Regional differences in predictive biomarker testing rates for patients with metastatic NSCLC in the Netherlands.

BACKGROUND: Predictive biomarker testing has a key role in the treatment decision-making for patients with non-small cell lung cancer (NSCLC) and is mandated by (inter)national guidelines. The aim of this study was to establish guideline-adherent biomarker testing rates in the Netherlands in 2019 and to examine associations of demographical, clinical, and environmental factors with guideline-adherent testing. METHODS: This study involved the integration of clinical data of the Netherlands Cancer Registry with pathology reports of the Dutch Nationwide Pathology Databank. Data extracted from these reports included sample type, diagnosis, and molecular testing status of predictive biomarkers. The study population comprised all patients diagnosed with metastatic non-squamous NSCLC in the Netherlands in 2019. RESULTS: In the cohort of 3877 patients with metastatic non-squamous NSCLC under investigation, overall molecular testing rates for non-fusion predictive biomarkers (EGFR, KRAS, BRAF, ERBB2, MET) ranged from 73.9 to 89.0 %, while molecular testing for fusion-drivers (ALK, ROS1, RET, NTRK) ranged from 12.6 % to 63.9 %. Guideline-adherent testing of EGFR, KRAS, and ALK was performed in 85.2 % of patients, with regional rates spanning from 76.0 % to 90.8 %. Demographical and clinical factors associated with guideline-adherent biomarker testing included lower age (OR = 1.05 per one year decrease; p < 0.001), female sex (OR = 1.36; p = 0.002), diagnosis of adenocarcinoma (OR = 2.48; p < 0.001), availability of histological tumor material (OR = 2.46; p < 0.001), and clinical stage of metastatic disease (p = 0.002). Other factors associated with guideline-adherent biomarker testing included diagnosis at academic center (OR = 1.87; p = 0.002) and patient's region of residence (p < 0∙001). CONCLUSION: Optimization of the chain-of-care of predictive biomarker testing in patients with NSCLC in the Netherlands is needed to provide adequate care for these patients.

Tailoring the properties of (catalytically)-active inclusion bodies.

BACKGROUND: Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. RESULTS: We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. CONCLUSION: We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.

Angiogenesis and healing with non-shrinking, fast degradeable PLGA/CaP scaffolds in critical-sized defects in the rabbit femur with or without osteogenically induced mesenchymal stem cells.

Cost effective and safely to apply tissue engineered constructs of big volume bone transplants for the reconstruction of critical sized defects (CSD) are still not available. Key problems with synthetic scaffold materials are shrinkage and fast degradation of the scaffolds, a lack of blood supply and nutrition in the central scaffold volume and the absent or the scarce development of bone tissue along the scaffold to bridge the bone defect. The use of composite scaffolds made of biopolymers like polylactidglycolid acid (PLGA) coated and loaded with calcium phosphates (CaP) revealed promising therapeutical options for the regeneration of critical sized bone defects. In this study interconnectively macroporous PLGA scaffolds loaded with microporous and coated with nanoporous calcium phosphates were either seeded in fixed bed bioreactors with allogenic osteogenically induced mesenchymal stem cells and implanted or implanted unseeded into critical sized femoral bone defects. As CSD a 12 mm long segment of the chinchilla femur was excised where the proximal and distal parts of the femur were fixed and stabilized by the use of an eight-hole linear reconstruction plate and secured with three bicortical screws (2.7 mm diameter) on every side of the osteotomy. Aim of the study was if we could find a way to load and coat PLGA scaffolds with CaP so that shrinkage of scaffolds could be avoided, which would favour angiogenesis, blood supply and nutrition in the construct and thus avoid central necroses regularly observed so far in transplants not vascularized and which would be inhabited by cells of he bone lineage forming new bone and healing the defect. Four weeks, at least, a notable shrinkage of the scaffolds was avoided and scaffolds were practically not degraded. Both scaffolds, loaded and loaded and coated, revealed blood vessels in all parts of the implants after 4 weeks. Only in scaffolds seeded with allogenic mesenchymal stem cells the development of bridging bone constructs between proximal and distal edges of the femur was observed after four weeks without further supplementation of growth factors. In case of the implantation of non-seeded scaffolds no obvious scaffold bound bone development could be shown.

Bone formation in trabecular bone cell seeded scaffolds used for reconstruction of the rat mandible.

This study tested whether different in vitro cultivation techniques for tissue-engineered scaffolds seeded with human trabecular bone cells affect in vivo bone formation when implanted into critical-size defects in rat mandibles. Human trabecular cells were isolated and seeded into three types of scaffolds (porous CaCO(3), mineralized collagen, porous tricalcium phosphate). Four in vitro groups were produced: empty control scaffolds incubated with cell culture medium for 24 h; scaffolds seeded with trabecular bone cells, cultivated under static conditions for 24 h; scaffolds seeded with trabecular bone cells, cultivated for 14 days under static conditions; scaffolds seeded with trabecular bone cells, cultivated for 14 days in a continuous flow perfusion bioreactor. The scaffolds were implanted press fit into non-healing defects, 5 mm diameter, in rat mandibles. After 6 weeks the presence of human cells was assessed; none were detected. Histomorphometric evaluation showed that neither seeding human trabecular bone cells nor the culturing technique increased the amount of early bone formation compared with the level provided by osteoconductive bone ingrowth from the defect edges. It is concluded that human bone marrow stroma cells in tissue-engineered scaffolds and associated in vitro technology are difficult to test in the mandible in animal models.

Effect of seeding technique and scaffold material on bone formation in tissue-engineered constructs.

The aim of the present study was to test the hypothesis that both scaffold material and the type of cell culturing contribute to the results of in vivo osteogenesis in tissue-engineered constructs in an interactive manner. CaCO3 scaffolds and mineralized collagen scaffolds were seeded with human trabecular bone cells at a density of 5 x 10(6) cells/cm(3) and were left to attach under standard conditions for 24 h. Subsequently, they were submitted to static and dynamic culturing for 14 days (groups III and IV, respectively). Dynamic culturing was carried out in a continuous flow perfusion bioreactor. Empty scaffolds and scaffolds that were seeded with cells and kept under standard conditions for 24 h served as controls (groups I and II, respectively). Five scaffolds of each biomaterial and from each group were implanted into the gluteal muscles of rnu rats for 6 weeks. Osteogenesis was assessed quantitatively by histomorphometry and expression of osteocalcin (OC) and vascular endothelial growth factor (VEGF) was determined by immunohistochemistry. CaCO3 scaffolds exhibited 15.8% (SD 3.1) of newly formed bone after static culture and 22.4% (SD 8.2) after dynamic culture. Empty control scaffolds did not show bone formation, and scaffolds after 24 h of standard conditions produced 8.2% of newly formed bone (SD 4.0). Differences between the controls and the scaffolds cultured for 14 days were significant, but there was no significant difference between static and dynamic culturing. Mineralized collagen scaffolds did not show bone formation in any group. There was a significant difference in the expression of OC within the scaffolds submitted to static versus dynamic culturing in the CaCO3 scaffolds. VEGF expression did not show significant differences between static and dynamic culturing in the two biomaterials tested. It is concluded that within the limitations of the study the type of biomaterial had the dominant effect on in vivo bone formation in small tissue-engineered scaffolds. The culture period additionally affected the amount of bone formed, whereas the type of culturing may have had a positive effect on the expression of osteogenic markers but not on the quantity of bone formation.

PET-TC en Endocrinología / PET-CT in Endocrinology

El examen PET-TC ha ganado un lugar en el estudio de los tumores de origen endocrino. El marcador metabólico 18F-FDG es el más empleado internacionalmente y el único por el momento en nuestro medio. Las principales limitaciones del método en Endocrinología incluyen la alta diferenciación y baja agresividad de la mayoría de los tumores endocrinos, dificultad en la identificación de lesiones de escasa celularidad y el pequeño tamaño. Las indicaciones para su empleo deben ser precisas debido a que no todos los tumores presentan sustancial avidez por este compuesto por una parte y poder extraer la máxima eficacia diagnóstica del método con adecuadas indicaciones por la otra. La indicación más importante es en pacientes con cáncer diferenciado de tiroides (CDT) con valores de Tg elevados y barridos con 131I negativos. Es aconsejable su indicación con valores de Tg mayores a los 10 ng/ml y con TSH estimulada (endógena o exógena). El objetivo es la localización de las recidivas y metástasis para su exéresis o el empleo de otras terapias alternativas al 131I. Tiene alto valor pronóstico ya que es mayor la fijación de FDG en las lesiones más agresivas. Un paciente con Tg elevada, barrido con 131I negativo y FDG positivo obliga al clínico a actuar más agresivamente para eliminar los focos patológicos, mientras que con FDG negativo puede tener una conducta expectante con controles posteriores. La introducción de otros marcadores emisores de positrones específicos como el 124I, isótopo del Iodo, seguramente aportarán mejores imágenes y diagnósticos. En los tumores neuroendocrinos la FDG tiene limitada aplicación, salvo cuando hay un grado significativo de desdiferenciación. En el cáncer medular de tiroides (CMT) es conveniente indicarlo cuando los niveles de calcitonina superan los 1000 pg/ml con el objeto de localizar el/los sitios de su producción. Con la introducción de radiofármacos más específicos de las diferentes líneas celulares que componen el espectro de los tumores neuroendocrinos con emisores de positrones, tales como 18F-DOPA, 68Ga DOTA, 11C metomidato, 11C-5-hidroxitriptofano, etc., se podrá estudiar con mayor precisión el comportamiento metabólico-molecular de estos tumores.

PET/CT scans have reached an important place in the evaluation of endocrine tumors. The metabolic marker 18F-FDG is the most widespread over the world, and, for the time being, it is the only one available in our country. The limitations of this technique in Endocrinology include high differentiation and low aggressiveness of most endocrine tumors, and low detection rate for low cellularity and/or small lesions. Indications for PET/CT scan in these tumors should be precise, due to the fact that not all of these lesions are significantly glucose-avid and to extract the maximum diagnostic efficacy of this modality to achieve the optimum diagnostic accuracy. The most important indication is DTC with high Tg levels and negative 131I scans. It is advisable to indicate a PET/CT scan in patients with Tg > 10 ng/ml and stimulated TSH (endogenous or exogenous). The aim is to locate recurrencies and metastases in order to remove them, either surgically or by any other therapy alternative to 131I. Due to higher uptake in more aggressive lesions, this study has a high prognostic value. In patients with high Tg levels, negative I-131 scan, and abnormal FDG uptake, the practitioner must act more aggressively in order to remove the pathologic foci, while with a negative FDG -PET scan, the conduct can be expectant, with periodic follow-up. The introduction of other positron-emitting tracers like 124-Iodine, is likely to yield superior quality images and provide better diagnoses. FDG has a limited efficiency in neuroendocrine tumors, unless they show a significant level of desdiffer-entiation. The scan is indicated in MTC, when calcytonin levels are above 1000 pg/ml, in order to locate the tumor sites. With the introduction of more specific positron-emitting radiopharmaceuticals, such as 18F-DOPA, 68Ga DOTA, 11C metomidate, 11C-hidroxytriptophan and others, it will be possible to study the metabolic-molecular behavior of these tumors with a more accurate approach.

Tomografía con emisión de positrones (TEP) fusionado con tomografía computada (TC) versus TC helicoidal para la detección de metástasis: análisis retrospectivo de 85 casos / Positron-emission tomography and computed tomography versus helicoidal computed tomography: comparative study for neoplasms metastasis retrospective study of 85 cases

Hasta el presente, la TC es el examen por imágenes más aceptado y utilizado en oncología. El objetivo del presente trabajo fue evaluar la capacidad diagnóstica del PET fusionado con TC versus la TC previa sola para la detección de metástasis en pacientes (ptes.) oncológicos. Se efectuó un análisis retrospectivo de los exámenes PET-TC y de TC previa de 85 ptes., 49 hombres y 36 mujeres (edad media 56 años, raqngo 3-96 años) con diversos antecedentes oncológicos. Las variables registradas en los estudios fueron presencia, número y localización de metástasis basándose en criterios morfológicos y metabólicos. La diferencia en la performance entre métodos se estableció utilizando un test de McNemar y un Wilcoxon signed-rank test...(AU)

Tomografía con emisión de positrones (TEP) fusionado con tomografía computada (TC) versus TC helicoidal para la detección de metástasis: análisis retrospectivo de 85 casos / Positron-emission tomography and computed tomography versus helicoidal computed tomography: comparative study for neoplasms metastasis retrospective study of 85 cases

Hasta el presente, la TC es el examen por imágenes más aceptado y utilizado en oncología. El objetivo del presente trabajo fue evaluar la capacidad diagnóstica del PET fusionado con TC versus la TC previa sola para la detección de metástasis en pacientes (ptes.) oncológicos. Se efectuó un análisis retrospectivo de los exámenes PET-TC y de TC previa de 85 ptes., 49 hombres y 36 mujeres (edad media 56 años, raqngo 3-96 años) con diversos antecedentes oncológicos. Las variables registradas en los estudios fueron presencia, número y localización de metástasis basándose en criterios morfológicos y metabólicos. La diferencia en la performance entre métodos se estableció utilizando un test de McNemar y un Wilcoxon signed-rank test...

Nutritional intervention in pressure ulcer guidelines: an inventory.

Nutritional data from the literature and the high prevalence of malnutrition in patients at risk of pressure ulcers (PUs) or with established PU mandate structural nutritional actions in these patients. Guidelines can help to improve nutritional alertness in professionals and promote structural nutritional assessment and nutritional intervention in PU-prone or PU patients. PU guidelines from 13 countries were compared with regard to nutritional management of PU patients. The attention paid to nutritional prevention and treatment in PU patients varied considerably across guidelines. Recommendations with regard to nutritional intervention should be incorporated transparently into PU guidelines and should be complete, specific, testable, and cover the entire nutritional cycle.

Different mRNA expression profile during tumor progression in a well-differentiated liposarcoma--A microdissection approach.

Like malignant fibrous histiocytoma (MFH), dedifferentiated liposarcoma represents a distinct subtype of liposarcoma and is characterized by an abrupt transition from well-differentiated liposarcoma (WDL) to highgrade dedifferentiated liposarcoma (DDL) . In addition, specific cytogenetic aberrations support the close biological relationship between WDL and DDL. Recent observations indicated the significance of cell cycle aberrations in tumor progression from the low-malignant, well differentiated to its dedifferentiated form, the prognosis of which is poor. Thus, alterations of mdm2 and p53 genes belong to the most frequently reported alterations in these two subtypes of liposarcoma. In previous investigations, we reported that loss of heterozygosity at the Rb gene locus, telomerase activity, hTERT, and c-Myc expression were associated with tumor progression in liposarcomas. In this study, we report on a case of a WD/DDL, in which both tumor components were separated using laser microdissection (P.A.L.M.) for the investigation of hTERT mRNA expression on a LightCycler. Macroscopically selected and histologically proven cryosections of low malignant and highly malignant tumor areas were cytogenetically investigated to confirm the diagnosis and to find additional chromosomal alterations with tumor progression.

Distinction between lipoma and liposarcoma by MDM2 alterations: a case report of simultaneously occurring tumors and review of the literature.

We investigated a lipoma and a well-differentiated/dedifferentiated liposarcoma (WD/DDL), occurring simultaneously in one patient for the possible role of p53 and mdm2 in the molecular oncogenesis of liposarcoma and tumor progression. The hypothesis tested was if there is a continuum in the development from lipoma to liposarcoma. Lipoma was characterized by a lack of p53 mutation, p53 LOH and p53 protein expression, as well as by mdm2 amplification and mdm2 protein expression. p53 mutation and p53 LOH were found neither in the well-differentiated nor in the dedifferentiated parts of the liposarcoma. In contrast, mdm2 amplification and an increase in mdm2 protein expression were found to be associated with malignancy and dedifferentiation, whereas p53 protein expression was only slightly increased. These findings indicate that mdm2 constitutes one of the most common targets for molecular aberration in WD/DDL. We suggest that mdm2 is a marker distinguishing between ordinary lipoma and well-differentiated liposarcoma, and that the genesis of these tumors is different.

Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: a method to restore cell-specific beta-trace glycoprotein productivity at high cell density.

The impact of different cultivation-infection strategies on the productivity of baculovirus-infected BTI-Tn-5B1-4 (High Five) cells was investigated. Using beta-trace protein as the recombinant glycoprotein, the effects of multiplicity of infection (MOI) and time of infection (TOI) were studied on growth after infection as well as the degree of infection and recombinant protein productivity in batch culture. The highest productivities were found when infecting Tn5 cells at early exponential growth phase (EGP) (low cell density) using a high MOI. To increase the productive cell density of Tn5 cells after beta-trace-baculovirus infection, we performed studies infecting cells in the range of 1 to 5 x 10(6) cells/mL in fresh medium. Although the protein production was increased twofold, a strong negative cell density effect was still observed when maximal productive cell density exceeded 1 x 10(6) cells/mL. To verify whether the changing cell environment of the batch experiments was responsible for the decrease in protein productivity at increasing cell density at infection, several perfusion experiments were designed by infecting Tn5 cells at cell densities over 2 x 10(6) cells/mL under more steady-state conditions. The use of this experimental setup enabled successful infections at high cell densities with volumetric productivities of up to 1.2 g L(-1) day(-1) of beta-trace protein, which is very high for a glycoprotein expressed with the baculovirus expression vector system (BEVS). The cell specific protein productivity observed after infections at higher cell densities in perfusion mode was the same as in batch experiments at low cell concentrations, which clearly demonstrates that the cell density effect could be completely overcome with perfusion cultivation.

Perfusion bioreactors for the production of recombinant proteins in insect cells.

CONCLUSION: High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.

Influence of cell- and media-derived factors on the integrity of a human monoclonal antibody after secretion into serum-free cell culture supernatants.

To investigate the effects of factors secreted by different cell lines on human monoclonal antibody (MAb) integrity, 600 mg of a human MAb, which specifically binds to human erythrocytes, were produced in a perfusion process. After purification by protein A affinity chromatography, the MAb was used for integrity testing in supernatants of several cell lines to investigate their potential to degrade the antibody in the extracellular environment. One insect cell line (IPLB-SF-21 AE) and four mammalian cell lines [CHO K1, BHK-21 (C13), C1271, P3-X63-Ag8.653], all of them commonly used for the production of recombinant proteins, and the human-human-mouse heterohybridoma cell line itself (H-CB-hahE), were adapted to serum-free culture media. For integrity testing all cell lines were cultivated in spinner flasks using serum-free media supplemented with 30 mug mL(-1) of purified MAb. MAb integrity was assayed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, both followed by Western blotting, and an antigen binding assay. None of the mammalian cells showed any detectable effects on antibody stability and integrity during exponential growth, whereas isoelectric focusing of monoclonal antibody taken from IPLB-SF-21 AE culture supernatants revealed a new band indicating a partial modification of the MAb by secreted factors of these cells. This observation did not correlate with the total proteolytic activity, which was measured in all supernatants and found to be lowest in the insest cell cultures. For mammalian cell cultures, it could be concluded from these findings that shifts of the antibody microheterogeneity pattern, which can be found normally as a result of variations in different production parameters, are not caused by extracellular factors once the product has been secreted into the supernatant. In addition to their well-known advantages in posttranslational modifications (e.g., formation of complex type N-glycans), mammalian cells appear to be more suitable as expression systems for human monoclonal antibodies to be used in vivo when compared with baculovirus-infected insect cells. (c) 1995 John Wiley & Sons, Inc.

Optimization of the growth conditions of Sf21 insect cells for high-density perfusion culture in stirred-tank bioreactors.

Spodoptera frugiperda insect cells (IPLB-Sf21-AE) (Sf21), infected with baculovirus expression vectors during their exponential growth phase, are commonly used to produce a variety of heterologous recombinant proteins. In the present study the culture conditions of these insect cells were studied to establish high-density suspension cultures with prolonged exponential growth phases. The Sf21 cells were grown in 125-ml spinner flasks using five different culture media supplemented with 5% fetal calf serum and four protein-free or low-protein culture media. The best results were achieved in EX-CELL 401 (protein-free media) and in IPL-41 modified with 2.5 g l-1 tryptose phosphate broth (serum-supplemented media), respectively. The latter was used for further batch and continuous cultivation of Sf21 cells in a perfused 1.4-l stirred-tank bioreactor with special attention to the oxygen requirement of these cells. Optimal growth was found at an oxygen concentration of 70% air saturation, resulting in a prolonged exponential growth phase that could be maintained for more than 16 days. A maximum cell density of 5.5 x 10(7) viable cells ml-1 was achieved.