RNA-Based Next-Generation Sequencing in Non-Small-Cell Lung Cancer in a Routine Setting: an Experience from an Italian Referral Center

Luca, Caterina De; Pepe, Francesco; Pisapia, Pasquale; Iaccarino, Antonino; Righi, Luisella; Listì, Angela; Russo, Gianluca; Campione, Severo; Pagni, Fabio; Nacchio, Mariantonia; Conticelli, Floriana; Russo, Maria; Fabozzi, Teresa; Vigliar, Elena; Bellevicine, Claudio; Rocco, Danilo; Laudati, Stefano; Iannaci, G.; Daniele, Bruno; Gridelli, Cesare; Novello, Silvia; Molina-Vila, Miguel Ángel; Rosell, Rafael; Troncone, Giancarlo; Malapelle, Umberto

doi:10.2217/pme-2022-0020

Cited by 3 publications

(2 citation statements)

References 19 publications

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“…We analyzed a total of 210 NSCLC samples by FISH, and we succeeded in characterizing 200 of these samples (95%) by targeted RNA NGS and 198 samples (94%) by RT-PCR. The results of our analyses, together with already published data [ 17 , 18 , 19 , 20 ], showed an excellent concordance rate (91%) of the targeted RNA NGS results with the so-far considered gold standard FISH technique, underlining the essential role of the NGS testing approach in the molecular pathology diagnostics laboratory.…”

Section: Discussionsupporting

confidence: 73%

Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

Pecciarini

Brunetto

Grassini

et al. 2023

Cells

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The ability to identify the broadest range of targetable gene fusions is crucial to facilitate personalized therapy selection for advanced lung adenocarcinoma (LuADs) patients harboring targetable receptor tyrosine kinase (RTK) genomic alterations. In order to evaluate the most effective testing approach for LuAD targetable gene fusion detection, we analyzed 210 NSCLC selected clinical samples, comparing in situ (Fluorescence In Situ Hybridization, FISH, and ImmunoHistoChemistry, IHC) and molecular (targeted RNA Next-Generation Sequencing, NGS, and RealTime-PCR, RT-PCR) approaches. The overall concordance among these methods was high (>90%), and targeted RNA NGS was confirmed to be the most efficient technique for gene fusion identification in clinical practice, allowing the simultaneous analysis of a large set of genomic rearrangements at the RNA level. However, we observed that FISH was useful to detect targetable fusions in those samples with inadequate tissue material for molecular testing as well as in those few cases whose fusions were not identified by the RNA NGS panel. We conclude that the targeted RNA NGS analysis of LuADs allows accurate RTK fusion detection; nevertheless, standard methods such as FISH should not be dismissed, as they can crucially contribute to the completion of the molecular characterization of LuADs and, most importantly, the identification of patients as candidates for targeted therapies.

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Section: Discussionsupporting

confidence: 73%

Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

Pecciarini

Brunetto

Grassini

et al. 2023

Cells

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“…Overall, NGS may represent a more impacting diagnostic method than FISH in the detection of FGFR2 fusions and allows the detection of multiple genetic alterations in tissue samples with scarce material ( Fig. 2 ) 59 , 80 .…”

Section: Fgfr2 Fusion Testing In Cca: a Practical Approachmentioning

confidence: 99%

FGFR2 testing in cholangiocarcinoma: translating molecular studies into clinical practice

et al. 2023

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Summary Cholangiocarcinoma (CCA) is a heterogeneous group of neoplasms burdened by a dismal prognosis. Several studies have investigated the genomic profile of CCA and identified numerous druggable genetic alterations, including FGFR2 fusions/rearrangements. Approximately 5-7% of CCAs and 10-20% of intrahepatic iCCAs harbor FGFR2 fusions. With the recent advent of FGFR-targeting therapies into clinical practice, a standardization of molecular testing for FGFR2 alterations in CCA will be necessary. In this review, we describe the technical aspects and challenges related to FGFR2 testing in routine practice, focusing on the comparison between Next-Generation Sequencing (NGS) and FISH assays, the best timing to perform the test, and on the role of liquid biopsy.

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Digital counting of tissue cells for molecular analysis: the QuANTUM pipeline

L’Imperio,

Cazzaniga,

Mannino

et al. 2024

Virchows Arch

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The estimation of tumor cellular fraction (TCF) is a crucial step in predictive molecular pathology, representing an entry adequacy criterion also in the next-generation sequencing (NGS) era. However, heterogeneity of quantification practices and inter-pathologist variability hamper the robustness of its evaluation, stressing the need for more reliable results. Here, 121 routine histological samples from non-small cell lung cancer (NSCLC) cases with complete NGS profiling were used to evaluate TCF interobserver variability among three different pathologists (pTCF), developing a computational tool (cTCF) and assessing its reliability vs ground truth (GT) tumor cellularity and potential impact on the final molecular results. Inter-pathologist reproducibility was fair to good, with overall Wk ranging between 0.46 and 0.83 (avg. 0.59). The obtained cTCF was comparable to the GT (p = 0.129, 0.502, and 0.130 for surgical, biopsies, and cell block, respectively) and demonstrated good reliability if elaborated by different pathologists (Wk = 0.9). Overall cTCF was lower as compared to pTCF (30 ± 10 vs 52 ± 19, p < 0.001), with more cases < 20% (17, 14%, p = 0.690), but none containing < 100 cells for the algorithm. Similarities were noted between tumor area estimation and pTCF (36 ± 29, p < 0.001), partly explaining variability in the human assessment of tumor cellularity. Finally, the cTCF allowed a reduction of the copy number variations (CNVs) called (27 vs 29, − 6.9%) with an increase of effective CNVs detection (13 vs 7, + 85.7%), some with potential clinical impact previously undetected with pTCF. An automated computational pipeline (Qupath Analysis of Nuclei from Tumor to Uniform Molecular tests, QuANTUM) has been created and is freely available as a QuPath extension. The computational method used in this study has the potential to improve efficacy and reliability of TCF estimation in NSCLC, with demonstrated impact on the final molecular results.

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RNA-Based Next-Generation Sequencing in Non-Small-Cell Lung Cancer in a Routine Setting: an Experience from an Italian Referral Center

Cited by 3 publications

References 19 publications

Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

FGFR2 testing in cholangiocarcinoma: translating molecular studies into clinical practice

Digital counting of tissue cells for molecular analysis: the QuANTUM pipeline

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