Evaluating Targeted Next-Generation Sequencing Assays and Reference Materials for NTRK Fusion Detection

Abstract

Neurotrophic tyrosine receptor kinase (NTRK1/2/3) fusions are oncogenic drivers in approximately 0.3% of solid tumors. High-quality testing to identify patients with NTRK fusion-positive tumors who could benefit from tropomyosin receptor kinase inhibitors is recommended, but the current NTRK testing landscape, including next-generation sequencing (NGS), is fragmented and availability of assays varies widely. The analytical and clinical performance of four commonly available RNA-based NGS assays, Archer's FusionPlex Lung panel (AFL), Illumina's TruSight Oncology 500 (TSO500), Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA), were evaluated. Experiments were conducted using contrived samples [formalin-fixed, paraffin-embedded cell lines and SeraSeq formalin-fixed, paraffin-embedded reference material], NTRK fusion-negative clinical samples, and NTRK fusion-positive clinical samples, according to local assays. Estimated limit of detection varied across the four assays: 30 to 620 fusion copies for AFL (cell lines), versus approximately 30 to 290 copies for TSO500 and approximately 1 to 28 copies for OFA and Oncomine Precision Assay. All assays showed 100% specificity for NTRK fusions detection, but quality control pass rate was variable (AFL, 43%; TSO500, 77%; and OFA, 83%). The NTRK fusion detection rate in quality control–validated clinical samples was 100% for all assays. This comparison of the strengths and limitations of four RNA-based NGS assays will inform physicians and pathologists regarding optimal assay selection to identify patients with NTRK fusion-positive tumors. Neurotrophic tyrosine receptor kinase (NTRK1/2/3) fusions are oncogenic drivers in approximately 0.3% of solid tumors. High-quality testing to identify patients with NTRK fusion-positive tumors who could benefit from tropomyosin receptor kinase inhibitors is recommended, but the current NTRK testing landscape, including next-generation sequencing (NGS), is fragmented and availability of assays varies widely. The analytical and clinical performance of four commonly available RNA-based NGS assays, Archer's FusionPlex Lung panel (AFL), Illumina's TruSight Oncology 500 (TSO500), Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA), were evaluated. Experiments were conducted using contrived samples [formalin-fixed, paraffin-embedded cell lines and SeraSeq formalin-fixed, paraffin-embedded reference material], NTRK fusion-negative clinical samples, and NTRK fusion-positive clinical samples, according to local assays. Estimated limit of detection varied across the four assays: 30 to 620 fusion copies for AFL (cell lines), versus approximately 30 to 290 copies for TSO500 and approximately 1 to 28 copies for OFA and Oncomine Precision Assay. All assays showed 100% specificity for NTRK fusions detection, but quality control pass rate was variable (AFL, 43%; TSO500, 77%; and OFA, 83%). The NTRK fusion detection rate in quality control–validated clinical samples was 100% for all assays. This comparison of the strengths and limitations of four RNA-based NGS assays will inform physicians and pathologists regarding optimal assay selection to identify patients with NTRK fusion-positive tumors. Fusions of the neurotrophic tyrosine receptor kinase genes (NTRK1/2/3) are important oncogenic drivers across a large range of tumor types.1Amatu A. Sartore-Bianchi A. Bencardino K. Pizzutilo E.G. Tosi F. Siena S. Tropomyosin receptor kinase (TRK) biology and the role of NTRK gene fusions in cancer.Ann Oncol. 2019; 30: viii5-viii15Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,2Rolfo C. NTRK gene fusions: a rough diamond ready to sparkle.Lancet Oncol. 2020; 21: 472-474Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Although their prevalence is generally low (approximately 0.3%) across all solid tumors, they are found at high frequency (>90%) in rare tumor types, such as mammary analogue secretory carcinoma and secretory breast carcinoma.2Rolfo C. NTRK gene fusions: a rough diamond ready to sparkle.Lancet Oncol. 2020; 21: 472-474Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 3Lassen U. How I treat NTRK gene fusion-positive cancers.ESMO Open. 2019; 4: e000612Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 4Cocco E. Scaltriti M. Drilon A. NTRK fusion-positive cancers and TRK inhibitor therapy.Nat Rev Clin Oncol. 2018; 15: 731-747Crossref PubMed Scopus (516) Google Scholar NTRK fusions are promising molecular targets for new therapies, and several tropomyosine receptor kinase (TRK) inhibitors have recently been approved, which provide effective targeted treatment options for patients presenting with these genomic rearrangements. Larotrectinib and entrectinib were the first two TRK inhibitors approved in the United States, Europe, and other countries for the treatment of NTRK fusion-positive advanced solid tumors5Garrido P. Hladun R. de Alava E. Alvarez R. Bautista F. Lopez-Rios F. Colomer R. Rojo F. Multidisciplinary consensus on optimising the detection of NTRK gene alterations in tumours.Clin Transl Oncol. 2021; 23: 1529-1541Crossref PubMed Scopus (5) Google Scholar; a companion diagnostic to larotrectinib (FoundationOne CDx; Foundation Medicine, Cambridge, MA) was also subsequently approved (https://www.fda.gov/drugs/fda-approves-companion-diagnostic-identify-ntrk-fusions-solid-tumors-vitrakvi, last accessed November 15, 2021). Both entrectinib and larotrectinib demonstrated strong clinical efficacy in patients with NTRK fusion-positive tumors enrolled in basket trials (response rates of 64% and 78%, respectively), supporting the relevance of NTRK fusions as treatment targets and, therefore, the importance of identifying patients harboring these gene rearrangements.6Rolfo C.D. De Braud F.G. Doebele R.C. Drilon A.E. Siena S. Patel M. Cho B.C. Liu S.V. Ahn M.-J. Chiu C.-H. Farago A.F. Goto K. Lee J. Bazhenova L. John T. Fakih M. Simmons B.P. Pitcher B. Huang X. Demetri G.D. Efficacy and safety of entrectinib in patients (pts) with NTRK-fusion positive (NTRK-fp) solid tumors: an updated integrated analysis.J Clin Oncol. 2020; 38: 3605Crossref Google Scholar,7McDermott R. van Tilburg C.M. Farago A.F. Kummar S. Tan D.S.W. Albert C.M. Berlin J. Lassen U.N. Doz F. Geoerger B. Mascarenhas L. Federman N. Reeves J.A. Dima L. Brega N. De La Cuesta E. Laetsch T.W. Hong D.S. Drilon A. Survival benefits of larotrectinib in an integrated dataset of patients with TRK fusion cancer.Ann Oncol. 2020; 31: S1101-S1102Abstract Full Text Full Text PDF Google Scholar Moreover, a third inhibitor, repotrectinib, has shown promising early efficacy8Cho B.C. Doebele R.C. Lin J.J. Nagasaka M. Baik C. van der Wekken A.J. Velcheti V. Lee K.H. Liu S.V. Solomon B. Kao S. Krebs M.G. Zhu V. Stopatschinskaja S. Camidge D.R. Drilon A. Phase 1/2 TRIDENT 1 study of repotrectinib in patients with ROS1+ or NTRK+ advanced solid tumors. World Conference on Lung Cancer 2020, Singapore2021Abstract Full Text Full Text PDF Google Scholar; and recently received a fast track designation in NTRK fusion–positive, tyrosine kinase inhibitor–pretreated advanced solid tumors from the US Food and Drug Administration (https://www.targetedonc.com/view/fda-grants-fast-track-to-repotrectinib-in-ntrk-positive-advanced-solid-tumors, last accessed November 15, 2021); other inhibitors are currently in development.9Jiang T. Wang G. Liu Y. Feng L. Wang M. Liu J. Chen Y. Ouyang L. Development of small-molecule tropomyosin receptor kinase (TRK) inhibitors for NTRK fusion cancers.Acta Pharm Sin B. 2021; 11: 355-372Crossref PubMed Scopus (20) Google Scholar Thus, it is essential to provide broad global access to high-quality testing to ensure patients with NTRK fusion–positive tumors who could benefit from these targeted agents are identified. However, the current testing landscape is fragmented and available assay options vary widely. Multiple technologies, such as immunohistochemistry, RT-PCR, fluorescence in situ hybridization, and next-generation sequencing (NGS), are capable of detecting NTRK fusions. Among these, NGS is preferred because of its high specificity, high-to-moderate sensitivity, and ability to simultaneously interrogate multiple actionable cancer gene mutations. Although DNA-based NGS can detect gene fusions by also targeting intronic regions, RNA-based NGS allows direct detection of exon-exon junctions indicative of a fusion. Previous studies10Kirchner M. Neumann F. M. J. E. R. M. J. T. A. C. P. R. V. A. RNA-based detection of gene fusions in and paraffin-embedded solid cancer 2019; 11: Scopus Google Scholar, C. A. S. for detecting gene fusions by next-generation Full Text Full Text PDF PubMed Scopus Google Scholar, C. U. L. of cancer in with for a by next-generation sequencing PubMed Scopus Google Scholar showed targeted sequencing of formalin-fixed, paraffin-embedded tumor samples was by detecting a range of gene including and in a range of solid tumor The for Oncology on NTRK fusion detection the of RNA-based sequencing for tumor NTRK fusions are and it can as a in tumor these fusions are mammary analogue secretory C. Scaltriti M. M. A.J. F. M. T. Lopez-Rios F. F. on the to detect NTRK fusions in and clinical Oncol. 2019; 30: Full Text Full Text PDF PubMed Scopus Google Scholar a of the currently available for NTRK the analytical performance of four commonly available RNA-based NGS assays was Archer's FusionPlex Lung panel TruSight Oncology 500 Oncomine Precision Assay Thermo and Oncomine Focus Assay Thermo a to the of clinical samples, which it to or NGS performance for these the were with contrived NTRK fusion-positive samples, cell and SeraSeq NTRK reference Although contrived samples are a of clinical samples, and all of the they are to they are for the of analytical assay performance and assay the ability of these assays to detect NTRK fusions in clinical tumor samples to NTRK fusion positive by local testing was all assays, samples were using Thermo Fisher's or Assay and a of the cell lines and reference samples were of for and using Thermo Fisher's assays assay and with assay The positive control was with copies of the and the were and 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sensitivity, of NTRK fusion-positive samples in NTRK fusion-negative samples were samples were first to on for the cell lines harboring an or SeraSeq NTRK were from was in in a of the limit of detection for as the at which all were two from were on by for OFA, and to the of the four assays. with for were on the SeraSeq reference as were cell samples for The 30 NTRK fusion-negative tumor samples as NTRK fusion by an NGS in the specificity for TSO500, and OFA were from and and from a large range of solid tumors: breast and The of TSO500, and OFA were by of the 30 NTRK fusion-negative samples on of the assays and the rate of assay for the quality as clinical samples by of de and as NTRK fusion positive by local assays, were the clinical comparison samples from several tumor tumor mammary analogue secretory carcinoma and carcinoma and and cancers were according to at of the and to with the The clinical NTRK fusion–positive samples were on of the four assays using their with the of for 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NTRK fusions at of and by fusion the AFL assay an limit of detection of 30 to 620 fusion copies for cell lines and to fusion copies for the SeraSeq NTRK versus 30 to 290 fusion copies for TSO500 OFA and fusion showed a low limit of detection of approximately 1 to 28 fusion copies and of FusionPlex Lung panel (AFL), TruSight Oncology 500 (TSO500), Oncomine Precision Assay and Oncomine Focus Assay for cell lines and SeraSeq neurotrophic tyrosine receptor kinase reference according to fusion by The ability of assay to detect the and fusions was in cell lines and the NTRK reference are on the the fusion was or on the the assay to detect the fusion. the of assay across samples, two were for the at which all were positive on was in SeraSeq two at the limit of detection, the for the TSO500, and OFA assays was with or fusions across AFL was the limit of detection for SeraSeq NTRK was at to fusions. All of the assays showed high specificity, with NTRK fusions in of the NTRK fusion-negative clinical However, pass for the assays for specificity were TSO500 and OFA a high rate and respectively), of samples on AFL all assay quality The of samples on AFL using the in samples samples on AFL using with the samples The were (AFL), fusion (OFA), and is the for the of AFL clinical samples was the for of the TSO500, and OFA in 30 NTRK samples on showed NTRK fusion in samples samples for specificity, samples were with AFL using with AFL quality control were quality and quality control were gene and samples an the to quality control were fusion panel and other fusion quality control FusionPlex Lung neurotrophic tyrosine receptor OFA, Oncomine Focus Oncomine Precision TSO500, TruSight Oncology samples were with AFL using with AFL quality control were quality and TSO500 quality control were gene and samples an the to quality control were fusion panel and other fusion quality control in a new FusionPlex Lung neurotrophic tyrosine receptor OFA, Oncomine Focus Oncomine Precision TSO500, TruSight Oncology Although specificity assays were with showed NTRK fusions in a of samples the NTRK gene fusions by the four assays in clinical samples NTRK fusions by local assays samples, for which of the NGS assays were to detect these were from the of the by the four assays (AFL, TSO500, and OFA, and fusions were by the assay OFA, which panel two of the fusions. these samples, NTRK fusion detection rates for samples were 100% for all assays on the targets were in the showed an NTRK fusion detection rate of several pass also showed the NTRK fusion been of NTRK Fusions by the TSO500, and OFA on NTRK tumor were of NTRK fusion sequencing with from and Illumina's fusion with was to with was to from panel has the for gene sequencing with sequencing fusions on samples were from the because of the next-generation sequencing assays were to detect these it was local have Oncomine fusion detection on fusions were local FusionPlex Lung mammary analogue secretory neurotrophic tyrosine receptor OFA, Oncomine Focus Oncomine Precision quality TSO500, TruSight Oncology tumor were was to OFA panel has the for gene samples were from the because of the next-generation sequencing assays were to detect these it was local have Oncomine in a new NTRK fusions were local assays. FusionPlex Lung mammary analogue secretory neurotrophic tyrosine receptor OFA, Oncomine Focus Oncomine Precision quality TSO500, TruSight Oncology The and of TRK such as entrectinib and as as other targeted for rare and 1 testing for oncogenic gene fusions is in the diagnostic F. for of patients with TRK fusion Clin 2019; PubMed Scopus Google Scholar as companion diagnostic assay for NTRK fusion detection (FoundationOne is approved by the US Food and Drug clinical a range of NGS assays to detect NTRK fusions. a of the options are available for NTRK testing in clinical the analytical performance of the four commonly RNA-based NGS assays was for the detection of NTRK fusions. clinical samples for of a new NTRK fusion detection assay is contrived options cell lines and SeraSeq reference were in to clinical samples, they are a to NTRK fusion testing by NGS to clinical samples in to contrived samples, to to clinical samples tumor tumor and and the from performance the The in (AFL, TSO500, or and OFA, and the four assays are by their the OFA and an which is and E. R. J. E. S. J. S. of versus for PubMed Scopus Google N. M. U. J. S. J. M. F. A. M. J. A. V. M. T. E. Lassen U. R. M. P. A. NTRK and in comparison of RNA-based targeted sequencing 2020; PubMed Scopus Google Scholar However, OFA and can detect fusions with in the assay and can samples with fusion This is by the of the OFA several of the NTRK fusions in its the updated panel was to detect NTRK fusions. Although the can also detect fusions by of the gene of demonstrated a low as it of NTRK fusions in the clinical samples the was to detect NTRK fusion in the NTRK fusion-positive reference This across the NTRK gene versus a direct of the NTRK gene it is to NTRK from the tumor to has been for R. G. sequencing for gene fusion in a 2020; Scopus Google Scholar is can the low of the assay or other of also an are to the of the is a for fusion detection in clinical the other the TSO500 which was to the four detecting supporting for in contrived samples, the of NTRK fusion-positive tumors by assay because at multiple the gene the AFL using the the other and a assay have been with of to E. R. J. E. S. J. S. of versus for PubMed Scopus Google N. M. U. J. S. J. M. F. A. M. J. A. V. M. T. E. Lassen U. R. M. P. A. NTRK and in comparison of RNA-based targeted sequencing 2020; PubMed Scopus Google Scholar The to with a of can in with the ability to at low and to to or clinical sensitivity, and the assay for All NGS assays showed high specificity, with but the AFL assay was with a high rate of is these were by samples and quality of the from these samples, the or the AFL assay the of AFL to and of quality has been C. S. R. J. J. F. A. K. T. N. T. A. J. R. S. of gene fusions using targeted next-generation a 2021; PubMed Scopus Google Scholar, R. M. K. P. K. P. R. J. C.M. A. M.G. Drilon A. M. of sequencing for kinase fusions in with by sequencing and low tumor Cancer 2019; PubMed Scopus Google Scholar, J. K. M. Doebele R.C. of molecular testing for detection of in a of positive Oncol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar, C. V. R. S. A. U. R. S. J. J. R. S. of in situ and for the detection of in solid 2019; 21: Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. B.P. J.A. Lee J. J. J. gene in a diagnostic of by next-generation PubMed Scopus Google Scholar for and in a clinical the of clinical samples because of NTRK gene fusions could samples sequencing This the importance of and local quality NGS as However, quality and an as NTRK fusion as rare with The SeraSeq NTRK reference showed variable across and as as The SeraSeq NTRK for versus and a is The SeraSeq reference also fusion the it is was to the for by the of the to fusion in of was of several samples the these were to quality and were by the of the clinical such as have an on received the regarding and to with of contrived the OFA and a limit of detection the by Thermo (approximately 1 to 28 fusion copies for assays versus the for OFA and to for for Oncomine Focus Assay and Thermo Oncomine Precision Assay This by a limit of detection by the and the were using an for which the assay with NTRK fusions. The limit of detection for the TSO500 assay was as on the of by (approximately 30 to 290 versus fusion TruSight Oncology 500 and TruSight Oncology 500 fusion have been by for the AFL the from the study could with by the The four assays were to the from local with NTRK fusion detection rates of for the of the of NTRK fusions. on clinical samples, on fusion to fusion were The all NTRK fusions were by the assays are as high of NTRK in the could a such as the fusion in cancer and fusion in in which of the assays were to with high the local have been a these two samples were from the of clinical other the of NTRK fusion in these could to a low including samples, as or of the tumor in This study samples, which can for RNA-based using to for NTRK fusion However, such to are to provide with in a clinical is essential to to identify NTRK fusions in patients with solid tumors to the as these patients have to with the tumor but a gene fusion. The NTRK fusion-positive cell lines were samples for study but low reference for NTRK fusions were to contrived samples are an important to NGS assay performance for NTRK fusion all four NGS assays are to detect NTRK assay its strengths and limitations The AFL assay a high but is to detect gene fusions at The TSO500 assay a has sensitivity, and can detect fusions. the and OFA of and at a high sensitivity, but they fusions are on the the NTRK fusions the OFA, is for low has a and can detect the the and specificity of of the of the TSO500, and OFA for the of NTRK Fusions in and for with and study NTRK fusions NTRK fusions NTRK fusions NTRK fusions NTRK fusions NTRK fusions they were in sequencing NTRK fusions NTRK fusions were in panel NTRK fusion-negative fusion copies at all all at to to to to samples performance FusionPlex Lung limit of neurotrophic tyrosine receptor OFA, Oncomine Focus Oncomine Precision TSO500, TruSight Oncology in a new FusionPlex Lung limit of neurotrophic tyrosine receptor OFA, Oncomine Focus Oncomine Precision TSO500, TruSight Oncology the ability of available NGS assays to detect rare gene such as or across tumor will pathologists and physicians to the of and testing for This will access to high-quality molecular testing and of patients with solid tumors harboring gene who benefit from treatment to the genomic of their and for their and the is the of and, as the of all in the study and for the of the and the of the with with

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