Detection of ALK fusion transcripts in FFPE lung cancer samples by NanoString technology

Abstract

Background ALK-rearranged lung cancers exhibit specific pathologic and clinical features and are responsive to anti-ALK therapies. Therefore, the detection of ALK-rearrangement is fundamental for personalized lung cancer therapy. Recently, new molecular techniques, such as NanoString nCounter, have been developed to detect ALK fusions with more accuracy and sensitivity.MethodsIn the present study, we intended to validate a NanoString nCounter ALK-fusion panel in routine biopsies of FFPE lung cancer patients. A total of 43 samples were analyzed, 13 ALK-positive and 30 ALK-negative, as previously detected by FISH and/or immunohistochemistry.ResultsThe NanoString panel detected the presence of the EML4-ALK, KIF5B-ALK and TFG-ALK fusion variants. We observed that all the 13 ALK-positive cases exhibited genetic aberrations by the NanoString methodology. Namely, six cases (46.15%) presented EML-ALK variant 1, two (15.38%) presented EML-ALK variant 2, two (15.38%) presented EML-ALK variant 3a, and three (23.07%) exhibited no variant but presented unbalanced expression between 5’/3’ exons, similar to other positive samples. Importantly, for all these analyses, the initial input of RNA was 100 ng, and some cases displayed poor RNA quality measurements.ConclusionsIn this study, we reported the great utility of NanoString technology in the assessment of ALK fusions in routine lung biopsies of FFPE specimens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-017-0428-0) contains supplementary material, which is available to authorized users.

Detection of known and novel ALK fusion transcripts in lung cancer patients using next-generation sequencing approaches

“…The nCounter Vantage ™ Lung Fusion Panel included junction probes specific to the fusion breakpoint, and probes upstream and downstream of a potential fusion junction for detection of gene-expression imbalance. Comparisons of NanoString performance with IHC and FISH have clearly shown showed a high degree of concordance with these gold standard techniques 17 , 20 , 35 , 36 . Amplicon-based NGS fusion panels have also been developed by different suppliers, and two main methods are available: the target enrichment-based (e.g.…”

“…This point has been addressed recently by Evangelista et al . that implemented NanoString panel for ALK fusion detection and demonstrated its applicability in series of 43 lung cancer biopsies using up to 100 ng of RNA with only 7% of sample failure 36 .…”

ALK in Non-Small Cell Lung Cancer (NSCLC) Pathobiology, Epidemiology, Detection from Tumor Tissue and Algorithm Diagnosis in a Daily Practice

“…A multiplexed transcript-based assay (nCounter, nanoString, Seattle, WA, USA) has been developed for simultaneous detection of multiple gene fusions. In particular, this approach has been developed for detection of an ALK rearrangement [ 83 ]. Briefly, this assay is based on the dual hybridization of a capture and a molecularly bar-codes reporter probe complementary to a contiguous target sequence [ 84 ].…”

New Approaches for Successful Identification of Several Gene Fusion Oncogenes in Paraffin-Embedded Tissue Samples from Advanced Non- Small-Cell Lung Cancer Patients

“…Our work [8] and the work of others [9] demonstrate that nCounter platform needs a reduced amount of genetic material for optimum analysis. Specifically, a thickness of four µm with a tumor area of 1.1 mm 2 and 10% tumor content with a total amount of 25-200 ng RNA are sufficient to obtain satisfactory results [8].…”