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W4313379654 abstract "The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements. The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements. Rearrangement involving the neurotrophic tyrosine receptor kinase (NTRK) family genes (NTRK1, NTRK2, and NTRK3) generate fusion proteins that are oncogenic drivers in many cancer types across adult and pediatric populations.1Kummar S. Lassen U.N. TRK inhibition: a new tumor-agnostic treatment strategy.Target Oncol. 2018; 13: 545-556Crossref PubMed Scopus (66) Google Scholar, 2Hsiao S.J. Zehir A. Sireci A.N. Aisner D.L. Detection of tumor NTRK gene fusions to identify patients who may benefit from tyrosine kinase (TRK) inhibitor therapy.J Mol Diagn. 2019; 21: 553-571Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 3Vaishnavi A. Le A.T. Doebele R.C. TRKing down an old oncogene in a new era of targeted therapy.Cancer Discov. 2015; 5: 25-34Crossref PubMed Scopus (443) Google Scholar Recently, larotrectinib and entrectinib have received clinical approval in the United States, Canada, Europe, and other countries for the treatment of adult patients with metastatic or locally advanced unresectable disease, and in some countries, entrectinib also in pediatric patients (≥12 years old), with solid tumors bearing the NTRK fusion.4Scott L.J. Larotrectinib: first global approval.Drugs. 2019; 79: 201-206Crossref PubMed Scopus (147) Google Scholar, 5Al-Salama Z.T. Keam S.J. Entrectinib: first global approval.Drugs. 2019; 79: 1477-1483Crossref PubMed Scopus (83) Google Scholar, 6Delgado J. Pean E. Melchiorri D. Migali C. Josephson F. Enzmann H. Pignatti F. The European Medicines Agency review of entrectinib for the treatment of adult or paediatric patients with solid tumours who have a neurotrophic tyrosine receptor kinase gene fusions and adult patients with non-small-cell lung cancer harbouring ROS1 rearrangements.ESMO Open. 2021; 6: 100087Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 7Harada G. Santini F.C. Wilhelm C. Drilon A. NTRK fusions in lung cancer: from biology to therapy.Lung Cancer. 2021; 161: 108-113Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Health Canada approved larotrectinib in 2019 and entrectinib in 2020 for adult and pediatric patients. Many methods may be used to screen for the presence of NTRK fusions in the tumor, including immunohistochemistry (IHC), fluorescent in situ hybridization, quantitative real-time PCR, and massively parallel next-generation sequencing (NGS). However, with three NTRK genes and >80 possible fusion partners to consider for testing, IHC and NGS have emerged as the preferred methods for screening and/or diagnosis of NTRK fusion tumors and patients in the clinic.2Hsiao S.J. Zehir A. Sireci A.N. Aisner D.L. Detection of tumor NTRK gene fusions to identify patients who may benefit from tyrosine kinase (TRK) inhibitor therapy.J Mol Diagn. 2019; 21: 553-571Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,8Penault-Llorca F. Rudzinski E.R. Sepulveda A.R. Testing algorithm for identification of patients with TRK fusion cancer.J Clin Pathol. 2019; 72: 460-467Crossref PubMed Scopus (117) Google Scholar,9Marchiò C. Scaltriti M. Ladanyi M. Iafrate A.J. Bibeau F. Dietel M. Hechtman J.F. Troiani T. López-Rios F. Douillard J.-Y. Andrè F. Reis-Filho J.S. ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research.Ann Oncol. 2019; 30: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar Screening using IHC prior to NGS is especially applicable for cancers with a low incidence of NTRK gene fusions.2Hsiao S.J. Zehir A. Sireci A.N. Aisner D.L. Detection of tumor NTRK gene fusions to identify patients who may benefit from tyrosine kinase (TRK) inhibitor therapy.J Mol Diagn. 2019; 21: 553-571Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,8Penault-Llorca F. Rudzinski E.R. Sepulveda A.R. Testing algorithm for identification of patients with TRK fusion cancer.J Clin Pathol. 2019; 72: 460-467Crossref PubMed Scopus (117) Google Scholar, 9Marchiò C. Scaltriti M. Ladanyi M. Iafrate A.J. Bibeau F. Dietel M. Hechtman J.F. Troiani T. López-Rios F. Douillard J.-Y. Andrè F. Reis-Filho J.S. ESMO recommendations on the standard methods to detect NTRK fusions in daily practice and clinical research.Ann Oncol. 2019; 30: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 10Bebb D.G. Banerji S. Blais N. Desmeules P. Gill S. Grin A. Feilotter H. Hansen A.R. Hyrcza M. Krzyzanowska M. Melosky B. Noujaim J. Purgina B. Ruether D. Simmons C.E. Soulieres D. Torlakovic E.E. Tsao M.-S. Canadian consensus for biomarker testing and treatment of TRK fusion cancer in adults.Curr Oncol. 2021; 28: 523-548Crossref PubMed Scopus (24) Google Scholar, 11Perreault S. Chami R. Deyell R.J. El Demellawy D. Ellezam B. Jabado N. Morgenstern D.A. Narendran A. Sorensen P.H.B. Wasserman J.D. Yip S. Canadian consensus for biomarker testing and treatment of TRK fusion cancer in pediatric patients.Curr Oncol. 2021; 28: 346-366Crossref PubMed Scopus (21) Google Scholar NGS panel testing is now a standard platform in clinical molecular diagnostic laboratories within Canada.12Yip S. Christofides A. Banerji S. Downes M.R. Izevbaye I. Lo B. MacMillan A. McCuaig J. Stockley T. Yousef G.M. Spatz A. A Canadian guideline on the use of next-generation sequencing in oncology.Curr Oncol. 2019; 26: e241-e254Crossref PubMed Scopus (30) Google Scholar However, multiple NGS sequencing platforms and commercially developed test panels are available. Except for large referral centers or commercial laboratories, most institutional laboratories adopt one NGS platform and one or a small number of commercial targeted panels to validate and use in their routine clinical work. In this context, the Canadian NTRK (CANTRK) ring study was initiated with the goal to optimize and enable testing protocols to screen for NTRK fusion cancers in pathology and molecular diagnostic laboratories across Canada, using pan-TRK IHC for screening and RNA-based NGS targeted panels for confirming the presence of NTRK gene fusions as currently used in Canada. The current article describes the design, execution, and results of the NGS part of the CANTRK project and discusses issues and possible solutions that may be useful for institutions and/or laboratories that also plan to establish NTRK testing programs. The CANTRK study included two components, one evaluating pan-TRK IHC and the other evaluating NGS-based testing for NTRK gene fusions (NTRK1, NTRK2, and NTRK3). Details on the IHC study will be reported separately. In the NGS study, 16 clinical molecular diagnostic laboratories from seven provinces (Alberta, British Columbia, Manitoba, Nova Scotia, Ontario, Quebec, and Saskatchewan) participated. Two additional laboratories were invited but were unable to complete testing. Laboratories were selected based on accreditation and licensing as a clinical molecular diagnostic laboratory in their province, as well as on existing ability to perform NGS using methods that detected fusions in NTRK1, NTRK2, or NTRK3 genes. All laboratories used various methods based on RNA sequencing for gene fusion detection. Results were returned to the project leader, and meetings were held to review and discuss results and discrepancies. The project and study protocol received approval by the Research Ethics Board at the University Health Network (19-5438). Before the start of the ring study, a commercial formalin-fixed, paraffin-embedded (FFPE) control sample containing multiple gene fusions including four NTRK gene fusions (Seraseq FFPE Tumor Fusion RNA Reference Material v4, DMark Biosciences, Toronto, ON, Canada) was distributed to all participating molecular laboratories as an aid to NGS validation. Twelve FFPE patient tumor samples with known NTRK1, NTRK2, or NTKR3 gene fusions, as well as two non-NTRK fusion cases (ALK and ROS1), were used as study materials (Table 1 and Supplemental Table S1). The NTRK fusion cases were collected from CANTRK participant laboratories following approval by local institutions as required. Tumor sites included lung adenocarcinoma, atypical nevus, papillary thyroid carcinoma (follicular variant, classical variant, metastatic), infantile fibrosarcoma, salivary gland secretory carcinoma, glioneuronal tumor, and breast secretory carcinoma. Pathology review and assessment of the tumor cellularity were performed by board-certified study pathologists (M.S.T. and S.N.M.F.). All samples had tumor cellularity >90% in the tumor-rich regions. The presence of the NTRK gene fusions and two control non-NTRK gene fusions (ALK and ROS1) were confirmed at two reference laboratories by study participants (B.L., The Ottawa Hospital, Ottawa, ON, Canada; T.L.S., University Health Network, Toronto, ON, Canada). Reference laboratories were chosen based on their use of two different NGS library methods, one using multiplex PCR with primers designed to detect known fusions [Oncomine Comprehensive Assay v3 (OCAv3), Thermo Fisher Scientific, Waltham, MA; used at University Health Network] and one using anchored multiplex PCR that can detect NTRK gene fusions in a partner-agnostic manner [FusionPlex Lung Panel (FPL) ArcherDX, Boulder, CO; used at The Ottawa Hospital].Table 1Validation Samples Used in the CANTRK StudySampleGene fusion (exon)Disease site or diagnosisCTRK-02TPR(21)::NTRK1(10)Lung adenocarcinomaCTRK-06ALK(20)::EML4(20)Lung adenocarcinomaCTRK-07TPM3(7)::NTRK1(12)Lung adenocarcinomaCTRK-09LMNA(3)::NTRK1(11)Atypical nevusCTRK-12TPM3(7)::NTRK1(10)PTC, follicular variantCTRK-13ETV6(5)::NTRK3(15)Salivary gland, MASCCTRK-14WNK2(23)::NTRK2(16)Glioneuronal tumorCTRK-15STRN3(7)::NTRK2(16)Glioneuronal tumorCTRK-18ROS1(34)::CD74(6)Lung adenocarcinomaCTRK-20ETV6(4)::NTRK3(14)PTC, classical variantCTRK-22Not testedInfantile fibrosarcomaCTRK-23ETV6(4)::NTRK3(14)Salivary glandCTRK-24ETV6(5)::NTRK3(15)Breast secretory carcinomaCTRK-28TPM3(7)::NTRK1(10)PTC, metastaticThe expected fusion result for each sample was determined by using the Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific) next-generation sequencing panel. Numbers in parentheses represent exons determined by reference laboratory testing [FusionPlex Lung (ArcherDX) or Oncomine Comprehensive Assay v3] and confirmed by consensus among other laboratory exon results. The disease site/diagnosis was determined by one of the study board-certified pathologists. One sample (CTRK-22) of infantile fibrosarcoma was withdrawn from analysis due to poor sample quality.CANTRK, Canadian NTRK; MASC, mammary analogue secretory carcinoma; PTC, papillary thyroid carcinoma. Open table in a new tab The expected fusion result for each sample was determined by using the Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific) next-generation sequencing panel. Numbers in parentheses represent exons determined by reference laboratory testing [FusionPlex Lung (ArcherDX) or Oncomine Comprehensive Assay v3] and confirmed by consensus among other laboratory exon results. The disease site/diagnosis was determined by one of the study board-certified pathologists. One sample (CTRK-22) of infantile fibrosarcoma was withdrawn from analysis due to poor sample quality. CANTRK, Canadian NTRK; MASC, mammary analogue secretory carcinoma; PTC, papillary thyroid carcinoma. For the ring study, each participating laboratory received three 7 μm thick sections for each study case. Laboratories also received an image of a hematoxylin and eosin–stained slide with the tumor-rich area marked with ink, for use in macro-dissection if required by standard practice for the participating laboratories. Laboratories were blinded to the known gene fusion in each sample. Each laboratory used their own validated protocols for RNA extraction and NTRK fusion detection (Supplemental Table S2).13Chen S. Zhou Y. Chen Y. Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor.Bioinformatics. 2018; 34: i884-i890Crossref PubMed Scopus (6625) Google Scholar, 14Langmead B. Salzberg S.L. Fast gapped-read alignment with Bowtie 2.Nat Methods. 2012; 9: 357-359Crossref PubMed Scopus (28880) Google Scholar, 15Danecek P. Bonfield J.K. Liddle J. Marshall J. Ohan V. Pollard M.O. Whitwham A. Keane T. McCarthy S.A. Davies R.M. Li H. Twelve years of SAMtools and BCFtools.Gigascience. 2021; 10: giab008Crossref PubMed Scopus (1840) Google Scholar, 16Quinlan A.R. Hall I.M. BEDTools: a flexible suite of utilities for comparing genomic features.Bioinformatics. 2010; 26: 841-842Crossref PubMed Scopus (12967) Google Scholar, 17Haas B.J. Dobin A. Stransky N. Li B. Yang X. Tickle T. Bankapur A. Ganote C. Doak T.G. Pochet N. Sun J. Wu C.J. Gingeras T.R. Regev A. STAR-Fusion: fast and accurate fusion transcript detection from RNA-seq.bioRxiv. 2017; ([Preprint] doi:10.1101/120295)Google Scholar, 18Davidson N.M. Majewski I.J. Oshlack A. JAFFA: high sensitivity transcriptome-focused fusion gene detection.Genome Med. 2015; 7: 43Crossref PubMed Scopus (100) Google Scholar The NGS panels used were capable of detecting gene fusions in NTRK1, NTRK2, and NTRK3. NGS panels included OCAv3 (four laboratories), Oncomine Focus Assay (OCF; Thermo Fisher Scientific; two laboratories), Oncomine Precision Assay (OPA; Thermo Fisher Scientific; one laboratory), AmpliSeq for Illumina Focus Panel (ASIFP: Illumina, San Diego, CA; four laboratories), TruSight RNA Pan-Cancer Panel (TS; Illumina; two laboratories), FPL (two laboratories), and QIAseq Multimodal Lung (QMML; Qiagen, Germantown, MD; one laboratory). RNA input amounts were validated by each laboratory, with 10 to 40 ng RNA input used for libraries for the OCAv3, OCF, OPA, and ASIFP panels, and 30 to 250 ng RNA for the TS, FPL, and QMML panels. Laboratories used their own bioinformatics tools for analysis, ranging from analysis tools supplied by panel manufacturers to custom laboratory-developed analyses (Supplemental Table S2). Participating laboratories reported back the results to the study lead and reference laboratory lead (M.S.T. and T.L.S.) who assessed the reported fusions against expected results. Discussions were held with participating laboratories to present results and discuss any discordant findings. For the two-way sample exchange between the two laboratories using the TS panel and the reference laboratory (University Health Network), extracted RNA from CTRK-20 containing the ETV6::NTRK3 fusion was sent from the reference laboratory to each laboratory using TS. In return, each laboratory using the TS panel sent extracted RNA to the reference laboratory. All three laboratories tested the received samples, and results of all exchanged samples were reported to the reference laboratory lead for comparison. The average RNA yield of each sample is shown in Supplemental Table S3. In the confirmation of NTRK fusion cases by the reference laboratories, two cases, CTRK-14 (WNK2::NTRK2) and CTRK-15 (STRN3::NTRK2), were detected by one reference laboratory using the anchored multiplex PCR-based FPL assay but not by the second reference laboratory using the amplicon-based OCAv3 due to the absence of primers for the partner genes WNK2 and STRN3 in the panel designs. One sample (CTRK-22; infantile fibrosarcoma with ETV6::NTRK3 fusion) was withdrawn from analysis due to a quality issue with the FFPE tumor tissue block (no laboratories generated data for the CTRK-22 sample). Histology review of the sample revealed evidence of autolysis and poor tissue preservation, which could contribute to the inadequacy for NGS. Of the 13 remaining samples, 8 of the fusions were detected by all 16 laboratories, including 6 samples with NTRK gene fusions (6 of 11 NTRK fusion samples, 55%) and 2 control samples with ALK or ROS1 fusions (Figure 1). Amplicon panels have the limitation of detecting only fusions for which primers are included in the panel design. Of the four amplicon library panels used in this ring study, one panel detected all fusions (OPA). Two NTRK fusions in glioneuronal tumor, WNK2::NTRK2 (CTRK-14) and STRN3::NTRK2 (CTRK-15), were not detected by the 10 laboratories using the OCF, ASIFP, or OCAv3 panels; this was due to the absence of primers for the partner genes WNK2 and STRN3 in the panel designs for the OCF and ASIFP assays (includes a total of 13 isoforms present for 9 partner genes, does not include WNK2 and STRN3; information supplied by manufacturer) and in the OCAv3 assay (includes a total of 18 isoforms present for 13 partner genes; does not include WNK2 and STRN3; information supplied by manufacturer). The more recent OPA panel from the same manufacturer includes primers for a larger number of NTRK2 gene partners (total of 89 isoforms present for 37 partner genes, including WNK2 and STRN3), and the WNK2::NTRK2 and STRN3::NTRK2 fusions were both detectable on the OPA panel. Of the six laboratories using the Focus panel from either of the two manufacturers (four laboratories using ASIFP, Illumina; two laboratories using OFA, Thermo Fisher Scientific), three laboratories (laboratories 1, 2, and 3) (Figure 1) were not able to detect the expected fusion in two lung adenocarcinomas samples, TPM3::NTRK1 (CTRK-07) and LMNA::NTRK1 (CTRK-09). The three laboratories that did not detect these fusions used ASIFP. One of the three laboratories using ASIFP obtained a partial result detecting a fusion involving NTRK1, without identifying the fusion partner, by using an analysis for an imbalance in the difference in expression between the 5′ assay and the 3′ assay of each gene in the fusion (no other laboratories assessed the expression imbalance assay results in routine use). Because the ability of three other laboratories to detect these fusions using the ASIFP or OFA ruled out an issue with primers in the panel design (as both ASIFP and OFA panels use the same design), it suggested the issue was due to either a lack of tumor in the FFPE material sent to these three laboratories (could not be investigated further due to lack of residual material) or due to a difference in the bioinformatics analysis in these laboratories. Further analysis identified that using a different bioinformatics tool (STAR-Fusion version 1.9.1)17Haas B.J. Dobin A. Stransky N. Li B. Yang X. Tickle T. Bankapur A. Ganote C. Doak T.G. Pochet N. Sun J. Wu C.J. Gingeras T.R. Regev A. STAR-Fusion: fast and accurate fusion transcript detection from RNA-seq.bioRxiv. 2017; ([Preprint] doi:10.1101/120295)Google Scholar (Supplemental Table S2) with the ability to align against a broader range of possible fusion targets enabled the detection of the TPM3::NTRK1 and LMNA::NTRK1 gene fusions in these two samples. Use of library methods that can select gene fusions for sequencing without prior knowledge of each partner gene or the specific fusion isoform can theoretically improve detection of rare or novel fusions. In the ring study, five laboratories used partner-agnostic methods (ie, able to detect NTRK fusions partners without prior knowledge of the partner gene), including two laboratories using hybridization capture libraries (TS, Illumina), two laboratories using anchored multiplex PCR libraries (FPL), and one laboratory using a single primer extension and amplification library (QMML, Qiagen). Both laboratories using the FPL panel detected all fusions. For the two laboratories using the TS panel, an ETV6::NTRK3 fusion in a papillary thyroid carcinoma (CTRK-20) was not detected, although a similar ETV6::NTRK3 fusion in a salivary duct carcinoma sample (CTRK-23) was detected by both laboratories (Figure 1). It was also noted that the results of CTRK-20 testing by other laboratories using amplicon panels gave a low number of fusion reads, suggesting that the fusion in this sample was present at a low level that may not be detectable by the TS panel but that was detectable by other panel methods. To investigate, a sample swap was arranged in which one reference laboratory (University Health Network) that detected the ETV6::NTRK3 fusion in CTRK-20 sent a sample of extracted RNA to the two laboratories using TS, who in return sent extracted RNA back to the reference laboratory for retesting. All three samples tested at the reference laboratory were confirmed to have reads supporting the presence of the ETV6::NTRK3 fusion (albeit at very low read levels), and both laboratories using TS detected the ETV6::NTRK3 fusion on RNA extracted at the reference laboratory (Supplemental Table S4). The sample exchange thus indicated that the TS panel was able to detect the ETV6::NTRK3 fusion but that the low fusion level in the sample was challenging to detect by using the TS panel. There is an emerging group of targeted tumor-agnostic therapies in which drug selection is based on tumor molecular features found in a variety of tumor types and at various ages of cancer onset.19Thein K.Z. Lemery S.J. Kummar S. Tissue-agnostic drug development: a new path to drug approval.Cancer Discov. 2021; 11: 2139-2144Crossref PubMed Scopus (11) Google Scholar Since 2015, Health Canada has approved pembrolizumab in 19 tumor indications for patients with microsatellite instability–high or mismatch repair–deficient cancers. In 2019 and 2020, larotrectinib and entrectinib, respectively, were approved by Health Canada for the treatment of adult and pediatric patients with solid tumors carrying NTRK gene fusions. Because these tumor-agnostic molecular features are rare in certain cancer types, molecular methods that capture the most variants are ideal for clinical testing, thus providing drug eligibility to the largest group of patients. However, the rarity of these variants poses a problem for validation by clinical molecular laboratories, which require positive control material. This is particularly true for NTRK gene fusions, which have pathognomonic tumor sites where certain NTRK gene fusions are common (eg, ETV6::NTRK3 fusions in salivary gland secretory carcinoma, secretory breast carcinoma, infantile fibrosarcoma, and congenital mesoblastic nephroma) yet other tumor sites where NTRK1, NTRK2 or NTRK3 fusions might exist but are very rare.2Hsiao S.J. Zehir A. Sireci A.N. Aisner D.L. Detection of tumor NTRK gene fusions to identify patients who may benefit from tyrosine kinase (TRK) inhibitor therapy.J Mol Diagn. 2019; 21: 553-571Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,20Xia H. Xue X. Ding H. Ou Q. Wu X. Nagasaka M. Shao Y.W. Hu X. Ou S.-H.I. Evidence of NTRK1 fusion as resistance mechanism to EGFR TKI in EGFR+ NSCLC: results from a large-scale survey of NTRK1 fusions in Chinese patients with lung cancer.Clin Lung Cancer. 2020; 21: 247-254Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 21Zhao X. Kotch C. Fox E. Surrey L.F. Wertheim G.B. Baloch Z.W. Lin F. Pillai V. Luo M. Kreiger P.A. Pogoriler J.E. Linn R.L. Russo P.A. Santi M. Resnick A.C. Storm P.B. Hunger S.P. Bauer A.J. Li M.M. NTRK fusions identified in pediatric tumors: the frequency, fusion partners, and clinical outcome.JCO Precis Oncol. 2021; 1 (PO.20.00250)PubMed Google Scholar, 22Roosen M. Odé Z. Bunt J. Kool M. The oncogenic fusion landscape in pediatric CNS neoplasms.Acta Neuropathol. 2022; 143: 427-451Crossref PubMed Scopus (13) Google Scholar, 23Westphalen C.B. Krebs M.G. Le Tourneau C. Sokol E.S. Maund S.L. Wilson T.R. Jin D.X. Newberg J.Y. Fabrizio D. Veronese L. Thomas M. de Braud F. Genomic context of NTRK1/2/3 fusion-positive tumours from a large real-world population.NPJ Precis Oncol. 2021; 5: 69Crossref PubMed Scopus (57) Google Scholar, 24Rosen E.Y. Goldman D.A. Hechtman J.F. Benayed R. Schram A.M. Cocco E. Shifman S. Gong Y. Kundra R. Solomon J.P. Bardelli A. Scaltriti M. Drilon A. Iasonos A. Taylor B.S. Hyman D.M. TRK fusions are enriched in cancers with uncommon histologies and the absence of canonical driver mutations.Clin Cancer Res. 2020; 26: 1624-1632Crossref PubMed Scopus (88) Google Scholar, 25Hong D.S. DuBois S.G. Kummar S. Farago A.F. Albert C.M. Rohrberg K.S. van Tilburg C.M. Nagasubramanian R. Berlin J.D. Federman N. Mascarenhas L. Geoerger B. Dowlati A. Pappo A.S. Bielack S. Doz F. McDermott R. Patel J.D. Schilder R.J. Tahara M. Pfister S.M. Witt O. Ladanyi M. Rudzinski E.R. Nanda S. Childs B.H. Laetsch T.W. Hyman D.M. Drilon A. Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials.Lancet Oncol. 2020; 21: 531-540Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar NTRK gene fusions are also best detected by using RNA-based NGS, a methodology still emergent in Canadian clinical laboratories. Of note, no Canadian laboratories in this study used DNA-based NGS for detection of NTRK gene fusions. Compared with RNA-based NGS for detection of gene fusions, DNA-based NGS is more challenging due to the larger intronic regions that need to be effectively covered by the NGS panel and the lack of evidence of expressed gene fusions evident when using RNA-based NGS. For these reasons, DNA-based NGS reportedly has reduced sensitivity for gene fusion detection compared with RNA-based NGS due to false-negative results.26Solomon J.P. Hechtman J.F. Detection of NTRK fusions: merits and limitations of current diagnostic platforms.Cancer Res. 2019; 79: 3163-3168Crossref PubMed Scopus (121) Google Scholar,27Benayed R. Offin M. Mullaney K. Sukhadia P. Rios K. Desmeules P. Ptashkin R. Won H. Chang J. Halpenny D. Schram A.M. Rudin C.M. Hyman D.M. Arcila M.E. Berger M.F. Zehir A. Kris M.G. Drilon A. Ladanyi M. High yield of RNA sequencing for targetable kinase fusions in lung adenocarcinomas with no mitogenic driver alteration detected by DNA sequencing and low tumor mutation burden.Clin Cancer Res. 2019; 25: 4712-4722Crossref PubMed Scopus (253) Google Scholar Comparison of NGS testing for NTRK gene fusions in clinical laboratories in other countries, including Germany28Pfarr N. Kirchner M. Lehmann U. Leichsenring J. Merkelbach-Bruse S. Glade J. Hummel M. Stögbauer F. Lehmann A. Trautmann M. Kumbrink J. Jung A. Dietmaier W. Endris V. Kazdal D. Evert M. Horst D. Kreipe H. Kirchner T. Wardelmann E. Lassen U. Büttner R. Weichert W. Dietel M. Schirmacher P. Stenzinger A. Testing NTRK testing: wet-lab and in silico comparison of RNA-based targeted sequencing assays.Genes Chromosomes Cancer. 2020; 59: 178-188Crossref PubMed Scopus (45) Google Scholar and South Korea,29Lee S.E. Lee M.-S. Jeon Y.K. Shim H.S. Kang J. Kim J. Choi Y.-L. Interlaboratory comparison study (ring test) of next-generation sequencing‒based NTRK fusion detection in South Korea.Cancer Res Treat. 2023; 55: 28-40Crossref Scopus (1) Google Scholar have shown that comprehensive detection of NTRK gene fusions is highly dependent on NGS library designs and on bioinformatics approaches for data analysis. Different diagnostic sensitivity is noted between NGS using amplicon libraries, which rely on knowledge of specific breakpoints with partner genes and thus may have reduced sensitivity for rare fusions, versus partner-agnostic library approaches (eg, hybrid capture, anchored multiplex PCR) in which prior knowledge of the fusion gene partner is not required.30Park H.J. Baek I. Cheang G. Solomon J.P. Song W. Comparison of RNA-based next-generation sequencing assays for the detection of NTRK gene fusions.J Mol Diagn. 2021; 23: 1443-1451Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar,31Bormann Chung C. Lee J. Barritault M. Bringuier P.-P. Xu Z. Huang W.-Y. Beharry A. Castillo J. Christiansen J. Lin J.C. Sheffield B.S. Evaluating targeted next-generation sequencing assays and reference materials for NTRK fusion detection.J Mol Diagn. 2022; 24: 18-32Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar A clinical laboratory external quality assessment scheme in Germany, Austria, and Switzerland, which assessed both fluorescent in situ hybridization and NGS by RNA sequencing in 27 centers, also showed detection challenges with certain NGS commercial panels, particularly amplicon panels in which rare targets were not included in design although with high sensitivity of 95.3% overall for RNA-based NGS analysis.32Kirchner M. Glade J. Lehmann U. Merkelbach-Bruse S. Hummel M. Lehmann A. Trautmann M. Kumbrink J. Jung A. Dietmaier W. Endris V. Kazdal D. Ploeger C. Evert M. Horst D. Kreipe H. Kirchner T. Wardelmann E. Büttner R. Weichert W. Dietel M. Schirmacher P. Stenzinger A. Pfarr N. NTRK testing: first results of the QuiP-EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA-based NGS assays.Genes Chromosomes Cancer. 2020; 59: 445-453Crossref PubMed Scopus (21) Google Scholar Because health care delivery in Canada is funded and regulated provincially, decisions by clinical molecular diagnostic laboratories regarding specific laboratory technologies to implement for clinical testing are region specific and based on local expertise or equipment access, with minimal harmonization across the country. Although this diversity of methods is acceptable under a strong laboratory accreditation program that exists within Canada (eg, by Accreditation Canada Diagnostics) and includes molecular-specific requirements and external quality assessment participation, it remains valuable for Canadian laboratories to assess their own test approaches compared with others as a way of identifying test-specific issues or to confirm test equivalency. Through the CANTRK study multicenter collaboration, tumor samples from multiple tumor sites with NTRK1, NTRK2, or NTRK3 fusions, with six different fusion gene partners, were shared across 16 Canadian clinical laboratories from seven provinces, overcoming a major barrier of individual laboratories sourcing sufficient positive control samples for validation. Canadian laboratories in this study used seven different RNA-based NGS commercial panels for detection of NTRK fusions. The NGS panels that detected all fusions (11 NTRK fusions) included the FPL, TS, OPA, and QMML panels (all partner-agnostic, except for OPA, which is amplicon based), whereas the panel detecting the least fusions was ASIFP (6 of 11 NTRK fusions detected, amplicon based). Although certain NGS library methods can allow selection of fusions without a priori knowledge of the fusion partners, including three library methods used by participants in this study (TS, FPL, and QMML), these methods may require longer workflows and higher amounts of RNA. The TruSight hybridization capture library used in the CANTRK study encountered a challenge in detecting low levels of fusions on one sample by both laboratories using this panel, which may indicate a reduced sensitivity of this panel at low tumor or variant content. It has also been reported in a comparison of RNA-based NGS methods for NTRK gene fusion detection that the hybridization-capture NGS TruSight Oncology 500 assay (Illumina) also exhibited reduced sensitivity for low-level fusion variants.29Lee S.E. Lee M.-S. Jeon Y.K. Shim H.S. Kang J. Kim J. Choi Y.-L. Interlaboratory comparison study (ring test) of next-generation sequencing‒based NTRK fusion detection in South Korea.Cancer Res Treat. 2023; 55: 28-40Crossref Scopus (1) Google Scholar In this study, two issues came forward as having a larger impact within Canadian laboratories. The first was the lack of primers in commercial panel amplicon libraries (panels OCAv3, OFA, and ASIFP) for two NTRK2 fusion partner genes (WNK2 and STRN3). Although amplicon library methods can allow use of small amounts of RNA, by design, prior knowledge of both partner genes and the specific exons involved in the fusion are required. Although NTRK2 fusions are the least frequent NTRK gene–related fusions (reported mainly in pediatric central nervous system tumors21Zhao X. Kotch C. Fox E. Surrey L.F. Wertheim G.B. Baloch Z.W. Lin F. Pillai V. Luo M. Kreiger P.A. Pogoriler J.E. Linn R.L. Russo P.A. Santi M. Resnick A.C. Storm P.B. Hunger S.P. Bauer A.J. Li M.M. NTRK fusions identified in pediatric tumors: the frequency, fusion partners, and clinical outcome.JCO Precis Oncol. 2021; 1 (PO.20.00250)PubMed Google Scholar, 22Roosen M. Odé Z. Bunt J. Kool M. The oncogenic fusion landscape in pediatric CNS neoplasms.Acta Neuropathol. 2022; 143: 427-451Crossref PubMed Scopus (13) Google Scholar, 23Westphalen C.B. Krebs M.G. Le Tourneau C. Sokol E.S. Maund S.L. Wilson T.R. Jin D.X. Newberg J.Y. Fabrizio D. Veronese L. Thomas M. de Braud F. Genomic context of NTRK1/2/3 fusion-positive tumours from a large real-world population.NPJ Precis Oncol. 2021; 5: 69Crossref PubMed Scopus (57) Google Scholar, 24Rosen E.Y. Goldman D.A. Hechtman J.F. Benayed R. Schram A.M. Cocco E. Shifman S. Gong Y. Kundra R. Solomon J.P. Bardelli A. Scaltriti M. Drilon A. Iasonos A. Taylor B.S. Hyman D.M. TRK fusions are enriched in cancers with uncommon histologies and the absence of canonical driver mutations.Clin Cancer Res. 2020; 26: 1624-1632Crossref PubMed Scopus (88) Google Scholar, 25Hong D.S. DuBois S.G. Kummar S. Farago A.F. Albert C.M. Rohrberg K.S. van Tilburg C.M. Nagasubramanian R. Berlin J.D. Federman N. Mascarenhas L. Geoerger B. Dowlati A. Pappo A.S. Bielack S. Doz F. McDermott R. Patel J.D. Schilder R.J. Tahara M. Pfister S.M. Witt O. Ladanyi M. Rudzinski E.R. Nanda S. Childs B.H. Laetsch T.W. Hyman D.M. Drilon A. Larotrectinib in patients with TRK fusion-positive solid tumours: a pooled analysis of three phase 1/2 clinical trials.Lancet Oncol. 2020; 21: 531-540Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar), in the absence of frequent updates by the manufacturer to commercial NGS panel target gene content, laboratories can only increase their detection of rare fusions by moving to a library preparation with more isoform detection or switching to a fusion partner–agnostic method, all of which require extensive clinical validations. As shown in this study, the diversity of NTRK gene fusions leads to false-negative findings in testing in which fusions for rare or novel partner genes or isoforms are not detectable due to panel design, particularly if existing commercial panels are not updated by the manufacturer as new partner genes are identified (as is the case with the OFA and OCAv3 panels, compared with the more recent OPA panel from the same manufacturer). The findings are also supported by a recent in silico analysis of the bed files of current assays and coverage of various NTRK gene fusions28Pfarr N. Kirchner M. Lehmann U. Leichsenring J. Merkelbach-Bruse S. Glade J. Hummel M. Stögbauer F. Lehmann A. Trautmann M. Kumbrink J. Jung A. Dietmaier W. Endris V. Kazdal D. Evert M. Horst D. Kreipe H. Kirchner T. Wardelmann E. Lassen U. Büttner R. Weichert W. Dietel M. Schirmacher P. Stenzinger A. Testing NTRK testing: wet-lab and in silico comparison of RNA-based targeted sequencing assays.Genes Chromosomes Cancer. 2020; 59: 178-188Crossref PubMed Scopus (45) Google Scholar in which the OCAv3 and OFA panels had the lowest detection of NTRK fusions among nine NGS methods. The second issue identified was bioinformatics analysis on data from the ASIFP panel. Because the manufacturer does not supply a custom bioinformatics analysis for this panel, laboratories must develop their own bioinformatics analysis. For three laboratories, their initial choice of bioinformatics analysis did not detect two NTRK1 fusions in lung adenocarcinomas. However, one of these laboratories confirmed that these two fusions were identifiable by using an alternate fusion detection software (STAR-Fusion version 1.9.117Haas B.J. Dobin A. Stransky N. Li B. Yang X. Tickle T. Bankapur A. Ganote C. Doak T.G. Pochet N. Sun J. Wu C.J. Gingeras T.R. Regev A. STAR-Fusion: fast and accurate fusion transcript detection from RNA-seq.bioRxiv. 2017; ([Preprint] doi:10.1101/120295)Google Scholar). This finding suggests that laboratories require appropriate control materials to optimize bioinformatics detection of rare events in their gene fusion detection panel data. During the course of this Canada-wide study, several questions related to quality assurance for clinical laboratories using NGS for RNA sequencing for fusion gene testing were identified. These included the quality criteria to report a positive result for a fusion gene from RNA sequencing, the challenges for bioinformatics analysis, and the best practices for reporting gene fusions. The information gathered from this study will be used to formulate a best practice guideline for analysis and reporting of gene fusions detected by NGS methods. We thank Haya Sarras and Josh Silvertown, Bayer Inc., for study support. M.S.T. and E.T. secured funding and designed the CANTRK study; T.L.S. and B.L. performed reference laboratory analysis; T.L.S. wrote the manuscript; and T.L.S., B.L., A.B., A.G.C., J.D., P.D., H.F., D.G., W.G., C.H., W.Y.H., I.I., G.L., S.N.M.F., A.I.P., P.C.P., J.-B.R., B.S.S., A.S., E.S., D.T-T., S.Y., and T.Z. performed experiments and analyzed the data. Download .docx (.02 MB) Help with docx files Supplemental Table S1 Download .docx (.02 MB) Help with docx files Supplemental Table S2 Download .docx (.01 MB) Help with docx files Supplemental Table S3 Download .docx (.01 MB) Help with docx files Supplemental Table S4" @default.
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