Localized Single Transcript Detection of Eml4‐Alk in NSCLC using Co‐localization Quantum Dot Fluorescent In Situ Hybridization (CoQFISH)

Abstract

Lung cancer remains one of the most common forms of cancer worldwide with more than 80% being non-small cell lung cancer (NSCLC). Two primary clinical tests are used to identify NSCLC subtypes through the detection of EGFR mutation and oncogenic gene fusions such as EML4-ALK. Since the discovery of EML4-ALK, there has been a multitude of targeted therapies such as ALK inhibitors alectinib, ceritinib, and crizotinib, which has shown great success in treatment. However, the methods for clinical detection of gene fusions remains constant through either RT-PCR or Break Apart FISH. By observing co-localization of signal in oligonucleotide conjugated quantum dots, we designed a novel co-localization based quantum dot fluorescent in situ hybridization (CoQFISH) technology capable of detecting sensitively and specifically single localized fusion transcripts using as little as two oligonucleotide probes in cell lines and tissue. We found that CoQFISH can detect EML4-ALK transcripts in a positive control NSCLC cell line H2228 using the co-localization of Eml4 and Alk targeted probe sets, which was verified through qPCR and targeted RNA-seq. Furthermore, we found that CoQFISH can positively identify EML4-ALK transcripts in human NSCLC tissue biopsies at a sensitivity that is higher than qPCR and RNA sequencing and offer information on the spatial location of each transcript. Looking further, quantum dot probes in CoQFISH are uniquely suited for resolving smaller areas on transcripts for identification of targets such as gene isoform transcripts. This novel technology would not only aid in research on understanding fusion genes and small RNA transcripts, but more importantly allow more sensitive diagnostics of potential cancer targets meaning earlier detection and better therapeutic response monitoring. Detection of CoQFISH probes targeting GAPDH. Positive signal was detected in human lung tissue for the positive control containing GAPDH (left). Human tissue was treated with RNAse treatment in the negative control (right) and showed no detected signal. CoQFISH detects Eml4-Alk fusion transcripts. Differential interference contrast (DIC) shows human non-small cell lung cancer tissue structure on the right. Co-localized signal from Eml4 (middle) and Alk (right) is indicated with the corresponding arrows. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.