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.

Evaluation of precision medicine assessment reports of the Belgian healthcare payer to inform reimbursement decisions.

INTRODUCTION: Precision medicines rely on companion diagnostics to identify patient subgroups eligible for receiving the pharmaceutical product. Until recently, the Belgian public health payer, RIZIV-INAMI, assessed precision medicines and companion diagnostics separately for reimbursement decisions. As both components are considered co-dependent technologies, their assessment should be conducted jointly from a health technology assessment (HTA) perspective. As of July 2019, a novel procedure was implemented accommodating for this joint assessment practice. The aim of this research was to formulate recommendations to improve the assessment in the novel procedure. METHODS: This study evaluated the precision medicine assessment reports of RIZIV-INAMI of the last 5 years under the former assessment procedure. The HTA framework for co-dependent technologies developed by Merlin et al. for the Australian healthcare system was used as a reference standard in this evaluation. Criteria were scored as either present or not present. RESULTS: Thirteen assessment reports were evaluated. Varying scores between reports were obtained for the domain establishing the co-dependent relationship between diagnostic and pharmaceutical. Domains evaluating the clinical utility of the biomarker and the cost-effectiveness performed poorly, whereas the budget impact and the transfer of trial data to the local setting performed well. RECOMMENDATIONS: Based on these results we recommend three amendments for the novel procedure. (i) The implementation of the linked evidence approach when direct evidence of clinical utility is not present, (ii) incorporation of a bias assessment tool, and (iii) further specify guidelines for submission and assessment to decrease the variability of reported evidence between assessment reports.

Roadbook for the implementation of next-generation sequencing in clinical practice in oncology and hemato-oncology in Belgium.

In the field of oncology research, next-generation sequencing has contributed significantly to the discovery of DNA mutations associated with diagnosis and prognosis. It also aids in the development of targeted therapies to specific mutations and the rise of personalized medicine. As part of molecular diagnostics in cancer patients, analysis by next-generation sequencing is becoming part of routine clinical practice. The introduction of this complex technology in a healthcare system comes with multiple challenges and requires a clear action plan. Such an action plan, as outlined in this paper, was developed in Belgium and includes steps in ensuring the quality and indications of NGS testing, installing data registration and tackling ethical issues. A final step is to perform a pilot study to control the access, quality, harmonization and expertise in DNA testing. This action plan can serve as a guide for similar initiatives by other countries to facilitate NGS implementation in clinical practice.

Role of metallothioneins as danger signals in the pathogenesis of colitis.

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients.

Absence of placental growth factor blocks dextran sodium sulfate-induced colonic mucosal angiogenesis, increases mucosal hypoxia and aggravates acute colonic injury.

Angiogenesis has recently been described as a component of inflammatory bowel disease. Placental growth factor (PlGF), a vascular endothelial growth factor (VEGF) homologue, establishes its angiogenic capacity under pathophysiological conditions. We investigated the function of PlGF in experimental models of acute colitis. Acute colonic damage was induced in PlGF knock-out ((-/-)) mice and PlGF wild-type ((+/+)) mice by dextran sodium sulfate (DSS) and trinitrobenzenesulfonic acid (TNBS). The concentrations of PlGF and VEGF were measured in distal colonic lysates using an enzyme-linked immunosorbent assay. Colonic injury was evaluated by assessing colon length, colonocyte apoptosis (by terminal dUTP nick-end labeling), colonic cytokine production and histological score. Infiltration of polymorphonuclear cells was determined by assaying myeloperoxidase (MPO) activity. In a separate experiment, recombinant PlGF was administered to PlGF(-/-) mice by adenoviral transfer before DSS administration. Mucosal vascularization was quantified by computerized morphometric analysis of CD31-stained distal colonic sections. Colonic mucosal hypoxia was visualized by pimonidazole staining. Both VEGF and PlGF were upregulated during acute colitis. In addition, compared with PlGF(+/+) controls, PlGF(-/-) mice showed a significant increase in weight loss and colonic shortening during both DSS and TNBS colitis. This correlated with enhanced colonocyte apoptosis, elevated colonic cytokine levels and increased histological damage score, but not with enhanced inflammatory cell infiltration (MPO activity). The increased morbidity of PlGF(-/-) mice during DSS colitis was preventable by adenovirus (Ad)-mediated overexpression of PlGF. After the administration of DSS, strongly reduced mucosal angiogenesis was observed in PlGF(-/-) mice compared with PlGF(+/+) mice. This was associated with an early increase in intestinal epithelial pimonidazole accumulation in PlGF(-/-) mice, suggesting a function of enhanced epithelial hypoxia in the observed differences between the two groups. In summary, our data show that the absence of PlGF strongly inhibits mucosal intestinal angiogenesis in acute colitis, which is associated with an early increase in intestinal epithelial hypoxia and aggravation of the course of the disease.

Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model.

Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F(1)) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., and Van Coster, R. (2004) J. Med. Genet. 41, 120-124) have reported that a homozygous missense mutation in the gene for human Atp12p (HuAtp12p), which replaces Trp-94 with Arg, was linked to the death of a 14-month-old patient. We have investigated the impact of the pathogenic W94R mutation on Atp12p structure/function. Plasmid-borne wild type human Atp12p rescues the respiratory defect of a yeast ATP12 deletion mutant (Deltaatp12). The W94R mutation alters the protein at the most highly conserved position in the Pfam sequence and renders HuAtp12p insoluble in the background of Deltaatp12. In contrast, the yeast protein harboring the corresponding mutation, ScAtp12p(W103R), is soluble in the background of Deltaatp12 but not in the background of Deltaatp12Deltafmc1, a strain that also lacks Fmc1p. Fmc1p is a yeast mitochondrial protein not found in higher eukaryotes. Tryptophan 94 (human) or 103 (yeast) is located in a positively charged region of Atp12p, and hence its mutation to arginine does not alter significantly the electrostatic properties of the protein. Instead, we provide evidence that the primary effect of the substitution is on the dynamic properties of Atp12p.

Human metallothionein expression under normal and pathological conditions: mechanisms of gene regulation based on in silico promoter analysis.

Metallothioneins (MTs) are ubiquitous metal-binding proteins that have been highly conserved throughout evolution. Although their physiological function is not completely understood, they are involved in diverse processes including metal homeostasis and detoxification, the oxidative stress response, inflammation, and cell proliferation. Te human MT gene family consists of at least 18 isoforms, containing pseudogenes as well as genes encoding functional proteins. Most of the MT isoforms can be induced by a wide variety of substances, such as metals, cytokines, and hormones. Different cell types express discrete MT isoforms, which reflects the specifically adapted functions of MTs and a divergence in their regulation. Te aberrant expression of MTs has been described in a number of diseases, including Crohn's disease, cancer, Alzheimer's disease, amyotrophic lateral sclerosis, Menkes disease, and Wilson's disease. Therefore, a thorough understanding of MT gene regulation is imperative. To date, the transcriptional regulation of MTs has primarily been studied in mice. While only four murine MT isoforms exist, the homology between murine and human MTs allows for the evaluation of the regulatory regions in their respective promoters. Here, we review the aberrant expression of MTs in human diseases and the mechanisms that regulate MT1 expression based on an in silico evaluation of transcription factor binding sites.

Evidence for a potential role of metallothioneins in inflammatory bowel diseases.

Inflammatory bowel diseases (IBDs) are a group of chronic, relapsing, immune-mediated disorders of the intestine, including Crohn's disease and ulcerative colitis. Recent studies underscore the importance of the damaged epithelial barrier and the dysregulated innate immune system in their pathogenesis. Metallothioneins (MTs) are a family of small proteins with a high and conserved cysteine content that are rapidly upregulated in response to an inflammatory stimulus. Herein, we review the current knowledge regarding the expression and potential role of MTs in IBD. MTs exert a central position in zinc homeostasis, modulate the activation of the transcription factor nuclear factor (NF)-kappaB, and serve as antioxidants. In addition, MTs could be involved in IBD through their antiapoptotic effects or through specific immunomodulating extracellular effects. Reports on MT expression in IBD are contradictory but clearly demonstrate a deviant MT expression supporting the idea that these aberrations in IBD require further clarification.

Unconditioned adult-derived neurosphere cells mainly differentiate towards astrocytes upon transplantation in sclerotic rat hippocampus.

PURPOSE: Cell transplantation is being investigated as an alternative treatment for medically refractory temporal lobe epilepsy (TLE). In this study the fate of adult-derived neurosphere cells was evaluated after transplantation in the lesioned hippocampus of the intrahippocampal kainic acid (KA) model for TLE. METHODS: Neurosphere-forming cells were derived from the subventricular zone (SVZ) of transgenic green fluorescent protein (GFP) reporter mice and expanded in culture. After 10 passages in vitro neurosphere-derived cells were transplanted in the hippocampus three days (KA3d group) and three weeks (KA3w group) after intrahippocampal KA injection. Survival and differentiation of neurosphere cells were evaluated three and six weeks after transplantation. RESULTS: A fraction (about 1%) of GFP-expressing neurosphere cells survived for at least six weeks after transplantation with a higher and more robust survival rate in the KA3d compared to the KA3w group. Although a small fraction of the cells expressed the neuronal marker NeuN, neurosphere cells mainly differentiated towards astrocytes. DISCUSSION: Our results indicate that adult-derived neurosphere cells are able to survive upon transplantation in the sclerotic hippocampus. The transplanted cells do not or hardly contribute to neuronal replacement and mainly adopt an astrogliotic fate.

Human mast cells express leptin and leptin receptors.

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.

Staphylococcus aureus enterotoxin B regulates prostaglandin E2 synthesis, growth, and migration in nasal tissue fibroblasts.

BACKGROUND: Superantigens and eicosanoids are important amplifiers and regulators of inflammation in airway diseases. We therefore studied the possible influence of Staphylococcus aureus enterotoxin B (SEB) on the cyclooxygenase (COX) pathway and basic functions of airway structural cells. METHODS: Fibroblasts were isolated from nasal inferior turbinate tissue and cultured in the presence of different concentrations of SEB. Preincubation with interferon (IFN)-gamma was performed to induce expression of major histocompatibility complex (MHC) class II receptors. Prostaglandin E2 (PGE(2)) production was assayed by enzyme-linked immunosorbent assay, and levels of COX-2 and prostanoid E receptors 1-4 (EP(1-4)) were assayed by real-time polymerase chain reaction. Migration and growth tests were performed, and SEB was localized within the cells by confocal microscopy. RESULTS: Stimulation with IFN-gamma and SEB significantly down-regulated PGE(2), COX-2, and EP(2) expression but not EP(1), EP(3), or EP(4) expression. The enterotoxin blocked cell growth but increased the fibroblast migration rate. SEB was localized within the cell in the presence and absence of MHC-II, suggesting that mechanisms other than conventional binding may allow the enterotoxin to enter the cell. CONCLUSIONS: These findings may have major implications for our understanding of the role played by bacterial superantigens in regulating the inflammatory and remodeling mechanisms of upper airway diseases and hence may help elucidate the pathophysiology of these diseases.

Paracellular entry of interleukin-10 producing Lactococcus lactis in inflamed intestinal mucosa in mice.

BACKGROUND: Genetically modified Lactococcus lactis secreting interleukin-10 (IL-10) has been demonstrated to provide localized delivery of a therapeutic agent through active in situ synthesis in murine colitis. At present, many aspects of the exact mechanism by which the beneficial effect of the IL-10-producing L. lactis on the mucosa is mediated remain to be clarified. METHODS: Our aim was to determine the interaction of L. lactis with the intestinal mucosa. Therefore, we administered IL-10-producing L. lactis to healthy mice and in 2 mouse models of chronic colitis. Paraffin sections of ileum and colon samples were examined with confocal and transmission electron microscopy. Ileum and colon homogenates were prepared after flushing and after removal of mucus layer and epithelium. These homogenates and homogenates of mesenteric lymph nodes and spleen were plated on agar and immunoblotting for L. lactis and IL-10 was performed. RESULTS: Both confocal and electron microscopy showed the presence of lactococci in inflamed intestinal mucosa of mice with colitis. We recovered viable bacteria that could still produce IL-10 from homogenates of inflamed ileum and colon of which mucous and epithelial layers were removed. We did not find lactococci in mesenteric lymph nodes or in the spleen of mice with colitis. CONCLUSIONS: This study demonstrates uptake of IL-10-secreting L. lactis by the paracellular route in inflamed mucosal tissue. We suggest that IL-10 production by L. lactis residing inside the mucosa in the vicinity of responsive cells can improve the local action of interleukin-10 in inflamed tissue and the efficiency of the treatment.

A dual chamber model of female cervical mucosa for the study of HIV transmission and for the evaluation of candidate HIV microbicides.

A dual chamber system was established to model heterosexual HIV transmission. Cell-associated, but not cell-free HIV, added to a confluent layer of cervical epithelial cells in the apical chamber, reproducibly infected monocyte-derived dendritic cells (MO-DC) and CD4(+) T cells in the basal compartment. Only minimal epithelial transmigration of HIV-infected mononuclear cells (HIV-PBMCs) was observed. Most evidence points to transepithelial migration of virus, released from HIV-PBMCs after their activation by epithelial cells. We used this model for evaluation of the therapeutic index of various potentially preventive antiviral compounds, including non-nucleoside reverse transcriptase inhibitors (NNRTIs, including UC781 and various diaryltriazines and diarylpyrimidines), poly-anionic entry inhibitors (including PRO2000, cellulose sulphate, dextrane sulphate 5000 and polystyrene sulphonate) and the fusion inhibitor T-20. The epithelium was pre-treated with compound and incubated with HIV-PBMCs for 24 h. Afterwards the apical chamber was removed and MO-DC/CD4(+) T cell co-cultures were further cultured without compound. NNRTIs, including a TMC120 gel, blocked infection of the sub-epithelial targets at sub-micromolar concentrations. Polyanionic entry inhibitors (up to 100 microg/ml) and T-20 (up to 449 microg/ml) failed to inhibit transmission. Moreover, whereas the NNRTIs used interfered with epithelial integrity with cervical epithelium only at very high concentrations, the evaluated entry inhibitors showed toxicity at concentrations that did not prevent infection.

Differentiation assays of bone marrow-derived Multipotent Adult Progenitor Cell (MAPC)-like cells towards neural cells cannot depend on morphology and a limited set of neural markers.

There are accumulating studies that report a neurogenic potential of bone marrow-derived cells both in vitro as well as in vivo. Most claims of neural "transdifferentiation" have based their conclusions on morphology and neural gene expression. Recently, doubts have been raised about the validity of both outcome parameters since non-neural cells can extend neurites and show aberrant neural gene expression as a response to stress inducing factors. In this study, we compared bone marrow-derived Multipotent Adult Progenitor Cell (MAPC)-like cells and neural stem cells (NSC) in their morphology and neural gene expression profile after neural differentiation using three differentiation protocols. We evaluated the expression of five neuroglial antigens [neurofilament 200 (NF200); beta III tubulin (beta3 tub); tau; Glial Fibrillary Acidic Protein (GFAP); Myelin Basic Protein (MBP) and RIP antigen] using real-time PCR (RT-PCR) and immunocytochemistry (ICC). MAPC-like cells adopted a neural-like morphology in one protocol but a fibroblast-like morphology in the two other protocols. RT-PCR and ICC show that MAPC-like cells already express the neural antigens beta III tubulin and NF200 at baseline, but no upregulation of these genes after exposure to three distinct differentiation protocols was seen. In contrast, NSC adopt neural and glial morphologies with a clear increase in expression of all neuroglial genes in all differentiation protocols used. In conclusion, our data demonstrate that neural-like morphology and expression of a limited set of neural marker genes by MAPC-like cells after differentiation are not absolute proof of neural transdifferentiation because MAPC-like cells only partially meet the criteria which are fulfilled by NSC after neural differentiation.

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria.

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.

Caspase inhibition causes hyperacute tumor necrosis factor-induced shock via oxidative stress and phospholipase A2.

Dysregulated apoptotic cell death contributes to many pathological conditions, including sepsis, prompting the suggestion that caspase inhibition to block apoptosis could have useful therapeutic applications. Because the cytokine tumor necrosis factor (TNF, also known as TNF-alpha) is both pro-apoptotic and pro-inflammatory and is involved in septic shock, we tested whether caspase inhibition would alleviate TNF-induced toxicity in vivo. General caspase inhibition by the protease inhibitor zVAD-fmk exacerbated TNF toxicity by enhancing oxidative stress and mitochondrial damage, resulting in hyperacute hemodynamic collapse, kidney failure and death. Thus, survival of TNF toxicity depends on caspase-dependent processes. Our results demonstrated the pathophysiological relevance of caspase-independent, ROS-mediated pathways in response to lethal TNF-induced shock in mice. In addition, survival of TNF toxicity seemed to require a caspase-dependent protective feedback on excessive reactive oxygen species (ROS) formation and phospholipase A2 activation.

The role of heregulin-alpha as a motility factor and amphiregulin as a growth factor in wound healing.

Wound healing is a complex process of which growth and motility are essential features. The aim of this study was to search for keratinocyte-derived secreted factors that may play a role in these mechanisms, and their corresponding receptors. Growth and motility factors were purified from conditioned medium from cultured primary keratinocytes. Receptor and growth factor expression profiles were investigated by immunohistochemical, western blotting, and in situ hybridization analysis on cultured keratinocytes and tissue sections derived from chronic wounds. The most potent autocrine growth factor for keratinocytes, which it was possible to purify and sequence from keratinocyte-conditioned medium, is amphiregulin. Its receptor HER-1 is up-regulated on the membranes of keratinocytes lining the edge of the wound. From the same keratinocyte-conditioned medium, heregulin-alpha was purified as a potent motility factor for keratinocytes. Its receptor is HER-3, which is up-regulated on the membranes of keratinocytes lining the edge of the wound and on keratinocytes that had migrated towards the centre of the wound. HER-4 - another receptor for heregulin-alpha - is weakly present in occasional cells near the edge of the wound. The co-receptor for HER-3 and HER-4 is HER-2/neu, which is also present in epidermal cells but not overexpressed. This study shows that heregulin-alpha is a potent motility factor for normal epithelial cells and that it plays a central role in the process of wound healing of stratified epithelia. Heregulin-alpha has already been shown to be the motility factor leading to migration of HER-2/neu-overexpressing breast cancer cells. The role of amphiregulin as a growth factor and of heregulin-alpha as a motility factor for keratinocytes in epidermal and mucosal wound healing parallels their motility and growth induction in carcinogenesis.