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Technical validation of a fully integrated NGS platform in real-world practice of Italian referral institution

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26 September 2023

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28 September 2023

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Abstract
Aims: To date, precision medicine plays a pivotal role in the clinical administration of solid tumor patients. In this scenario, a rapidly increasing number of predictive biomarkers have been approved in diagnostic practice or are currently investigated in clinical trials. A pitfall in the molecular tests is the diagnostic routine sample available to analyze predictive biomarkers; scant tissue sample often represents the only diagnostical source of nucleic acids to assess molecular analysis. At the sight of these critical issues, Next Generation Sequencing (NGS) platforms emerged as referral testing strategy for molecular analysis of predictive biomarkers in routine practice but high-skilled personnel, extensive working-time drastically impact on the widespread diffusion of this technology in diagnostic setting. Here, we technically validate a fully integrated NGS platform on diagnostic routine tissue samples previously tested with NGS based diagnostic workflow by a referral institution. Methods: A retrospective series of n=64 samples (n=32 DNA, n=32 RNA samples), previously tested using a customized NGS assay (SiRe™ and SiRe fusion) were retrieved from internal archive of University of Naples Federico II. Each sample was tested by adopting Oncomine Precision Assay (OPA), able to detect 2769 molecular actionable alterations [hot spot mutations, copy number variations (CNV) and gene fusions on fully integrated NGS platform (Genexus, Thermofisher Scientifics. (26,27) Concordance rate between these technical approaches was carried out. Results: Genexus system successfully carried out molecular analysis in all instances. A concordance rate of 96.9% (31 out of 32) was observed between OPA and SiRe™ panel both for DNA and RNA based analysis. A negative predictive value of 100% and a positive predictive value of 96.9% (62 out of 64) was assessed. Conclusions: Fully automatized Genexus system combined with OPA (Thermofisher Scientifics) may be considered a technically valuable, saving time sequencing platform to test predictive biomarkers in diagnostic routine practice.
Keywords: 
Subject: Medicine and Pharmacology  -   Oncology and Oncogenics

1. Introduction

In the last decades, personalized medicine lay the basis for a novel therapeutical option for solid tumor patients. (1,2) To date, target therapy is routinely available for the clinical administration of several solid tumor patients, including metastatic colorectal cancer (mCRC), melanoma (MM), non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor (GIST), breast cancer (BC) patients. (3-9) Particularly, an increasing number of predictive biomarkers was approved in clinical practice to select lung cancer patients diagnosed with NSCLC type to the best therapeutical option. (8,9) In this evolving scenario, the minimal request in terms of predictive biomarkers to clinically administrate solid tumor patients has been regulated by international societies. (10-14) The most common diagnostic sample available to approach diagnosis and molecular tests in advanced tumor stage consists in a “scant sample” with low abundance of neoplastic cells to successfully carry out mandatory gene testing. (15-17) In this scenario, cytological specimens and small biopsies represent the most common biological source to accurately perform molecular analysis. In addition, cell block (CB), a hybrid preparation where the aspirated material is processed following standardized formalin fixation and paraffin embedding (FFPE), represents an alternative source of neoplastic cells affected by lowest quality and quantity of nucleic acids adopted in molecular tests. (18-19) Despite tissue specimens is considered “gold standard” for molecular testing, a not negligible percentage of patients does not access to molecular tests due to insufficient diagnostic material. (16-17) In this scenario, liquid biopsy becomes an integrating biological source to successfully perform molecular analysis when tissue is not available. Particularly, circulating tumor DNA (ctDNA) isolated from peripheral blood withdrawn consists in a reliable source to detect target molecular alterations. (21) At the sight of these aspects, single plex technology result inadequate to successfully analyze minimum gene panel established for each solid tumor. In this heterogenous landscape of biological sources, next generation sequencing (NGS) platforms play a crucial role in the molecular analysis of predictive biomarkers. (22-24) This technology allows to simultaneously analyze very low frequency clinically relevant biomarkers from very low amount of nucleic acids in a single run. (22,23) Remarkably, NGS systems are scalable decreasing reaction cost in accordance with the number of samples processed in each run. (24) On the other hand, adequate number of samples saving technical costs may be collected in more than 30 days for a not negligible number of small-medium institutions involved in molecular tests. This aspect drastically impacts on turnaround -time (TAT) resulting in a delay for the clinical administration of tumor patients. (24,25) In this scenario, Ion Torrent™ Genexus™ Integrated Sequencer (Genexus; Thermofisher Scientifics, Waltham Massachusetts) was designed to automatically carry out entire NGS workflow (from tissue and liquid biopsy derived nucleic acids extraction to data analysis) without other manual operations. (26-28) This technology allows to successfully carry out molecular analysis of a small batch of diagnostic specimens (1- 8) without impacting on Turn-around Time (TAT) of diagnostic workflow. We aimed to evaluate the concordance rate between Genexus system and Ion Torrent S5™ plus (Thermofisher Scientifics, Waltham Massacchusetts) on a retrospective series of extracted genomic DNA (gDNA) from solid tumor patients previously tested in our diagnostic routine.

2. Study design

A retrospective series of n=64 previously extracted DNA and RNA specimens from solid tumor patients [n=16 CRC, n=13 NSCLC, n=2 BC and n=1 MM and n=32 NSCLC cases for DNA and RNA related molecular analysis, respectively) was retrieved from internal archive of Predictive molecular pathology laboratory of University of Naples Federico II. Clinical pathological data were listed in Table 1 and Table 2.
Each sample was previously tested by adopting a customized NGS assay (SiRe™ and SiRe fusion), that covers n=568 clinically relevant alterations in BRAF, EGFR, KRAS, NRAS, PIK3CA, c-KIT, PDGFRA and ALK, ROS1, RET, and NTRK gene fusions, as well as and MET exon 14 skipping alterations, routinely employed in molecular testing of solid tumor patients. (29) The Oncomine Precision Assay (OPA), able to detect 2769 molecular actionable alterations [hot spot mutations, copy number variations (CNV) and gene fusions, was combined with Genexus (Thermofisher Scientifics) platform to assess molecular profile of selected samples. (26,27) Concordance rate of OPA on Genexus system with SiRe™ on S5 plus platform was investigated. All information regarding human material will be managed using anonymous numerical codes, and all samples will be handled in compliance with the Helsinki Declaration (http://www.wma.net/ en/30publications/10policies/b3/).

3. Material and methods

3.1. Routine sample processing startegy

Nucleic acids were previously purified from n=4 representative slides of neoplastic area (>10%). Particularly, QIAamp DNA Mini Kit (Qiagen, Crawley, West Sussex, UK) was adopted following manufacturer instructions. DNA quantification was successfully carried out in all instances according to standardized procedures. Conversely, RNA volume was maximized for cDNA synthesis. Selected samples were routinely analyzed with SiRe™ and SiRe fusion panel on Ion S5™ plus (Thermofisher Scientifics) to assess mutational status in clinically relevant biomarkers for NSCLC patients. (29-31) Briefly, 15 μl of extracted DNA/cDNA was dispensed on Ion Chef system (Thermofisher Scientifics) for library preparation. A total of n= 8 samples were simultaneously processed following previously validated thermal condition. After pooling, templating procedure was carried out for n=16 libraries by using Ion 510™ & Ion 520™ & Ion 530™ Kit Chef (Thermofischer Scientifics) according to manufacturer instructions on 520 chip (Thermofisher Scientifics). Data were inspected by adopting designed bed files on proprietary Torrent Suite [v.5.0.2]. In details, variant inspection was performed with variant caller plug-in (v.5.0.2.1) able to filter variants with ⩾5X allele coverage and a quality score ⩾20, within an amplicon that covered at least 500X alleles.

3.2. Genexus analysis

A series of n=64 extracted gDNA and gRNA from solid tumor patients were retrospectively tested on Genexus (Thermofisher Scientifics) system. The platform enables entire NGS workflows (from library preparation to data interpretation) within 24 hours. OPA assay includes most clinically relevant actionable genes (EGFR, BRAF, KRAS, ALK, ROS1, NTRK, and RET) for NSCLC patients. (27,28) Briefly, samples were created on dedicated server and assigned to a new run. Genexus platform was loaded with OPA primers, strip solutions, strip reagents and supplies according to manufacturer instructions. A total of 10 ng was required by OPA assay on Genexus platform. Accordingly, each sample was diluted and immediately dispensed on 96-well plate, following manufacturer instructions. Finally, nucleic acids were sequenced on GX5TM chip that allows simultaneous processing of n=8 samples in a single line with OPA assay. Data analysis was performed on proprietary Genexus software. Particularly, detected alterations were annotated by adopting Oncomine Knowledgebase Reporter Software (Oncomine Reporter 5.0).

4. Results

4.1. Hot spot mutations

Overall, Genexus system successfully carried out molecular analysis in all DNA series. In details, a median number of total reads, mapped reads, mean read length, percent reads on target, mean depth, uniformity of amplicon coverage of 1134878.2 (ranging from 424900.0 to 1791041.0), 1074345.7 (ranging from 365139.0 to 1756414.0), 90.9 bp (ranging from 71 to 103 bp), 88.3% (ranging from 77.7 to 93.7%), 3602.9 (ranging from 994.00 to 6097.0) and 98.2% (ranging from 96.7 to 99.4%) were detected, respectively. (Table 3).
Remarkably, n=29 out of 32 (90.6%) patients [n=16 CRC, n= 10 NSCLC, n=2 BC and n=1 MM) showed molecular alterations covered by OPA reference genes. Of note, 24 out of 29 (82.7%) cases highlighted clinically relevant molecular alterations referenced by SiRe™ panel. In particular, n=3 out 29 EGFR mutations [n=1 exon 19 c.2300_2308dup p.A767_V769dup; n=1 exon 21 c.2573T>G p.L858R and a concomitant EGFR exon 20 c.2369C>T p.T790M+ exon 21 c.2573T>G p.L858R; n=13 out of 29 KRAS molecular alterations [n=3 exon 2 c.35G>A p.G12D; n=2 exon 2 c.34G>T p.G12C; n=2 exon 2 c.35G>A p.G12V; n=1 exon 2 c.38G>A p.G13D; n=1 exon 3 c.182A>T p.Q61L; n=1 exon 3 c.181C>A p.Q61K; n=1 exon 4 c.436G>A p.A146T and n=2 concomitant KRAS exon 2 c.35G>A p.G12D+ c.38G>A p.G13D; KRAS exon 2 c.38G>A p.G13D+ c.38_39delinsAA p.G13E]; n=3 out of 29 BRAF mutations [n=2 exon 15 c.1799T>A p.V600E and n=1 exon 15 c.1801A>G p.K601E]; n=4 out of 29 PIK3CA hot spot mutations [n=2 exon 9 c.1633G>A p.E545K and n=2 exon 20 c.3140A>G p.H1047R]; n=3 out 29 NRAS mutations [n=2 exon 3 c.181C>A p.Q61K and n=1 exon 3 c.182A>G p.Q61R]; n=1 out of 29 c-KIT molecular alterations [exon 11 c.1727T>C p.L576P] were detected. (Table 4).
Molecular profile detected by OPA on Genexus platform matched with Sire panel on S5 plus system in 31 out of 32 patients (96.9%). Remarkably, positive results previously identified adopting SiRe panel were confirmed in 23 out of 24 (95.8%) patients. Particularly, ID#19 showed exon 9 PIK3CA p.E545K hot spot mutation not observed by using S5 system with standardized clinical cut-off. (Figure 1)
No significant variations in accordance with histological groups, mutation type and mutant allele fraction levels between Genexus and previously tested samples on S5 platform were identified. In addition, OPA assay also identified n= 16 out of 32 (50.0%) DNA based molecular alterations in other genes not covered by SiRe panel. As regards, 12 out of 16, 1 out of 16 and 1 out of 16 highlighted TP53, CTNNB1 and MTOR hotspot molecular altercations, respectively. Moreover, a concomitant TP53 (exon 7 p.G279E plus exon 5 p.V197M) and TP53 (exon 4 p.R175H) in association with CTNNB1 (exon 3 p.S45F) hotspot mutations were identified in ID#2 and ID#16 cases. (Table 5).

4.2. Fusions rearrangements

Regarding RNA samples, Genexus platform successfully analyzed all retrieved cases. Briefly, a median number of total reads, mapped reads and mean read length of 1721491.0 (ranging from 1471817.00 to 2462555.00), 158230.4 (ranging from 37387.0 to 1029745.00), 98.8 bp (ranging from 91 to 104 bp) were identified, respectively. (Table 6).
Of note, 10 out of 32 (31.2%) patients highlighted aberrant transcripts by using Genexus platform. Among them, 5 out of 10 and 2 out of 10 patients showed ALK and RET rearrangements, respectively. Moreover, three patients were positive for ROS1, NTRK aberrant transcripts and MET Δ 14 skipping mutation, respectively. (Table 7) Interestingly, rearranged genes were identified by OPA on Genexus platform in 9 out of 10 (90.0%) retrieved cases showing a concordance rate of 96.9% (31 out of 32 cases) with SiRe panel on S5 system. Particularly, ID#1 was positive for NTRK3-KANK1 fusion transcript not previously detected with SiRe panel on S5 platform. No significant variations were observed in accordance with histological groups, rearranged genes, fusion partners, and mapped reads levels between Genexus and previously tested samples on S5 platform.

5. Discussion

In the era of personalized medicine, the rapidly increasing number of predictive biomarkers yet approved in clinical practice have revolutionized the treatment strategy for solid tumor patients. (1-2,32) Although the widespread diffusion of single-gene testing platforms in the vast majority of laboratories involved in molecular tests, low multiplexing biomarker’s analysis discouraging their implementation as pivotal diagnostic platform in clinical practice (23-24). As regards, NGS techniques allows to simultaneously cover clinically relevant molecular alterations from a plethora of diagnostic routine specimens saving technical costs and maintaining adequate TAT (33). Moreover, NGS platforms may also benefit of automatized technical procedures that allows accurate and reproducible analysis spending low bench-working time (33). Genexus system consists in a scalable, versatile and fully automatized sequencer able to carry out each technical procedure without manual operations (34). This system is built to integrate analytical procedures (nucleic acids extraction, libraries preparation, template generation, sequencing) with data analysis by adopting pre-customized pipeline analysis. Here, we have validated Genexus system in our diagnostic routine by comparing its analytical performance on a retrospective series of clinical cases previously analyzed with a custom NGS panel on S5 system. As expected, all diagnostic specimens (n=64) were successfully analyzed by using this fully automatized system. Overall, a concordance rate of 96.9% (62 out of 64) was reached by adopting Sire panel on S5 system as reference standard. Interestingly, molecular analysis unmatched with previously archived data in only two cases (DNA-ID#19 and RNA-ID#1). Of note, DNA-ID#19 sample derived from a BC patient resulted positive for PIK3CA exon 9 p.E545K hotspot alteration on Genexus system with a mutant allele fraction (MAF) of 7.2%. Following manufacturer clinical cut-off (MAF ≥5%), previous analysis did not show any clinically relevant molecular alteration. By approaching visual inspection of raw data, the same alteration at 0.9% was detected. Similarly, RNA-ID#1 showed NTRK3 (ex14) - KANK1 (ex3) aberrant transcript not previously detected with the standard reference approach. In this case NTRK3 was not covered by reference range of SiRe fusion panel.
In a not negligible percentage of cases, synchronous lesions may be observed in CRC patients. In this scenario, NGS may be considered an affordable technical strategy to comprehensively evaluate molecular assessment of CRC patients where heterogeneous specimens are clinically available (28). DNA-ID#11 and DNA-ID#2 represent synchronous lesions of a CRC elected to molecular test. Interestingly, both S5 and Genexus systems revealed KRAS exon 2 p.G12C and PIK3CA exon 20 p.H1047R hot spot mutations demonstrating a common origin of these lesions. Moreover, NGS systems overcome technical issues from the analysis of “complex” molecular alteration. DNA-ID#22 case confirmed two concomitant KRAS exon 2 hotspot mutations p.G13D+p.G13E on Genexus platform previously detected by reference technology. Although this study provides encouraging results for the implementation of Genexus system in clinical routine setting of solid tumor patients, some limitations may be identified. Firstly, this technical report aims to compare analytical parameters of two NGS-based technologies on a series of diagnostic routine specimens without any clinical considerations. Secondly, this retrospective study is based on the analysis of a small group of cases retrieved from internal archive of University of Naples Federico II. All these crucial points warrant further analysis, but this preliminary data may suggest that fully automatized Genexus system integrated with commercially available OPA (Thermofisher Scientifics) represent a technically affordable, saving time sequencing platform enable to analyze clinically relevant molecular alterations in diagnostic routine specimens.

Author Contributions

“Conceptualization, CDL, FP, GT and UM.; methodology, all the authors.; software, CDL, FP, GT and UM; validation, all the authors.; formal analysis, all the authors; data curation, CDL, FP, GT and UM.; writing—original draft preparation, CDL, FP; writing—review and editing, GT and UM.; visualization, all the authors; supervision, GT and UM.; project administration, GT and UM All authors have read and agreed to the published version of the manuscript.”

Funding

1. Monitoraggio ambientale, studio ed approfondimento della salute della popolazione residente in aree a rischio—In attuazione della D.G.R. Campanian.180/2019. 2. POR Campania FESR 2014–2020 Progetto “Sviluppo di Approcci Terapeutici Innovativi per patologie Neoplastiche resistenti ai trattamenti—SATIN”. 3. This work has been partly supported by a grant from the Italian Health Ministry’s research program (ID: NET-2016-02363853). National Center for Gene Therapy and Drugs based on RNA Technology MUR-CN3 CUP E63C22000940007 to DS.

Patient consent for publication

Not applicable

Ethics approval

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Competing interests

Pasquale Pisapia has received personal fees as speaker bureau from Novartis for work performed outside of the current study. 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.

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Figure 1. PIK3CA p.E545K hotspot mutations manually inspected with Golden Helix Genome Browser v.2.0.7 (Bozeman, MT, USA) (A) and automatically annotated on on proprietary Genexus software (B).
Figure 1. PIK3CA p.E545K hotspot mutations manually inspected with Golden Helix Genome Browser v.2.0.7 (Bozeman, MT, USA) (A) and automatically annotated on on proprietary Genexus software (B).
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Table 1. Clinical characteristics of archival cases and corresponding requests on DNA-based molecular alterations.
Table 1. Clinical characteristics of archival cases and corresponding requests on DNA-based molecular alterations.
ID Sex Age Sample Type Tumour N.C. Clinical Request
DNA 1* M 78 Resection CRC 70.0% RAS, BRAF
DNA 2* M 78 Resection CRC 70.0% RAS, BRAF
DNA 3 M 89 Biopsy CRC 50.0% RAS, BRAF
DNA 4 F 68 Resection NSCLC 70.0% EGFR, KRAS, BRAF
DNA 5 M 73 Resection CRC 50.0% RAS, BRAF
DNA 6 M 53 Biopsy NSCLC 30.0% EGFR, KRAS, BRAF
DNA 7 M 66 Resection CRC 40.0% RAS, BRAF
DNA 8 F 78 Resection CRC 40.0% RAS, BRAF
DNA 9 F 67 Resection NSCLC 60.0% EGFR, KRAS, BRAF
DNA 10 F 51 Resection CRC 30.0% RAS, BRAF
DNA 11 M 50 Resection CRC 80.0% c-KIT, PDGFRA
DNA 12 F 50 Biopsy NSCLC 50.0% EGFR, KRAS, BRAF
DNA 13 M 70 Biopsy NSCLC 20.0% EGFR, KRAS, BRAF
DNA 14 F 59 Resection NSCLC 40.0% EGFR, KRAS, BRAF
DNA 15 M 66 Biopsy NSCLC 30.0% EGFR, KRAS, BRAF
DNA 16 M 56 Resection CRC 50.0% RAS, BRAF
DNA 17 M 66 Resection NSCLC 60.0% EGFR, KRAS, BRAF
DNA 18 F 51 Biopsy CRC 50.0% RAS, BRAF
DNA 19 F 41 Biopsy BC 30.0% PIK3CA
DNA 20 F 82 Biopsy CRC 30.0% RAS, BRAF
DNA 21 M 67 Biopsy CRC 50.0% RAS, BRAF
DNA 22 M 82 Resection NSCLC 80.0% EGFR, KRAS, BRAF
DNA 23 M 74 Resection NSCLC 70.0% EGFR, KRAS, BRAF
DNA 24 M 74 Resection CRC 40.0% RAS, BRAF
DNA 25 F 44 Biopsy CRC 40.0% RAS, BRAF
DNA 26 F 69 Biopsy NSCLC 60.0% EGFR, KRAS, BRAF
DNA 27 M 54 Resection CRC 30.0% RAS, BRAF
DNA 28 F 74 Resection MM 90.0% BRAF, NRAS
DNA 29 F 63 Biopsy NSCLC 40.0% EGFR, KRAS, BRAF
DNA 30 M 56 Resection NSCLC 50.0% EGFR, KRAS, BRAF
DNA 31 F 52 Resection CRC 60.0% RAS, BRAF
DNA 32 F 45 Resection BC 60.0% PIK3CA
* Same patient, different lesions. Abbreviations: BC (Breast Cancer); BRAF (Murine Sarcoma Viral Oncogene Homolog B); c-KIT (KIT Proto-Oncogene); CRC (Colorectal Cancer); DNA (Deoxyribonucleic Acid); EGFR (Epidermal Growth Factor Receptor); F (Female); ID (Identifier); KRAS (Kirsten Rat Sarcoma Virus); M (Male); MM (Malignant Melanoma); N.C. (Neoplastic Cellularity); NSCLC (Non-Small-Cell Lung Cancer); PIK3CA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase, Catalytic Subunit Alpha); RAS (Rat Sarcoma Virus).
Table 2. Clinical characteristics of archival cases and corresponding requests on RNA-based molecular alterations.
Table 2. Clinical characteristics of archival cases and corresponding requests on RNA-based molecular alterations.
ID Sex Age Sample Type Tumour N.C. Clinical Request
RNA 1 M 56 Resection NSCLC 60.0% ALK, ROS1, RET, MET, NTRK
RNA 2 F 58 Biopsy NSCLC 70.0% ALK, ROS1, RET, MET, NTRK
RNA 3 M 77 Biopsy NSCLC 25.0% ALK, ROS1, RET, MET, NTRK
RNA 4 M 79 Resection NSCLC 70.0% ALK, ROS1, RET, MET, NTRK
RNA 5 M 79 Biopsy NSCLC 30.0% ALK, ROS1, RET, MET, NTRK
RNA 6 M 59 Biopsy NSCLC 30.0% ALK, ROS1, RET, MET, NTRK
RNA 7 F 70 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 8 M 62 Biopsy NSCLC 25.0% ALK, ROS1, RET, MET, NTRK
RNA 9 M 61 Biopsy NSCLC 40.0% ALK, ROS1, RET, MET, NTRK
RNA 10 M 66 Resection NSCLC 60.0% ALK, ROS1, RET, MET, NTRK
RNA 11 M 68 Biopsy NSCLC 40.0% ALK, ROS1, RET, MET, NTRK
RNA 12 M 64 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 13 F 65 Biopsy NSCLC 60.0% ALK, ROS1, RET, MET, NTRK
RNA 14 M 58 Biopsy NSCLC 20.0% ALK, ROS1, RET, MET, NTRK
RNA 15 F 79 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 16 M 52 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 17 M 67 Resection NSCLC 60.0% ALK, ROS1, RET, MET, NTRK
RNA 18 M 87 Biopsy NSCLC 40.0% ALK, ROS1, RET, MET, NTRK
RNA 19 M 25 Biopsy NSCLC 60.0% ALK, ROS1, RET, MET, NTRK
RNA 20 F 60 Biopsy NSCLC 30.0% ALK, ROS1, RET, MET, NTRK
RNA 21 M 60 Resection NSCLC 60.0% ALK, ROS1, RET, MET, NTRK
RNA 22 F 36 Biopsy NSCLC 30.0% ALK, ROS1, RET, MET, NTRK
RNA 23 M 66 Biopsy NSCLC 60.0% ALK, ROS1, RET, MET, NTRK
RNA 24 F 47 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 25 M 67 Biopsy NSCLC 30.0% ALK, ROS1, RET, MET, NTRK
RNA 26 F 64 Biopsy NSCLC 10.0% ALK, ROS1, RET, MET, NTRK
RNA 27 M 54 Biopsy NSCLC 40.0% ALK, ROS1, RET, MET, NTRK
RNA 28 F 37 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 29 M 79 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 30 F 71 Biopsy NSCLC 30.0% ALK, ROS1, RET, MET, NTRK
RNA 31 M 68 Biopsy NSCLC 50.0% ALK, ROS1, RET, MET, NTRK
RNA 32 F 72 Biopsy NSCLC 70.0% ALK, ROS1, RET, MET, NTRK
Abbreviations: ALK (Anaplastic Lymphoma Kinase); F (Female); ID (Identifier); M (Male); MET (Tyrosine-Protein Kinase Met); N.C. (Neoplastic Cellularity); NSCLC (Non-Small-Cell Lung Cancer); NTRK (Neurotrophic Tyrosine Receptor Kinase); RET (RET Proto-Oncogene); RNA (Ribonucleic Acid); ROS1 (Proto-Oncogene Tyrosine-Protein Kinase ROS).
Table 3. Technical parameters from DNA-based analysis by using S5 plus and Genexus systems.
Table 3. Technical parameters from DNA-based analysis by using S5 plus and Genexus systems.
DNA Analysis Technical Parameters - S5 Plus (SiRe™ Panel) vs Genexus (OPA Panel)
ID Platform Total Reads Mean Read Length Mapped Reads On Target Reads Mean Depth Uniformity
DNA 1* S5 Plus 254212 126 253622 94.6% 5712 100%
Genexus 872831 76 736530 77.7% 2044 99.1%
DNA 2* S5 Plus 215464 128 215047 92.6% 4740 100%
Genexus 732691 84 663064 83.9% 2034 98.8%
DNA 3 S5 Plus 298541 135 297999 93.9% 6662 100%
Genexus 1143038 91 1076855 88.8% 3528 98.1%
DNA 4 S5 Plus 524926 155 523086 92.3% 11489 100%
Genexus 1419289 101 1393603 92.9% 5210 98.1%
DNA 5 S5 Plus 361148 137 360373 91.3% 7830 100%
Genexus 1094620 98 1064051 91.5% 3810 98.6%
DNA 6 S5 Plus 314176 128 313706 99.2% 7406 100%
Genexus 1090358 98 1049935 90.8% 3837 99,0%
DNA 7 S5 Plus 635201 142 634226 92.1% 13911 100%
Genexus 1002231 92 946318 88.9% 3150 98.9%
DNA 8 S5 Plus 524182 131 523608 93.0% 11591 100%
Genexus 1262760 95 1208543 90.9% 4176 98.9%
DNA 9 S5 Plus 942781 161 940605 94.6% 21192 100%
Genexus 1791041 97 1756414 93,0% 6097 97.9%
DNA 10 S5 Plus 393979 126 393371 89.5% 8381 100%
Genexus 989635 60 717385 64.9% 1459 98.9%
DNA 11 S5 Plus 451494 139 450779 94.4% 10127 100%
Genexus 776893 78 679358 80.4% 1863 96.7%
DNA 12 S5 Plus 88915 129 88784 98.0% 2072 92.9%
Genexus 1297992 91 1263558 92.7% 3996 93.9%
DNA 13 S5 Plus 296845 143 296434 96.2% 6790 100%
Genexus 1196122 99 1174442 92.7% 4258 98.5%
DNA 14 S5 Plus 37206 133 37173 95.2% 842,7 97.6%
Genexus 1125616 97 1093531 91.8% 3824 98.6%
DNA 15 S5 Plus 782397 150 780894 95.2% 17703 100%
Genexus 1465786 92 1423741 91.9% 4574 95.3%
DNA 16 S5 Plus 378978 140 378373 93.3% 8402 100%
Genexus 1084647 87 1012693 87.6% 3054 98.2%
DNA 17 S5 Plus 520304 135 519653 91.5% 11317 100%
Genexus 1048030 98 1016324 91.4% 3617 98.8%
DNA 18 S5 Plus 49127 138 49055 95.3% 1113 97.6%
Genexus 1294194 97 1256161 91.9% 4435 98.9%
DNA 19 S5 Plus 486407 147 485652 96.6% 11165 97.6%
Genexus 1343529 97 1311776 92.3% 4658 99.4%
DNA 20 S5 Plus 346019 131 345464 97.4% 8010 97.6%
Genexus 974476 71 759420 75.7% 2023 98.8%
DNA 21 S5 Plus 67488 130 67417 95.9% 1540 97.6%
Genexus 1150249 90 1094010 90.3% 3519 98.8%
DNA 22 S5 Plus 52080 170 51956 90.4% 1119 100%
Genexus 1494337 100 1470085 92.3% 5451 97.9%
DNA 23 S5 Plus 614960 141 613813 96.2% 14059 97.6%
Genexus 1574234 91 1510266 91.2% 4865 97.7%
DNA 24 S5 Plus 188967 136 188623 98.1% 4407 97.6%
Genexus 1093646 103 1071141 92.2% 4072 99.1%
DNA 25 S5 Plus 140163 145 139930 95.5% 3183 97.6%
Genexus 949852 94 911448 90,0% 3064 99.4%
DNA 26 S5 Plus 40233 142 40180 96.7% 925,4 97.6%
Genexus 1497022 99 1476425 93.7% 5365 98.3%
DNA 27 S5 Plus 153378 133 153236 96.0% 3501 97.6%
Genexus 1059772 95 1021186 90.2% 3498 98.7%
DNA 28 S5 Plus 155154 118 154695 96.5% 3553 92.8%
Genexus 424900 75 365139 79.3% 994 97.4%
DNA 29 S5 Plus 358001 160 356995 95.2% 8095 100%
Genexus 1165795 98 1134969 92.2% 4075 98.4%
DNA 30 S5 Plus 275579 149 274340 98.4% 6428 100%
Genexus 1080846 92 1034348 90.3% 3392 98.4%
DNA 31 S5 Plus 259364 130 258623 92.6% 5702 100%
Genexus 1109488 92 1054465 89.9% 3457 98.9%
DNA 32 S5 Plus 263420 126 262682 93.4% 5841 97.6%
Genexus 710181 82 631880 82.5% 1893 96.7%
*Same patient with different lesions. Abbreviations: DNA (Deoxyribonucleic Acid); ID (Identifier).
Table 4. Comparison of DNA-related molecular alterations between S5 plus and Genexus platforms.
Table 4. Comparison of DNA-related molecular alterations between S5 plus and Genexus platforms.
ID S5Plus (SiRe™ Panel) Genexus (OPA Panel)
DNA 1* KRAS p.G12C 27.6%
PIK3CA p.H1047R 35.0%
KRAS p.G12C 32.9%
PIK3CA p.H1047R 33.2%
DNA 2* KRAS p.G12C 37.2%
PIK3CA p.H1047R 42.2%
KRAS p.G12C 32.7%
PIK3CA p.H1047R 36.4%
DNA 3 KRAS p.G12D 20.7% KRAS p.G12D 18.9%
DNA 4 EGFR p.L858R 27.7% EGFR p.L858R 18.9%
DNA 5 KRAS p.G12V 34.5% KRAS p.G12V 33.0%
DNA 6 WT WT
DNA 7 KRAS p.G12D 57.2% KRAS p.G12D 60.8%
DNA 8 KRAS p.Q61K 16.8% KRAS p.Q61K 19.3%
DNA 9 WT WT
DNA 10 KRAS p.G12D 50.6% KRAS p.G12D 55.3%
DNA 11 c-KIT p.L576P 68.0% c-KIT p.L576P 63.8%
DNA 12 EGFR p.A767_V769dup 67.2% EGFR p.A767_V769dup 72.8%
DNA 13 WT WT
DNA 14 WT WT
DNA 15 BRAF p.K601E 16.3% BRAF p.K601E 16.1%
DNA 16 KRAS p.G12D 9.3%
KRAS p.G13D 14.1%
KRAS p.G12D 8.2
KRAS p.G13D 12.1%
DNA 17 KRAS p.Q61L 32.7% KRAS p.Q61L 36.3%
DNA 18 NRAS p.Q61K 19.3% NRAS p.Q61K 18.2%
DNA 19 PIK3CA E545K 0.8%** PIK3CA E545K 7.2%
DNA 20 BRAF p.V600E 30.5% BRAF p.V600E 30.0%
DNA 21 NRAS p.Q61K 46.7% NRAS p.Q61K 36.2%
DNA 22 KRAS p.G13D 47.4%***
KRAS p.G13E 47.9%***
KRAS p.G13D 41.9%***
KRAS p.G13E 42.0%***
DNA 23 WT WT
DNA 24 KRAS p.A146T 30.80% KRAS p.A146T 26.4%
DNA 25 WT WT
DNA 26 BRAF p.V600E 27.3% BRAF p.V600E 30.3%
DNA 27 KRAS p.G13D 14.9% KRAS p.G13D 12.2%
DNA 28 NRAS p.Q61R 34.3% NRAS p.Q61R 28.2%
DNA 29 EGFR p.L858R 9.7%
EGFR p.T790M 9.5%
EGFR p.L858R 9.3%
EGFR p.T790M 11.0%
DNA 30 WT WT
DNA 31 KRAS p.G12V 51.2%
PIK3CA p.E545K 32.2%
KRAS p.G12V 59.2%
PIK3CA p.E545K 31.0%
DNA 32 WT WT
* Different lesion of same patient. ** Below 5%; *** Concominant SNV. Abbreviations: BRAF (Murine Sarcoma Viral Oncogene Homolog B); c-KIT (KIT Proto-Oncogene); DNA (Deoxyribonucleic Acid); EGFR (Epidermal Growth Factor Receptor); ID (Identifier); KRAS (Kirsten Rat Sarcoma Virus); PIK3CA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase, Catalytic Subunit Alpha); RAS (Rat Sarcoma Virus); WT (Wild-Type).
Table 5. Expanded list of molecular alterations covered by OPA on Genexus platform.
Table 5. Expanded list of molecular alterations covered by OPA on Genexus platform.
ID Other Mutations (OPA Panel)
DNA 1* MTOR p.R2217W 4.5%
DNA 2* TP53 p.G279E 4.8%
TP53 p.V197M 4.0%
DNA 7 TP53 p.H179Y 75.8%
DNA 9 TP53 p.R273H 35.0%
DNA 12 TP53 p.V197M 77.7%
DNA 14 TP53 p.R273H 10.0%
DNA 16 CTNNB1 p.S45F 41.1%
TP53 p.R175H 13.2%
DNA 18 TP53 p.Y220C 19.7%
DNA 19 TP53 p.L194F 9.9%
DNA 20 TP53 p.P151S 54.7%
DNA 21 TP53 p.K132R 51.4%
DNA 23 TP53 p.C238S 25.3%
DNA 27 CTNNB1 p.S45F 21.8%
DNA 30 TP53 p.H179Y 24.6%
DNA 31 TP53 p.Y220C 56.1%
DNA 32 TP53 p.E285K 4.8%
*Same patient, different lesion. Abbreviations: CTNNB1 (Catenin Beta 1); DNA (Deoxyribonucleic Acid); ID (Identifier); MTOR (Mammalian Target Of Rapamycin); TP53 (Tumor Protein P53).
Table 6. Technical parameters from RNA-based analysis by using S5 plus and Genexus systems.
Table 6. Technical parameters from RNA-based analysis by using S5 plus and Genexus systems.
RNA analysis Technical Parameters - S5 Plus (SiRe Fusion Panel) vs Genexus (OPA Panel)
ID Platform Total Reads Mean Read Length Mapped Reads
RNA 1 S5 Plus 503832 92 489474
Genexus 2355408 99 170105
RNA 2 S5 Plus 829380 124 823978
Genexus 1748261 99 140327
RNA 3 S5 Plus 641591 89 348169
Genexus 2462555 104 54529
RNA 4 S5 Plus 254394 93 242076
Genexus 1667488 100 37387
RNA 5 S5 Plus 234803 67 176276
Genexus 1755508 91 111713
RNA 6 S5 Plus 357284 89 319350
Genexus 1542252 101 72995
RNA 7 S5 Plus 1070656 111 1067615
Genexus 1571469 100 150711
RNA 8 S5 Plus 535701 103 526127
Genexus 1737696 96 1029745
RNA 9 S5 Plus 494550 87 421901
Genexus 1634624 103 72104
RNA 10 S5 Plus 161964 100 153003
Genexus 1815512 96 51505
RNA 11 S5 Plus 190170 98 187044
Genexus 1597727 98 386493
RNA 12 S5 Plus 677654 91 513093
Genexus 1554237 101 171919
RNA 13 S5 Plus 765186 129 753177
Genexus 1777747 100 178846
RNA 14 S5 Plus 222717 103 217972
Genexus 1503566 102 48005
RNA 15 S5 Plus 490208 125 483482
Genexus 1523971 99 61024
RNA 16 S5 Plus 20405 91 17060
Genexus 1878041 97 42572
RNA 17 S5 Plus 367743 117 346142
Genexus 1769313 97 80920
RNA 18 S5 Plus 191027 99 189336
Genexus 1513615 97 365130
RNA 19 S5 Plus 240954 126 239481
Genexus 1744270 100 133226
RNA 20 S5 Plus 203214 86 195547
Genexus 1284559 94 173554
RNA 21 S5 Plus 195912 91 185689
Genexus 1940917 96 60947
RNA 22 S5 Plus 464854 119 462638
Genexus 1715374 98 294552
RNA 23 S5 Plus 258734 93 251939
Genexus 1644449 99 141394
RNA 24 S5 Plus 287598 104 284682
Genexus 1573653 103 68184
RNA 25 S5 Plus 297871 114 294124
Genexus 1587686 99 111160
RNA 26 S5 Plus 428858 118 426903
Genexus 1682103 100 185977
RNA 27 S5 Plus 173120 98 171187
Genexus 1471817 98 252247
RNA 28 S5 Plus 187176 145 185591
Genexus 1903859 98 126388
RNA 29 S5 Plus 311784 84 262726
Genexus 1839064 102 45998
RNA 30 S5 Plus 416422 93 393110
Genexus 1727113 101 57972
RNA 31 S5 Plus 240891 112 239186
Genexus 1598494 99 133522
RNA 32 S5 Plus 156106 63 97917
Genexus 1965363 93 52222
Abbreviations: ID (Identifier); RNA (Ribonucleic Acid).
Table 7. Comparison of RNA-related molecular alterations between S5 plus and Genexus platforms.
Table 7. Comparison of RNA-related molecular alterations between S5 plus and Genexus platforms.
ID S5Plus (SiRe Fusion Panel) Genexus (OPA Panel)
RNA 1 No Fusion NTRK3 (ex14) - KANK1 (ex3) 1571 reads *
RNA 2 No Fusion No Fusion
RNA 3 No Fusion No Fusion
RNA 4 No Fusion No Fusion
RNA 5 No Fusion No Fusion
RNA 6 No Fusion No Fusion
RNA 7 ALK (ex20) - EML4 (ex6) 601 reads ALK (ex20) - EML4 (ex6) 353 reads
RNA 8 No Fusion No Fusion
RNA 9 No Fusion No Fusion
RNA 10 No Fusion No Fusion
RNA 11 No Fusion No Fusion
RNA 12 No Fusion No Fusion
RNA 13 ALK (ex20) - unknown partner 149 reads ALK (ex20) - DCTN1 (ex26) 2268 reads
RNA 14 No Fusion No Fusion
RNA 15 No Fusion No Fusion
RNA 16 No Fusion No Fusion
RNA 17 No Fusion No Fusion
RNA 18 No Fusion No Fusion
RNA 19 ROS1 (ex34) - CD74 (ex6) 2208 reads ROS1 (ex34) - CD74 (ex6) 1992 reads
RNA 20 ALK (ex20) - EML4 (ex6) 43 reads ALK (ex20) - EML4 (ex6) 1040 reads
RNA 21 No Fusion No Fusion
RNA 22 ALK (ex20) - EML4 (ex13) 11335 reads ALK (ex20) - EML4 (ex13) 7212 reads
RNA 23 No Fusion No Fusion
RNA 24 RET (ex12) - KIF5B (ex15) 4063 reads RET (ex12) - KIF5B (ex15) 2417 reads
RNA 25 No Fusion MET (ex13) - MET (ex15) 9638 reads
RNA 26 No Fusion No Fusion
RNA 27 No Fusion No Fusion
RNA 28 ALK (ex20) - EML4 (ex20) 6293 reads ALK (ex20) - EML4 (ex20) 1140 reads
RNA 29 No Fusion No Fusion
RNA 30 No Fusion No Fusion
RNA 31 No Fusion No Fusion
RNA 32 RET (ex12) - CCDC6 (ex1) 494 reads RET (ex12) - CCDC6 (ex1) 172 reads
*Not covered from SiRe Fusion Panel. Abbreviations: ALK (Anaplastic Lymphoma Kinase); CCDC6 (Coiled-Coil Domain-Containing Protein 6); CD74 (HLA Class II Histocompatibility Antigen Gamma Chain); DCTN1 (Dynactin Subunit 1); EML4 (Echinoderm Microtubule-Associated Protein-Like 4); EX (Exon); ID (Identifier); KANK1 (KN Motif And Ankyrin Repeat Domains 1); KIF5B (Kinesin Family Member 5B); MET (Tyrosine-Protein Kinase Met); NTRK (Neurotrophic Tyrosine Receptor Kinase); RET (RET Proto-Oncogene); RNA (Ribonucleic Acid); ROS1 (Proto-Oncogene Tyrosine-Protein Kinase ROS).
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