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Objectives To evaluate a testing algorithm for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that includes the use of PCR-based targeted single nucleotide polymorphism (SNP) detection assays preceded by a multiplex PCR sensitive to S-Gene Target Failure (SGTF). Methods PCR SNP assays targeting SARS-CoV-2 S-gene mutations ΔH69–V70, L452R, E484K, N501Y, H655Y and P681R using melting curve analysis were performed on 567 samples in which SARS-CoV-2 viral RNA was detected by a multiplex PCR. Viral whole-genome sequencing (WGS) was performed to confirm the presence of SNPs and to identify the Pangolin lineage. Additionally, 1133 SARS-CoV-2 positive samples with SGTF were further assessed by WGS to determine the presence of ΔH69–V70. Results The N501Y-specific assay (n = 567) had an overall percentage agreement (OPA) of 98.5%. The ΔH69-V70-specific (n = 178) and E484K-specific (n = 401) assays had OPA of 96.6% and 99.7%, respectively. Assessment of H655Y (n = 139) yielded a 100.0% concordance when applied in the proposed algorithm. The L452R-specific (n = 67) and P681R-specific (n = 62) assays had an OPA of 98.2% and 98.1%, respectively. The proposed algorithm identified six variants of concern/interest (VOC/VOI)—Alpha (n = 149), Beta (n = 65), Gamma (n = 86), Delta (n = 49), Eta (n = 6), Kappa (n = 6)—and 205 non-VOC/VOI strains—including the variants under monitoring B.1.214.2 (n = 43) and B.1.1.318 (n = 18) and Epsilon (n = 1). An excellent concordance was observed for the identification of all SARS-CoV-2 lineages evaluated. Conclusions We present a flexible testing algorithm for the rapid detection of current and emerging SARS-CoV-2 VOC/VOIs, which can be easily adapted based on the local endemicity of specific variants.

International guidelines made RAS (KRAS and NRAS) status a prerequisite for the use of anti-EGFR agents for metastatic colorectal cancer (CRC) patients. Daily, new data emerges on the theranostic and prognostic role of molecular biomarkers; this is a strong incentive for a validated, sensitive, and broadly available molecular screening test. Next-generation sequencing (NGS) has begun to supplant other technologies for genomic profiling. We report here our 2 years of clinical practice using NGS results to guide therapeutic decisions. The Ion Torrent AmpliSeq colon/lung cancer panel, which allows mutation detection in 22 cancer-related genes, was prospectively used in clinical practice (BELAC ISO 15189 accredited method). The DNA of 741 formalin-fixed paraffinembedded CRC tissues, including primary tumors and metastasis, was obtained from 14 different Belgian institutions and subjected to targeted NGS. Of the tumors tested, 98% (727) were successfully sequenced and 89% (650) harbored at least one mutation. KRAS, BRAF and NRAS mutations were found in 335 (46%), 78 (11%) and 32 (4%) samples, respectively. These mutation frequencies were consistent with those reported in public databases. Moreover, mutations and amplifications in potentially actionable genes were identified in 464 samples (64%), including mutations in PIK3CA (14%), ERBB2 (0.4%), AKT1 (0.6%), and MAP2K1 (0.1%), as well as amplifications of ERBB2 (0.3%) and EGFR (0.3%). The median turnaround time between reception of the sample in the laboratory and report release was 8 calendar days. Overall, the AmpliSeq colon/lung cancer panel was successfully applied in daily practice and provided reliable clinically relevant information for CRC patients., SCOPUS: ar.j, info:eu-repo/semantics/published

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. ispartof: CANCERS vol:11 issue:12 ispartof: location:Switzerland status: published

Objective: International efforts to catalogue mutations for multiple forms of cancer coupled with the successes of targeted agents in patients with molecularly defined tumors and improvements in genomic technology have generated enthusiasm for incorporating genomic profiling into clinical cancer practice. Molecular testing represents a paradigm shift in lung cancer diagnosis and has now become a standard of care. International guidelines recommend testing for EGFR mutations to guide patient selection for therapy. However, different biomarkers for which potentially active agents are being evaluated, such as PIK3CA, BRAF or ERBB2 mutations, have been proposed as valuable for managing patients with lung cancer. The increase in the number of genes to test is associated with a decrease in the sample size. The pathologist is facing a new challenge: optimization of available tumor tissue. As the number of clinically significant genetic variants has increased, clinical testing has evolved, moving from single mutations to multiplex hotspot evaluations in multiple cancer genes. Recently, next generation sequencing (NGS) has begun to supplant other technologies for gene panel sequencing that is now required for targeted therapies. In the present study we evaluate the clinical applicability of targeted NGS for patients with lung cancer. Methods: After initial validation of the Ion Torrent AmpliSeq colon/lung cancer panel using commercial reference standards, the panel which interrogates 1850 hotspots in 22 cancer-related genes was prospectively applied to clinical practice. The DNA of 234 samples from 2 different institutions was obtained from formalin-fixed paraffin-embedded material (including 69 surgical specimens, 83 biopsies and 82 cell blocks) and subjected to targeted NGS with the Ion Torrent AmpliSeq colon/lung cancer panel using the Ion Torrent Personal Genome Machine. Results: Our validation study showed that sensitivity and accuracy for detecting variants at an allelic frequency >4% was 100% for commercial reference standards. The set of 234 samples included 120 primary tumors and 114 metastatic lesions. 88% of tested samples were adenocarcinomas. For one case only, the sequencing was not performed due to an insufficient quantity of available tissue. Among the 233 cases sequenced, 223 (95.7%) samples were successfully sequenced. The number of mutations per tumor ranged from 0 to 9. The most frequent mutations were found in TP53 (42.1%) and KRAS (35.9%). Of successfully sequenced cases, 57 potentially actionable mutations were identified in 54 patients (24.4%), including 26 EGFR mutations, 8 PIK3CA mutations, 14 BRAF mutations, 3 PTEN mutations, 2 ERBB2 insertions, 2 NRAS mutations and 2 MEK1 mutations. The frequencies of these variants detected by NGS were consistent with frequencies reported in public databases. Conclusions: Overall, the AmpliSeq colon/lung cancer panel can be applied in daily practice even for small samples, such as lung biopsies or cell blocks. Moreover, it provides clinically relevant information for lung cancer patients. Citation Format: Nicky D'Haene, Marie Le Mercier, Nancy De Neve, Oriane Blanchard, Myriam Remmelink, Birgit Weynand, Isabelle Salmon. Clinical application of targeted next-generation sequencing for lung cancer patients: A Belgian experience. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-39.

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the rapid and uncontrolled clonal growth of myeloid lineage cells in the bone marrow. The advent of oral, selective inhibitors of the B-cell leukemia/lymphoma-2 (BCL-2) apoptosis pathway, such as venetoclax, will likely induce a paradigm shift in the treatment of AML. However, the high cost of this treatment and the risk of additive toxicity when used in combination with standard chemotherapy represent limitations to its use and underscore the need to identify which patients are most&mdash, and least&mdash, likely to benefit from incorporation of venetoclax into the treatment regimen. Bone marrow specimens from 93 newly diagnosed AML patients were collected in this study and evaluated for BCL-2 protein expression by immunohistochemistry. Using this low-cost, easily, and readily applicable analysis method, we found that 1 in 5 AML patients can be considered as BCL-2&minus, In addition to a lower bone marrow blast percentage, this group exhibited a favorable molecular profile characterized by lower WT1 expression and underrepresentation of FLT3 mutations. As compared to their BCL-2+ counterparts, the absence of BCL-2 expression was associated with a favorable response to standard chemotherapy and overall survival, thus potentially precluding the necessity for venetoclax add-on.

Non‑coding RNAs (ncRNAs) have been shown to serve important roles in carcinogenesis via complex mechanisms, including transcriptional and post‑transcriptional regulation, and chromatin interactions. Urothelial carcinoma‑associated 1 (UCA1), a long ncRNA, was recently shown to have tumorigenic properties in urothelial bladder cancer (UBC), as demonstrated by enhanced proliferation, migration, invasion and therapy resistance of UBC cell lines in vitro. These in vitro findings suggested that UCA1 is associated with aggressive tumor behavior and could have prognostic implications in UBC. The aims of the present study were to therefore to investigate the statistical associations between UCA1 RNA expression and UBC pathological features, patient prognosis and p53 and Ki‑67 expression. Chromogenic in situ hybridization and immunohistochemistry were performed on UBC tissue microarrays to characterize UCA1 RNA, and p53 and Ki‑67 expression in 208 UBC cases, including 145 non‑muscle‑invasive and 63 muscle‑invasive cases. UCA1 was observed in the tumor cells of 166/208 (80%) UBC cases tested. No expression was noted in normal stromal and endothelium cells. Patients with UBC that overexpressed UCA1 (35%) had a significantly higher survival rate (P=0.006) compared with that in patients with UBC that did not overexpress UCA1. This prognostic factor was independent of tumor morphology, concomitant carcinoma in situ, tumor grade and tumor stage. In addition, the absence of UCA1 overexpression was significantly associated with a high Ki‑67 proliferative index (P=0.008) and a p53 'mutated' immunoprofile (strong nuclear expression or complete absence of staining; P=0.003). In conclusion, the present results identified UCA1 as potentially being a novel independent prognostic marker in UBC that was associated with a better patient prognosis and that could serve a pivotal role in bladder cancer carcinogenesis., SCOPUS: ar.j, info:eu-repo/semantics/published

The large screening of exons 18 to 21 of the epidermal growth factor receptor (EGFR) gene may lead to the discovery of rare, atypical molecular alterations for which the sensitivity to tyrosine kinase inhibitors (TKIs) remains uncertain. We are reporting a rare exon 18 EGFR mutation (p.E709_710 > D) that confers sensitivity to second-generation EGFR TKI (afatinib), lasting for 1 year. Tumor progression biopsy showed small cell lung cancer transformation, associated with a SMAD4 mutation. Key Points A rare exon 18 epidermal growth factor receptor mutation with sensitivity to afatinib is reported. Small cell transformation was observed at tumor progression. Acquisition of a SMAD4 mutation was observed at tumor progression.

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2-GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2-GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.

Promising targeted therapy and personalized medicine are making molecular profiling of tumours a priority. For colorectal cancer (CRC) patients, international guidelines made RAS (KRAS and NRAS) status a prerequisite for the use of anti-epidermal growth factor receptor agents (anti-EGFR). Daily, new data emerge on the theranostic and prognostic role of molecular biomarkers, which is a strong incentive for a validated, sensitive and broadly available molecular screening test in order to implement and improve multi-modal therapy strategy and clinical trials. Next generation sequencing (NGS) has begun to supplant other technologies for genomic profiling. Targeted NGS is a method that allows parallel sequencing of thousands of short DNA sequences in a single test offering a cost-effective approach for detecting multiple genetic alterations with a minimum amount of DNA. In the present review, we collected data concerning the clinical application of NGS technology in the setting of colorectal cancer., SCOPUS: re.j, info:eu-repo/semantics/published

A large number of drugs are known to bind with high affinity to sigma receptors (sigma-Rs) and have been used in the clinic to treat mental disorders for many years. However, recent publications highlighting sigma-R overexpression in many cancer tissues suggest potential applications for sigma-R ligands in cancer diagnosis and therapy. The present review focuses on the involvement of sigma-Rs in cancer biology and the potential therapeutic contributions of their pharmacologic ligands in oncology. After summarizing the current and general knowledge regarding sigma-Rs, we detail data reported in the particular context of oncology. We then investigate the potential and specific signal transduction pathways and mechanisms involved in the actions of sigma-R ligands in cancer biology. These processes include modulations of (1) the plasma membrane and lipid raft components, (2) intracellular calcium levels, (3) cytoskeletal protein functions, and (4) endoplasmic reticulum stress. Finally, we conclude by speculating on the roles of sigma-R overexpression and sigma-R ligands in cancer biology and offer perspectives on cancer therapy.

Gliomas account for more than 50% of all primary brain tumors. The worst prognosis is associated with gliomas of astrocytic origin, whereas gliomas with an oligodendroglial origin offer higher sensitivity to chemotherapy, especially when oligodendroglioma cells display 1p19q deletions. Temozolomide (TMZ) provides therapeutic benefits and is commonly used with radiotherapy in highly malignant astrocytic tumors, including glioblastomas. The actual benefits of TMZ during long-term treatment in oligodendroglioma patients have not yet been clearly defined. In this study, we have investigated the effects of such a long-term TMZ treatment in the unique Hs683 oligodendroglioma model. We have observed increased TMZ sensitivity of Hs683 orthotopic tumors that were previously treated in vitro with months of progressive exposure to increasing TMZ concentrations before being xenografted into the brains of immunocompromised mice. Whole-genome and proteomic analyses have revealed that this increased TMZ sensitivity of Hs683 oligodendroglioma cells previously treated for long periods with TMZ can be explained, at least partly, by a TMZ-induced p38-dependant dormancy state, which in turn resulted in changes in amino acid metabolism balance, in growth delay, and in a decrease in Hs683 oligodendroglioma cell-invasive properties. Thus, long-term TMZ treatment seems beneficial in this Hs683 oligodendroglioma model, which revealed itself unable to develop resistance against TMZ., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Cell motility and resistance to apoptosis characterize glioblastoma (GBM) growth and malignancy. In our current work we report that galectin-1, a homodimeric adhesion molecule and carbohydrate-binding protein with affinity for β-galactosides, is linked with cell surface expression of integrin β1 and the process of integrin trafficking. Using immunofluorescence, depletion of galectin-1 through both stable knockdown and transient-targeted small interfering RNA (siRNA) treatment induces an intracellular accumulation of integrin-β1 coincident with a diminution of integrin-β1 at points of cellular adhesion at the cell membrane. Galectin-1 depletion does not alter the gene expression level of integrin-β1. Transient galectin-1 depletion effectuates as well the perinuclear accumulation of protein kinase C epsilon (PKCe) and the intermediate filament vimentin, both of which have been shown to mediate integrin recycling in motile cells. Our results argue for the involvement of galectin-1 in the PKCe/vimentin-controlled trafficking of integrin-β1. The understanding of molecular mediators such as galectin-1 and the pathways through which they drive the cell invasion so descriptive of GBM is anticipated to reveal potential therapeutic targets that promote glioma malignancy.

Malignant gliomas, especially glioblastomas, are associated with a dismal prognosis. Despite advances in diagnosis and treatment, glioblastoma patients still have a median survival expectancy of only 14 months. This poor prognosis can be at least partly explained by the fact that glioma cells diffusely infiltrate the brain parenchyma and exhibit decreased levels of apoptosis, and thus resistance to cytotoxic drugs. Galectins are a family of mammalian beta-galactoside-binding proteins characterized by a shared characteristic amino acid sequence. They are expressed differentially in normal vs. neoplastic tissues and are known to play important roles in several biological processes such as cell proliferation, death and migration. This review focuses on the role played by galectins, especially galectin-1 and galectin-3, in glioma biology. The involvement of these galectins in different steps of glioma malignant progression such as migration, angiogenesis or chemoresistance makes them potentially good targets for the development of new drugs to combat these malignant tumors.

Osteopontin (OPN), a member of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) family, is overexpressed in human glioblastoma. Higher levels of OPN expression correlate with increased tumor grade and enhanced migratory capacity of tumor cells. Based on these observations, we explored the possibility that knocking down OPN expression in glioblastoma cells could exert an anti-tumoral activity using an avian in vivo glioblastoma model that mimics closely human gliobastoma. Human U87-MG glioma cells transfected with specific anti-OPN small interfering RNAs (siRNAs) were grafted onto the chicken chorio-allantoic membrane (CAM). OPN-deficient U87-MG cells gave rise to tumors that were significantly smaller than tumors formed from untransfected cells (paired t-test, p < 0.05). Accordingly, the amount of proliferating cells in OPN-deficient tumors showed a six-fold reduction when compared to control tumors. However, OPN inhibition did not affect significantly tumor-associated angiogenesis. In vitro, OPN-silenced U87-MG and U373-MG cells showed decreased motility and migration. This is the first demonstration that OPN inhibition blocks glioma tumor growth, making this invasion-related protein an attractive target for glioma therapy.

Aromatic 3,3'-diesters of thiodigalactoside were synthesized in a rapid three-step sequence from commercially available thiodigalactoside and evaluated as inhibitors of cancer- and immunity-related galectins. For each of galectins-1, -3, -7, and -9N-terminal domain, aromatic 3,3'-diesters of thiodigalactoside were found to have affinities in the low micromolar range, which represents a 7-70 fold enhancement over thiodigalactoside itself. No significant improvement was found for galectin-8 N-terminal domain. Two of the compounds were selected for testing in cell culture and were shown to have potent antimigratory effects on human PC-3 prostate and human A549 nonsmall-cell lung cancer cells.

Aneuploidy is found in most solid tumours, but it remains unclear whether it is the cause or the consequence of tumorigenesis. Using Plk4 overexpression (PLK4OE) during epidermal development, we assess the impact of centrosome amplification and aneuploidy on skin development and tumorigenesis. PLK4OE in the developing epidermis induced centrosome amplification and multipolar divisions, leading to p53 stabilization and apoptosis of epidermal progenitors. The resulting delayed epidermal stratification led to skin barrier defects. Plk4 transgene expression was shut down postnatally in the surviving mice and PLK4OE mice never developed skin tumours. Concomitant PLK4OE and p53 deletion (PLK4OE/p53cKO) rescued the differentiation defects, but did not prevent the apoptosis of PLK4OE cells. Remarkably, the short-term presence of cells with supernumerary centrosomes in PLK4OE/p53cKO mice was sufficient to generate aneuploidy in the adult epidermis and triggered spontaneous skin cancers with complete penetrance. These results reveal that aneuploidy induced by transient centrosome amplification can accelerate tumorigenesis in p53-deficient cells.

To evaluate the efficiency of the UCA1 test as a diagnostic tool for the detection of bladder cancer., SCOPUS: ar.j, info:eu-repo/semantics/published

Objective: Recently, Next Generation Sequencing (NGS) has begun to supplant other technologies for gene mutation testing that is now required for targeted therapies. However, transfer of NGS technology to clinical daily practice requires validation. Methods: We validated the Ion Torrent AmpliSeq Colon and Lung cancer panel interrogating 1850 hotspots in 22 genes using the Ion Torrent Personal Genome Machine. First, we used commercial reference standards that carry mutations at defined allelic frequency (AF). Then, 51 colorectal adenocarcinomas (CRC) and 39 non small cell lung carcinomas (NSCLC) were retrospectively analyzed. Results: Sensitivity and accuracy for detecting variants at an AF >4% was 100% for commercial reference standards. Among the 90 cases, 89 (98.9%) were successfully sequenced. Among the 86 samples for which NGS and the reference test were both informative, 83 showed concordant results between NGS and the reference test; i.e. KRAS and BRAF for CRC and EGFR for NSCLC, with the 3 discordant cases each characterized by an AF, SCOPUS: ar.j, info:eu-repo/semantics/published

Galectins are a family of carbohydrate-binding proteins with an affinity for beta-galactosides. Galectin-1 (Gal-1) is differentially expressed by various normal and pathological tissues and appears to be functionally polyvalent, with a wide range of biological activity. The intracellular and extracellular activity of Gal-1 has been described. Evidence points to Gal-1 and its ligands as one of the master regulators of such immune responses as T-cell homeostasis and survival, T-cell immune disorders, inflammation and allergies as well as host-pathogen interactions. Gal-1 expression or overexpression in tumors and/or the tissue surrounding them must be considered as a sign of the malignant tumor progression that is often related to the long-range dissemination of tumoral cells (metastasis), to their dissemination into the surrounding normal tissue, and to tumor immune-escape. Gal-1 in its oxidized form plays a number of important roles in the regeneration of the central nervous system after injury. The targeted overexpression (or delivery) of Gal-1 should be considered as a method of choice for the treatment of some kinds of inflammation-related diseases, neurodegenerative pathologies and muscular dystrophies. In contrast, the targeted inhibition of Gal-1 expression is what should be developed for therapeutic applications against cancer progression. Gal-1 is thus a promising molecular target for the development of new and original therapeutic tools.

Despite advances in diagnosis and treatment, the overall outcomes for patients with brain tumors remain unpredictable. New prognostic markers are still needed to identify high-risk patients for whom the standard treatment has poor outcomes and would thus be well suited for more aggressive therapies. Neovascularization has long been implicated as a salient feature of glioma progression. In fact, high-grade gliomas are among the most vascular of all solid tumors, and vascular proliferation is a pathological hallmark of glioblastomas. Galectins are known to play important roles in cancer biology, including cancer cell migration, tumor immune escape or tumor angiogenesis. Moreover, galectins were reported to be involved in glioma progression. Given the key role of angiogenesis in brain tumors, the expression of galectins in tumor-associated endothelial cells (EC) and the implication of galectins in angiogenesis, the present review will focus on the expression of galectins in ECs of normal brain and brain tumors.

Aims The assessment of thyroid nodules is a common clinical challenge. Fine-needle aspiration (FNA) is the standard pre-operative tool for thyroid nodule diagnosis. However, up to 30% of the samples are classified as indeterminate. This often leads to unnecessary surgery. In this study, we evaluated the added value of next-generation sequencing (NGS) for helping in the diagnosis of FNA samples. Methods and results We analysed retrospectively 34 indeterminate FNA samples for which surgical resection was performed. DNA was obtained from cell blocks or from stained smears and subjected to NGS to analyse mutations in 50 genes. Mutations in BRAF, NRAS, KRAS and PTEN, that are known to be involved in thyroid cancer biology, were detected in seven FNA samples. The presence of a mutation in these genes was a strong indicator of cancer because five (71%) of the mutation-positive FNA samples had a malignant diagnosis after surgery. Moreover, there was only an 8% cancer risk in nodules with an indeterminate cytological diagnosis but with a negative molecular test. Conclusion This study demonstrates that thyroid FNA can be analysed successfully by NGS. The detection of mutations known to be involved in thyroid cancer improves the sensitivity of thyroid FNA diagnosis.

Les gliomes representent les tumeurs cerebrales primitives les plus frequentes et regroupent differentes entites au pronostic tres different. L’examen anatomopathologique est le gold standard pour le diagnostic de ces tumeurs. Cependant, les pathologistes peuvent rencontrer des difficultes diagnostiques dues entre autres a l’heterogeneite tumorale ou a la petite taille des prelevements. Nous avons assiste, ces dernieres annees, a des avancees majeures dans la decouverte des alterations moleculaires de ces cancers qui ont mene au developpement de nouveaux marqueurs moleculaires, certains avec un role diagnostic, d’autres avec un impact pronostic et/ou predictif de la reponse therapeutique. Dans la pratique quotidienne, il est donc devenu primordial de tester la presence de differentes alterations moleculaires telles que la codeletion 1p/19q, les mutations des genes IDH, la deletion de p16, l’amplification du gene EGFR ou la methylation du promoteur du gene MGMT, afin de confirmer le diagnostic, d’evaluer le pronostic des patients ainsi que d’orienter les choix therapeutiques.

Aim:Accumulating evidence suggests that extracellular galectin-1 and galectin-3 promote angiogenesis. Increased expression of galectin-1 and/or galectin-3 has been reported to be associated with tumour progression. Thus, it is critical to identify their influence on angiogenesis.Methods:We examined the individual and combined effects of galectin-1 and galectin-3 on endothelial cell (EC) growth and tube formation using two EC lines, EA.hy926 and HUVEC. The activation of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) was determined by ELISA and Western blots. We evaluated the VEGFR1 and VEGFR2 levels in endosomes by proximity ligation assay.Results:We observed different responses to exogenous galectins depending on the EC line. An enhanced effect on EA.hy926 cell growth and tube formation was observed when both galectins were added together. Focusing on this enhanced effect, we observed that together galectins induced the phosphorylation of both VEGFR1 and VEGFR2, whereas galectin-1 and -3 alone induced VEGFR2 phosphorylation only. In the same way, the addition of a blocking VEGFR1 antibody completely abolished the increase in tube formation induced by the combined addition of both galectins. In contrast, the addition of a blocking VEGFR2 antibody only partially inhibited this effect. Finally, the addition of both galectins induced a decrease in the VEGFR1 and VEGFR2 endocytic pools, with a significantly enhanced effect on the VEGFR1 endocytic pool. These results suggest that the combined action of galectin-1 and galectin-3 has an enhanced effect on angiogenesis via VEGFR1 activation, which could be related to a decrease in receptor endocytosis. © 2013 D'Haene et al., SCOPUS: ar.j, info:eu-repo/semantics/published

2038Background: vascular endothelial growth factor receptor (VEGFR) signal transduction mediates glioblastoma (GB) associated neo-angiogenesis. Axitinib, a small molecule TKI with high affinity and specificity for the VEGFRs, has demonstrated anti-tumor activity in patients with recurrent GB (J Clin Oncol 32:5s, 2014 [suppl; abstr 2018]). We investigated whether the combination of axitinib with lomustine (LOM) improves the outcome of pts with rGB as opposed to axitinib monotherapy. Methods: pts with rGB were randomized between single agent axitinib (AXI) vs. axitinib in combination with LOM (AXILOM) in an open label, randomized, phase II clinical trial. Pts in the AXI arm were allowed to cross over to AXILOM at progression. Six-month PFS served as the primary endpoint. Results: between February 2014 and July 2015, 56 pts were randomized 1:1 to AXI and AXILOM. Baseline characteristics were well balanced between both study arms (median age 56y [range 18-75]; 35M/21F; 15, 19, 11 and 11 pts had a WHO-PS of 0,...

Objective: Glioblastomas (GBMs) are the most common malignant primary brain tumours in adults. These tumours are resistant to conventional treatment approaches including surgical resection, radiotherapy and chemotherapy. International efforts to catalogue mutations for multiple forms of cancer coupled with the successes of targeted agents in patients with molecularly defined tumors and improvements in genomic technology have generated enthusiasm for incorporating genomic profiling into clinical cancer practice. The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Therefore, the number of biomarkers that will need to be assessed is expected to increase rapidly. Recently, next generation sequencing (NGS) has begun to supplant other technologies for gene panel sequencing. However, few studies have validated the use of targeted NGS for GBM patients. In the present study we evaluate the clinical applicability of targeted NGS for patients with GBM. Methods: DNA from 50 GBM samples (formalin-fixed paraffin-embedded tissue) was retrospectively subjected to targeted NGS with the Ampliseq Cancer Hotspot Panel, using the Ion Torrent Personal Genome Machine, which allowed us to analyze 2850 known cancer-related mutations in 50 genes. In addition, MGMT methylation status, EGFR amplification and 1p19q deletion were evaluated by Methylation Specific PCR (MSP), chromogenic in situ hybridisation (ISH) and fluorescence ISH, respectively. Results: Preliminary results on 28 patients, all successfully sequenced, showed that the most frequent mutations were found in TP53 (25%) and EGFR (18%). Potentially actionable mutations were identified in 9 patients (32%), including 5 EGFR mutations, 2 PIK3CA mutations, 1 PTEN mutation, 1 PDGFRA mutation and 1 IDH1 mutation. The frequencies of these variants detected by NGS were consistent with frequencies reported in public databases. Moreover, PDGFRA and EGFR amplifications were detected by coverage analysis for 10 (36%) and 2 (7%) patients respectively. Conclusions: Overall, the AmpliSeq Cancer Hotspot Panel can be applied in daily practice for GBM samples. Moreover, it can provide clinically relevant information for GBM patients. Citation Format: Anne-Laure Trépant, Marie Le Mercier, Calliope Maris, Nancy De Nève, Oriane Blanchard, Nicky D'Haene, Isabelle Salmon. Clinical validation of targeted next-generation sequencing for glioblastoma patients. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B10.

BACKGROUND: Caveolin-1 is a protein that displays promotive versus preventive roles in cancer progression according to circumstances. Temozolomide (TMZ) is the standard chemotherapeutic to treat glioma patients. The present work aims to characterizeTMZ-induced effects on caveolin-1 expression in glioma cells. METHODS: Human astroglioma (U373 and T98G) and oligodendroglioma (Hs683) cell lines were used in vitro as well as in vivo orthotopic xenografts (Hs683 and U373) into the brains of immunocompromisedmice. In vitro TMZ-induced effects on protein expression and cellular localization were determined by Western blot analysis and on the actin cytoskeleton organization by means of immunofluorescence approaches. In vivo TMZ-induced effects in caveolin-1 expression in human glioma xenografts were monitored by means of immunohistochemistry. RESULTS: TMZ modified caveolin-1 expression and localization in vitro and in vivo after an administration schedule that slightly, if at all, impaired cell growth characteristics in vitro . Caveolin-1 by itself (at a 100-ng/ml concentration) was able to significantly reduce invasiveness (Boyden chambers) of the three human glioma cell lines. The TMZ-inducedmodification in caveolin-1 expression in flotation/raft compartments was paralleled by altered Cyr61 and β 1 integrin expression, two elements that have already been reported to collaborate with caveolin-1 in regulating glioma cell biology, and all these features led to profound reorganization of the actin cytoskeleton. An experimental Src kinase inhibitor, AZD0530, almost completely antagonized the TMZ-induced modulation in caveolin-1 expression. CONCLUSION: TMZ modifies caveolin-1 expression in vitro and in vivo in glioma cells, a feature that directly affects glioma cell migration properties.

Glioblastoma (GBM) is the most frequent malignant glioma. Treatment of GBM patients is multimodal with maximum surgical resection, followed by concurrent radiation and chemotherapy with the alkylating drug temozolomide (TMZ). The present study aims to identify genes implicated in the acquired resistance of two human GBM cells of astrocytic origin, T98G and U373, to TMZ. Resistance to TMZ was induced by culturing these cells in vitro for months with incremental TMZ concentrations up to 1 mM. Only partial resistance to TMZ has been achieved and was demonstrated in vivo in immunocompromised mice bearing orthotopic U373 and T98G xenografts. Our data show that long-term treatment of human astroglioma cells with TMZ induces increased expression of facilitative glucose transporter/solute carrier GLUT/SLC2A family members, mainly GLUT-3, and of the AKR1C family of proteins. The latter proteins are phase 1 drug-metabolizing enzymes involved in the maintenance of steroid homeostasis, prostaglandin metabolism, and metabolic activation of polycyclic aromatic hydrocarbons. GLUT-3 has been previously suggested to exert roles in GBM neovascularization processes, and TMZ was found to exert antiangiogenic effects in experimental gliomas. AKR1C1 was previously shown to be associated with oncogenic potential, with proproliferative effects similar to AKR1C3 in the latter case. Both AKR1C1 and AKR1C2 proteins are involved in cancer pro-proliferative cell chemoresistance. Selective targeting of GLUT-3 in GBM and/or AKR1C proteins (by means of jasmonates, for example) could thus delay the acquisition of resistance to TMZ of astroglioma cells in the context of prolonged treatment with this drug., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

A large number of drugs are known to bind with high affinity to sigma receptors (sigma-Rs) and have been used in the clinic to treat mental disorders for many years. However, recent publications highlighting sigma-R overexpression in many cancer tissues suggest potential applications for sigma-R ligands in cancer diagnosis and therapy. The present review focuses on the involvement of sigma-Rs in cancer biology and the potential therapeutic contributions of their pharmacologic ligands in oncology. After summarizing the current and general knowledge regarding sigma-Rs, we detail data reported in the particular context of oncology. We then investigate the potential and specific signal transduction pathways and mechanisms involved in the actions of sigma-R ligands in cancer biology. These processes include modulations of (1) the plasma membrane and lipid raft components, (2) intracellular calcium levels, (3) cytoskeletal protein functions, and (4) endoplasmic reticulum stress. Finally, we conclude by speculating on the roles of sigma-R overexpression and sigma-R ligands in cancer biology and offer perspectives on cancer therapy.

Osteopontin (OPN), a member of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) family, is overexpressed in human glioblastoma. Higher levels of OPN expression correlate with increased tumor grade and enhanced migratory capacity of tumor cells. Based on these observations, we explored the possibility that knocking down OPN expression in glioblastoma cells could exert an anti-tumoral activity using an avian in vivo glioblastoma model that mimics closely human gliobastoma. Human U87-MG glioma cells transfected with specific anti-OPN small interfering RNAs (siRNAs) were grafted onto the chicken chorio-allantoic membrane (CAM). OPN-deficient U87-MG cells gave rise to tumors that were significantly smaller than tumors formed from untransfected cells (paired t-test, p0.05). Accordingly, the amount of proliferating cells in OPN-deficient tumors showed a six-fold reduction when compared to control tumors. However, OPN inhibition did not affect significantly tumor-associated angiogenesis. In vitro, OPN-silenced U87-MG and U373-MG cells showed decreased motility and migration. This is the first demonstration that OPN inhibition blocks glioma tumor growth, making this invasion-related protein an attractive target for glioma therapy.

Cell motility and resistance to apoptosis characterize glioblastoma (GBM) growth and malignancy. In our current work we report that galectin-1, a homodimeric adhesion molecule and carbohydrate-binding protein with affinity for beta-galactosides, is linked with cell surface expression of integrin beta1 and the process of integrin trafficking. Using immunofluorescence, depletion of galectin-1 through both stable knockdown and transient-targeted small interfering RNA (siRNA) treatment induces an intracellular accumulation of integrin-beta1 coincident with a diminution of integrin-beta1 at points of cellular adhesion at the cell membrane. Galectin-1 depletion does not alter the gene expression level of integrin-beta1. Transient galectin-1 depletion effectuates as well the perinuclear accumulation of protein kinase C epsilon (PKCepsilon) and the intermediate filament vimentin, both of which have been shown to mediate integrin recycling in motile cells. Our results argue for the involvement of galectin-1 in the PKCepsilon/vimentin-controlled trafficking of integrin-beta1. The understanding of molecular mediators such as galectin-1 and the pathways through which they drive the cell invasion so descriptive of GBM is anticipated to reveal potential therapeutic targets that promote glioma malignancy.

Galectin (Gal) 1 is a hypoxia-regulated proangiogenic factor that also directly participates in glioblastoma cell migration. To determine how Gal-1 exerts its proangiogenic effects, we investigated Gal-1 signaling in the human Hs683 glioblastoma cell line. Galectin 1 signals through the endoplasmic reticulum transmembrane kinase/ribonuclease inositol-requiring 1alpha, which regulates the expression of oxygen-regulated protein 150. Oxygen-regulated protein 150 controls vascular endothelial growth factor maturation. Galectin 1 also modulates the expression of 7 other hypoxia-related genes (i.e. CTGF, ATF3, PPP1R15A, HSPA5, TRA1, and CYR61) that are implicated in angiogenesis. Decreasing Gal-1 expression in Hs683 orthotopic xenografts in mouse brains by siRNA administration impaired endoplasmic reticulum stress and enhanced the therapeutic benefits of the proautophagic drug temozolomide. These results suggest that decreasing Gal-1 expression (e.g. through brain delivery of nonviral infusions of anti-Gal-1 siRNA in patients) can represent an additional therapeutic strategy for glioblastoma.

Glioblastomas (GBMs) are resistant to apoptosis but less so to autophagy; a fact that may at least partly explain the therapeutic benefits of the pro-autophagic drug temozolomide in the treatment of GBM patients. Galectin-1 (Gal1) whose expression is stimulated by hypoxia is a potent modulator of GBM cell migration and a pro-angiogenic molecule. Hypoxia is also known to confer cancer cells with resistance to chemotherapy and radiotherapy and to modulate the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. The present study investigates whether decreasing Gal1 expression (by means of a siRNA approach) in human Hs683 GBM cells increases their sensitivity to pro-autophagic or pro-apoptotic drugs. The data reveal that temozolomide, the standard treatment for glioma patients, increases Gal1 expression in Hs683 cells both in vitro and in vivo. However, reducing Gal1 expression in these cells by siRNA increases the anti-tumor effects of various chemotherapeutic agents, in particular temozolomide both in vitro and in vivo. This decrease in Gal1 expression in Hs683 cells does not induce apoptotic or autophagic features, but is found to modulate p53 transcriptional activity and decrease p53-targeted gene expression including DDIT3/GADD153/CHOP, DUSP5 ATF3 and GADD45A. The decrease in Gal1 expression also impairs the expression levels of seven other genes implicated in chemoresistance: ORP150, HERP, GRP78/Bip, TRA1, BNIP3L, GADD45B and CYR61, some of which are located in the ER and whose expression is also known to be modified by hypoxia. This novel facet of Gal1 involvement in glioblastoma biology may be amenable to therapeutic manipulation.

Le next generation sequencing (NGS) est une technologie qui permet de sequencer simultanement des millions de courts fragments d’ADN. Cette technologie offre un grand avantage par rapport aux techniques de sequencage traditionnelles de part son debit, son cout reduit, sa rapidite et sa grande sensibilite pour la detection de mutations dans l’ADN. Le NGS permet differentes applications comme le sequencage de genomes entiers, d’exome, de transcriptome ou le sequencage cible de multiples regions. De plus ce sequencage peut etre effectue a partir de seulement 10 ng d’ADN permettant ainsi de sequencer de tres petits echantillons FFPE tels que ceux le plus couramment disponible dans la pratique clinique. De nombreuses etudes ont ainsi demontre la faisabilite et les avantages du sequencage cible par NGS pour la caracterisation moleculaire de differents types de cancers tel que les cancers colorectaux et pulmonaires, et l’interet d’integrer ces methodes a la pratique clinique.