Breaking Down RET Breakpoints in Lung Adenocarcinoma
Authors: Hideo Watanabe, Angela N. Brooks, Matthew Meyerson
Journal: Journal of Thoracic Oncology (2014)

In 1960, the first recurrent chromosomal rearrangement in human malignancies was identified by Nowell and Hungerford in chronic myelogenous leukemia and described as the Philadelphia chromosome, which is currently recognized as the BCR-ABL translocation. The activated ABL tyrosine kinase, the fusion protein product from this rearrangement, also became the first defined molecular target against which a tyrosine kinase inhibitor was successfully developed.1Druker BJ Talpaz M Resta DJ et al.Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.N Engl J Med. 2001; 344: 1031-1037Crossref PubMed Scopus (4445) Google Scholar Since the introduction of chromosomal banding techniques in the 1970s, which enabled easier detection of these chromosomal rearrangements, numerous recurrent rearrangements have been reported across many types of hematopoietic malignancies and in solid tumors. In fact, recurrent RET rearrangements in papillary thyroid carcinomas were the first discovered chromosomal translocations involving a tyrosine kinase in solid tumors.2Takahashi M Ritz J Cooper GM Activation of a novel human transforming gene, RET, by DNA rearrangement.Cell. 1985; 42: 581-588Abstract Full Text PDF PubMed Scopus (606) Google Scholar,3Fusco A Grieco M Santoro M et al.A new oncogene in human thyroid papillary carcinomas and their lymph-nodal metastases.Nature. 1987; 328: 170-172Crossref PubMed Scopus (352) Google Scholar In lung cancer, there has been a recent profusion of new activating gene fusion discoveries, beginning with the discoveries of EML4-ALK fusion genes and ROS1 fusions.4Soda M Choi YL 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 (4302) Google Scholar,5Rikova K Guo A Zeng Q et al.Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.Cell. 2007; 131: 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (1914) Google Scholar Recently, with the advent of systematic genome-wide sequencing technologies, chromosomal rearrangements of other tyrosine kinases such as RET,6Takeuchi K Soda M Togashi Y et al.RET, ROS1 and ALK fusions in lung cancer.Nat Med. 2012; 18: 378-381Crossref PubMed Scopus (1037) Google Scholar, 7Lipson D Capelletti M Yelensky R et al.Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies.Nat Med. 2012; 18: 382-384Crossref PubMed Scopus (714) Google Scholar, 8Kohno T Ichikawa H Totoki Y et al.KIF5B-RET fusions in lung adenocarcinoma.Nat Med. 2012; 18: 375-377Crossref PubMed Scopus (666) Google Scholar, 9Ju YS Lee WC Shin JY et al.A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing.Genome Res. 2012; 22: 436-445Crossref PubMed Scopus (408) Google Scholar NTRK1,10Vaishnavi A Capelletti M Le AT et al.Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer.Nat Med. 2013; 19: 1469-1472Crossref PubMed Scopus (462) Google Scholar FGFR3,11Kim Y Hammerman PS Kim J et al.Integrative and comparative genomic analysis of lung squamous cell carcinomas in East Asian patients.J Clin Oncol. 2014; 32: 121-128Crossref PubMed Scopus (153) Google Scholar, 12Majewski IJ Mittempergher L Davidson NM et al.Identification of recurrent FGFR3 fusion genes in lung cancer through kinome-centred RNA sequencing.J Pathol. 2013; 230: 270-276Crossref PubMed Scopus (97) Google Scholar, 13Wu YM Su F Kalyana-Sundaram S et al.Identification of targetable FGFR gene fusions in diverse cancers.Cancer Discov. 2013; 3: 636-647Crossref PubMed Scopus (541) Google Scholar and NRG1 ligand fusions14Fernandez-Cuesta L, Plenker D, Osada H, et al. CD74-NRG1 fusions in lung adenocarcinoma. Cancer Discov. In pressGoogle Scholar have been identified. With many ongoing preclinical and clinical studies to test the efficacy of kinase inhibitors for such aberrantly activated molecules, a rapid introduction of accurate and sensitive diagnosis of those chromosomal rearrangements has become increasingly important to identify patients who may benefit from these therapeutics. Current techniques to detect these chromosomal rearrangements include a break-apart fluorescence in situ hybridization (FISH) assay, a fusion FISH assay on unstained tissue slides, and reverse-transcriptase polymerase chain reaction (RT-PCR) on RNA samples from fresh frozen tissue specimens. In addition, targeted sequencing using hybrid capture followed by next-generation sequencing on DNA samples has been used as a detection method (See Table 1 for details).TABLE 1Description of Methods Used to Discover Chromosomal Rearrangements Between Two Hypothetical Genes, Gene A and Gene BMethodTarget Nucleic AcidApproach to Detect a RearrangementBreak-apart fluorescence in situ hybridization (FISH)DNAHybridization probes corresponding to the 5'-end of gene A and the 3'-end of gene A are detected in separate chromosomal locationsFusion FISHDNAA red-color probe corresponding to gene A and a green-color probe corresponding to gene B are detected at the same locus and visualized as a merged yellow colorRT-PCRRNAA forward primer corresponding to the 5'-end of a transcript for gene A and a reverse primer corresponding to the 3'-end of a transcript for gene B will only generate a PCR product when the fusion existsCapture followed by next-generation sequencingDNACustomized probes corresponding to targeted exons and introns of gene A and/or gene B are used to capture DNA only from these segments and then subjected to next-generation sequencing. Rearrangements are detected computationally from sequencing readsRT-PCR, reverse-transcriptase polymerase chain reaction. Open table in a new tab RT-PCR, reverse-transcriptase polymerase chain reaction. The diagnosis of RET fusions is of increasing potential clinical importance because response to cabozantinib has already been reported in some patients,15Drilon A Wang L Hasanovic A et al.Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas.Cancer Discov. 2013; 3: 630-635Crossref PubMed Scopus (410) Google Scholar and numerous small molecule RET inhibitors are currently under investigation. Analyzing fusion breakpoints not only can lead to a better understanding of the molecular mechanisms that generate these rearrangements but also can help in the design of better detection methods. The study by Mizukami et al.16Mizukami T Shiraishi K Shimada Y et al.Molecular mechanisms underlying oncogenic RET fusion in lung adenocarcinoma.J Thorac Oncol. 2014; 9: 622-630Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar in this issue describes the structures of breakpoint junctions involving the RET oncogene of 16 lung adenocarcinoma samples. Breakpoints from 14 samples with documented KIF5B-RET fusion transcripts by RT-PCR were identified using targeted genomic PCR followed by Sanger sequencing. Breakpoints from two samples with documented CCDC6-RET fusions by break-apart and fusion FISH assays were identified using targeted next-generation sequencing, with capture probes targeting the exons and introns spanning across exons 7 to 12 of the RET gene. This study also analyzed two previously published DNA sequences from lung adenocarcinomas with rearrangements involving the RET gene. The authors’ findings are in concordance with previous studies that observed genomic breakpoints in a few confined genomic regions spanning kilobases, but not at specific genomic loci, and without significant enrichment of nucleotide motifs or chromatin features that would make them susceptible to breaks.17Reiter A Saussele S Grimwade D et al.Genomic anatomy of the specific reciprocal translocation t(15;17) in acute promyelocytic leukemia.Genes Chromosomes Cancer. 2003; 36: 175-188Crossref PubMed Scopus (53) Google Scholar, 18Chen W Kalscheuer V Tzschach A et al.Mapping translocation breakpoints by next-generation sequencing.Genome Res. 2008; 18: 1143-1149Crossref PubMed Scopus (111) Google Scholar, 19Xiao Z Greaves MF Buffler P et al.Molecular characterization of genomic AML1-ETO fusions in childhood leukemia.Leukemia. 2001; 15: 1906-1913Crossref PubMed Scopus (41) Google Scholar, 20Wiemels JL Leonard BC Wang Y et al.Site-specific translocation and evidence of postnatal origin of the t(1;19) E2A-PBX1 fusion in childhood acute lymphoblastic leukemia.Proc Natl Acad Sci U S A. 2002; 99: 15101-15106Crossref PubMed Scopus (96) Google Scholar Efforts to analyze these breakpoints at the sequence level have identified relatively consistent reciprocal rearrangements with small insertion or deletion of a few base pairs in chemotherapy-related leukemia or radiation-induced papillary thyroid carcinomas, whereas broader range stretches of DNA insertion, deletion, or duplication have been observed in spontaneous cancers.21Bongarzone I Butti MG Fugazzola L et al.Comparison of the breakpoint regions of ELE1 and RET genes involved in the generation of RET/PTC3 oncogene in sporadic and in radiation-associated papillary thyroid carcinomas.Genomics. 1997; 42: 252-259Crossref PubMed Scopus (48) Google Scholar,22Nikiforov YE Koshoffer A Nikiforova M Stringer J Fagin JA Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas.Oncogene. 1999; 18: 6330-6334Crossref PubMed Scopus (90) Google Scholar Mizukami et al. further analyzed the breakpoint sequences to infer which DNA repair mechanisms were involved to illegitimately rejoin the DNA ends of fusion partners and found that while many of the events are reciprocal inversions, there are cases of nonreciprocal rearrangements. Unfortunately, the authors were unable to analyze sequences on the other end of nonreciprocal breakpoint junctions, as captured-based sequencing data for these cases were not available. Nevertheless, these findings have significant implication to the interpretation of results from break-apart FISH assays in a clinical setting. Similar to the cases of EML4-ALK fusions,23Dai Z Kelly JC Meloni-Ehrig A et al.Incidence and patterns of ALK FISH abnormalities seen in a large unselected series of lung carcinomas.Mol Cytogenet. 2012; 5: 44Crossref PubMed Scopus (32) Google Scholar their results suggest it is of positive diagnostic value to only detect a probe corresponding to the 3′ end of the RET gene. This study identified diverse DNA repair mechanisms for breaks. In most cases, a lack of sequence homology at breakpoint junctions implicated nonhomologous end joining as the repair mechanism. In four cases with reciprocal rearrangements, duplication of sequences at both ends of the breakpoint junctions implicated break-induced replication repair. This is in contrast to repair mechanisms observed in papillary thyroid carcinomas, where nonhomologous end joining has only been observed.21Bongarzone I Butti MG Fugazzola L et al.Comparison of the breakpoint regions of ELE1 and RET genes involved in the generation of RET/PTC3 oncogene in sporadic and in radiation-associated papillary thyroid carcinomas.Genomics. 1997; 42: 252-259Crossref PubMed Scopus (48) Google Scholar,22Nikiforov YE Koshoffer A Nikiforova M Stringer J Fagin JA Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas.Oncogene. 1999; 18: 6330-6334Crossref PubMed Scopus (90) Google Scholar Although the exact factors that cause DNA breaks or choice of DNA repair pathways are unknown, the difference in repair mechanisms between lung adenocarcinoma and papillary thyroid carcinomas may be explained by a difference in carcinogenic insults in these two cohorts. Evidence of break-induced replication repair in lung adenocarcinoma suggests single-stranded breaks occur in addition to double-stranded breaks; however, there was no difference in features of breaks or DNA repair mechanisms between never-smokers and ever-smokers; therefore, the role of smoking-related carcinogens in the genesis of RET fusions is unclear. The work by Mizukami et al. provides rich information on breakpoints involving the RET oncogene. The information provided by Mizukami et al. should aid in the design of more sensitive molecular detection of these rearrangements in lung adenocarcinoma samples. The development of sensitive methods to detect rearrangements for DNA samples is particularly important for cases where RNA is not available or is of low quality. However, while many rearrangements are observed within relatively confined genomic regions, this study and other studies have shown that rearrangements do not occur at recurrent breakpoint positions nor retain specific sequence features characteristics of a single repair mechanism. This highlights the challenge for designing genomic PCR-based methods to cover all probable breakpoints, unless highly multiplexed. Therefore, this study implies that unbiased approaches using next-generation sequencing, including whole genome sequencing, sequencing after capture of selected regions of RNA or DNA encompassing the relevant breakpoints in RET, or transcriptome sequencing of RNA may be the best methodologies for the detection of RET chromosomal rearrangements in lung adenocarcinoma.