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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

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.

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.

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.

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

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

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.

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,...

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., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published