Nancy De Nève

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.

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

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.

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.

Neurotensin is a versatile modulator of in vitro human pancreatic ductal adenocarcinoma cell (PDAC) migration.

Background: While the neurotensin (NT) roles in pancreatic cancer growth are well documented, its effects on pancreatic cancer cell migration have not been described. Methods: The NT-induced effects on the migration process of human pancreatic ductal adenocarcinoma cells (PDACs) were characterized by means of various assays including computer-assisted video-microscopy, fluorescence microscopy, ELISA-based, small GTPase pull-down and phosphorylation assays. Results: The NT-induced modifications on in vitro PDACs migration largely depended on the extra-cellular matrix environment and cell propensity to migrate collectively or individually. While NT significantly reduced the level of migration of collectively migrating PDACs on vitronectin, it significantly increased the level of individually migrating PDACs. These effects were mainly mediated through the sortilin/NTR3 receptor. Neurotensin both induced altered expression of αV and β5 integrin subunits in PDACs cultured on vitronectin resulting in modified adhesion abilities, and caused modifications to the organization of the actin cytoskeleton through the NT-mediated activation of small Rho GTPases. While the NT effects on individually migrating PDACs were mediated at least through the EGFR/ERK signaling pathways, those on collectively migrating PDACs appeared highly dependent on the PI 3-kinase pathway. Conclusion: This study strongly suggests the involvement of neurotensin in the modulation of human PDAC migration.

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

PurposeTo evaluate the efficiency of the UCA1 test as a diagnostic tool for the detection of bladder cancer.MethodsBetween 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.ResultsThe 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.ConclusionThe 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.

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.

Abstract A1-39: Clinical application of targeted next-generation sequencing for lung cancer patients: A Belgian experience

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 D9Haene, 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.

Abstract B10: Clinical validation of targeted next-generation sequencing for glioblastoma patients

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 Trepant, Marie Le Mercier, Calliope Maris, Nancy De Neve, Oriane Blanchard, Nicky D9Haene, 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.