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Brief Report

Technical Validation of a Fully Integrated NGS Platform in the Real-World Practice of Italian Referral Institutions

by
Caterina De Luca
†,
Francesco Pepe
†,
Gianluca Russo
,
Mariantonia Nacchio
,
Pasquale Pisapia
,
Maria Russo
,
Floriana Conticelli
,
Lucia Palumbo
,
Claudia Scimone
,
Domenico Cozzolino
,
Gianluca Gragnano
,
Antonino Iaccarino
,
Giancarlo Troncone
and
Umberto Malapelle
*
Department of Public Health, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Mol. Pathol. 2023, 4(4), 259-274; https://doi.org/10.3390/jmp4040022
Submission received: 14 September 2023 / Revised: 23 October 2023 / Accepted: 25 October 2023 / Published: 31 October 2023
Browse Figure
Review Reports Versions Notes

Abstract

:
Aims: To date, precision medicine has played 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 being investigated in clinical trials. A pitfall in molecular testing is the diagnostic routine sample available to analyze predictive biomarkers; a scant tissue sample often represents the only diagnostical source of nucleic acids with which to conduct molecular analysis. At the sight of these critical issues, next-generation sequencing (NGS) platforms emerged as referral testing strategies for the molecular analysis of predictive biomarkers in routine practice, but the need for highly skilled personnel and extensive working time drastically impacts the widespread diffusion of this technology in diagnostic settings. Here, we technically validate a fully integrated NGS platform on diagnostic routine tissue samples previously tested with an 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), was retrieved from the internal archive of the University of Naples Federico II. Each sample was tested by adopting an Oncomine Precision Assay (OPA), which is able to detect 2769 molecular actionable alterations [hotspot mutations, copy number variations (CNV) and gene fusions] on fully integrated NGS platforms (Genexus, Thermo Fisher Scientific (Waltham, MA, USA). The concordance rate between these technical approaches was determined. Results: The Genexus system successfully carried out molecular analysis in all instances. A concordance rate of 96.9% (31 out of 32) was observed between the OPA and SiRe™ panels 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) were assessed. Conclusions: A fully automatized Genexus system combined with OPA (Thermo Fisher Scientific) may be considered a technically valuable, time-saving sequencing platform to test predictive biomarkers in diagnostic routine practice.

1. Introduction

In recent decades, personalized medicine has laid the basis for a novel therapeutical option for solid-tumor patients [1,2]. Currently, 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), and breast cancer (BC) patients [3,4,5,6,7,8,9]. In particular, an increasing number of predictive biomarkers are being approved in clinical practice to provide lung cancer patients diagnosed with the NSCLC type with 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 [10,11,12,13,14]. The most common diagnostic sample available to approach diagnosis and molecular tests in the advanced tumor stage consists of a “scant sample” with a low abundance of neoplastic cells to successfully carry out mandatory gene testing [15,16,17]. 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 the lowest quality and quantity of nucleic acids adopted in molecular tests [18,19]. Despite tissue specimens being considered the “gold standard” for molecular testing, a non-negligible percentage of patients do not have access to molecular tests due to insufficient diagnostic material [16,17]. In this scenario, liquid biopsy becomes an integrating biological source for successfully performing molecular analysis when tissue is not available. Moreover, circulating tumor DNA (ctDNA) isolated from peripheral blood is a reliable source for detecting target molecular alterations [20,21]. At the sight of these aspects, single plex technology results are inadequate to successfully analyze the minimum gene panel established for each solid tumor. In this heterogeneous landscape of biological sources, next-generation sequencing (NGS) platforms play a crucial role in the molecular analysis of predictive biomarkers [22,23,24]. This technology allows us to simultaneously analyze very low-frequency clinically relevant biomarkers using very low amounts of nucleic acids in a single run [22,23]. Remarkably, NGS systems are scalable, decreasing reaction costs in accordance with the number of samples processed in each run [24]. On the other hand, an adequate number of samples may be collected in more than 30 days for a non-negligible number of small–medium institutions involved in molecular tests, thereby saving on technical costs. This aspect drastically impacts turnaround time (TAT), resulting in a delay in the clinical administration of tumor patients [24,25]. In this scenario, the Ion Torrent™ Genexus™ Integrated Sequencer (Genexus; Thermo Fisher Scientific, Waltham, MA, USA) was designed to automatically carry out the entire NGS workflow (from tissue and liquid biopsy-derived nucleic acids extraction to data analysis) without other manual operations [26,27,28]. This technology allows us to successfully carry out the molecular analysis of a small batch of diagnostic specimens [1,2,3,4,5,6,7,8] without impacting the turnaround time (TAT) of the diagnostic workflow. We aimed to evaluate the concordance rate between the Genexus system and Ion Torrent S5™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) 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 the internal archive of the predictive molecular pathology laboratory of the University of Naples Federico II. Clinical pathological data are 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 MET exon 14 skipping alterations, which is routinely employed in the molecular testing of solid-tumor patients [29]. The Oncomine Precision Assay (OPA), able to detect 2769 molecular actionable alterations [hotspot mutations, copy number variations (CNV) and gene fusions], was combined with the Genexus (Thermo Fisher Scientific) platform to assess the molecular profile of selected samples [26,27]. The concordance rate of the OPA in the Genexus system with SiRe™ on the S5 Plus platform was investigated. All information regarding human material were managed using anonymous numerical codes, and all samples were handled in compliance with the Helsinki Declaration (http://www.wma.net/en/30publications/10policies/b3/, accessed on 1 September 2023).

3. Material and Methods

3.1. Routine Sample Processing Strategy

Nucleic acids were previously purified from n = 4 representative slides of neoplastic area (>10%). Specifically, a QIAamp DNA Mini Kit (Qiagen, Crawley, West Sussex, UK) was utilised following manufacturer instructions. DNA quantification was successfully carried out in all cases, adopting a Qubit fluorimeter (Thermo Fisher) or a TapeStation 4200 microfluidic platform (Agilent Technologies, Santa Clara, CA, USA) following manufacturer instructions. In the instance of an inadequate amount of nucleic acids, we maximized for volume input. Conversely, RNA volume was maximized for cDNA synthesis. Selected samples were routinely analyzed with SiRe™ and SiRe fusion panels using the Ion S5™ Plus software (Thermo Fisher Scientific) to assess mutational status in clinically relevant biomarkers for NSCLC patients [29,30]. Briefly, 15 μL of extracted DNA/cDNA was dispensed into the Ion Kit-Chef system (Thermo Fisher Scientific) for library preparation. A total of n = 8 samples was simultaneously processed following previously validated thermal conditions. After pooling, a templating procedure was carried out for n = 16 libraries by using the Ion 510™, Ion 520™ and Ion 530™ Kit-Chef (Thermo Fisher Scientific) according to manufacturer instructions on a 520 chip (Thermo Fisher Scientific). Data were inspected by adopting designed bed files on proprietary Torrent Suite software [v.5.0.2]. In detail, variant inspection was performed with a variant caller plug-in (v.5.0.2.1), which is able to filter variants with ≥5× allele coverage and a quality score ≥20, within an amplicon that covered at least 500× alleles.

3.2. Genexus Analysis

A series of n = 64 extracted gDNA and gRNA samples from solid-tumor patients was retrospectively tested in the Genexus (Thermo Fisher Scientific) system. The platform enables entire NGS workflows (from library preparation to data interpretation) within 24 h. The OPA assay includes the most clinically relevant actionable genes (EGFR, BRAF, KRAS, ALK, ROS1, NTRK, and RET) for NSCLC patients [27,28]. Briefly, samples were created on a dedicated server and assigned to a new run. The 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 the OPA assay on the Genexus platform. Accordingly, each sample was diluted and immediately dispensed on a 96-well plate, following manufacturer instructions. Finally, nucleic acids were sequenced on a GX5TM chip that allows for the simultaneous processing of n = 8 samples in a single line with an OPA assay. Data analysis was performed using proprietary Genexus software (1.0). Particularly, detected alterations were annotated by adopting Oncomine Knowledgebase Reporter Software (Oncomine Reporter 5.0). In addition, BAM files were also visually inspected with the Golden Helix Genome Browser v.2.0.7 (Bozeman, MT, USA) in hotspot regions in EGFR, KRAS, and BRAF lung cancer-addicted molecular alterations.

4. Results

4.1. Hotspot Mutations

Overall, the Genexus system successfully carried out molecular analysis in all DNA series. In detail, a median number of total reads, mapped reads, mean read length, percent reads on target, mean depth, uniformity of amplicon coverage of 1,134,878.2 (ranging from 424,900.0 to 1,791,041.0), 1,074,345.7 (ranging from 365,139.0 to 1,756,414.0), 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 the 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 hotspot 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]; and n = 1 out of 29 c-KIT molecular alterations [exon 11 c.1727T>C p.L576P] were detected (Table 4).
The molecular profile detected by OPA on the Genexus platform matched with the Sire panel on the S5 Plus system in 31 out of 32 patients (96.9%). Remarkably, positive results previously identified adopting the SiRe panel were confirmed in 23 out of 24 (95.8%) patients. Particularly, ID#19 showed an exon 9 PIK3CA p.E545K hotspot mutation not observed by using the S5 system with a standardized clinical cut-off (MAF = ≥5.0%) (Figure 1).
No significant variations in accordance with histological groups, mutation type and mutant allele fraction levels between Genexus and the previously tested samples on the S5 platform were identified. In addition, the OPA assay also identified n = 16 out of 32 (50.0%) DNA-based molecular alterations in other genes not covered by the SiRe panel. Moreover, 12 out of 16, 1 out of 16, and 1 out of 16 highlighted TP53, CTNNB1 and MTOR hotspot molecular alterations, respectively. Moreover, 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, the Genexus platform successfully analyzed all retrieved cases. Briefly, a median number of total reads, mapped reads and mean read length of 1,721,491.0 (ranging from 1,471,817.00 to 2,462,555.00), 158,230.4 (ranging from 37,387.0 to 1,029,745.00), 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 the 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 mutations, respectively (Table 7). Interestingly, rearranged genes were identified by OPA on the Genexus platform in 9 out of 10 (90.0%) retrieved cases, showing a concordance rate of 96.9% (31 out of 32 cases) with the SiRe panel in the S5 system. Particularly, ID#1 was positive for a NTRK3–KANK1 fusion transcript not previously detected with the SiRe panel on the S5 platform. No significant variations were observed in accordance with histological groups, rearranged genes, fusion partners, and mapped read levels between Genexus and previously tested samples on the S5 platform.

5. Discussion

In the era of personalized medicine, the rapidly increasing number of predictive biomarkers approved in clinical practice has revolutionized the treatment strategy for solid-tumor patients [1,2,9]. Although there is a widespread diffusion of single-gene testing platforms in the vast majority of laboratories involved in molecular tests, low multiplexing biomarker analysis discourages their implementation as pivotal diagnostic platforms in clinical practice [23,24]. As regards NGS techniques, they allow us to simultaneously cover clinically relevant molecular alterations from a plethora of diagnostic routine specimens, saving technical costs and maintaining adequate TAT [31]. Moreover, NGS platforms may also benefit from automatized technical procedures that allow for accurate and reproducible analysis, resulting in low bench-working time [31]. The Genexus system consists of a scalable, versatile, and fully automatized sequencer that is able to carry out each technical procedure without manual operations [32]. This system is built to integrate analytical procedures (nucleic acid extraction, library preparation, template generation, sequencing) with data analysis by adopting pre-customized pipeline analysis. Accordingly, automatized data analysis carried out by proprietary software supports healthcare professional figures involved in molecular testing. This approach allows us to save time by accurately interpreting molecular records, in comparison with semi-automatized procedures. As regards the NGS-based multiplexing strategy, it is considered a reliable technical approach that is able to decrease technical costs in molecular tests. Here, we have validated the Genexus system in our diagnostic routine by comparing its analytical performance in a retrospective series of clinical cases previously analyzed with a custom NGS panel in the 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 the Sire panel in the S5 system as the reference standard. Interestingly, molecular analysis was unmatched with previously archived data in only two cases (DNA-ID#19 and RNA-ID#1). Of note, sample DNA-ID#19 derived from a BC patient had a positive result for PIK3CA exon 9 p.E545K hotspot alteration in the Genexus system, with a mutant allele fraction (MAF) of 7.2%. Following the manufacturer’s clinical cut-off (MAF ≥ 5%), previous analysis did not show any clinically relevant molecular alteration. By conducting a visual inspection of raw data, the same alteration at 0.9% was detected. This event may occur in residual scant samples where mutated alleles may encounter decreasing VAF levels [33]. Similarly, RNA-ID#1 showed NTRK3 (ex14)—KANK1 (ex3), an aberrant transcript not previously detected with the standard reference approach. In this case, NTRK3 was not covered by reference range of the SiRe fusion panel.
In a non-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 conduct the 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 testing. Interestingly, both S5 and Genexus systems revealed KRAS exon 2 p.G12C and PIK3CA exon 20 p.H1047R hotspot mutations, demonstrating a common origin of these lesions. Moreover, NGS systems overcome technical issues from the analysis of “complex” molecular alteration. Case DNA-ID#22 confirmed two concomitant KRAS exon 2 hotspot mutations (p.G13D+p.G13E) on the Genexus platform, previously detected by reference technology. Although this study provides encouraging results for the implementation of the Genexus system in the clinical routine setting of solid-tumor patients, some limitations may be identified. Firstly, this technical report aims to compare the analytical parameters of two NGS-based technologies using 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 the internal archive of the University of Naples Federico II. All these crucial points warrant further analysis, but this preliminary data may suggest that a fully automatized Genexus system integrated with commercially available OPA (Thermo Fisher Scientific) represents a technically affordable, time-saving sequencing platform that enables us to analyze clinically relevant molecular alterations in diagnostic routine specimens.

Author Contributions

Conceptualization, C.D.L., F.P., G.T. and U.M.; methodology, C.D.L., F.P., G.R., M.N., P.P., M.R., F.C., L.P., C.S., D.C., G.G., A.I., G.T. and U.M.; software, C.D.L., F.P., G.T. and U.M.; validation, C.D.L., F.P., G.R., M.N., P.P., M.R., F.C., L.P., C.S., D.C., G.G., A.I., G.T. and U.M.; formal analysis, C.D.L., F.P., G.R., M.N., P.P., M.R., F.C., L.P., C.S., D.C., G.G., A.I., G.T. and U.M.; data curation, C.D.L., F.P., G.T. and U.M.; writing—original draft preparation, C.D.L. and F.P.; writing—review and editing, G.T. and U.M.; visualization, C.D.L., F.P., G.R., M.N., P.P., M.R., F.C., L.P., C.S., D.C., G.G., A.I., G.T. and U.M.; supervision, G.T. and U.M.; project administration, G.T. and U.M. 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.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement

Not applicable.

Conflicts of Interest

Pasquale Pisapia has received personal fees as speaker bureau from Novartis for work performed outside of the curr ent 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. The remaining authors declare no conflict of interest.

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Jmp 04 00022 g001
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 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 proprietary Genexus software (B).
Jmp 04 00022 g001
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.
IDSexAgeSample TypeTumorN.C.DNA Amount (ng/μL)DINClinical Request
DNA 1 *M78ResectionCRC70.0%11.8NARAS, BRAF
DNA 2 *M78ResectionCRC70.0%47.7NARAS, BRAF
DNA 3M89BiopsyCRC50.0%12.9NARAS, BRAF
DNA 4F68ResectionNSCLC70.0%54.16.8EGFR, KRAS, BRAF
DNA 5M73ResectionCRC50.0%60.0NARAS, BRAF
DNA 6M53BiopsyNSCLC30.0%6.05.6EGFR, KRAS, BRAF
DNA 7M66ResectionCRC40.0%35.6NARAS, BRAF
DNA 8F78ResectionCRC40.0%20.2NARAS, BRAF
DNA 9F67ResectionNSCLC60.0%5.023.1EGFR, KRAS, BRAF
DNA 10F51ResectionCRC30.0%23.5NARAS, BRAF
DNA 11M50ResectionCRC80.0%39.1NAc-KIT, PDGFRA
DNA 12F50BiopsyNSCLC50.0%9.81.6EGFR, KRAS, BRAF
DNA 13M70BiopsyNSCLC20.0%15.93.7EGFR, KRAS, BRAF
DNA 14F59ResectionNSCLC40.0%47.36.5EGFR, KRAS, BRAF
DNA 15M66BiopsyNSCLC30.0%2.83.3EGFR, KRAS, BRAF
DNA 16M56ResectionCRC50.0%55.0NARAS, BRAF
DNA 17M66ResectionNSCLC60.0%115.04.9EGFR, KRAS, BRAF
DNA 18F51BiopsyCRC50.0%37.0NARAS, BRAF
DNA 19F41BiopsyBC30.0%35.13.7PIK3CA
DNA 20F82BiopsyCRC30.0%29.8NARAS, BRAF
DNA 21M67BiopsyCRC50.0%27.2NARAS, BRAF
DNA 22M82ResectionNSCLC80.0%39.96.9EGFR, KRAS, BRAF
DNA 23M74ResectionNSCLC70.0%45.54.3EGFR, KRAS, BRAF
DNA 24M74ResectionCRC40.0%2.2NARAS, BRAF
DNA 25F44BiopsyCRC40.0%7.3NARAS, BRAF
DNA 26F69BiopsyNSCLC60.0%14.84.7EGFR, KRAS, BRAF
DNA 27M54ResectionCRC30.0%22.6NARAS, BRAF
DNA 28F74ResectionMM90.0%11.4NABRAF, NRAS
DNA 29F63BiopsyNSCLC40.0%8.56.2EGFR, KRAS, BRAF
DNA 30M56ResectionNSCLC50.0%3.94.5EGFR, KRAS, BRAF
DNA 31F52ResectionCRC60.0%37.9NARAS, BRAF
DNA 32F45ResectionBC60.0%25.2NAPIK3CA
* 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 Viral Oncogene Homolog); M (Male); MM (Malignant Melanoma); NA (Not Assessable N.C. (Neoplastic Cellularity); NSCLC (Non-Small-Cell Lung Cancer); PIK3CA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase, Catalytic Subunit Alpha); RAS (Rat Sarcoma Viral Oncogene Homolog).
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.
IDSexAgeSample TypeTumorN.C.Clinical Request
RNA 1M56ResectionNSCLC60.0%ALK, ROS1, RET, MET, NTRK
RNA 2F58BiopsyNSCLC70.0%ALK, ROS1, RET, MET, NTRK
RNA 3M77BiopsyNSCLC25.0%ALK, ROS1, RET, MET, NTRK
RNA 4M79ResectionNSCLC70.0%ALK, ROS1, RET, MET, NTRK
RNA 5M79BiopsyNSCLC30.0%ALK, ROS1, RET, MET, NTRK
RNA 6M59BiopsyNSCLC30.0%ALK, ROS1, RET, MET, NTRK
RNA 7F70BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 8M62BiopsyNSCLC25.0%ALK, ROS1, RET, MET, NTRK
RNA 9M61BiopsyNSCLC40.0%ALK, ROS1, RET, MET, NTRK
RNA 10M66ResectionNSCLC60.0%ALK, ROS1, RET, MET, NTRK
RNA 11M68BiopsyNSCLC40.0%ALK, ROS1, RET, MET, NTRK
RNA 12M64BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 13F65BiopsyNSCLC60.0%ALK, ROS1, RET, MET, NTRK
RNA 14M58BiopsyNSCLC20.0%ALK, ROS1, RET, MET, NTRK
RNA 15F79BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 16M52BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 17M67ResectionNSCLC60.0%ALK, ROS1, RET, MET, NTRK
RNA 18M87BiopsyNSCLC40.0%ALK, ROS1, RET, MET, NTRK
RNA 19M25BiopsyNSCLC60.0%ALK, ROS1, RET, MET, NTRK
RNA 20F60BiopsyNSCLC30.0%ALK, ROS1, RET, MET, NTRK
RNA 21M60ResectionNSCLC60.0%ALK, ROS1, RET, MET, NTRK
RNA 22F36BiopsyNSCLC30.0%ALK, ROS1, RET, MET, NTRK
RNA 23M66BiopsyNSCLC60.0%ALK, ROS1, RET, MET, NTRK
RNA 24F47BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 25M67BiopsyNSCLC30.0%ALK, ROS1, RET, MET, NTRK
RNA 26F64BiopsyNSCLC10.0%ALK, ROS1, RET, MET, NTRK
RNA 27M54BiopsyNSCLC40.0%ALK, ROS1, RET, MET, NTRK
RNA 28F37BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 29M79BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 30F71BiopsyNSCLC30.0%ALK, ROS1, RET, MET, NTRK
RNA 31M68BiopsyNSCLC50.0%ALK, ROS1, RET, MET, NTRK
RNA 32F72BiopsyNSCLC70.0%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 (Ion Reporter 5.2.0.1) and Genexus systems.
Table 3. Technical parameters from DNA-based analysis by using S5 Plus (Ion Reporter 5.2.0.1) and Genexus systems.
DNA Analysis Technical Parameters—S5 Plus (SiRe™ Panel) vs. Genexus (OPA Panel)
IDPlatformTotal ReadsMean Read LengthMapped ReadsOn Target ReadsMean DepthUniformity
DNA 1 *S5 Plus254,212126253,62294.6%5712100%
Genexus872,83176736,53077.7%204499.1%
DNA 2 *S5 Plus215,464128215,04792.6%4740100%
Genexus732,69184663,06483.9%203498.8%
DNA 3S5 Plus298,541135297,99993.9%6662100%
Genexus1,143,038911,076,85588.8%352898.1%
DNA 4S5 Plus524,926155523,08692.3%11,489100%
Genexus1,419,2891011,393,60392.9%521098.1%
DNA 5S5 Plus361,148137360,37391.3%7830100%
Genexus1,094,620981,064,05191.5%381098.6%
DNA 6S5 Plus314,176128313,70699.2%7406100%
Genexus1,090,358981,049,93590.8%383799.0%
DNA 7S5 Plus635,201142634,22692.1%13,911100%
Genexus1,002,23192946,31888.9%315098.9%
DNA 8S5 Plus524,182131523,60893.0%11,591100%
Genexus1,262,760951,208,54390.9%417698.9%
DNA 9S5 Plus942,781161940,60594.6%21,192100%
Genexus1,791,041971,756,41493,0%609797.9%
DNA 10S5 Plus393,979126393,37189.5%8381100%
Genexus989,63560717,38564.9%145998.9%
DNA 11S5 Plus451,494139450,77994.4%10,127100%
Genexus776,89378679,35880.4%186396.7%
DNA 12S5 Plus88,91512988,78498.0%207292.9%
Genexus1,297,992911,263,55892.7%399693.9%
DNA 13S5 Plus296,845143296,43496.2%6790100%
Genexus1,196,122991,174,44292.7%425898.5%
DNA 14S5 Plus37,20613337,17395.2%842.797.6%
Genexus1,125,616971,093,53191.8%382498.6%
DNA 15S5 Plus782,397150780,89495.2%17,703100%
Genexus1,465,786921,423,74191.9%457495.3%
DNA 16S5 Plus378,978140378,37393.3%8402100%
Genexus1,084,647871,012,69387.6%305498.2%
DNA 17S5 Plus520,304135519,65391.5%11,317100%
Genexus1,048,030981,016,32491.4%361798.8%
DNA 18S5 Plus49,12713849,05595.3%111397.6%
Genexus1,294,194971,256,16191.9%443598.9%
DNA 19S5 Plus486,407147485,65296.6%11,16597.6%
Genexus1,343,529971,311,77692.3%465899.4%
DNA 20S5 Plus346,019131345,46497.4%801097.6%
Genexus974,47671759,42075.7%202398.8%
DNA 21S5 Plus67,48813067,41795.9%154097.6%
Genexus1,150,249901,094,01090.3%351998.8%
DNA 22S5 Plus52,08017051,95690.4%1119100%
Genexus14,94,3371001,470,08592.3%545197.9%
DNA 23S5 Plus614,960141613,81396.2%14,05997.6%
Genexus1,574,234911,510,26691.2%486597.7%
DNA 24S5 Plus188,967136188,62398.1%440797.6%
Genexus1,093,6461031,071,14192.2%407299.1%
DNA 25S5 Plus140,163145139,93095.5%318397.6%
Genexus949,85294911,44890,0%306499.4%
DNA 26S5 Plus40,23314240,18096.7%925.497.6%
Genexus1,497,022991,476,42593.7%536598.3%
DNA 27S5 Plus153,378133153,23696.0%350197.6%
Genexus1,059,772951,021,18690.2%349898.7%
DNA 28S5 Plus155,154118154,69596.5%355392.8%
Genexus424,90075365,13979.3%99497.4%
DNA 29S5 Plus358,001160356,99595.2%8095100%
Genexus1,165,795981,134,96992.2%407598.4%
DNA 30S5 Plus275,579149274,34098.4%6428100%
Genexus1,080,846921,034,34890.3%339298.4%
DNA 31S5 Plus259,364130258,62392.6%5702100%
Genexus1,109,488921,054,46589.9%345798.9%
DNA 32S5 Plus263,420126262,68293.4%584197.6%
Genexus710,18182631,88082.5%189396.7%
* 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.
IDS5Plus (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 3KRAS p.G12D 20.7%KRAS p.G12D 18.9%
DNA 4EGFR p.L858R 27.7%EGFR p.L858R 18.9%
DNA 5KRAS p.G12V 34.5%KRAS p.G12V 33.0%
DNA 6WTWT
DNA 7KRAS p.G12D 57.2%KRAS p.G12D 60.8%
DNA 8KRAS p.Q61K 16.8%KRAS p.Q61K 19.3%
DNA 9WTWT
DNA 10KRAS p.G12D 50.6%KRAS p.G12D 55.3%
DNA 11c-KIT p.L576P 68.0%c-KIT p.L576P 63.8%
DNA 12EGFR p.A767_V769dup 67.2%EGFR p.A767_V769dup 72.8%
DNA 13WTWT
DNA 14WTWT
DNA 15BRAF p.K601E 16.3%BRAF p.K601E 16.1%
DNA 16KRAS p.G12D 9.3%
KRAS p.G13D 14.1%		KRAS p.G12D 8.2%
KRAS p.G13D 12.1%
DNA 17KRAS p.Q61L 32.7%KRAS p.Q61L 36.3%
DNA 18NRAS p.Q61K 19.3%NRAS p.Q61K 18.2%
DNA 19PIK3CA E545K 0.8% **PIK3CA E545K 7.2%
DNA 20BRAF p.V600E 30.5%BRAF p.V600E 30.0%
DNA 21NRAS p.Q61K 46.7%NRAS p.Q61K 36.2%
DNA 22KRAS p.G13D 47.4% ***
KRAS p.G13E 47.9% ***		KRAS p.G13D 41.9% ***
KRAS p.G13E 42.0% ***
DNA 23WTWT
DNA 24KRAS p.A146T 30.80%KRAS p.A146T 26.4%
DNA 25WTWT
DNA 26BRAF p.V600E 27.3%BRAF p.V600E 30.3%
DNA 27KRAS p.G13D 14.9%KRAS p.G13D 12.2%
DNA 28NRAS p.Q61R 34.3%NRAS p.Q61R 28.2%
DNA 29EGFR p.L858R 9.7%
EGFR p.T790M 9.5%		EGFR p.L858R 9.3%
EGFR p.T790M 11.0%
DNA 30WTWT
DNA 31KRAS p.G12V 51.2%
PIK3CA p.E545K 32.2%		KRAS p.G12V 59.2%
PIK3CA p.E545K 31.0%
DNA 32WTWT
* Different lesion of same patient. ** Below 5%; *** Concomitant 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 the Genexus platform.
Table 5. Expanded list of molecular alterations covered by OPA on the Genexus platform.
IDOther Mutations (OPA Panel)
DNA 1 *MTOR p.R2217W 4.5%
DNA 2 *TP53 p.G279E 4.8%
TP53 p.V197M 4.0%
DNA 7TP53 p.H179Y 75.8%
DNA 9TP53 p.R273H 35.0%
DNA 12TP53 p.V197M 77.7%
DNA 14TP53 p.R273H 10.0%
DNA 16CTNNB1 p.S45F 41.1%
TP53 p.R175H 13.2%
DNA 18TP53 p.Y220C 19.7%
DNA 19TP53 p.L194F 9.9%
DNA 20TP53 p.P151S 54.7%
DNA 21TP53 p.K132R 51.4%
DNA 23TP53 p.C238S 25.3%
DNA 27CTNNB1 p.S45F 21.8%
DNA 30TP53 p.H179Y 24.6%
DNA 31TP53 p.Y220C 56.1%
DNA 32TP53 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)
IDPlatformTotal ReadsMean Read LengthMapped Reads
RNA 1S5 Plus503,83292489,474
Genexus2,355,40899170,105
RNA 2S5 Plus829,380124823,978
Genexus1,748,26199140,327
RNA 3S5 Plus641,59189348,169
Genexus2,462,55510454,529
RNA 4S5 Plus254,39493242,076
Genexus1,667,48810037,387
RNA 5S5 Plus234,80367176,276
Genexus1,755,50891111,713
RNA 6S5 Plus357,28489319,350
Genexus1,542,25210172,995
RNA 7S5 Plus1,070,6561111,067,615
Genexus1,571,469100150,711
RNA 8S5 Plus535,701103526,127
Genexus1,737,696961,029,745
RNA 9S5 Plus494,55087421,901
Genexus1,634,62410372,104
RNA 10S5 Plus161,964100153,003
Genexus1,815,5129651,505
RNA 11S5 Plus190,17098187,044
Genexus1,597,72798386,493
RNA 12S5 Plus677,65491513,093
Genexus1,554,237101171,919
RNA 13S5 Plus765,186129753,177
Genexus1,777,747100178,846
RNA 14S5 Plus222,717103217,972
Genexus1,503,56610248,005
RNA 15S5 Plus490,208125483,482
Genexus1,523,9719961,024
RNA 16S5 Plus20,4059117,060
Genexus1,878,0419742,572
RNA 17S5 Plus367,743117346,142
Genexus1,769,3139780,920
RNA 18S5 Plus191,02799189,336
Genexus1,513,61597365,130
RNA 19S5 Plus240,954126239,481
Genexus1,744,270100133,226
RNA 20S5 Plus203,21486195,547
Genexus1,284,55994173,554
RNA 21S5 Plus195,91291185,689
Genexus1,940,9179660,947
RNA 22S5 Plus464,854119462,638
Genexus1,715,37498294,552
RNA 23S5 Plus258,73493251,939
Genexus1,644,44999141,394
RNA 24S5 Plus287,598104284,682
Genexus1,573,65310368,184
RNA 25S5 Plus297,871114294,124
Genexus1,587,68699111,160
RNA 26S5 Plus428,858118426,903
Genexus1,682,103100185,977
RNA 27S5 Plus173,12098171,187
Genexus1,471,81798252,247
RNA 28S5 Plus187,176145185,591
Genexus1,903,85998126,388
RNA 29S5 Plus311,78484262,726
Genexus1,839,06410245,998
RNA 30S5 Plus416,42293393,110
Genexus1,727,11310157,972
RNA 31S5 Plus240,891112239,186
Genexus1,598,49499133,522
RNA 32S5 Plus156,1066397,917
Genexus1,965,3639352,222
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.
IDS5Plus (SiRe Fusion Panel)Genexus (OPA Panel)
RNA 1No FusionNTRK3 (ex14)—KANK1 (ex3) 1571 reads *
RNA 2No FusionNo Fusion
RNA 3No FusionNo Fusion
RNA 4No FusionNo Fusion
RNA 5No FusionNo Fusion
RNA 6No FusionNo Fusion
RNA 7ALK (ex20)—EML4 (ex6) 601 readsALK (ex20)—EML4 (ex6) 353 reads
RNA 8No FusionNo Fusion
RNA 9No FusionNo Fusion
RNA 10No FusionNo Fusion
RNA 11No FusionNo Fusion
RNA 12No FusionNo Fusion
RNA 13ALK (ex20)—unknown partner 149 readsALK (ex20)—DCTN1 (ex26) 2268 reads
RNA 14No FusionNo Fusion
RNA 15No FusionNo Fusion
RNA 16No FusionNo Fusion
RNA 17No FusionNo Fusion
RNA 18No FusionNo Fusion
RNA 19ROS1 (ex34)—CD74 (ex6) 2208 readsROS1 (ex34)—CD74 (ex6) 1992 reads
RNA 20ALK (ex20)—EML4 (ex6) 43 readsALK (ex20)—EML4 (ex6) 1040 reads
RNA 21No FusionNo Fusion
RNA 22ALK (ex20)—EML4 (ex13) 11,335 readsALK (ex20)—EML4 (ex13) 7212 reads
RNA 23No FusionNo Fusion
RNA 24RET (ex12)—KIF5B (ex15) 4063 readsRET (ex12)—KIF5B (ex15) 2417 reads
RNA 25MET (ex13)—MET (ex15) 46,929 readsMET (ex13)—MET (ex15) 9638 reads
RNA 26No FusionNo Fusion
RNA 27No FusionNo Fusion
RNA 28ALK (ex20)—EML4 (ex20) 6293 readsALK (ex20)—EML4 (ex20) 1140 reads
RNA 29No FusionNo Fusion
RNA 30No FusionNo Fusion
RNA 31No FusionNo Fusion
RNA 32RET (ex12)—CCDC6 (ex1) 494 readsRET (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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MDPI and ACS Style

De Luca, C.; Pepe, F.; Russo, G.; Nacchio, M.; Pisapia, P.; Russo, M.; Conticelli, F.; Palumbo, L.; Scimone, C.; Cozzolino, D.; et al. Technical Validation of a Fully Integrated NGS Platform in the Real-World Practice of Italian Referral Institutions. J. Mol. Pathol. 2023, 4, 259-274. https://doi.org/10.3390/jmp4040022

AMA Style

De Luca C, Pepe F, Russo G, Nacchio M, Pisapia P, Russo M, Conticelli F, Palumbo L, Scimone C, Cozzolino D, et al. Technical Validation of a Fully Integrated NGS Platform in the Real-World Practice of Italian Referral Institutions. Journal of Molecular Pathology. 2023; 4(4):259-274. https://doi.org/10.3390/jmp4040022

Chicago/Turabian Style

De Luca, Caterina, Francesco Pepe, Gianluca Russo, Mariantonia Nacchio, Pasquale Pisapia, Maria Russo, Floriana Conticelli, Lucia Palumbo, Claudia Scimone, Domenico Cozzolino, and et al. 2023. "Technical Validation of a Fully Integrated NGS Platform in the Real-World Practice of Italian Referral Institutions" Journal of Molecular Pathology 4, no. 4: 259-274. https://doi.org/10.3390/jmp4040022

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