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.

NGS for (Hemato-) Oncology in Belgium: Evaluation of Laboratory Performance and Feasibility of a National External Quality Assessment Program.

Next-generation sequencing (NGS) is being integrated into routine clinical practice in the field of (hemato-) oncology to search for variants with diagnostic, prognostic, or therapeutic value at potentially low allelic frequencies. The complex sequencing workflows used require careful validation and continuous quality control. Participation in external quality assessments (EQA) helps laboratories evaluate their performance and guarantee the validity of tests results with the ultimate goal of ensuring high-quality patient care. Here, we describe three benchmarking trials performed during the period 2017-2018 aiming firstly at establishing the state-of-the-art and secondly setting up a NGS-specific EQA program at the national level in the field of clinical (hemato-) oncology in Belgium. DNA samples derived from cell line mixes and artificially mutated cell lines, designed to carry variants of clinical relevance occurring in solid tumors, hematological malignancies, and BRCA1/BRCA2 genes, were sent to Belgian human genetics, anatomic pathology, and clinical biology laboratories, to be processed following routine practices, together with surveys covering technical aspects of the NGS workflows. Despite the wide variety of platforms and workflows currently applied in routine clinical practice, performance was satisfactory, since participating laboratories identified the targeted variants with success rates ranging between 93.06% and 97.63% depending on the benchmark, and few false negative or repeatability issues were identified. However, variant reporting and interpretation varied, underlining the need for further standardization. Our approach showcases the feasibility of developing and implementing EQA for routine clinical practice in the field of (hemato-) oncology, while highlighting the challenges faced.

Standardization of Somatic Variant Classifications in Solid and Haematological Tumours by a Two-Level Approach of Biological and Clinical Classes: An Initiative of the Belgian ComPerMed Expert Panel.

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software.

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.

microRNA expression in autonomous thyroid adenomas: Correlation with mRNA regulation.

The objective of the study was to identify the deregulated miRNA in autonomous adenoma and to correlate the data with mRNA regulation. Seven autonomous adenoma with adjacent healthy thyroid tissues were investigated. Twelve miRNAs were downregulated and one was upregulated in the tumors. Combining bioinformatic mRNA target prediction and microarray data on mRNA regulations allowed to identify mRNA targets of our deregulated miRNAs. A large enrichment in mRNA encoding proteins involved in extracellular matrix organization and different phosphodiesterases were identified among these putative targets. The direct interaction between miR-101-3p and miR-144-3p and PDE4D mRNA was experimentally validated. The global miRNA profiles were not greatly modified, confirming the definition of these tumors as minimal deviation tumors. These results support a role for miRNA in the regulation of extracellular matrix proteins and tissue remodeling occurring during tumor development, and in the important negative feedback of the cAMP pathway, which limits the consequences of its constitutive activation in these tumors.

MiRNA expression may account for chronic but not for acute regulation of mRNA expression in human thyroid tumor models.

BACKGROUND: For thyroid tumorigenesis, two main human in vitro models are available: primary cultures of human thyrocytes treated with TSH or EGF/serum as models for autonomous adenomas (AA) or papillary thyroid carcinomas (PTC) respectively, and human thyroid tumor derived cell lines. Previous works of our group have assessed properties of those models, with a special emphasis on mRNA regulations. It is often assumed that miRNA may be one of the primary events inducing these mRNA regulations. METHODS: The purpose of this study was to investigate the representativity of those models to study microRNA regulations and their relation with mRNA expression. To achieve this aim, the miRNA expressions profiles of primary cultures treated with TSH or EGF/serum and of 6 thyroid cancer cell lines were compared to the expression profiles of 35 tumor tissues obtained by microarrays. RESULTS: Our data on primary cultures have shown that the TSH or EGF/serum treatment did not greatly modify the microRNA expression profiles, which is contrary to what is observed for mRNA expression profiles, although they still evolved differently according to the treatment. The analysis of miRNA and mRNA expressions profiles in the cell lines has shown that they have evolved into a common, dedifferentiated phenotype, closer to ATC than to the tumors they are derived from. CONCLUSIONS: Long-terms TSH or EGF/serum treatments do not mimic AA or PTC respectively in terms of miRNA expression as they do for mRNA, suggesting that the regulations of mRNA expression induced by these physiological agents occur independently of miRNA. The general patterns of miRNA expression in the cell lines suggest that they represent a useful model for undifferentiated thyroid cancer. Mirna probably do not mediate the rapid changes in gene expression in rapid cell biology regulation.

miRNA expression in anaplastic thyroid carcinomas.

Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents an end stage of thyroid tumor progression. No effective treatment exists so far. In this study, we analyzed the miRNA expression profiles of 11 ATC by microarrays and their relationship with the mRNA expression profiles of the same 11 ATC samples. ATC show distinct miRNA expression profiles compared to other less aggressive thyroid tumor types. ATC show 18 commonly deregulated miRNA compared to normal thyroid tissue (17 downregulated and 1 upregulated miRNA). First, the analysis of a combined approach of the mRNA gene expression and of the bioinformatically predicted mRNA targets of the deregulated miRNA suggested a role for these regulations in the epithelial to mesenchymal transition (EMT) process in ATC. Second, the direct interaction between one of the upregulated mRNA target, the LOX gene which is an EMT key player, and a downregulated miRNA, the miR-29a, was experimentally validated by a luciferase assay in HEK cell. Third, we confirmed that the ATC tissue is composed of about 50% of tumor associated macrophages (TAM) and suggested, by taking into account our data and published data, their most likely direct or paracrine intercommunication between them and the thyroid tumor cells, amplifying the tumor aggressiveness. Finally, we demonstrated by in situ hybridization a specific thyrocyte localization of 3 of the deregulated miRNA: let-7g, miR-29a and miR-30e and we pointed out the importance of identifying the cell type localization before drawing any conclusion on the physiopathological role of a given gene.

Cancer stem cells, a fuzzy evolving concept: a cell population or a cell property?

The cancer stem cells (CSC) hypothesis represents a pathological extrapolation of the physiological concept of embryonic and somatic stem cells. In its initial definition, it encompassed the hypothesis of a qualitatively distinct population of immortal cancer cells originating from somatic stem cells, which generate in xenotransplants by a deterministic irreversible process, the hierarchy of more differentiated finite lifespan derived cells, which constitute, themselves, the bulk of the cancer. These CSC would express specific biomarkers and gene expressions related to chemo- and radioresistance, stemness, epithelial-mesenchymal transition, etc. No convincing congruence of several of these properties in one cell population has been demonstrated. The concept has greatly evolved with time and with different authors ("the plasticity of cancer stem cells"), leading to a minimal definition of cells generating a hierarchy of derived cells. In this article these concepts are analyzed. It is proposed that stemness is a property, more or less reversible, a hallmark of some cells at some time in a cancer cell population, as immortality, dormancy, chemo- or radioresistance, epithelial-mesenchymal transition etc. These phenotypic properties represent the result of independent, linked, or more or less congruent, genetic, epigenetic, or signaling programs.

5-aza-2'-deoxycytidine has minor effects on differentiation in human thyroid cancer cell lines, but modulates genes that are involved in adaptation in vitro.

BACKGROUND: In thyroid cancer, the lack of response to specific treatment, for example, radioactive iodine, can be caused by a loss of differentiation characteristics of tumor cells. It is hypothesized that this loss is due to epigenetic modifications. Therefore, drugs releasing epigenetic repression have been proposed to reverse this silencing. METHODS: We investigated which genes were reinduced in dedifferentiated human thyroid cancer cell lines when treated with the demethylating agent 5-aza-2'-deoxycytidine (5-AzadC) and the histone deacetylase inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid, by using reverse transcriptase-polymerase chain reaction and microarrays. These results were compared to the expression patterns in in vitro human differentiated thyrocytes and in in vivo dedifferentiated thyroid cancers. In addition, the effects of 5-AzadC on DNA quantities and cell viability were investigated. RESULTS: Among the canonical thyroid differentiation markers, most were not, or only to a minor extent, re-expressed by 5-AzadC, whether or not combined with TSA or forskolin, an inducer of differentiation in normal thyrocytes. Furthermore, 5-AzadC-modulated overall mRNA expression profiles showed only few commonly regulated genes compared to differentiated cultured primary thyrocytes. In addition, most of the commonly strongly 5-AzadC-induced genes in cell lines were either not regulated or upregulated in anaplastic thyroid carcinomas. Further analysis of which genes were induced by 5-AzadC showed that they were involved in pathways such as apoptosis, antigen presentation, defense response, and cell migration. A number of these genes had similar expression responses in 5-AzadC-treated nonthyroid cell lines. CONCLUSIONS: Our results suggest that 5-AzadC is not a strong inducer of differentiation in thyroid cancer cell lines. Under the studied conditions and with the model used, 5-AzadC treatment does not appear to be a potential redifferentiation treatment for dedifferentiated thyroid cancer. However, this may reflect primarily the inadequacy of the model rather than that of the treatment. Moreover, the observation that 5-AzadC negatively affected cell viability in cell lines could still suggest a therapeutic opportunity. Some of the genes that were modulated by 5-AzadC were also induced in nonthyroid cancer cell lines, which might be explained by an epigenetic modification resulting in the adaptation of the cell lines to their culture conditions.

Thyroid cancer cell lines: an overview.

Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution.

mRNA expression in papillary and anaplastic thyroid carcinoma: molecular anatomy of a killing switch.

Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents the end stage of thyroid tumor progression. No effective treatment exists so far. ATC frequently derive from papillary thyroid carcinomas (PTC), which have a good prognosis. In this study, we analyzed the mRNA expression profiles of 59 thyroid tumors (11 ATC and 48 PTC) by microarrays. ATC and PTC showed largely overlapping mRNA expression profiles with most genes regulated in all ATC being also regulated in several PTC. 43% of the probes regulated in all the PTC are similarly regulated in all ATC. Many genes modulations observed in PTC are amplified in ATC. This illustrates the fact that ATC mostly derived from PTC. A molecular signature of aggressiveness composed of 9 genes clearly separates the two tumors. Moreover, this study demonstrates gene regulations corresponding to the ATC or PTC phenotypes like inflammatory reaction, epithelial to mesenchymal transition (EMT) and invasion, high proliferation rate, dedifferentiation, calcification and fibrosis processes, high glucose metabolism and glycolysis, lactate generation and chemoresistance. The main qualitative differences between the two tumor types bear on the much stronger EMT, dedifferentiation and glycolytic phenotypes showed by the ATC.

Role of Epac and protein kinase A in thyrotropin-induced gene expression in primary thyrocytes.

cAMP pathway activation by thyrotropin (TSH) induces differentiation and gene expression in thyrocytes. We investigated which partners of the cAMP cascade regulate gene expression modulations: protein kinase A and/or the exchange proteins directly activated by cAMP (Epac). Human primary cultured thyrocytes were analysed by microarrays after treatment with the adenylate cyclase activator forskolin, the protein kinase A (PKA) activator 6-MB-cAMP and the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (007) alone or combined with 6-MB-cAMP. Profiles were compared to those of TSH. Cultures treated with the adenylate cyclase- or the PKA activator alone or the latter combined with 007 had profiles similar to those induced by TSH. mRNA profiles of 007-treated cultures were highly distinct from TSH-treated cells, suggesting that TSH-modulated gene expressions are mainly modulated by cAMP and PKA and not through Epac in cultured human thyroid cells. To investigate whether the Epac-Rap-RapGAP pathway could play a potential role in thyroid tumorigenesis, the mRNA expressions of its constituent proteins were investigated in two malignant thyroid tumor types. Modulations of this pathway suggest an increased Rap pathway activity in these cancers independent from cAMP activation.

Extracellular superoxide dismutase is a thyroid differentiation marker down-regulated in cancer.

Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H(2)O(2) synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H(2)O(2)-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C-Ca(2+) and cAMP-protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.

Thyroid gene expression in familial nonautoimmune hyperthyroidism shows common characteristics with hyperfunctioning autonomous adenomas.

CONTEXT: Dominant activating mutations of the TSH receptor are the cause of familial nonautoimmune hyperthyroidism (FNAH) (inherited mutations affecting the whole gland since embryogenesis) and the majority of hyperfunctioning autonomous adenomas (AAs) (somatic mutations affecting only one cell later in the adulthood). OBJECTIVE: The objective of the study was defining the functional and molecular phenotypes of FNAH and comparing them with the ones of AA. DESIGN: Functional phenotypes were determined in vitro and molecular phenotypes by hybridization on microarray slides. PATIENTS: Nine patients with FNAH were investigated, six for functional in vitro study of the tissue and five for gene expression. RESULTS: Iodide metabolism, H(2)O(2), cAMP, and inositol phosphate generation in FNAH slices stimulated or not with TSH were normal. The mitogenic response of cultured FNAH thyrocytes to TSH was normal but more sensitive to the hormone. Gene expression profiles of FNAH and AAs showed that among 474 genes significantly regulated in FNAH, 93% were similarly regulated in AAs. Besides, 783 genes were regulated only in AAs. Bioinformatic analysis pointed out common down-regulations of genes involved in immune response, cell/cell and cell/matrix adhesions, and apoptosis. Pathways up-regulated only in AAs mainly involve diverse biosyntheses. These results are consonant with the larger growth of AAs than FNAH tissues. CONCLUSIONS: Whether hereditary or somatic after birth, activating mutations of the TSH receptor have the same qualitative consequences on the thyroid cell phenotype, but somatic mutations in AAs have a much stronger effect than FNAH mutations. Both are variants of one disease: genetic hyperthyroidism.

Long-term EGF/serum-treated human thyrocytes mimic papillary thyroid carcinomas with regard to gene expression.

Constitutive activation of the RAS/RAF/MAPK pathway has been found in different tumor types including papillary thyroid carcinomas (PTCs). To get more insight into genes primarily regulated in the human tumor cells, an in vitro model was developed in which primary cultures of human thyrocytes were treated for different times with epidermal growth factor and serum (EGF/serum), which stimulate the MAPK cascade. Gene expression profiles were obtained by microarrays and compared to the expression profiles of PTCs. An evolution from short-term to long-term EGF/serum-treated cells was found, i.e., a program change showing a distinction between gene expression profiles of short-term and long-term EGF/serum-treated cells. The late pattern of EGF/serum stimulated cells converges to the pattern of PTCs. Comparison of these two types of cells with cAMP activated cells, from thyroid-stimulating hormone-treated thyrocytes and autonomous adenomas, showed distinct gene expression profiles for the two pathways. For the two models, an overlap was found in a number of genes which were early induced in vitro but down-regulated later in vitro and in the in vivo tumors. Thus, long-term stimulated human primary cultures demonstrate a clear relation with the tumor in vivo and could therefore be used as models for the disease.

ACLAME: a CLAssification of Mobile genetic Elements.

The ACLAME database (http://aclame.ulb.ac.be) is a collection and classification of prokaryotic mobile genetic elements (MGEs) from various sources, comprising all known phage genomes, plasmids and transposons. In addition to providing information on the full genomes and genetic entities, it aims to build a comprehensive classification of the functional modules of MGEs at the protein, gene and higher levels. This first version contains a comprehensive classification of 5069 proteins from 119 DNA bacteriophages into over 400 functional families. This classification was produced automatically using TRIBE-MCL, a graph-theory-based Markov clustering algorithm that uses sequence measures as input, and then manually curated. Manual curation was aided by consulting annotations available in public databases retrieved through additional sequence similarity searches using Psi-Blast and Hidden Markov Models. The database is publicly accessible and open to expert volunteers willing to participate in its curation. Its web interface allows browsing as well as querying the classification. The main objectives are to collect and organize in a rational way the complexity inherent to MGEs, to extend and improve the inadequate annotation currently associated with MGEs and to screen known genomes for the validation and discovery of new MGEs.