Rapid EGFR evaluation from used H&E, IHC and FISH diagnostic slides with the Idylla platform.

AIMS: Diagnostic tumour samples are mandatory for morphologic and molecular diagnosis of non-small cell lung cancer (NSCLC) to establish the best therapeutic approach. In the presence of small tumour tissue sample, the pathologist needs to make responsible choices to achieve a correct diagnosis and save material for subsequent molecular evaluations. Nevertheless, in some instances, the diagnostic process can lead to tissue depletion. The automated Idylla epidermal growth factor receptor (EGFR) mutation test has been developed to rapidly process formalin-fixed paraffin-embedded (FFPE) pathologic material, without previous DNA extraction. This study aimed to test whether this platform is suitable for the reuse of H&E, immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) diagnostic slides. METHODS: A training set of 19 FFPE tissues with known EGFR status was revaluated on H&E slides. Fourteen of them were also tested using IHC and FISH treated specimens. An additional series of 25 H&E, IHC or FISH slides of NSCLC cases tested for EGFR mutation at an external institution was blindly assessed as a validation cohort. RESULTS: Combining the two sets, 32 of 32 classical ex19dels and p.L858R were correctly identified. Three uncommon mutations (p.G719X, p.L861Q and ex20ins) were also detected. Four discrepancies were related to rare ex19del/ins not included in the Idylla list of detectable mutations. Two p.T790M variants were missed on one FFPE and two H&E slides but were detected using IHC and FISH sections from the same FFPE blocks. CONCLUSIONS: The Idylla EGFR mutation test is highly reliable using differently treated tumour specimens and should be validated in larger studies.

Robust Performance of the Novel Research-Use-Only Idylla GeneFusion Assay Using a Diverse Set of Pathological Samples with a Proposed 1-Day Workflow for Advanced NSCLC Evaluation.

A range of different techniques are available for predictive biomarker testing for non-small-cell lung cancer (NSCLC) clinical management. International guidelines suggest next-generation sequencing (NGS) as the preferred procedure, but other reverse transcriptase-polymerase chain reaction (RT-PCR)-based methods are rapidly evolving. In this study, we evaluated the reliability and accuracy of the IdyllaTM GeneFusion assay, a rapid and fully automated platform able to simultaneously detect ALK, ROS1, RET and NTRK1/2/3 and MET ex14 skipping mutations and compared its performance with routine reference methods. The cohort included thirty-seven NSCLCs plus two parotid gland carcinomas, previously characterized for the above alterations through either IHC, FISH, RT-PCR or NGS. In 36 of 39 cases, the Idylla GeneFusion assay and the reference methods were concordant (overall agreement: 92.3%). Tumor sections stored at room temperature for up to 60 days and 17 cases older than 2 years were successfully characterized. Our results suggest that the Idylla GeneFusion assay is a reliable tool to define gene fusion status and may be a valuable stand-alone diagnostic test when time efficiency is needed or NGS is not feasible.

Intrapatient Molecular and Histologic Heterogeneity After First-generation or Second-generation TKI Therapy of NSCLC Patients: Potential Clinical Impact on Subsequent third-generation TKI Treatment.

OBJECTIVES: The discovery of tyrosine kinase inhibitors (TKI) has remarkably improved the clinical course of patients with non-small cell lung cancer driven by Epidermal Growth Factor Receptor (EGFR) mutations. However, virtually in all cases, the disease resurfaces in a TKI-resistant form that is mainly linked to an acquired EGFR-T790M mutation, a MET amplification, or small cell lung cancer (SCLC) transformation. Third-generation TKIs are able to block tumor growth through an irreversible binding to the T790M-mutated receptor. Such new treatments require the diagnostic analysis of new pathologic tissue or a liquid biopsy to detect the presence of the T790M mutation. MATERIALS AND METHODS: Pre-TKI and post-TKI biopsies from 27 patients with an activating EGFR mutation were collected and analyzed for EGFR-T790M mutation, MET amplification, and SCLC transformation. RESULTS: The T790M mutation was found in 16 patients (59%) whereas MET gene amplification was found in 2 (10.5%) of 19 evaluated cases. The histologic transformation from adenocarcinoma (ADC) to SCLC was identified in 3 patients (11%). In one of them reversal from SCLC back to adenocarcinoma was observed. One patient had the T790M mutation concordantly detected in 2 synchronous lesions whereas another patient showed T790M positivity only in one of 2 specimens. In 4 patients longitudinal biopsies revealed T790M gains and losses not always according to biological expectations. CONCLUSIONS: Intrapatient molecular or histologic heterogeneity may be frequently found during routine treatment of non-small cell lung cancer patients. This biological aspect may have profound repercussions on subsequent therapeutic decisions, and therefore requires in-depth investigation.

Reaction of peroxynitrite with hyaluronan and related saccharides.

The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although 1H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.