ORIGINAL ARTICLE Multiplex Diagnosis of Oncogenic Fusion and MET Exon Skipping by Molecular Counting Using Formalin-Fixed Paraffin Embedded Lung Adenocarcinoma Tissues Kuniko Sunami, MD,a,g Koh Furuta, MD, PhD,b Koji Tsuta, MD, PhD,c Shinji Sasada, MD, PhD,d Takehiro Izumo, MD, PhD,d Takashi Nakaoku, MD,a Yoko Shimada, MFSc,a Motonobu Saito, MD, PhD,a Hiroshi Nokihara, MD, PhD,e Shun-ichi Watanabe, MD, PhD,f Yuichiro Ohe, MD, PhD,e,g Takashi Kohno, PhDa,* aDivision of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan bDivision of Clinical Laboratory, National Cancer Center Hospital, Tokyo, Japan cDivision of Pathology, National Cancer Center Research Institute, Tokyo, Japan dDepartment of Endoscopy, Respiratory Endoscopy Division, National Cancer Center Research Institute, Tokyo, Japan eDepartment of Thoracic Oncology, National Cancer Center Research Institute, Tokyo, Japan fDepartment of Thoracic Surgery, National Cancer Center Hospital, Tokyo, Japan gCourse of Advanced Clinical Research of Cancer, Juntendo University Graduate School of Medicine, Tokyo, Japan Received 31 July 2015; revised 23 September 2015; accepted 13 October 2015 ABSTRACT detected oncogenic fusions in bronchial lavage fluid and transbronchial biopsy samples. Introduction: Fusions of the anaplastic lymphoma receptor tyrosine kinase gene (ALK), ret proto-oncogene (RET), ROS Conclusions: The MC assay allows multiplex detection of proto-oncogene 1, receptor tyrosine kinase gene (ROS1), B- oncogenic fusion and exon-skipped transcripts in tumor Raf proto-oncogene, serine/threonine kinase gene (BRAF), samples, including in formalin-fixed paraffin-embedded and neuregulin 1 gene (NRG1) and intronic MMNG HOS samples obtained in the clinic. Transforming gene (MET) mutations are druggable onco- Ó 2015 International Association for the Study of Lung gene alterations in lung adenocarcinoma that cause Cancer. Published by Elsevier Inc. All rights reserved. expression of aberrant transcripts. Because these aberrant transcripts are both infrequent (incidence <5%) and mutually exclusive, multiplex assays are required to detect Keywords: Personalized medicine; Molecular counting; them in tumor samples. Oncogene fusion; MET exon skipping; Genetic diagnosis Methods: Aberrant transcripts of the six aforementioned oncogenes (36 transcripts in total) were examined in a Introduction molecular counting (MC) assay, which counts RNA mole- Analyses of lung adenocarcinoma (LADC) genomes cules by simultaneous hybridization of several probes. fi Forty-one samples of surgically resected lung adenocarci- have identi ed several oncogene alterations that cause noma tissue found to express one of these aberrant onco- expression of aberrant transcripts in tumor cells. A genic transcripts upon whole transcriptome sequencing representative example is the fusion of oncogenes to (test cohort: n ¼ 22) or reverse transcription polymerase chain reaction (validation cohort: n ¼ 19) analyses were subjected to MC, after which biopsies were performed on *Corresponding author. tumor tissue samples. Disclosure: The authors declare no conflicts of interest. Results: Address for correspondence: Takashi Kohno, PhD, Division of Genome Threshold values for the diagnosis of each of the Biology, National Cancer Center Research Institute, 1-1 Tsukiji 5- 36 transcripts were determined in frozen and formalin- chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail: [email protected] fixed paraffin-embedded samples from the test cohort. On ª 2015 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. the basis of these threshold values, the MC assay diagnosed ISSN: 1556-0864 expression of oncogenic transcripts in the validation cohort http://dx.doi.org/10.1016/j.jtho.2015.10.005 samples with 100% accuracy. The assay also accurately Journal of Thoracic Oncology Vol. 11 No. 2: 203-212 204 Sunami et al Journal of Thoracic Oncology Vol. 11 No. 2 other partner genes, which causes constitutive activation multiplex assays allowing simultaneous identification of of protein kinases encoded by these oncogenes.1,2 These several alterations are required. Furthermore, in contrast alterations are a mutually exclusive driver mutation in to hot spot mutations such as EGFR, Kirsten rat sarcoma lung carcinogenesis and are a target for therapies based viral gene homolog gene (KRAS), and BRAF (which acti- on protein tyrosine kinase inhibitors (TKIs). A well- vate oncogenes in tumor cell), the presence of highly known example is the oncogenic epidermal growth diverse genomic fusion points located within intron factor receptor gene (EGFR) mutation, which is a target sequences hampers the detection of gene fusions by for therapy using EGFR-TKIs. Indeed, LADCs harboring multiplex genome polymerase chain reaction (PCR)- the anaplastic lymphoma receptor tyrosine kinase gene based assays using next-generation sequencers.20 In (ALK) oncogene fusion (in 3%–5% of all cases of LADC) addition, identifying the intronic mutations responsible respond very well to ALK inhibitors such as crizotinib,3 for MET exon skipping using genomic DNA is difficult ceritinib,4 and alectinib.5 Ret proto-oncogene (RET) and because of their highly diverse locations and the occur- ROS proto-oncogene 1, receptor tyrosine kinase gene rence of passenger mutations.21 Multiplex reverse tran- (ROS1) oncogene fusions are also rare (each occurring in scription (RT)-PCR–based assays using tumor RNA 1%–3% of cases of LADC6–10), but they are a promising are often used to detect aberrant transcripts caused by target for therapy with TKIs. Several clinical trials gene fusions and exon skipping because there are focusing on RET and ROS1 fusion–positive LADC have few aberrant transcript patterns, despite the diversity been undertaken, and the results of these studies have with respect to the location of the responsible mutations. recently been reported.11,12 In addition, there is a report However, the poor quality of the RNA obtained from of single patient with RET fusion–positive LADC who FFPE tumor tissues, particularly from archived tissues, responded to RET TKI.13 These reports indicate the makes it difficult to develop reliable RT-PCR–based therapeutic effects of RET and ROS1 TKIs. Recently, we multiplex assays. and others identified B-Raf proto-oncogene, serine/ Molecular counting (MC) using the nCounter system threonine kinase gene (BRAF) and neuregulin 1 gene (NanoString Technologies, Seattle, WA) is a method of (NRG1) fusions as another therapeutic target in quantifying RNA that is not based on RT and PCR; this approximately 10% of patients with invasive mucinous method directly counts RNA molecules by means of LADC.14,15 Recent studies have also identified another simultaneous hybridization of multiple probes. Indeed, type of oncogene alteration that generates aberrant previous studies have demonstrated the feasibility of oncogenic transcripts in approximately 3% of LADCs: this method for quantifying RNA expression and skipping of the coding exon 14 in MMNG HOS Trans- detecting gene fusions.22,23 Here, we used this method to forming gene (MET) oncogene transcripts on account of detect 36 oncogenic transcripts, 35 of which were aberrant splicing, which is associated mainly with derived from five oncogenes (ALK, RET, ROS1, NRG1, and intronic mutations.16,17 The skipped transcript produces BRAF) fused to partner genes and the aforementioned a constitutively active MET protein lacking the E3 MET exon–skipped transcript above. To examine the ubiquitin protein ligase (CBL) binding domain that feasibility of this method, RNA samples obtained from negatively regulates MET kinase. Indeed, in recent re- 62 LADC tissue samples from 41 patients (39 FFPE and ports, a MET TKI showed antitumor activity in patients 23 snap-frozen tissue samples in total; Fig. 1B) were with LADCs expressing exon 14–skipped MET tran- subjected to MC. These samples were selected from scripts,18,19 thus suggesting that this alteration is also a 608 consecutive patients with LADC patients because promising therapeutic target. whole RNA sequencing or RT-PCR analyses of snap- Precision medicine for patients with LADC on the frozen tissues revealed that they either did or did not basis of the TKIs described earlier requires the diagnosis express aberrant oncogenic transcripts. of multiplex oncogene aberrations in tumor cells. Because the tumors of most patients subjected to TKI therapy are inoperable, diagnosis must be performed by using small Methods amounts of fixed formalin-fixed and paraffin-embedded Subjects (FFPE) biopsy samples or archived surgical specimens Consecutive patients with LADC who underwent for histopathological examination. In fact, techniques surgical resection between 1997 and 2008 at the Na- such as fluorescence in situ hybridization, which detects tional Cancer Center Hospital in Tokyo (n ¼ 608) were gross chromosome rearrangements, are often used to screened for EGFR, KRAS, BRAF, and HER2 hot spot diagnose gene fusions in such clinical specimens. How- mutations by the high-resolution melting method using ever, the recent discovery of a number of uncommon but genomic DNA obtained from snap-frozen tumor tis- druggable oncogene fusions in LADC, along with the sues.24 RT-PCR of RNA obtained from snap-frozen tumor finding that they are mutually exclusive, means that tissues as described previously was used to examine the February 2016 Multiplex Diagnosis of Oncogenic Fusions 205 Figure 1. (A) Selection of study subjects. From 608 consecutive patients