Precision cancer medicine: What has translated into clinical use in Belgium?

RATIONALE: In 2016, Belgium launched the Next Generation Sequencing (NGS) Roadbook, consisting in 10 Actions, across the health care system, to facilitate the uptake of NGS in routine clinical practice. We compiled feedback on deployment of the NGS Roadbook from governmental stakeholders and beneficiaries: health policy makers, insurance providers, pathologists, geneticists, and oncologists. MAIN FINDINGS: The Roadbook ensured the establishment of a Commission on Personalized Medicine and NGS testing guidelines. A national benchmarking trial ensued, and 10 networks of hospitals and laboratories adopted a reimbursement convention with the Belgian Health Insurance Agency. The Healthdata.be platform centralizes the collection of NGS metrics, and citizens were consulted on their views about NGS and genomics. CONCLUSION: The Roadbook facilitated the implementation of NGS in routine (hemato-)oncology care in Belgium. Some challenges remain linked to data sharing and access by a wider range of stakeholders. Next steps include continuous monitoring of health outcomes and the budgetary impact.

Reduced humoral immune response after BNT162b2 coronavirus disease 2019 messenger RNA vaccination in cancer patients under antineoplastic treatment.

BACKGROUND: Cancer patients are at a higher risk of developing severe coronavirus disease 2019 (COVID-19). However, the safety and efficacy of COVID-19 vaccination in cancer patients undergoing treatment remain unclear. PATIENTS AND METHODS: In this interventional prospective multicohort study, priming and booster doses of the BNT162b2 COVID-19 vaccine were administered 21 days apart to solid tumor patients receiving chemotherapy, immunotherapy, targeted or hormonal therapy, and patients with a hematologic malignancy receiving rituximab or after allogeneic hematopoietic stem cell transplantation. Vaccine safety and efficacy (until 3 months post-booster) were assessed. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibody levels were followed over time (until 28 days after the booster) and in vitro SARS-CoV-2 50% neutralization titers (NT50) toward the wild-type Wuhan strain were analyzed 28 days after the booster. RESULTS: Local and systemic adverse events (AEs) were mostly mild to moderate (only 1%-3% of patients experienced severe AEs). Local, but not systemic, AEs occurred more frequently after the booster dose. Twenty-eight days after the booster vaccination of 197 cancer patients, RBD-binding antibody titers and NT50 were lower in the chemotherapy group {234.05 IU/ml [95% confidence interval (CI) 122.10-448.66] and 24.54 (95% CI 14.50-41.52), respectively} compared with healthy individuals [1844.93 IU/ml (95% CI 1383.57-2460.14) and 122.63 (95% CI 76.85-195.67), respectively], irrespective of timing of vaccination during chemotherapy cycles. Extremely low antibody responses were seen in hematology patients receiving rituximab; only two patients had RBD-binding antibody titers necessary for 50% protection against symptomatic SARS-CoV-2 infection (<200 IU/ml) and only one had NT50 above the limit of detection. During the study period, five cancer patients tested positive for SARS-CoV-2 infection, including a case of severe COVID-19 in a patient receiving rituximab, resulting in a 2-week hospital admission. CONCLUSION: The BNT162b2 vaccine is well-tolerated in cancer patients under active treatment. However, the antibody response of immunized cancer patients was delayed and diminished, mainly in patients receiving chemotherapy or rituximab, resulting in breakthrough infections.

Perspectives on the integration of Immuno-Oncology Biomarkers and drugs in a Health Care setting.

Immunotherapies, specifically checkpoint inhibitors, are becoming an important component in cancer care with the most application now in melanoma and lung cancer patients. Some drawbacks that converge with this new evolution are the rather low response rates to these drugs and their high cost with a significant economic impact on the health care system. These major challenges can likely be circumvented by implementing a "personalized immuno-oncology" approach to accomplish a selection of optimal responders based on biomarkers. In this paper we first discuss the legal framework for the development of valuable in vitro diagnostics. Based on a case study in lung cancer, the clinical validity and utility requirements of predictive immuno-oncology biomarkers is highlighted and an overview is given on the evolution towards multiplex or omics-based assays together with its challenges and pitfalls. Finally, some initiatives between the public and private sector are pinpointed to sustain the future access to innovative medicines in cancer therapy at a reasonable cost.

Enhanced analysis of real-time PCR data by using a variable efficiency model: FPK-PCR.

Current methodology in real-time Polymerase chain reaction (PCR) analysis performs well provided PCR efficiency remains constant over reactions. Yet, small changes in efficiency can lead to large quantification errors. Particularly in biological samples, the possible presence of inhibitors forms a challenge. We present a new approach to single reaction efficiency calculation, called Full Process Kinetics-PCR (FPK-PCR). It combines a kinetically more realistic model with flexible adaptation to the full range of data. By reconstructing the entire chain of cycle efficiencies, rather than restricting the focus on a 'window of application', one extracts additional information and loses a level of arbitrariness. The maximal efficiency estimates returned by the model are comparable in accuracy and precision to both the golden standard of serial dilution and other single reaction efficiency methods. The cycle-to-cycle changes in efficiency, as described by the FPK-PCR procedure, stay considerably closer to the data than those from other S-shaped models. The assessment of individual cycle efficiencies returns more information than other single efficiency methods. It allows in-depth interpretation of real-time PCR data and reconstruction of the fluorescence data, providing quality control. Finally, by implementing a global efficiency model, reproducibility is improved as the selection of a window of application is avoided.

Use of pJANUS-02-001 as a calibrator plasmid for Roundup Ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment.

Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938-944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.

Metabolism and hepatotoxicity of N,N-dimethylformamide, N-hydroxymethyl-N-methylformamide, and N-methylformamide in the rat.

The metabolism and hepatotoxicity of N,N-dimethylformamide (DMF) and two of its metabolites, N-hydroxymethyl-N-methylformamide (HMMF) and N-methylformamide (NMF) were evaluated over a 4-day period in rats. DMF toxicity was dose dependent and delayed toxicity after the administration of a high DMF dose (13.7 mmol/kg) in comparison to a lower dose (4.1 mmol/kg) was observed. Treatment of rats with 13.7 mmol/kg DMF, HMMF, or NMF showed i) that DMF is more toxic than HMMF or NMF, and ii) that hepatotoxicity occurs later for DMF than for HMMF or NMF. Analysis of serum and urine samples demonstrated that DMF is first metabolized to HMMF, which is then partially converted to NMF. After HMMF administration, NMF was found both in serum and in urine. The time course of DMF and HMMF toxicity in relation to NMF formation fitted the hypothesis that the hepatotoxicity of DMF and HMMF is mediated via NMF. The degree of hepatotoxicity after HMMF and NMF treatment is similar. However, the degree of DMF hepatotoxicity is much higher than in the case of NMF or HMMF. The role of NMF as an obligatory intermediate in DMF and HMMF hepatotoxicity is discussed.

Differential effects of elicitors on the viability of rice suspension cells.

We have compared the effects of two elicitors of defense-related processes on rice (Oryza sativa L.) suspension cells. Both chitosan and salicylic acid induced the accumulation of extracellular chitinase, thickening of the cell wall, and a variety of cytological changes in treated cells. Chitosan also induced the production of a brown pigment and cell death. Both of these effects depended on the availability of reactive oxygen species, because the damage was greatly reduced by either catalase or free-radical scavengers. Pretreating cells with salicylic acid also protected them from the cytotoxic effects of chitosan. This type of induced tolerance persisted when salicylic acid was removed and was not simply due to the release of extracellular substances, because salicylic acid-treated cells did not protect untreated cells from chitosan-induced death. Salicylic acid also stimulated the production of a 10-kilodalton subtilisin inhibitor that was not produced by chitosan-treated cells. Most of these changes are associated with the hypersensitive response of many plant species, including monocotyledons, and may serve as an in vitro model for investigating the biochemistry of some diseases.

The interaction between reaction forces and stabilization systems during intrusion of the anterior teeth and its effect on the posterior unit.

The aim of this research is to attain a better understanding of the initial reaction forces induced by an intrusion mechanism (acting on the anterior teeth) on the posterior unit and to examine how these forces can be neutralized. The experiments were performed on the dentition of a dry human skull and initial tooth displacements were registered by means of two laser measuring techniques, namely holographic interferometry and the laser reflection technique. It was established that of all reaction forces induced by the intrusion arch, distal tipping of the first molars is the most pronounced. A transpalatal bar connecting the teeth does not counteract this movement. The stabilization of the posterior unit with a transpalatal bar, buccal sectionals, and high-pull headgear proved to be the most effective technique.

Two-dimensional gel electrophoresis, protein electroblotting and microsequencing: a direct link between proteins and genes.

We have used two-dimensional gel electrophoresis as a general "preparative" method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4-vinyl-N-methylpyridinium iodide)-coated glass fiber sheets. The major spots were excised and subjected to NH2-terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two-dimensional gels and the Coomassie Brilliant Blue-stained spots were excised from the gels. The combined spots were re-eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase-high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two-dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.

[Rapid maxillary suture expansion]. / Sutuurexpansie in de orthodontie.

Rapid maxillary expansion is a technique with an old but controversial history. Bio-mechanics and apparatus have largely involved over the years and the procedure is generally accepted. Discussion concerns only the patient age range on which the therapy is applicable. Fourteen to eighteen seems the accepted age limit. Three essential parameters determine the success of the expansion: degree of interlocking, synostosis and skeletal resistance from the surrounding osseous tissues.

Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.

Protein changes induced by salinity stress were investigated in the roots of the salt-sensitive rice cultivar Taichung native 1. We found eight proteins to be induced and obtained partial sequences of one with a molecular mass of 15 kilodaltons and an isoelectric point of 5.5. Using an oligonucleotide probe based on this information, a cDNA clone, salT, was selected and found to contain an open reading frame coding for a protein of 145 amino acid residues. salT mRNA accumulates very rapidly in sheaths and roots from mature plants and seedlings upon treatment with Murashige and Skoog salts (1%), air drying, abscisic acid (20 microM), polyethylene glycol (5%), sodium chloride (1%), and potassium chloride (1%). Generally, no induction was seen in the leaf lamina even when the stress should affect all parts of the plant uniformly. The organ-specific response of salT is correlatable with the pattern of Na+ accumulation during salt stress.

A plant manganese superoxide dismutase is efficiently imported and correctly processed by yeast mitochondria.

In the plant Nicotiana plumbaginifolia, manganese superoxide dismutase (MnSOD) is synthesized in the cytoplasm as a preprotein and is subsequently translocated to the mitochondrial matrix with corresponding cleavage of an NH2-terminal leader sequence. To determine whether the plant enzyme could replace the endogenous SOD activities of Escherichia coli and yeast, constructions have been made in appropriate vectors for expression of the preprotein and the mature MnSOD. These were introduced into SOD-deficient strains for complementation studies. In E. coli, both forms of the protein were shown to be active and able to complement SOD deficiency to different degrees. Expression of the preprotein in a yeast strain lacking a mitochondrial MnSOD resulted in a restoration of wild-type growth, only possible if the plant protein was being targeted to the mitochondria. Subsequent studies revealed that the protein was processed and that the leader sequence was cleaved at the identical position as recognized by the mitochondrial peptidase of plants. The components mediating mitochondrial import thus appear to be highly conserved between plants and yeast.