Published OnlineFirst September 28, 2016; DOI: 10.1158/1078-0432.CCR-16-1679 Personalized Medicine and Imaging Clinical Cancer Research RET Aberrations in Diverse Cancers: Next-Generation Sequencing of 4,871 Patients Shumei Kato1, Vivek Subbiah2, Erica Marchlik3, Sheryl K. Elkin3, Jennifer L. Carter3, and Razelle Kurzrock1 Abstract Purpose: Aberrations in genetic sequences encoding the tyrosine (52/88)], cell cycle–associated genes [39.8% (35/88)], the PI3K kinase receptor RET lead to oncogenic signaling that is targetable signaling pathway [30.7% (27/88)], MAPK effectors [22.7% with anti-RET multikinase inhibitors. Understanding the compre- (20/88)], or other tyrosine kinase families [21.6% (19/88)]. hensive genomic landscape of RET aberrations across multiple RET fusions were mutually exclusive with MAPK signaling cancers may facilitate clinical trial development targeting RET. pathway alterations. All 72 patients harboring coaberrations Experimental Design: We interrogated the molecular portfolio had distinct genomic portfolios, and most [98.6% (71/72)] of 4,871 patients with diverse malignancies for the presence of had potentially targetable coaberrations with either an FDA- RET aberrations using Clinical Laboratory Improvement Amend- approved or an investigational agent. Two cases with lung ments–certified targeted next-generation sequencing of 182 or (KIF5B-RET) and medullary thyroid carcinoma (RET M918T) 236 gene panels. thatrespondedtoavandetanib(multikinase RET inhibitor)- Results: Among diverse cancers, RET aberrations were iden- containing regimen are shown. tified in 88 cases [1.8% (88/4, 871)], with mutations being Conclusions: RET aberrations were seen in 1.8% of diverse the most common alteration [38.6% (34/88)], followed cancers, with most cases harboring actionable, albeit dis- by fusions [30.7% (27/88), including a novel SQSTM1-RET] tinct, coexisting alterations. The current report suggests that and amplifications [25% (22/88)]. Most patients had coexisting optimal targeting of patients with RET anomalies will aberrations in addition to RET anomalies [81.8% (72/88)], require customized combination strategies. Clin Cancer Res; with the most common being in TP53-associated genes [59.1% 23(8); 1988–97. Ó2016 AACR. Introduction RET aberrations can result in gain of function via amplifi- cation or mutations and rearrangements that result in ligand- The RET proto-oncogene encodes a transmembrane receptor independent kinase activation. These alterations have been tyrosine kinase composed of an extracellular cadherin domain, reported in different types of malignancies and in hereditary cysteine-rich region, transmembrane domain, and an intracel- conditions. lular kinase domain (1, 2). It functions as the receptor for the Mutations in RET have been reported in patients with med- growth factors of the glial cell line–derived neurotropic factor ullary thyroid carcinoma. They are seen in 43% to 71% of family (3). Binding of ligand facilitates RET kinase activation, sporadic cases, with the most common mutation being RET which leads to activation of multiple downstream effectors, M918T (5–8). Of note, germline mutations of RET are a hall- including MAPK and PI3K pathways (3). Physiologically, RET is mark of multiple endocrine neoplasia (MEN), including type crucial for neural crest development (1, 2); loss-of-function 2A, 2B, and familial medullary thyroid carcinoma, with all three mutations in RET are associated with aganglionic megacolon in subtypes being associated with a high risk of developing a Hirschsprung disease (4). medullary thyroid carcinoma (70%–100% risk by age 70 years; ref. 9). Clinically, MEN 2A is also associated with pheochro- mocytoma and parathyroid hyperplasia, whereas MEN 2B is associated with mucosal neuromas, pheochromocytomas, intes- 1Department of Medicine, Center for Personalized Cancer Therapy and Division tinal ganglioneuromas and marfanoid habitus; familial medul- of Hematology and Oncology, University of California, San Diego, Moores Cancer lary thyroid carcinoma is not associated with other conditions Center, San Diego, California. 2Department of Investigational Cancer Therapeu- RET 3 (9). Interestingly, different mutations are associated with tics, The University of Texas MD Anderson Cancer Center, Houston, Texas. N-of- distinct subtypes of MEN: (i) RET C634R, which leads to ligand- One, Inc., Lexington, Massachusetts. independent receptor dimerization, is most commonly associ- Note: Supplementary data for this article are available at Clinical Cancer ated with MEN 2A; (ii) RET M918T, which leads to decreased Research Online (http://clincancerres.aacrjournals.org/). auto-inhibition and increased kinase activity, as well as ATP Corresponding Author: Shumei Kato, UC San Diego Moores Cancer Center, binding, is associated with MEN 2B; and (iii) various mutations 3855 Health Sciences Drive, La Jolla, CA 92093. Phone: 858-822-2372; Fax: 858- at codons 609, 618, 620, 768, 804, and 891 are reported in both 822-6186; E-mail: [email protected] MEN 2A and familial medullary thyroid carcinoma (9, 10). doi: 10.1158/1078-0432.CCR-16-1679 These observations suggest that different RET-activating muta- Ó2016 American Association for Cancer Research. tions have dissimilar oncogenic effects. 1988 Clin Cancer Res; 23(8) April 15, 2017 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 28, 2016; DOI: 10.1158/1078-0432.CCR-16-1679 RET Aberrations in Cancer To facilitate the clinical trials targeting RET, a comprehensive Translational Relevance understanding of RET aberrations among diverse cancer types To optimize clinical trials targeting RET aberrations, is essential. Therefore, we examined the genomic landscape of understanding the relevant genomic landscape in diverse RET alterations using targeted next-generation sequencing cancersiscritical.Here,wereport the molecular portfolio, (NGS) in 4,871 patients with diverse malignancies, and we including coexisting alterations, of RET-altered cancers also show two illustrative cases of lung and medullary thyroid in 4,871 patients who were evaluated using clinical grade carcinoma with KIF5B-RET and RET M918T alterations, respec- next-generation sequencing. Among 88 cases with RET tively, who both responded to vandetanib (multikinase RET aberrations, the most common alterations included muta- inhibitor) containing regimen. tions [38.6% (34/88)], fusions [30.7% (27/88)], and ampli- fication [25% (22/88)]. In the 72 patients harboring coa- berrations along with RET alterations, there were no two Materials and Methods patients with identical molecular portfolios. Among 292 Patients coexisting molecular aberrations, 200 were molecularly We investigated the RET gene status of patients with distinct. The median number of coaberrations per patient diverse malignancies that were referred for NGS from October was three (range, 0–16). In 71 of the 72 tumors with more 2011 to November 2013 (N ¼ 4,871; Table 1 and Supplementary than one alteration, at least one coexisting genomic alter- Tables S2 and S3; Fig. 1). The submitting physicians provided ation was potentially pharmacologically tractable, suggest- specification of tumor types. The database was deidentified with ing the need for customized combination treatments. only diagnosis available. NGS data were collected and interpreted by N-of-One, Inc. The dataset of 4,871 sequenced tumors was queried for RET and coexisting gene alterations. Clinical impact was demonstrated by selected case studies. This study was per- formed in accordance with the guidelines of the UCSD and the On the other hand, fusions in RET have been described in MD Anderson Internal Review Board. patients with papillary thyroid carcinoma, accounting for approx- imately 20% to 40% of sporadic cases (2, 11), with a higher Tissue samples and mutational analysis frequency of RET fusions observed after radioiodine exposure We collected sequencing information from 4,871 cancers (60%; ref. 12). Diverse RET fusions have been identified, but more whose formalin-fixed, paraffin-embedded (FFPE) tumor samples than 90% of fusions involve the coiled–coil domain-containing were submitted to a Clinical Laboratory Improvement Amend- protein 6 (CCDC6)-RET or nuclear receptor coactivator 4 ments–certified laboratory for genomic profiling (Foundation (NCOA4)-RET (also referred in the literature as RET/PTC1 and Medicine). Samples were required to have a surface area 25 RET/PTC3, respectively; ref. 2). mm2, volume 1mm3, nucleated cellularity 80%, and tumor Although less frequent, several activating RET fusions have content 20% (28). The methods used in this assay have been been reported in 1% to 2% of patients with non–small cell lung validated and reported previously (28–30). In short, 50 to 200 ng cancer (NSCLC). These include CCDC6-RET (13, 14), NCOA4- of genomic DNA was extracted and purified from the submitted RET (14), kinesin family member 5b (KIF5B)-RET (13–18), and FFPE tumor samples. This whole-genome DNA was subjected to tripartite motif-containing 33 (TRIM33)-RET (15). shotgun library construction and hybridization-based capture Identification of RET aberrations is therapeutically important before paired-end sequencing on the Illumina HiSeq2000 plat- as they are targetable with several FDA-approved multikinase form. Hybridization selection is performed using individually inhibitors that have anti-RET activity, including vandetanib synthesized baits targeting the exons of