Monitoring the SARS-CoV-2 pandemic: screening algorithm with single nucleotide polymorphism detection for the rapid identification of established and emerging variants.

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.

SMAD4 Mutation in Small Cell Transformation of Epidermal Growth Factor Receptor Mutated Lung Adenocarcinoma.

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.

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.

UCA1 overexpression is associated with less aggressive subtypes of bladder cancer.

Non­coding RNAs (ncRNAs) have been shown to serve important roles in carcinogenesis via complex mechanisms, including transcriptional and post­transcriptional regulation, and chromatin interactions. Urothelial carcinoma­associated 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 Ki­67 expression. Chromogenic in situ hybridization and immunohistochemistry were performed on UBC tissue microarrays to characterize UCA1 RNA, and p53 and Ki­67 expression in 208 UBC cases, including 145 non­muscle­invasive and 63 muscle­invasive 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 Ki­67 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.

Clinical application of targeted next-generation sequencing for colorectal cancer patients: a multicentric Belgian experience.

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 paraffin-embedded 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.

Clinical Application of Targeted Next Generation Sequencing for Colorectal Cancers.

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.

Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer.

Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients.

Transient PLK4 overexpression accelerates tumorigenesis in p53-deficient epidermis.

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.

Clinical Validation of Targeted Next Generation Sequencing for Colon and Lung Cancers.

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 <10%. CONCLUSIONS: Overall, the AmpliSeq colon/lung cancer panel was specific and sensitive for mutation analysis of gene panels and can be incorporated into clinical daily practice.

Diagnostic value of the UCA1 test for bladder cancer detection: a clinical study.

PURPOSE: To evaluate the efficiency of the UCA1 test as a diagnostic tool for the detection of bladder cancer. METHODS: Between October 2009 and December 2011 the UCA1 test was performed on collected urine samples from 162 patients divided into screening and follow-up groups, based on the absence or presence of prior bladder cancer. The test performance was then evaluated in each group and compared to cystoscopy and urinary cytology. RESULTS: The overall sensitivity, specificity and positive and negative predictive values for the UCA1 test were 70, 70.7, 75.6 and 64.5%, respectively. We observed no difference in performance for tumours of higher grade or stage, but sensitivity was increased in the screening population compared to patients under follow-up (83.9 vs. 59%). The UCA1 test successfully detected all 7 cases of isolated carcinoma in situ and was more sensitive in this particular setting than cystoscopy or urinary cytology. CONCLUSION: The efficiency of the UCA1 test for the detection of primary and recurring bladder cancer in our study was lower than previously reported. We confirmed the role of UCA1 as a possible adjunct to cystoscopy and cytology when a primary bladder cancer is suspected, but its role in the follow-up of recurring tumours remains limited. Further studies are needed to investigate the role of the UCA1 test in the early detection of carcinoma in situ lesions.

Next-generation sequencing improves the diagnosis of thyroid FNA specimens with indeterminate cytology.

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.

SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma.

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.

Galectins and neovascularization in central nervous system tumors.

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.

VEGFR1 and VEGFR2 involvement in extracellular galectin-1- and galectin-3-induced angiogenesis.

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.

A simplified approach for the molecular classification of glioblastomas.

Glioblastoma (GBM) is the most common malignant primary brain tumors in adults and exhibit striking aggressiveness. Although GBM constitute a single histological entity, they exhibit considerable variability in biological behavior, resulting in significant differences in terms of prognosis and response to treatment. In an attempt to better understand the biology of GBM, many groups have performed high-scale profiling studies based on gene or protein expression. These studies have revealed the existence of several GBM subtypes. Although there remains to be a clear consensus, two to four major subtypes have been identified. Interestingly, these different subtypes are associated with both differential prognoses and responses to therapy. In the present study, we investigated an alternative immunohistochemistry (IHC)-based approach to achieve a molecular classification for GBM. For this purpose, a cohort of 100 surgical GBM samples was retrospectively evaluated by immunohistochemical analysis of EGFR, PDGFRA and p53. The quantitative analysis of these immunostainings allowed us to identify the following two GBM subtypes: the "Classical-like" (CL) subtype, characterized by EGFR-positive and p53- and PDGFRA-negative staining and the "Proneural-like" (PNL) subtype, characterized by p53- and/or PDGFRA-positive staining. This classification represents an independent prognostic factor in terms of overall survival compared to age, extent of resection and adjuvant treatment, with a significantly longer survival associated with the PNL subtype. Moreover, these two GBM subtypes exhibited different responses to chemotherapy. The addition of temozolomide to conventional radiotherapy significantly improved the survival of patients belonging to the CL subtype, but it did not affect the survival of patients belonging to the PNL subtype. We have thus shown that it is possible to differentiate between different clinically relevant subtypes of GBM by using IHC-based profiling, a method that is advantageous in its ease of daily implementation and in large-scale clinical application.

Sigma receptors and their ligands in cancer biology: overview and new perspectives for cancer therapy.

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.

Correlation between IDH1 gene mutation status and survival of patients treated for recurrent glioma.

Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been frequently found in low-grade glioma and secondary glioblastoma and are associated with a significantly younger age at diagnosis and a superior overall survival. We investigated the IDH1 gene mutation status by nested PCR and denaturing gradient gel electrophoresis (DGGE) on DNA extracted from archival tumor blocks of 63 glioma patients who were treated following recurrence with the epidermal growth factor receptor (EGFR)-targeted blocking monoclonal antibody cetuximab, or the vascular endothelial growth factor (receptor) (VEGF(R))-targeted agents sunitinib malate and bevacizumab. In our study population, IDH1 mutation was significantly correlated with a longer overall survival (OS) from the time of initial diagnosis. Patients with IDH1 mutation also had a superior OS from the time of recurrence when treated with sunitinib or bevacizumab but a worse OS when treated with cetuximab. Our observations support the hypothesis that IDH1 mutation may correlate with the benefit from VEGF(R)- versus EGFR-targeted therapy at the time of recurrence in glioma patients.

Temozolomide modifies caveolin-1 expression in experimental malignant gliomas in vitro and in vivo.

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 ß(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.

Long-term in vitro treatment of human glioblastoma cells with temozolomide increases resistance in vivo through up-regulation of GLUT transporter and aldo-keto reductase enzyme AKR1C expression.

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.