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

Abstract

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.

A narrative review of methods for the identification of ALK fusions in patients with non-small cell lung carcinoma

“…Tables 3,4 provide real-world considerations to avoid false negative and false positive results when using IHC, FISH, real-time PCR or NGS ( 4 , 21 - 23 , 55 , 56 , 59 , 62 , 63 , 78 - 81 ). Although the main potential solutions are shown in the tables and briefly discussed below, the key factor for success in ALK testing is a deep understanding of the concept of “molecular redundancy”.…”

“…FISH slides that only show the atypical pattern of positivity (isolated orange signals) should ideally be scored with an image analysis algorithm to rule out a suboptimal or a very low signal intensity of the green probe, an overlooked reason for ALK FISH false positive results ( 23 , 80 ). Regarding PCR-based methods (real-time PCR or NGS), when an ALK fusion is identified by expression imbalance or with a low number of reads, orthogonal testing is recommended to improve specificity ( 21 , 59 , 63 , 78 , 79 ).…”

“…RNA-based real-time PCR assays are very popular in regions with a very high prevalence of epidermal growth factor receptor ( EGFR ) mutations or when tumor content, turnaround time and/or cost preclude the use of NGS ( 21 , 43 , 45 , 58 , 59 ). Taking into consideration the molecular epidemiology of ALK fusions (see above), the design (i.e., breadth) of these kits is probably not as relevant for this discussion as it is for the detection of other actionable fusions ( 60 ).…”

“…Taking into consideration the molecular epidemiology of ALK fusions (see above), the design (i.e., breadth) of these kits is probably not as relevant for this discussion as it is for the detection of other actionable fusions ( 60 ). Nonetheless, users of these assays should be constantly aware that “ pseudo false negatives” (i.e., due to rare or uncommon fusion partners not included in the design of an assay) are unavoidable because the most widely used real-time PCR assays are designed to only identify some of the non- EML4-ALK fusion partners ( 21 , 43 , 45 , 58 , 59 ). Finally, it must be overemphasized that sometimes the fusions are detected by imbalance assessment, a strategy that is a potential source of confusion because its lower specificity (see below).…”

Update in Lung Cancer Molecular Pathology: Technological Advances and Clinical Practice

“…Of note, SRM has the possibility to quantitatively screen several protein targets, comprising ALK and MET, and FR-alpha, hENT1, RRM1, TUBB3, ERCC1, and XRCC1 for chemotherapy. Another original work by Leone et al [3] described a multicentric experience using a novel, fully integrated and automated cartridge-based assay providing deparaffinization and digestion of the tissue up to mRNA amplification using real-time-PCR (Idylla, Biocartis) with the novel GeneFusion assay. The assay simultaneously identifies ALK, ROS1, RET and NTRK1/2/3 gene fusions and MET ex14 skipping mutations, and the results were matched with different techniques routinely available for predictive biomarker testing, either immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time-PCR (RT-PCR) or next-generation sequencing (NGS).…”