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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.

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.

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.

In July 2020, an active smoker, 63-year old man was admitted to the oncology unit of A.O.R.N. Sant’Anna e San Sebastiano (Caserta, Italy). Chest radiology highlighted right pleural effusion. Total-body CT scanning revealed a solid lesion with lobulated contours in the apical segment of the upper right lobe. The patient’s oncologist requested a molecular assessment of EGFR, ALK, ROS1, BRAF, and KRAS, as well as an evaluation of PD-L1 expression level. To this end, we carried out NGS analysis, on DNA extracted from cytospins, by adopting a custom-designed NGS panel (SiRe®). Overall, no actionable mutations in the tested genes were identified. Conversely, concomitant BRAF exon 11 p.G469A and a KRAS exon 4 p.A146T mutations were detected. Owing to the limited data on the presence of KRAS exon 4 p.A146T point mutation in lung adenocarcinoma patients, a further molecular confirmatory analysis was carried out with a dedicated KRAS cartridge on a fully automated real time polymerase chain reaction. When DNA was extracted from the TTF-1 positive tumor cell slide, the same KRAS alteration was observed. Unfortunately, the patient died in August 2020 before having the chance to start any type of treatment.

AimsFollowing the development for liquid biopsies of the SiRe next-generation sequencing (NGS) panel that covers 568 clinical relevant mutations in EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRa genes, in this current study, we apply this small NGS panel on tissue samples of lung cancer.MethodsA total of 322 specimens were prospectively tested. Technical parameters were analysed on both cytological and histological samples. In a subset of 75 samples, the EGFR SiRe results were compared with those generated by the European Community (CE)–IVD EGFR assay on Idylla platform. Clinical outcomes of 11 patients treated, on the basis of SiRe results, were also evaluated.ResultsOnly 28 (8.7%) specimens failed to produce a library; out of the 294 remaining samples, a total of 168 somatic mutations were found. In nearly all instances (74/75–99%), the EGFR SiRe results were confirmed by Idylla. In general, SiRe analytical parameters were excellent. However, histological and cytological specimens differed in relation to average reads for sample, mean number of mapped reads, median read length and average reads for amplicon. Treatment outcome evaluation in 11 patients showed a partial response in 82 % (9/11) patients with a median progression-free survival of 340 days.ConclusionsThe small gene panel SiRe is a clinically relevant tool useful to widespread the adoption of NGS in predictive molecular pathology laboratories.

Molecular cytopathology is a rapidly evolving field embracing both conventional microscopy and molecular pathology. Its growing popularity stems from the fact that in many types of advanced cancers, including non small cell lung cancer (NSCLC), cytological samples often constitute the only available specimens for morphomolecular analysis. Indeed, non formalin fixed and paraffin embedded (FFPE) cytological samples feature a higher quality of extracted nucleic acids than histological specimens. However, because of the growing complexity of molecular testing, several efforts should be made to validate the analytical performance of the wide array of currently available molecular technologies, including next generation sequencing (NGS). This technology has the terrific advantage of allowing simultaneous detection of scores of predictive biomarkers even in low-input DNA/RNA specimens. Here, we briefly review the role of the modern cytopathologist in the morphomolecular diagnosing of advanced stage NSCLC and the adoption of NGS in conventional cytopreparations (cell blocks, direct smears, and liquid-based cytology) and supernatants.

AimsTo date, reverse transcriptase PCR (RT-PCR) on nasopharyngeal swabs is the ‘gold standard’ approach for the diagnosis of COVID-19. The need to develop easy to use, rapid, robust and with minimal hands-on time approaches are warranted. In this setting, the Idylla SARS-CoV-2 Test may be a valuable option. The aim of our study is to evaluate the analytical and clinical performance of this assay on previously tested SARS-CoV-2 people by conventional RT-PCR based approach in different settings, including initial diagnosis and clinical follow-up.MethodsTo evaluate the sensitivity and specificity of the Idylla SARS-CoV-2 Test, we retrieved 55 nasopharyngeal swabs, previously analysed by a fully validated assay, from symptomatic patients or from people who have been in close contact with COVID-19 positive cases. Discordant or high discrepant cases were further analysed by a third technique. In addition, a second subset of 14 nasopharyngeal swab samples with uncertain results (cycle threshold between 37 and 40), by using the fully validated assay, from patients with viral infection beyond day 21, were retrieved.ResultsOverall, Idylla showed a sensitivity of 93.9% and a specificity of 100.0%. In addition, in the additional 14 nasopharyngeal swab samples, only five (35.7%) featured a positive result by the Idylla SARS-CoV-2 Test.ConclusionsWe demonstrated that the Idylla SARS-CoV-2 Test may represent a valid, fast, highly sensitive and specific RT-PCR test for the identification of SARS-CoV-2 infection.

Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors.

Lung cancer is the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) representing its most commonly diagnosed sub-type. Despite the significant improvements in lung cancer biomarkers knowledge, accompanied by substantial technological advances in molecular tumor profiling, a considerable fraction (up to 30 %) of advanced NSCLC patient presents with major testing challenges or tissue unavailability for molecular analysis. In this context, liquid biopsy is on the rise, currently gaining considerable interest within the molecular pathology and oncology community. Molecular profiling of liquid biopsy specimens using next generation molecular biology methodologies is a rapidly evolving field with promising applications not exclusively limited to advanced stages but also more recently expanding to early stages cancer patients. Here, we offer an overview of some of the most consolidated and emerging applications of next generation sequencing technologies for liquid biopsy testing in NSCLC.

Although gene fusions occur rarely in non-small-cell lung cancer (NSCLC) patients, they represent a relevant target in treatment decision algorithms. To date, immunohistochemistry and fluorescence in situ hybridization are the two principal methods used in clinical trials. However, using these methods in routine clinical practice is often impractical and time consuming because they can only analyze single genes and the quantity of tissue material is often insufficient. Thus, novel technologies, able to test multiple genes in a single run with minimal sample input, are being under investigation. Here, we discuss the utility of next-generation sequencing and nCounter technologies in detecting simultaneous gene fusions in NSCLC patients.

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/&micro, L. 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.

Background Immune-checkpoint inhibitors (ICIs) represent an important treatment option for patients who have advanced stage non-small cell lung cancer (NSCLC). Currently, evaluation of the expression level of programmed death-ligand 1 (PD-L1) has proven highly successful as a positive predictive biomarker for ICIs. In addition to PD-L1, other promising predictive biomarkers are emerging, including high tumor mutational burden (TMB-H). However, measuring TMB-H remains challenging for several reasons, among which is the difficulty in obtaining adequate tissue material from NSCLC patients. There are no data in the current literature regarding the possibility of adopting cell blocks (CBs) for TMB evaluation; therefore, our goal was to evaluate the feasibility of analyzing TMB on CBs. Methods For evaluation of differences in run metric parameters, 8 pairs of histological and CB samples from patients with NSCLC were analyzed using the Oncomine Tumor Mutational Load Assay on Ion Torrent S5 GS next-generation sequencing (NGS) platform. Results Most CBs (6/8, 75.0%) were successfully analyzed by adopting the broad NGS panel approach. CBs provided results similar to those obtained on histological matched specimens in terms of median total reads (7207048.80 vs 7558817.80), median mapped reads (7075753.83 vs 7513822.00), median read lengths (115.50 vs. 113.00), median percentage of reads on-target (97.49% vs. 98.45%), median average reads per amplicon (454.67 vs 476.14), and median uniformity of amplicon coverage (83.52% vs 84.13%). Conclusion In this pilot study, we demonstrated the technical feasibility of assessing TMB on CBs.

AimsIn everyday practice, epidermal growth factor receptor (EGFR) testing is centralised in referral laboratories that receive paucicellular cytological specimens. Ideally, EGFR testing should be carried out in the centre where the patient is diagnosed such that the most cellular slide can be selected from in-house collected cytological material. However, available techniques are little standardised and difficult to be implemented in settings with little expertise in molecular testing. The Idylla EGFR prototype assay is a rapid and fully automated test which may easily be adopted by a wider number of pathological centres. This study assessed whether an Idylla EGFR prototype assay can be reliably applied to cytological lung cancer specimens.MethodsThe limit of detection (LOD) of the Idylla EGFR prototype assay was assessed by cell line dilution studies. A total of 10 ng was directly placed inside an Idylla EGFR prototype assay cartridge. Idylla results were compared with fragment length (exon 19 del) and Taqman assays.ResultsThe Idylla EGFR prototype assay showed an LOD of 1% mutant allele and yielded valid results in 74/76 (97.3%) samples, detecting all the mutant cases (n=32) identified by standard techniques; in addition, Idylla detected two low abundance EGFR exon 19 deletions and two G719X exon 18 point mutations, not covered by our standard reference method.ConclusionsIdylla EGFR prototype assay is sensitive on extracted DNA and can reliably be applied on cytological samples, enabling implementation of EGFR testing even in less experienced diagnostic units.