hepatectomy, nephrectomy, splenectomy, and jejunum segment resection. Following removal of the organ, small slices or abdominal organ "chips" were prepared. During multiorgan recipient operations, chips from each of these organs were transplanted into the omentum; in the control single-organ groups, only 1 organ was transplanted. All animals survived. Biopsies were taken for histology after 6 weeks. All organs were found to have developed a blood supply. In the liver chips, hypertrophied cells could be detected. In the margin of the kidney tissue, both the glomeruli and tubules were preserved. Lymphoid zone and red pulp were intact in spleen chips. All layers of the intestinal chips were identifiable and contained intraluminal mucinous substances. This model is a simple surgical intervention with the possibility of the investigation of 4 organs.

Hematological, hemorheological, immunological, and morphological studies of spleen autotransplantation in mice: preliminary results.

Using a spleen autotransplantation model, we conducted hematological, hemorheological, immunological, and morphological studies in mice 6 weeks after splenectomy. Sixty male and female A/J inbred mice were equally divided into 3 groups: 1) SE group, splenectomy was performed; 2) AU group, spleen chips were autotransplanted into the omentum without vascular anastomosis following splenectomy; and 3) C group (controls), no intervention in these mice. At postoperative week 6, the following studies were performed: 1) measurement of hematological parameters; 2) hemorheological studies, including relative cell transit time (RCTT) and fibrinogen levels; and 3) activity of peripheral phagocytes, measured by zymozan-induced chemiluminescence, which was calculated in stimulation index values (SI). In addition, histological investigations of autotransplants were conducted. Erythrocyte mean cell volume and platelet counts, RCTT, fibrinogen levels, and activity of phagocytes were significantly higher in the SE group, compared to those in the C group. In the AU group, these parameters were similar to those in the C group. Morphologically, the transplanted spleen showed normal histology. These data indicate that the transplanted spleens restored their function. We conclude that spleen autotransplantation reserves the normal morphology of spleen and restores most of the spleen's hematological, hemorheological, and immunological functions. Both SI index and erythrocyte deformability can be an informative detection of decreasing splenic function. These data suggest that spleen autotransplantation may provide a useful tool to prevent complications following splenectomy in a clinical setting.