Noncanonical Gene Fusions Detected at the DNA Level Necessitate Orthogonal Diagnosis Methods Before Targeted Therapy.

Abstract

Patients with NSCLC harboring oncogenic tyrosine kinase fusions involving ALK,1Soda M. Choi Y.L. Enomoto M. et al.Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4173) Google Scholar, 2Kwak E.L. Bang Y.J. Camidge D.R. et al.Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3743) Google Scholar, 3Shaw A.T. Kim D.W. Nakagawa K. et al.Crizotinib versus chemotherapy in advanced ALK -positive lung cancer.N Engl J Med. 2013; 368: 2385-2394Crossref PubMed Scopus (2753) Google Scholar, 4Ou S.I. Bartlett C.H. Mino-Kenudson M. Cui J. Iafrate A.J. Crizotinib for the treatment of ALK -rearranged non-small cell lung cancer: a success story to usher in the second decade of molecular targeted therapy in oncology.Oncologist. 2012; 17: 1351-1375Crossref PubMed Scopus (181) Google Scholar ROS1,5Shaw A.T. Ou S.H. Bang Y.J. et al.Crizotinib in ROS1-rearranged non-small-cell lung cancer.N Engl J Med. 2014; 371: 1963-1971Crossref PubMed Scopus (1341) Google Scholar RET,6Drilon A. Oxnard G.R. Tan D.S.W. et al.Efficacy of Selpercatinib in RET fusion–positive non–small-cell lung cancer.N Engl J Med. 2020; 383: 813-824Crossref PubMed Scopus (242) Google Scholar and NTRK7Mullard A. FDA approves landmark tissue-agnostic cancer drug.Nat Rev Drug Discov. 2018; 18: 7Google Scholar can benefit from targeted therapies. Accurate molecular diagnoses of these gene fusions are critical before receiving target-specific inhibitor treatments. Traditional methods for diagnosing gene fusions include fluorescence in situ hybridization and immunohistochemistry (IHC). In addition, the U.S. Food and Drug Administration (FDA) has approved next-generation sequencing (NGS)–based assays such as FoundationOne CDx8FoundationOne CDx.https://www.accessdata.fda.gov/cdrh_docs/pdf17/P170019a.pdfDate accessed: December 4, 2020Google Scholar (Foundation Medicine, Cambridge, MA) and MSK Solid Fusion9Food and Drug AdministrationMSK IMPACT FDA unveils a streamlined path for the authorization of tumor profiling tests alongside its latest product action.https://www.fda.gov/news-events/press-announcements/fda-unveils-streamlined-path-authorization-tumor-profiling-tests-alongside-its-latest-product-actionDate accessed: December 4, 2020Google Scholar,10New York State Department of HealthMemorial Hosp for Cancer and Allied Diseases Dept of Pathology.https://www.wadsworth.org/memorial-hosp-for-cancer-and-allied-diseases-dept-of-pathology-75Date accessed: December 4, 2020Google Scholar (Memorial Sloan Kettering Cancer Center, New York, NY) for the diagnosis of gene fusion using tissue specimens. More recently, the FDA-approved “liquid biopsy” NGS assays for gene fusion detection using DNA extracted from blood samples, including Guardant360 CDx11GuardantVol. 360:CDx.https://www.accessdata.fda.gov/cdrh_docs/pdf20/P200010C.pdfDate accessed: December 4, 2020Google Scholar (Guardant, Redwood City, CA) and FoundationOne Liquid CDx (Foundation Medicine).12Food and Drug AdministrationFoundationOne Liquid C. Dx FDA approves liquid biopsy NGS companion diagnostic test for multiple cancers and biomarkers.https://www.fda.gov/drugs/fda-approves-liquid-biopsy-ngs-companion-diagnostic-test-multiple-cancers-and-biomarkersDate accessed: December 4, 2020Google Scholar Furthermore, new NGS assays for fusion detection are on the horizon, such as the ArcherDX assay (ArcherDX, Boulder, CO), which uses the anchored multiplex polymerase chain reaction13Zheng Z. Liebers M. Zhelyazkova B. et al.Anchored multiplex PCR for targeted next-generation sequencing.Nat Med. 2014; 20: 1479-1484Crossref PubMed Scopus (549) Google Scholar method for enriching DNA and RNA from both blood and tissue samples and has received FDA Breakthrough Device designation.14Archer D.X. Receives breakthrough device designation to detect NTRK gene fusions.https://www.prnewswire.com/news-releases/archerdx-receives-breakthrough-device-designation-to-detect-ntrk-gene-fusions-301061603.htmlDate accessed: December 4, 2020Google Scholar Thus, DNA-based NGS assays for gene fusion detection are expected to be widely used. However, the increasingly reported uncommon gene fusions in the catalogs of ALK,15Ou S.H. Zhu V.W. Nagasaka M. Catalog of 5’ Fusion Partners in ALK-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100015Abstract Full Text Full Text PDF PubMed Google Scholar ROS1,16Ou S.H. Nagasaka M. A catalog of 5’ Fusion Partners in ROS1-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100048Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar and RET17Ou S.H. Zhu V.W. Catalog of 5′ fusion partners in RET+ NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100037Abstract Full Text Full Text PDF Scopus (20) Google Scholar fusions that are detected by DNA-based NGS assays have raised questions on their clinical significances and underlying mechanisms and called for the use of reliable molecular testing methods before initiating targeted therapy. In the current issue, Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar report the comprehensive molecular profiling of ALK, ROS1, and RET fusions in a total of 3787 NSCLC cases using targeted DNA NGS, targeted RNA NGS, whole transcriptome sequencing (WTS), and IHC; and address the clinic significance of uncommon gene fusions detected at the genomic DNA level. Among the 1171 cases in Cohort A, negative cases (n = 140) by conventional amplification-refractory mutation system–polymerase chain reaction and hybridization-based DNA NGS assays were subsequently subjected to targeted RNA NGS analyses, resulting in the identification of 10% (n = 14) of the cases harboring actionable genetic alterations. The percentage of positive targeted RNA NGS findings among DNA NGS–negative cases (10%) is consistent with an earlier study by Benayed et al.,19Benayed R. Offin M. Mullaney K. et al.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 (180) Google Scholar in which an RNA NGS assay on the basis of amplification-refractory mutation system–polymerase chain reaction13Zheng Z. Liebers M. Zhelyazkova B. et al.Anchored multiplex PCR for targeted next-generation sequencing.Nat Med. 2014; 20: 1479-1484Crossref PubMed Scopus (549) Google Scholar (MSK Solid Fusion20Zehir A. Benayed R. Shah R.H. et al.Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.Nat Med. 2017; 23: 703-713Crossref PubMed Scopus (1652) Google Scholar) identified actionable in-frame fusions in 14% (33 of 232) of targeted DNA NGS (MSK-IMPACT [Molecular Diagnostics Service and Marie-Josee and Herny R. Kravis Center for Molecular Oncology]21Cheng D.T. Mitchell T. Zehir A. et al.Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology.J Mol Diagn. 2015; 17: 251-264Abstract Full Text Full Text PDF PubMed Scopus (1063) Google Scholar)–negative cases. Interestingly, both studies found that ROS1 was the most frequently missed gene by DNA NGS assays, consisting of about 27% of all DNA NGS–negative and RNA NGS–positive cases (Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar: four of 14; Benayed et al.19Benayed R. Offin M. Mullaney K. et al.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 (180) Google Scholar: 10 of 33). The explanations for high false-negative results of ROS1 fusion associated with DNA NGS assays include the following: (1) a large size genomic region in which breakpoints occur recurrently (ROS1 introns 31 to 35: 15,777 bases; ALK intron 19: 1933 bases); hence, more capture probes are needed; (2) a low guanine and cytosine content of the capturing region (guanine and cytosine% ROS1 introns 31 to 35: 37.9%; ALK intron 19: 51.7%) that leads to suboptimal probe hybridization thermodynamics; hence, low sequencing data coverages and high failure rates in capturing probe design; and (3) a low sequence diversity (at least associated with the high AT repeats) at breakpoints associated with uncertain sequence alignments to reference genome, hence no calls. The challenges in ROS1 fusion detection might have led to underreported ROS1 gene fusions in NSCLC and contributed to the observation that fewer gene fusion partners have been cataloged for ROS1 than for ALK and RET.16Ou S.H. Nagasaka M. A catalog of 5’ Fusion Partners in ROS1-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100048Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar With the rapid adoption of NGS in clinic, an ever-expanding list of uncommon gene fusions involving ALK,15Ou S.H. Zhu V.W. Nagasaka M. Catalog of 5’ Fusion Partners in ALK-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100015Abstract Full Text Full Text PDF PubMed Google Scholar ROS1,16Ou S.H. Nagasaka M. A catalog of 5’ Fusion Partners in ROS1-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100048Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar and RET17Ou S.H. Zhu V.W. Catalog of 5′ fusion partners in RET+ NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100037Abstract Full Text Full Text PDF Scopus (20) Google Scholar fusions have been reported. It is a pressing question on the clinical significance of these uncommon fusions when they are detected at the DNA level, yet without accompanying common/recurrent driver gene fusions in the same tumors. Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar reported 16 such DNA NGS detected uncommon ALK fusion cases (two cases reported as reciprocal fusions and harbored dual 5′ ALK and 3′ ALK with uncommon fusion partners) having available RNA NGS data. Among them, five (31%) cases were negative by both RNA NGS and IHC; seven (44%) cases actually harbored the typical EML4-ALK fusions by RNA NGS; and the remaining four cases exhibited consistent fusion partners (KIF5B/KLC1/TPM3) between DNA and RNA NGS assays. For RET uncommon fusions, all six cases with available RNA NGS results exhibited typical RET fusion partners (KIF5B/CCDC6), whereas the DNA NGS all pointed to different fusion partners. For ROS1 uncommon fusions, one of four cases was negative by RNA NGS assay. Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar conducted WTS on cases with 5′ ALK reciprocal fusions. Consistent with their targeted RNA NGS results, WTS detected EML4-ALK transcripts and did not detect reciprocal ALK fusion transcripts. ALK and RET are not expressed in normal lung tissue (transcript per million: ALK 0.08, RET 0.47),22G.T. Ex portalhttps://gtexportal.org/home/gene/ALKDate accessed: December 4, 2020Google Scholar,23G.T. Ex portalhttps://gtexportal.org/home/gene/RETDate accessed: December 4, 2020Google Scholar whereas ROS1 is (transcript per million: 11.09).24G.T. Ex portalhttps://gtexportal.org/home/gene/ROS1Date accessed: December 4, 2020Google Scholar ALK kinase is expressed only when fused to an active promoter such as 5′ EML4, making IHC an effective approach for ALK fusion detection with an FDA-approved assay.25Ventana ALK (D5F3) Vol. CDx. Assay.https://www.accessdata.fda.gov/cdrh_docs/pdf14/P140025a.pdfDate accessed: December 5, 2020Google Scholar Given the high expression of ROS1 in normal lung tissue, no high-performing IHC assay for ROS1 fusion detection is available. WTS is a powerful discovery tool; however, the associated low specificities (e.g., 37% of the identified 25,664 fusions could not be validated in genomic data26Gao Q. Liang W.W. Foltz S.M. et al.Driver fusions and their implications in the development and treatment of human cancers.Cell Rep. 2018; 23 (e3): 227-238Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) requires functional validations and high-performing bioinformatics solutions before clinical translations. Furthermore, WTS requires high quality samples that might not be widely accessible clinically. For example, Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar reported that assay success rates for WTS and targeted RNA NGS were 37% (seven of 19) and 94% (85 of 90), respectively. It is interesting that 44% of uncommon (non-EML4 partner) ALK fusions detected at the DNA level expressed EML4-ALK transcripts. As Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar suggested, chromothripsis could be one explanation for the difference in fusion partners detected by DNA versus RNA NGS assays, after RNA splicing of complex gene rearrangements. It has been noted that most of the uncommon ALK and RET fusion partners reported by DNA NGS are in chromosomal proximity to ALK15Ou S.H. Zhu V.W. Nagasaka M. Catalog of 5’ Fusion Partners in ALK-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100015Abstract Full Text Full Text PDF PubMed Google Scholar and RET,17Ou S.H. Zhu V.W. Catalog of 5′ fusion partners in RET+ NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100037Abstract Full Text Full Text PDF Scopus (20) Google Scholar respectively, whereas ROS116Ou S.H. Nagasaka M. A catalog of 5’ Fusion Partners in ROS1-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100048Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar fusion partners do not follow the same pattern. Fusions preferentially clustered intrachromosome than interchromosome (Fig. 1A [top] and B [top]). In tumors with both primary (driver fusion retaining 3′ kinase domain) and reciprocal (retaining 5′ ALK/ROS1/RET) fusions, the reciprocal fusions are presumably “passenger fusions” yet remain predominantly intrachromosomal (Fig. 1A [bottom] and B [bottom]), reflecting likely direct joining of double strand breaks in close proximity by means of the nonhomologous end joining pathway.27Chiarle R. Translocations in normal B cells and cancers. Insights from new technical approaches.Adv Immunol. 2013; 117: 39-71Crossref Scopus (7) Google ScholarFigure 1(A) Chromosomal distribution of fusion partners in ALK-positive NSCLC. The pie charts represent proportions of different chromosomes in which distinct gene fusion partners are (top) in the fusion group with 3′ ALK kinase domain reported from Ou et al.15Ou S.H. Zhu V.W. Nagasaka M. Catalog of 5’ Fusion Partners in ALK-positive NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100015Abstract Full Text Full Text PDF PubMed Google Scholar and Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar and (bottom) in the reciprocal fusion group retaining 5′ ALK in tumors harboring both primary and reciprocal fusions. The p values were derived from chi-square tests for a nondifferential distribution of the fusion partners in Chr2 versus other chromosomes, weighted with chromosome sizes. The ideogram (right) depicts the locations of fusion partners in Chr2 of the above two groups. (B) Chromosomal distribution of fusion partners in RET-positive NSCLC. The pie charts represent proportions of different chromosomes in which distinct gene fusion partners are (top) in the fusion group with 3′ RET kinase domain reported from Ou et al.17Ou S.H. Zhu V.W. Catalog of 5′ fusion partners in RET+ NSCLC circa 2020.JTO Clin Res Rep. 2020; 1: 100037Abstract Full Text Full Text PDF Scopus (20) Google Scholar and Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar; and (bottom) in the reciprocal fusion group retaining 5′ RET in tumors harboring both primary and reciprocal fusions. P values were derived from chi-square tests for a nondifferential distribution of the fusion partners in Chr10 versus other chromosomes, weighted with chromosome sizes. The ideogram (right) depicts the locations of fusion partners in Chr10 of the above two groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar presented important data revealing that 31% (five of 16) of uncommon (non-EML4 partner) ALK fusions detected at the DNA level were fusion negative by targeted RNA NGS and IHC. Patients in this group had a significantly shorter progression-free survival after crizotinib treatment compared with patients in the RNA NGS or IHC–confirmed ALK fusion group (2.0 mo [95% confidence interval: 1.2–2.8] versus 11.0 mo [95% confidence interval: 8.9–13.1]). The same research group previously also reported the discrepancy in fusions with intergenic breakpoints identified by DNA sequencing versus RNA sequencing.28Li W. Liu Y. Li W. Chen L. Ying J. Intergenic breakpoints identified by DNA sequencing confound targetable kinase fusion detection in NSCLC.J Thorac Oncol. 2020; 15: 1223-1231Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar An earlier study reported that nonreciprocal/reciprocal ALK translocation, as compared with 3′ ALK fusion alone, was associated with worse progression-free survival and greater likelihood of baseline brain metastases in patients with ALK-rearranged NSCLC who received first-line crizotinib.29Zhang Y. Zeng L. Zhou C. et al.Detection of nonreciprocal/reciprocal ALK translocation as poor predictive marker in patients with first-line crizotinib-treated ALK-rearranged NSCLC.J Thorac Oncol. 2020; 15: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar However, as the authors acknowledged, one limitation was the lack of RNA confirmation for the DNA NGS fusion results. The data presented in this issue by Li et al.18Li W. Guo L. Liu Y. et al.Potential unreliability of uncommon ALK, ROS1, and RET genomic breakpoints in predicting the efficacy of targeted therapy in NSCLC.J Thorac Oncol. 2021; 16: 404-418Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar and previously published studies19Benayed R. Offin M. Mullaney K. et al.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 (180) Google Scholar,20Zehir A. Benayed R. Shah R.H. et al.Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.Nat Med. 2017; 23: 703-713Crossref PubMed Scopus (1652) Google Scholar,30Lindeman N.I. Cagle P.T. Aisner D.L. et al.Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.J Thorac Oncol. 2018; 13: 323-358Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar,31Marchiò C. Scaltriti M. Ladanyi M. et al.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 (175) Google Scholar unanimously call for orthogonal molecular diagnosis methods in addition to DNA-based assays in the detection of gene fusions in NSCLC. This work was supported by grants from The Swedish Research Council ( 2016-02830 to Z.Z.), the National Natural Science Foundation of China ( 81672098 to Z.Z. and to 81802276 Z.S.), and the Lau Grant, Ming Wai Lau Centre of Reparative Medicine, Karolinska Institutet (to Z.Z.). Potential Unreliability of Uncommon ALK, ROS1, and RET Genomic Breakpoints in Predicting the Efficacy of Targeted Therapy in NSCLCJournal of Thoracic OncologyVol. 16Issue 3PreviewVariable genomic breakpoints have been identified through the application of target-capture DNA next-generation sequencing (NGS) for ALK, ROS1, and RET fusion detection in NSCLC. We investigated whether ALK, ROS1, and RET genomic breakpoint location can predict matched targeted therapy efficacy. Full-Text PDF Open Access