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Validation, Implementation, and Clinical Impact of the Oncomine Myeloid Targeted-Amplicon DNA and RNA Ion Semiconductor Sequencing Assay

2021; Elsevier BV; Volume: 23; Issue: 10 Linguagem: Inglês

10.1016/j.jmoldx.2021.07.010

ISSN

1943-7811

Autores

Christina K. Ferrone, Henry Wong, Laura Semenuk, Barnaba Werunga, Brooke Snetsinger, Xiao Zhang, Grace Zhang, Janet Lui, Guillaume Richard‐Carpentier, Susan Crocker, David Good, Annette E. Hay, Graeme Quest, Nancy Carson, Harriet Feilotter, Michael J. Rauh,

Tópico(s)

Lymphoma Diagnosis and Treatment

Resumo

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics. The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics. Next-generation sequencing (NGS) technology permits simultaneous and efficient analysis of multiple clinically relevant genes that may harbor rare or novel variants.1Thomas M. Sukhai M.A. Zhang T. Dolatshahi R. Harbi D. Garg S. Misyura M. Pugh T. Stockley T.L. Kamel-Reid S. Integration of technical, bioinformatic, and variant assessment approaches in the validation of a targeted next-generation sequencing panel for myeloid malignancies.Arch Pathol Lab Med. 2017; 141: 759-775Crossref PubMed Scopus (18) Google Scholar In myeloid malignancies, these include epigenetic regulators, splicing factors, genes involved in signal transduction, and transcription factors, among others.2Murati A. Brecqueville M. Devillier R. Mozziconacci M.J. Gelsi-Boyer V. Birnbaum D. Myeloid malignancies: mutations, models and management.BMC Cancer. 2012; 12: 304Crossref PubMed Scopus (98) Google Scholar, 3Papaemmanuil E. Gerstung M. Malcovati L. Tauro S. Gundem G. Van Loo P.V. et al.Clinical and biological implications of driver mutations in myelodysplastic syndromes.Blood. 2013; 122: 3616-3627Crossref PubMed Scopus (1126) Google Scholar, 4Hofmann I. Elghetany T.M. Myelodysplastic syndromes and myeloproliferative disorders.2016Crossref Google Scholar, 5Arber D.A. Orazi A. 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Kronenberg Z. Sherman R.L. Tyner J.W. Dalley B.K. Dao K.H. Yandell M. Druker B.J. Gotlib J. O'Hare T. Deininger M.W. Age-related mutations and chronic myelomonocytic leukemia.Leukemia. 2016; 30: 906-913Crossref PubMed Scopus (83) Google Scholar, 11Grossmann V. Kohlmann A. Eder C. Haferlach C. Kern W. Cross N.C.P. Haferlach T. Schnittger S. Molecular profiling of chronic myelomonocytic leukemia reveals diverse mutations in >80% of patients with TET2 and EZH2 being of high prognostic relevance.Leukemia. 2011; 25: 877-879Crossref PubMed Scopus (110) Google Scholar The 2017 revised World Health Organization (WHO) classification of myeloid neoplasms also includes many new molecular features, necessitating higher-throughput approaches to detect these variants for diagnostic, prognostic, and therapeutic purposes.5Arber D.A. Orazi A. Hasserjian R. Borowitz M.J. Le Beau M.M. Bloomfield C.D. Cazzola M. Vardiman J.W. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia.Blood. 2016; 127: 2391-2406Crossref PubMed Scopus (4716) Google Scholar The identification of recurrent molecular genetic abnormalities in myeloid malignancies relies heavily on the precision and accuracy of the diagnostic approach. These parameters can vary between NGS assays, depending on required nucleic acid input amounts, library preparation/sequencing time, and sequencing technology (amplicon-based versus probe-mediated), among other features. Given our experience with Ion Torrent sequencing (locally and within networks),12Hume S. Nelson T.N. Speevak M. McCready E. Agatep R. Feilotter H. Parboosingh J. Stavropoulos D.J. Taylor S. Stockley T.L. CCMG practice guideline: laboratory guidelines for next-generation sequencing.J Med Genet. 2019; 56: 792-800Crossref PubMed Scopus (13) Google Scholar, 13Cook E.K. Izukawa T. Young S. Rosen G. Jamali M. Zhang L. Johnson D. Bain E. Hilland J. Ferrone C.K. Buckstein J. Francis J. Momtaz B. McNaughton A.J.M. Liu X. Snetsinger B. Buckstein R. Rauh M.J. Comorbid and inflammatory characteristics of genetic subtypes of clonal hematopoiesis.Blood Adv. 2019; 3: 2482-2486Crossref PubMed Scopus (37) Google Scholar, 14Sekeres M.A. Othus M. List A.F. Odenike O. Stone R.M. Gore S.D. Litzow M.R. Buckstein R. Fang M. Roulston D. Bloomfield C.D. Moseley A. Nazha A. Zhang Y. Velasco M.R. Gaur R. Atallah E. Attar E.C. Cook E.K. Cull A.H. Rauh M.J. Appelbaum F.R. Erba H.P. Randomized phase II study of azacitidine alone or in combination with lenalidomide or with vorinostat in higher-risk myelodysplastic syndromes and chronic myelomonocytic leukemia: North American Intergroup Study SWOG S1117.J Clin Oncol. 2017; 35: 2745-2753Crossref PubMed Scopus (133) Google Scholar and because we were granted early access to the Oncomine Myeloid Research Assay (or Panel; OMP), we thus aimed to validate this comprehensive DNA/RNA-based amplicon sequencing assay (Thermo Fisher, Waltham, MA). We began with the early-access version of OMP (eOMP) but, on release, focused on the commercial OMP version. Amplicon-based assays are rapid, require little starting material, and are well suited for conditions where rapid testing to support patient management is needed, such as AML. This panel offers simultaneous detection of gene alterations and fusions, an appealing feature that may potentially eliminate or streamline the need for separate laborious techniques. In addition to validating the OMP, we aimed to demonstrate the clinical utility of integrating this type of testing into clinical care for patients with hematological malignancies. The OMP is a commercially available amplicon-based sequencing assay that targets 40 genes and 29 fusion transcript drivers (using RNA as input material) known to be recurrently mutated in myeloid malignancies. The panel includes 3 primer pools (2 DNA and 1 RNA), 526 DNA amplicons, and 698 RNA amplicons. It targets complete exonic regions of 17 genes and 23 exonic hotspots (Table 1). The Ion AmpliSeq Myeloid Research Assay is an early-access version of the OMP provided by Thermo Fisher to Kingston Health Sciences Centre (KHSC) in Kingston, ON, Canada, and Saint John Regional Hospital (SJRH) in Saint John, NB, Canada, to validate the research assay and inform refinements, before its commercial release. The eOMP includes 3 primer pools (2 DNA and 1 RNA), with 360 DNA amplicons and 679 RNA amplicons. It targets 30 DNA genes and 22 fusion transcript drivers, including complete exonic regions of 9 genes and 21 exonic hotspots (Table 1).Table 1Gene Targets of the Commercial and Early-Access OMPCommercial OMPDNARNAHotspot genes (n = 23)Full genes (n = 17)Fusion driver genes (n = 29)ABL1IDH1NRASASXL1PRPF8ABL1FGFR2NTRK3BRAFIDH2PTPN11BCORRB1ALKFUSNUP214CBLJAK2SETBP1CALRRUNX1BCL2HMGA2PDGFRACSF3RKITSF3B1CEBPASH2B3BRAFJAK2PDGFRBDNMT3AKRASSRSF2ETV6STAG2CBFBKMT2A (MLL)RARAFLT3MPLU2AF1EZH2TET2CCND1MECOMRBM15GATA2MYD88WT1IKZF1TP53CREBBPMETRUNX1HRASNPM1NF1ZRSR2EGFRMLLT10TCF3PHF6ETV6MLLT3TFE3FGFR1MYBL1Early-Access OMPDNARNAHotspot genes (n = 21)Full genes (n = 9)Fusion driver genes (n = 22)ABL1GATA2NPM1CALRSH2B3ABL1JAK2NUP214ASXL1IDH1NRASCEBPATET2BCL2KMT2A (MLL)PDGFRABRAFIDH2PTPN11ETV6TP53BRAFMECOMPDGFRBCBLJAK2SETBP1EZH2ZRSR2CBFBMLLT10RARACSF3RKITSF3B1RUNX1CCND1MLLT3RBM15DNMT3AKRASSRSF2CREBBPNPM1RUNX1FLT3MPLU2AF1ETV6NTRK3TCF3FGFR1The OMP targets 40 DNA genes and 29 RNA fusion driver genes. The early-access OMP targets 30 DNA genes and 22 RNA fusion driver genes. Genes included in both panels are known to be recurrently mutated in myeloid malignancies.OMP, Oncomine Myeloid Panel. Open table in a new tab The OMP targets 40 DNA genes and 29 RNA fusion driver genes. The early-access OMP targets 30 DNA genes and 22 RNA fusion driver genes. Genes included in both panels are known to be recurrently mutated in myeloid malignancies. OMP, Oncomine Myeloid Panel. Ethics approval for use of patient samples was obtained from the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board. Informed consent was not required because NGS testing was performed as part of standard clinical procedure, and data were de-identified. A total of 46 samples were used in the validation and implementation of the OMP. These included: 36 samples derived from peripheral blood or bone marrow aspirates from patients diagnosed with a variety of myeloid malignancies from SJRH and KHSC; 6 College of American Pathologists control samples; and 4 SeraCare control samples (SeraCare, Milford, MA) (Supplemental Table S1). The prospective patient cohort involved in assessing actionability of OMP included 187 peripheral blood and bone marrow samples from 168 unique patients, obtained at KHSC between April 2018 and October 2019. This included all AML patients seen at KHSC (who qualified for funded NGS testing in Ontario as of April 2018), as well as other patients with known or suspected hematological malignancies who were recommended for NGS testing based on pathology and clinical consultation at our site. Genomic DNA (gDNA) was extracted at KHSC during the eOMP phase using the DNeasy Blood and Tissue kit (Qiagen, Venlo, the Netherlands; 69504), according to the manufacturer's protocol, with the substitution of proteinase K from Qiagen (19133) and low Tris-EDTA buffer (Thermo Fisher; 12090015) as the final suspension solution. During the OMP phase at KHSC, gDNA was extracted using the Puregene kit (Qiagen; 158389). At SJRH, gDNA was extracted using the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche, Indianapolis, IN; 03730964001). gDNA was later quantified by real-time quantitative PCR using the TaqMan RNase P Detection Reagents Kit (Thermo Fisher; 4316831) to assess quality and suitability for library preparation. The optimal input amount for proceeding to manual library preparation was 20 ng of gDNA and RNA, in a maximum of 3 μL. To proceed to automated library preparation using the Ion Chef Instrument (Thermo Fisher), the optimal input amount of gDNA and RNA was 10 ng, in up to 15 and 8 μL, respectively. At KHSC and SJRH, RNA was extracted using the QIAamp RNA Blood Mini Kit (Qiagen; 52304). Library preparation and templating were performed manually using Ion AmpliSeq Library Kits (Thermo Fisher) during the eOMP phase following the manufacturer's protocol, with final libraries resuspended in 50 μL of low Tris-EDTA. Library preparation and templating were completed using the Ion Chef, according to the manufacturer's protocol during the OMP phase. DNA and RNA/cDNA libraries were sequenced on the Ion PGM Sequencer using Ion 318 Chips at KHSC and on the Ion S5 XL Sequencer using Ion 520 Chips and Ion 530 Chip Kit (all Thermo Fisher) at SJRH during the eOMP phase. Libraries were sequenced on the Ion S5 XL Sequencer using Ion 530 Chips at KHSC and SJRH when using the commercial OMP. Barcoded sequences were uploaded to Torrent Suite version 5.8 and aligned to human genome assembly GRCh37 (hg19) using the Torrent Mapping Alignment Program. Reference transcripts are listed in Supplemental Table S2. Files were uploaded to Ion Reporter (IR) version 5.2-5.6, where variants were annotated using Torrent Variant Caller. Variant annotation was performed using SeqNext (JSI Medical Systems, New York, NY) at SJRH. The variant calling workflow is outlined in Supplemental Figure S1. During the eOMP phase at KHSC, each sample was independently filtered through our custom Strict filter to exclude University of California, Santa Cruz (UCSC), common SNPs (minor allele frequency ≥1%), to exclude nonexonic and synonymous variants, and to initially include only variants with an allele ratio of variant allele frequency (VAF) 0.2 to 1.0 and a depth coverage of >25. Next, we searched for lower-frequency variants (VAF, 0.05 to 0.19) or lower coverage variants reported in the Catalogue of Somatic Mutations in Cancer (COSMIC)15Forbes S.A. Beare D. Boutselakis H. Bamford S. Bindal N. Tate J. Cole C.G. Ward S. Dawson E. Ponting L. Stefancsik R. Harsha B. Kok C.Y. Jia M. Jubb H. Sondka Z. Thompson S. De T. Campbell P.J. COSMIC: somatic cancer genetics at high-resolution.Nucleic Acids Res. 2017; 45: D777-D783Crossref PubMed Scopus (1132) Google Scholar database and/or with IR P ≤ 0.0001. Candidate variants were visually inspected using the Integrative Genomics Viewer version 2.4-2.8 (Broad Institute, Cambridge, MA).16Robinson J.T. Thorvaldsdóttir H. Wenger A.M. Zehir A. Mesirov J.P. Variant review with the Integrative Genomics Viewer.Cancer Res. 2017; 77: e31-e34Crossref PubMed Scopus (337) Google Scholar Variants were excluded if they appeared only in the ends of short sequence reads, or consistently exhibited forward or reverse strand bias. During the OMP phase at KHSC, each sample was independently filtered through the Oncomine Filter supplied by Thermo Fisher (annotation criteria are available in Supplemental Table S3). Candidate variants were explored using Alamut software version 2.9.0 (Interactive Biosoftware, Rouen, France), which aids with variant interpretation and reporting. Variants were also inspected using COSMIC and Integrative Genomics Viewer. In addition, as validation of the bioinformatics pipeline is important,17Roy S. Coldren C. Karunamurthy A. Kip N.S. Klee E.W. Lincoln S.E. Leon A. Pullambhatla M. Temple-Smolkin R.L. Voelkerding K.V. Wang C. Carter A.B. Standards and guidelines for validating next-generation sequencing bioinformatics pipelines: a joint recommendation of the Association for Molecular Pathology and the College of American Pathologists.J Mol Diagn. 2018; 20: 4-27Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar we compared variants detected using the vendor-defined Oncomine filter with those detected using our own filters, as defined above. Finally, clinically relevant variants were reported following assessment by at least one hematopathologist and one clinical molecular geneticist. At KHSC, an orthogonal multiplex amplified fragment length assay was conducted to interrogate FLT3 and NPM1 genes within one assay, and JAK2, CALR, and MPL genes within another, to confirm the presence of variants detected by the OMP. These involved fluorescently labeled amplified products that were detected and analyzed by capillary electrophoresis. Sizes of the amplified products are specific to the wild-type sequence, a control sequence, or a known mutated sequence, as targeted by specific primer placement and digested by a restriction enzyme. Conventional cytogenetic/fluorescence in situ hybridization and PCR-based techniques were also used for confirmation of fusion drivers detected by the OMP, when samples were available. At SJRH, orthogonal verification for sensitivity and test accuracy assessment included real-time quantitative PCR, Sanger sequencing with Applied Biosystems (Thermo Fisher) minor variant finder software in some cases, traditional cytogenetic methods, and fluorescence in situ hybridization. Some samples were also sent to external laboratories for gene-specific tests (such as CALR or MPL). Variants in the prospective cohort were assessed for clinical actionability by electronic chart record, molecular board review, and local clinical input. 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