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

Abstract

Objective: Targeted therapy is an important part of the treatment of lung adenocarcinoma. Tests for EGFR mutation, ALK, ROS1, RET and NTRK gene fusions are needed to make a treatment decision. These gene fusions are traditionally detected by fluorescence in situ hybridisation (FISH) or immunohistochemistry. In this study, we investigated whether gene fusions in pulmonary adenocarcinoma could be accurately detected by RNA next-generation sequencing (RNA-NGS) and whether cytology cell blocks could be used effectively for this test.Methods: Archived cytological specimens of lung adenocarcinoma submitted for RNA sequencing between 2019 and 2022 at Fox Chase Cancer Center were retrospectively retrieved. Hybrid capture-based targeted RNA next generation sequencing was used, which covers 507 fusion genes, including ALK, ROS1, RET and NTRKs, irrespective of their partner genes. DNA NGS, FISH and chromosomal microarray analysis were used to confirm the results of the RNA-NGS.Results: A total of 129 lung adenocarcinoma cytology specimens were submitted for molecular testing. Eight of 129 (6.2%) cases were excluded from RNA sequencing as their cell blocks contained inadequate numbers of tumour cells. One case (0.8%) failed to yield adequate RNA. The overall success rate was 93% (120/129). Ten of 120 (8.3%) cytology cases were positive for gene fusions, including 7 ALK, 2 ROS1 fusion genes, and 1 RET fusion gene. Twenty-two cell block cases were also tested for ALK fusion genes using FISH. However, 11 of 22 (50%) failed the testing due to inadequate material.Conclusions: Cytology cell blocks can be used as the main source of material for molecular testing for lung cancer. Detection of gene fusions by RNA-based NGS on cell blocks is convenient and reliable in daily practice.

DLX1 acts as a novel prognostic biomarker involved in immune cell infiltration and tumor progression in lung adenocarcinoma

“…cg07936950, cg05938001, cg12308746, and cg17737681 were the four CpG sites with the highest DNA methylation. Targeted therapy is also an important component of lung cancer treatment, and treatment regimens require testing for EGFR, ALK, ROS1, RET, MET, BRAF, NTRK, HER2, NRG1, FGFR1, and PIK3CA ( Guo et al, 2019 ; Wei et al, 2023 ). When further testing the sensitivity of DLX1 gene mutations in LUAD, we found that the incidence of DLX1 gene mutations in LUAD tissues was only 1.5% by cBioPortal study.…”

Molecular testing of cytology specimens: Issues in specimen adequacy and clinical utility

“…7 Much of the literature regarding next generation sequencing has involved analysis of larger specimen types including core needle biopsies and resection specimens but cytology has been demonstrated to be a useful alternative when larger specimens are unavailable. [8][9][10][11][12][13][14] NGS testing of cytology specimens has been shown to be useful for analysis of malignancies from a variety of body sites including the lung, salivary gland, pancreas, and thyroid. 8,10,11,14 Cytologic specimens suitable for NGS testing include cell blocks, 11,12 smeared specimens, cytospins and liquid based preparations.…”

“…[8][9][10][11][12][13][14] NGS testing of cytology specimens has been shown to be useful for analysis of malignancies from a variety of body sites including the lung, salivary gland, pancreas, and thyroid. 8,10,11,14 Cytologic specimens suitable for NGS testing include cell blocks, 11,12 smeared specimens, cytospins and liquid based preparations. 8,9 When properly performed, NGS testing of cytology specimens appears to be as accurate as testing of larger surgical specimens.…”