Molecular Testing on Cytology for Gene Fusion Detection

Abstract

Cytology samples are suitable for the study of genotypic and phenotypic changes observed in different tumors. Being a minimally invasive technique, cytology sampling has been used as an acceptable alternative to track the alterations associated with tumor progression. Although the detection of gene mutations is well-established on cytology, in the last few years, gene fusion detections are becoming mandatory, especially in some tumor types such as lung cancer. Different technologies are available such as immunocytochemistry, fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and massive parallel sequencing approaches. Considering that many new drugs targeted fusion proteins, cytological samples can be of use to detect gene fusions in solid and lymphoproliferative tumor patients. In this article, we revised the use of several techniques utilized to check gene fusions in cytological material.

Cell block‐based RNA next generation sequencing for detection of gene fusions in lung adenocarcinoma: An institutional experience

“…Fusion genes can be detected by DNA‐based sequencing; however, the sensitivity is low if the fusion breakpoints are located in long intronic regions that are not covered by hybrid‐capture probes. In contrast, RNA‐based NGS detects only the exome‐encoded and expressed fusion genes 1 . Targeted RNA sequencing uses an amplicon‐based or hybrid‐capture approach to enrich the targeted regions.…”

“…Non‐small cell lung carcinomas, especially adenocarcinoma, are increasingly subjected to targeted therapies. Crizotinib and alectinib are used for carcinomas with the Anaplastic Lymphoma Kinase ( ALK ) fusion gene, selpercatinib is used for those with the Rearranged During Transfection ( RET ) fusion gene, and entrectinib is used for the ROS Proto‐Oncogene 1 Receptor Tyrosine Kinase ( ROS1 ) and Neurotrophic Receptor Kinase (NTRK) fusion genes 1 . Routine testing for these fusion genes is recommended by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology 2,3 .…”

Fusion Detection in Microsecretory Adenocarcinoma and Mucoepidermoid Carcinoma Using Chromogenic RNA In Situ Hybridization: A Promising Alternative to DNA-Based Fluorescence In-Situ Hybridization

“…Advantages of FISH over chromogenic RNA-ISH include 1) relative stability of DNA compared to RNA in archival FFPE tissue, and 2) the ability to assess for rearrangements using break-apart probes when break point are inconsistent and/or fusion partners are promiscuous. From a clinical application perspective, however, FISH assays cannot be used to identify small intrachromosomal rearrangements (e.g., NCOA4 :: RET ), and cannot confirm that detected fusions are expressed (7). Resources required for implementation and interpretation, such as a fluorescence microscope and experienced staff for FISH studies, are also limited, making RNA-ISH an attractive option for pathology laboratories.…”

“…FISH is considered a standard technique in many clinical molecular laboratories with the advantage of direct spatial localization of genetic abnormalities within tumor cells. Unfortunately, FISH is time consuming, requires the use of fluorescent probes (fluorescence gradually weakens over time), and can only be analyzed using specialized equipment (epifluorescence microscope with an adjusted set of filters) (7). Other less commonly used non-in situ methods include RT-PCR (reverse transcriptase-polymerase chain reaction) and NGS (next-generation sequencing).…”

“…Other less commonly used non-in situ methods include RT-PCR (reverse transcriptase-polymerase chain reaction) and NGS (next-generation sequencing). RT-PCR and NGS both face challenges related to the fragmentation of RNA in formalin-fixed paraffin-embedded (FFPE) tissue samples, and NGS has the additional barriers of higher cost, a longer workflow, and complex analysis (7).…”