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. 2024 Mar;12(1):73-95.
doi: 10.1007/s40487-023-00252-5. Epub 2024 Jan 11.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience

Francesco Pepe #  1 , Gianluca Russo #  1 , Alessandro Venuta  1 , Claudia Scimone  1 , Mariantonia Nacchio  1 , Pasquale Pisapia  1 , Gaia Goteri  2 , Francesca Barbisan  2 , Caterina Chiappetta  3 , Angelina Pernazza  4 , Domenico Campagna  5 , Marco Giordano  5 , Giuseppe Perrone  6   7 , Giovanna Sabarese  7 , Annalisa Altimari  8 , Dario de Biase  9 , Giovanni Tallini  8   9   10 , Daniele Calistri  11 , Elisa Chiadini  11 , Laura Capelli  11 , Alfredo Santinelli  12 , Anna Elisa Gulini  12 , Elisa Pierpaoli  12 , Manuela Badiali  13 , Stefania Murru  13 , Riccardo Murgia  14 , Elena Guerini Rocco  15   16 , Konstantinos Venetis  15 , Nicola Fusco  15   16 , Denise Morotti  17 , Andrea Gianatti  17 , Daniela Furlan  18 , Giulio Rossi  19 , Laura Melocchi  19 , Maria Russo  1 , Caterina De Luca  1 , Lucia Palumbo  1 , Saverio Simonelli  1 , Antonella Maffè  20 , Paola Francia di Celle  21 , Tiziana Venesio  22 , Maria Scatolini  23 , Enrico Grosso  23 , Sara Orecchia  24 , Matteo Fassan  25   26 , Mariangela Balistreri  27 , Elisabetta Zulato  27 , Daniela Reghellin  28 , Elena Lazzari  28 , Maria Santacatterina  28 , Maria Liliana Piredda  28 , Manuela Riccardi  29 , Licia Laurino  29 , Elena Roz  30 , Domenico Longo  31 , Daniela Petronilla Romeo  31 , Carmine Fazzari  31 , Andrea Moreno-Manuel  32   33   34 , Giuseppe Diego Puglia  35 , Andrey D Prjibelski  36 , Daria Shafranskaya  36 , Luisella Righi  37 , Angela Listì  37 , Domenico Vitale  1 , Antonino Iaccarino  1 , Umberto Malapelle  38 , Giancarlo Troncone  1
Affiliations

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience

Francesco Pepe et al. Oncol Ther. 2024 Mar.

Abstract

Introduction: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples.

Methods: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated.

Results: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation.

Conclusions: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Keywords: Lung; Molecular pathology; Tumor biomarker.

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Conflict of interest statement

Pasquale Pisapia has received personal fees as speaker bureau from Novartis for work performed outside of the current study. Giuseppe Perrone has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Diaceutics, Merck, AstraZeneca, Novartis, Daiichi Sankyo, Exact Sciences, Diatech Pharmacogenetics unrelated to the current work. Elena Guerini-Rocco has relevant relationship (advisory fees, honoraria, travel accommodation and expenses, grants and non-financial support) with AstraZeneca, Exact Sciences, GlaxoSmithKline (GSK), Novartis, Roche, Thermo Fisher Scientific unrelated to the current work. Nicola Fusco reports honoraria from Merck Sharp and Dohme (MSD), Boehringer Ingelheim, Novartis, AstraZeneca and Daiichi Sankyo unrelated to the current work. Matteo Fassan reports research funding (to the institution) from QED, Macrophage pharma, Astellas, Diaceutics; personal honoraria as invited speaker from Roche, Astellas, AstraZeneca, Incyte, Bristol Myers Squibb (BMS), Merck Serono, Pierre Fabre, GSK, Novartis, Amgen; participation in advisory board for Amgen, Astellas, Roche, Merck Serono, GSK, Novartis, Janssen unrelated to the current work. Umberto Malapelle has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientific, Eli Lilly, Diaceutics, GSK, Merck and AstraZeneca, Janssen, Diatech, Novartis and Hedera unrelated to the current work. Giancarlo Troncone reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer, Boehringer Ingelheim, Eli Lilly, BMS, GSK, Menarini, AstraZeneca, Amgen and Bayer, unrelated to the current work. Francesco Pepe, Gianluca Russo, Alessandro Venuta, Claudia Scimone, Mariantonia Nacchio, Gaia Goteri, Francesca Barbisan, Caterina Chiappetta, Angelina Pernazza, Domenico Campagna, Marco Giordano, Giovanna Sabarese, Annalisa Altimari, Dario de Biase, Giovanni Tallini, Daniele Calistri, Elisa Chiadini, Laura Capelli, Alfredo Santinelli, Anna Elisa Gulini, Elisa Pierpaoli, Manuela Badiali, Stefania Murru, Riccardo Murgia, Konstantinos Venetis, Denise Morotti, Andrea Gianatti, Daniela Furlan, Giulio Rossi, Laura Melocchi, Maria Russo, Caterina De Luca, Lucia Palumbo, Saverio Simonelli, Antonella Maffè, Paola Francia di Celle, Tiziana Venesio, Maria Scatolini, Enrico Grosso, Sara Orecchia, Mariangela Balistreri, Elisabetta Zulato, Daniela Reghellin, Elena Lazzari, Maria Santacatterina, Maria Liliana Piredda, Manuela Riccardi, Licia Laurino, Elena Roz, Domenico Longo, Daniela Petronilla Romeo, Carmine Fazzari, Andrea Moreno-Manuel, Giuseppe Diego Puglia, Andrey D. Prjibelski, Daria Shafranskaya, Luisella Righi, Angela Listì, Domenico Vitale, Antonino Iaccarino have nothing to disclose.

Figures

Fig. 1
Fig. 1
Schematic representation of study design. Briefly, after internal validation of standard reference sample at University of Naples Federico II, a set of eight slides were distributed to each center. Here, own diagnostic workflow (from nucleic acid extraction to molecular data interpretation) was applied. Data were shared with the coordinating center. MET MET proto-oncogene, receptor tyrosine kinase, ROS1 c-ros oncogene 1, SLC34A2 solute carrier family 34 member 2
Fig. 2
Fig. 2
DNA and RNA concentration by participating center. Scatterplots showing a DNA and b RNA concentration obtained by each participating center, colored by the extraction method (red, automatic procedure; blue, manual procedure)
Fig. 3
Fig. 3
Pie charts showing the different techniques used for analysis of DNA a KRAS and b EGFR hotspot mutations. NGS next-generation sequencing, PyroSeq pyrosequencing, KRAS Kirsten rat sarcoma viral oncogene homologue, EGFR epidermal growth factor receptor
Fig. 4
Fig. 4
Pie charts showing the different techniques used for analysis of RNA a ROS1 gene fusions and b MET exon 14 skipping. NGS next-generation sequencing, MET MET proto-oncogene, receptor tyrosine kinase, ROS1 c-ros oncogene 1
Fig. 5
Fig. 5
Bar chart showing the percentage of cases in which MET exon 14 skipping was detected using DNA-based technologies, RNA-based technologies, or both. MET MET proto-oncogene, receptor tyrosine kinase
See this image and copyright information in PMC

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