FULLY AUTOMATED REAL-TIME PCR IN MOLECULAR PATHOLOGY: NOVEL APLLICATIONS

A deeper knowledge of cancer biology, alongside advances in diagnostic technologies, has led to much more effective therapeutic strategies in Indeed, over the last decade or so, the incessant quest for new genetic alterations capable of predicting patient response to treatments, has given rise to the development of highly sophisticated molecular tests Accordingly, many sequencing platforms can nowadays detect multiple driver mutations simultaneously thanks to fully automated procedures which drastically reduce turnaround time and costs. A case in point is the Idylla™ system (Biocartis NV, Mechelen, Belgium. This system is a fully automated sample-to-result Real-Time PCR with all reagents integrated in a single-use cartridge; it consists of a console which displays results as either "mutation" or "no mutation" when DNA quality is adequate, or as "invalid" when it is. What is most striking about this platform is that the cartridge can run multiple sample types, including solid and liquid biopsies, yielding results within approximately two hours. Another advantage is its ease of use taking up only a couple of minutes of hands-on time to prepare samples and load the cartridge. This fascinating assay has been validated for the identification of many driver genes, both on tissue and on plasma specimens. In particular Idylla™ EGFR, Idylla™ BRAF, Idylla™ KRAS, Idylla™ NRAS-BRAF, and Idylla™ MSI have been validated on formalin fixed paraffin embedded (FFPE) samples; instead, Idylla™ ctKRAS and Idylla™ ctNRAS-BRAF have been validated on plasma samples. Many more are currently in the pipeline. The aim of my PhD project was to develop and validate novel clinical applications of the the Idylla™ system. I divided my thesis into four chapters. The first chapter focuses on the performance of the Idylla™ EGFR Assay on cytological non-small cell lung cancer samples. This test was specifically designed to process formalin-fixed, paraffin-embedded sections without requiring preliminary DNA extraction. On the other hand, my PhD work has demonstrated that this assay can also be used to process archival smears from patients with NSCLC by scraping the stained cellular material directly into the cartridge. The second chapter focuses on the viable application of Idylla™ NRAS-BRAF Assay to cytological thyroid fine-needle aspirates (FNAs) with undetermined morphology. Our research demonstrated that FNA needle rinses can be genotyped by the same cytopathologist who performs the FNA, a procedure that is commonly called rapid on-site molecular evaluation (ROME). The third chapter, instead, discusses the feasibility of using Idylla™ to analyze liquid biopsy specimens. Such application is highly important, given the scant availability of tissue specimens in advanced NSCLC patients. Our laboratory results have indeed demonstrated the efficiency of the Idylla™ ctKRAS Assay in detecting plasma the KRAS p.G12C mutation, a novel target in NSCLC patients. Finally, in the fourth chapter, I explore the analytical and clinical performance of the Idylla™ SARS-CoV-2 test on previously tested SARS-CoV-2 people by conventional RT-PCR based approach in different settings, including initial diagnosis and clinical follow-up. In this regard, I provide substantial evidence that this assay may represent a valid, fast, and highly sensitive and specific RT-PCR test for the identification of SARS-CoV-2 infection

An Italian Multicenter Perspective Harmonization Trial for the Assessment of MET Exon 14 Skipping Mutations in Standard Reference Samples

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice

RNA-Based Assay for Next-Generation Sequencing of Clinically Relevant Gene Fusions in Non-Small Cell Lung Cancer

Gene fusions represent novel predictive biomarkers for advanced non-small cell lung cancer (NSCLC). In this study, we validated a narrow NGS gene panel able to cover therapeutically-relevant gene fusions and splicing events in advanced-stage NSCLC patients. To this aim, we first assessed minimal complementary DNA (cDNA) input and the limit of detection (LoD) in different cell lines. Then, to evaluate the feasibility of applying our panel to routine clinical samples, we retrospectively selected archived lung adenocarcinoma histological and cytological (cell blocks) samples. Overall, our SiRe RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/µL. It also successfully analyzed 46 (95.8%) out of 48 samples. Among these, 43 (93.5%) out of 46 samples reproduced the same results as those obtained with conventional techniques. Intriguingly, the three discordant results were confirmed by a CE-IVD automated real-time polymerase chain reaction (RT-PCR) analysis (Easy PGX platform, Diatech Pharmacogenetics, Jesi, Italy). Based on these findings, we conclude that our new SiRe RNA fusion panel is a valid and robust tool for the detection of clinically relevant gene fusions and splicing events in advanced NSCLC

Liquid Biopsy Analysis in Clinical Practice: Focus on Lung Cancer

Lung cancer is the leading cause of cancer death worldwide. Despite the emergence of highly effective targeted therapies, up to 30% of advanced stage non-small cell lung cancer (NSCLC) patients do not undergo tissue molecular testing because of scarce tissue availability. Liquid biopsy, on the other hand, offers these patients a valuable opportunity to receive the best treatment options in a timely manner. Indeed, besides being much faster and less invasive than conventional tissue-based analysis, it can also yield specific information about the genetic make-up and evolution of patients’ tumors. However, several issues, including lack of standardized protocols for sample collection, processing, and interpretation, still need to be addressed before liquid biopsy can be fully incorporated into routine oncology practice. Here, we reviewed the most important challenges hindering the implementation of liquid biopsy in oncology practice, as well as the great advantages of this approach for the treatment of NSCLC patients

Idylla assay and next generation sequencing: an integrated EGFR mutational testing algorithm

Any reference laboratory testing non-small cell lung cancer samples for predictive biomarkers needs to develop and validate a wide range of different molecular techniques, each with a specific time requirement and application. Updated international guidelines suggest that next generation sequencing (NGS) to be the initial procedure. However, in a non-negligible subset of cases, library generation may fail or amplicon coverage may be insufficient. In these NGS 'invalid' cases, the Idylla system may represent a viable option for rapid epidermal growth factor receptor (EGFR) genotyping

Fully automated PCR detection of KRAS mutations on pancreatic endoscopic ultrasound fine-needle aspirates

Aims: In cystic and solid pancreatic lesions, KRAS mutational status refines the diagnosis of uncertain endoscopic ultrasound (EUS) aspirates. This test should have a fast turnaround time and ideally be performed at the centre where the patient is diagnosed. The Idylla KRAS Mutation Test enables standardisation even in units without molecular expertise. Methods: The Idylla test was designed for use with formalin-fixed paraffin-embedded (FFPE) sections. However, we directly pipetted 3 µL (corresponding to 1/10th of a DNA preparation from the aspirate sample) in the cartridge, which was automatically run as if an FFPE sample had been inserted. The performance was compared with Sanger sequencing, Allele Specific Locked Nucleic Acid PCR (ASLNAqPCR), and 454 Next Generation Sequencing (454-NGS) in light of clinicopathological end points. Results: Idylla yielded valid results in 49/52 (94.2%) cases, in 2 h. A total of 18/49 cases showed mutation either in KRAS exon 2 (14/18) or in exon 3 (4/18). Idylla KRAS test had 100% specificity and a sensitivity (55.1%) higher than Sanger sequencing (41.3%) and identical to ASLNAqPCR (55.1%). When the low-abundant mutant allele (<5%) cases were excluded from the analysis, the Idylla KRAS Mutation Test clinical sensitivity increased to 61.9% approaching that of 454-NGS (66.6%). Conclusions: This is the first study that applied the novel Idylla KRAS test to the clinical setting of pancreatic cancer. In particular, this system can be easily implemented in the routine assessment of pancreatic EUS-fine-needle aspiration-derived DNA samples to quickly provide information on KRAS mutational status to supplement cytological evaluatio