RNA-Based Multiplexing Assay for Routine Testing of Fusion and Splicing Variants in Cytological Samples of NSCLC Patients

Abstract

The detection of ALK receptor tyrosine kinase (ALK), ROS proto-oncogen1, receptor tyrosine kinase (ROS1), ret proto-oncogen (RET), and MET proto-oncogen exon 14 skipping (METΔex14) allows for the selection of specific kinase inhibitor treatment in patients with non-small cell lung cancer (NSCLC). Multiplex technologies are recommended in this setting. We used nCounter, a multiplexed technology based on RNA hybridization, to detect ALK, ROS1, RET, and METΔex14 in RNA purified from cytological specimens (n = 16) and biopsies (n = 132). Twelve of the 16 cytological samples (75.0%) were evaluable by nCounter compared to 120 out of 132 (90.9%) biopsies. The geometrical mean (geomean) of the housekeeping genes of the nCounter panel, but not the total amount of RNA purified, was significantly higher in biopsies vs. cytological samples. Among cytological samples, we detected ALK (n = 3), METΔex14 (n = 1) and very high MET expression (n = 1) positive cases. The patient with METΔex14 had a partial response to tepotinib, one of the patients with ALK fusions was treated with crizotinib with a complete response. Cell blocks and cytological extensions can be successfully used for the detection of fusions and splicing variants using RNA-based methods such as nCounter.

Fusion‐positive non‐small cell lung carcinoma: Biological principles, clinical practice, and diagnostic implications

“…161 Successful evaluation of the ALK fusion status from RNA samples purified from tissue but also from cytological specimens have been desribed. 162 An important advantage of RNA-based fusion detection over DNA-based approaches is the reduced complexity of the fusion product after transcription and splicing compared to the genomic rearrangement. In addition, DNA-based fusion detection requires much more sequencing capacity because entire intronic regions must be covered, whereas at the RNA level, exon boundary sequencing is sufficient.…”

“…Other RNA‐based detection methods may also be considered, such as the NanoString/nCounter technology, which employs multiplexed RNA hybridization to evaluate hundreds of gene fusions in a single reaction 161 . Successful evaluation of the ALK fusion status from RNA samples purified from tissue but also from cytological specimens have been desribed 162 …”

Digital multiplexed analysis of circular RNAs in FFPE and fresh non‐small cell lung cancer specimens

“…5 µg of total RNA was either treated or mock-treated with RNase-R (Lucigen, Madison, WI, USA). RNA RT-qPCR and Sanger sequencing of circRNA junction sites were performed as previously described [18]. 10 µL of total RNA was converted into cDNA using the M-MLV reverse transcriptase enzyme and random hexamers (Invitrogen).…”

What Is New in Biomarker Testing at Diagnosis of Advanced Non-Squamous Non-Small Cell Lung Carcinoma? Implications for Cytology and Liquid Biopsy

“…RNA NGS can be used from cytological samples for ALK status assessment and identification of the different partners of ALK fusion [75,80]. Moreover, other technologies, such as multiplex technology based on RNA hybridization, are able to detect ALK in RNA purified from tissue and also from the cytological specimen, in addition to being able to look for associated genomic alterations of interest [81,82]. ALK translocated lung cancers are a heterogeneous group of tumors, and it is now pivotal to evaluate ALK-positive NS-NSCLC at baseline in the context of their genomic background in order to more clearly predict the behavior of tumors before targeted therapy [72,82].…”

“…The molecular techniques include fluorescence in situ hybridization, targeted or multiplex RT-PCR, DNA-or RNA-based NGS, and analyses using NanoString technology on tissue sections [109,119,121]. Depending on the quality and quantity of the nucleic acids extracted from the sample, false negative results can be obtained [78,81,109,119,121]. Depending on the case, these approaches could be performed with cytological samples, but only a few studies in this area have been performed to date and studies comparing cytological and tissue samples are strongly needed [109,[112][113][114][115].…”