RET Aberrations in Diverse Cancers: Next-Generation Sequencing of 4,871 Patients Shumei Kato1, Vivek Subbiah2, Erica Marchlik3, Sheryl K

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. 2016 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 pheochromocytoma 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- 2016 American Association for Cancer Research. tions have dissimilar oncogenic effects.

1988 Clin Cancer Res; 23(8) April 15, 2017

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 amplification [25% (22/88)]. In the 72 patients harboring coaberrations 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 performed 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 approximately 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 182 or 236 cancer-related (RET/EGFR/VEGFR2 inhibitor, approved for medullary thyroid genes and the introns of 14 or 19 genes frequently rearranged in carcinoma), cabozantinib (RET/MET/VEGFR2 inhibitor, approv- cancer (Supplementary Table S4). Sequence data were processed ed for medullary thyroid carcinoma), lenvatinib (RET/FGFR/ using a customized analysis pipeline (28). Sequencing was per- VEGFR/KIT/PDGFR inhibitor, approved for differentiated thy- formed with an average sequencing depth of coverage greater than roid carcinoma and renal cell carcinoma), ponatinib (RET/ 250, with >100 at >99% of exons. This method of sequencing FGFR/VEGFR/KIT/PDGFR/BCR-ABL inhibitor, approved for allows for detection of copy number alterations, gene rearrange- chronic myeloid leukemia and Philadelphia chromosome–pos- ments, and somatic mutations with 99% specificity and >99% itive acute lymphoblastic leukemia), sunitinib [RET/VEGFR/KIT/ sensitivity for base substitutions at 5 mutant allele frequency PDGFR/FLT3 inhibitor, approved for renal cell carcinoma and and >95% sensitivity for copy number alterations. A threshold of imatinib-resistant gastrointestinal stromal tumor (GIST)], regor- 8 copies for gene amplification with 6 copies considered afenib (RET/VEGFR/KIT/BRAF/CRAF inhibitor, approved for equivocal (except for ERRB2, which is considered equivocally colorectal cancer and GIST), and sorafenib (RET/VEGFR/KIT/ amplified with 5 copies) was used. BRAF/CRAF inhibitor, approved for differentiated thyroid carcinoma, renal cell carcinoma and hepatocellular carcinoma; cBio Cancer Genomics Portal data refs. 19, 20). However, none of these inhibitors are FDA approv- For comparison purposes, we evaluated the RET aberra- ed on the basis of targeting RET aberrations. Meanwhile, there tion status using the cBio Cancer Genomics Portal data are a series of reports suggesting that matched therapies against (cBioPortal; http://cbioportal.org, accessed May 2016; refs. RET aberrations can yield significant responses (8, 15, 21–27). 31, 32), which provides access to publicly available datasets Further clinical trials with a focus on RET-aberrant advanced with genomic information from a diverse array of cancer cancer are being conducted to determine impact on outcome types (please refer to Supplementary Methods for additional (Supplementary Table S1). information).

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Results including RET R163Q, M255I, R525Q, V706M, A756V, M1109I, and SQSTM1-RET, fusion was unknown (Supplemen- Analysis of RET aberrations among diverse cancers (N ¼ 4,871) tary Table S3). Among the 4,871 diverse cancer patients, the most common diagnosis was breast carcinoma [10.4% (506/4,871)], followed by lung adenocarcinoma [8.5% (412/4,871)], and sarcoma Overview of cancer diagnoses and RET aberrations [7.1% (348/4,871); Table 1 and Supplementary Table S2]. RET aberrations were most commonly seen in patients with Overall, RET aberrations were identified in 88 cases [1.8% medullary thyroid carcinoma [80% (4/5)], followed by ana- (88/4,871)]. Among 88 cases with RET aberrations, 38.6% plastic thyroid carcinoma [16.7% (2/12)], lung carcinosarcoma (34/88) were mutations, 30.7% (27/88) were fusions (defined [16.7% (1/6)], and ureter urothelial carcinoma [16.7% (1/6)]; as RET rearrangement with known fusion partner. e.g., KIF5B- however, these cancer diagnoses were not reported in cBioPor- RET), 25% (22/88) were amplifications, and 3.4% (3/88) were tal, and thus, direct comparisons were not feasible (Table 1 and rearrangements without specificidentified fusion partner (e.g., Supplementary Table S5; Fig. 2 and Supplementary Fig. S1). RET rearrangement, exon 11). In addition, RET duplication Although there was only one patient each for hemangioper- and loss were each observed in 1 of 88 cases. (Fig. 1). According icytoma and pheochromocytoma, they both harbored RET to cBioPortal, RET aberrations have been reported in 3.0% aberrations (Table 1 and Supplementary Table S3). In some (181/6,011) of diverse cancers (Supplementary Table S5; cancer diagnoses, including cholangiocarcinoma (n ¼ 159), Supplementary Fig. S1). In the current report, most RET aber- neuroendocrine carcinoma (n ¼ 97), renal cell carcinoma rations were activating alterations [71.6% (63/88)] and (n ¼ 92), and glioblastoma (n ¼ 84), we did not observe RET one inactivating RET alteration was observed (RET loss). The aberrations (Supplementary Table S2). In contrast, data from functional significance of 27.3% (24/88) of RET aberrations, cBioPortal showed rare RET alterations in cholangiocarcinoma

Table 1. RET aberrations and associated cancer diagnosis (N ¼ 88) Any aberrations Fusiona Mutation Rearrangementa Amplification Duplication Loss Diagnosis n (%) n (%) n (%) n (%) n (%) n (%) n (%) Hemangiopericytoma (n ¼ 1) 1 (100) 0 1 (100) 0 0 0 0 Pheochromocytoma (n ¼ 1) 1 (100) 0 1 (100) 0 0 0 0 Medullary thyroid carcinoma (n ¼ 5) 4 (80.0) 0 4 (80.0) 0 0 0 0 Paraganglioma (n ¼ 4) 1 (25.0) 0 1 (25.0) 0 0 0 0 Anaplastic thyroid carcinoma (n ¼ 12) 2 (16.7) 0 2 (16.7) 0 0 0 0 Lung carcinosarcoma (n ¼ 6) 1 (16.7) 1 (16.7) 0 0 0 0 0 Ureter urothelial carcinoma (n ¼ 6) 1 (16.7) 0 1 (16.7) 0 0 0 0 Uterine carcinosarcoma (n ¼ 19) 3 (15.8) 0 1 (5.3) 0 1 (5.3) 1 (5.3) 0 Papillary thyroid carcinoma (n ¼ 23) 3 (13.0) 2 (8.7) 1 (4.3) 0 0 0 0 Basal cell carcinoma (n ¼ 8) 1 (12.5) 0 1 (12.5) 0 0 0 0 Merkel cell carcinoma (n ¼ 10) 1 (10) 0 1 (10.0) 0 0 0 0 Atypical lung carcinoid (n ¼ 11) 1 (9.1) 0 1 (9.1) 0 0 0 0 Fallopian tube adenocarcinoma (n ¼ 12) 1 (8.3) 0 0 0 1 (8.3) 0 0 Ovarian epithelial carcinoma (n ¼ 54) 4 (7.4) 1 (1.9) 2 (3.7) 0 1 (1.9) 0 0 Salivary gland adenocarcinoma (n ¼ 31) 2 (6.5) 1 (3.2) 0 0 1 (3.2) 0 0 Lung adenocarcinoma (n ¼ 412) 23 (5.6) 16 (3.9) 3 (0.7) 2 (0.5) 2 (0.5) 0 0 Meningioma (n ¼ 18) 1 (5.6) 0 1 (5.6) 0 0 0 0 Duodenal adenocarcinoma (n ¼ 20) 1 (5.0) 0 0 0 1 (5.0) 0 0 Cervical adenocarcinoma (n ¼ 24) 1 (4.2) 0 1 (4.2) 0 0 0 0 Adrenal carcinoma (n ¼ 27) 1 (3.7) 0 1 (3.7) 0 0 0 0 Gastroesophageal junction carcinoma (n ¼ 29) 1 (3.4) 0 0 0 1 (3.4) 0 0 GIST (n ¼ 30) 1 (3.3) 0 1 (3.3) 0 0 0 0 Non–small cell lung carcinoma (n ¼ 125) 4 (3.2) 4 (3.2) 0 0 0 0 0 Cutaneous squamous cell carcinoma (n ¼ 36) 1 (2.8) 0 0 0 0 0 1 (2.8) Hepatocellular carcinoma (n ¼ 44) 1 (2.3) 0 1 (2.3) 0 0 0 0 Pancreatic ductal adenocarcinoma (n ¼ 160) 3 (1.9) 0 1 (0.6) 1 (0.6) 1 (0.6) 0 0 Prostate adenocarcinoma (n ¼ 64) 1 (1.6) 0 0 0 1 (1.6) 0 0 Melanoma (n ¼ 136) 2 (1.5) 0 1 (0.7) 0 1 (0.7) 0 0 Esophageal adenocarcinoma (n ¼ 69) 1 (1.4) 0 1 (1.4) 0 0 0 0 Endometrial adenocarcinoma (n ¼ 79) 1 (1.3) 0 1 (1.3) 0 0 0 0 Ovarian serous carcinoma (n ¼ 169) 2 (1.2) 0 0 0 2 (1.2) 0 0 Carcinoma unknown primary (n ¼ 270) 3 (1.1) 2 (0.7) 0 0 1 (0.4) 0 0 Bladder urothelial (transitional cell) carcinoma (n ¼ 91) 1 (1.1) 0 0 0 1 (1.1) 0 0 Lung squamous cell carcinoma (n ¼ 93) 1 (1.1) 0 0 0 1 (1.1) 0 0 Colorectal adenocarcinoma (n ¼ 300) 3 (1.0) 0 2 (0.7) 0 1 (0.3) 0 0 HNSCC (n ¼ 108) 1 (0.9) 0 0 0 1 (0.9) 0 0 Sarcoma (n ¼ 348) 3 (0.9) 0 1 (0.3) 0 2 (0.6) 0 0 Gastric adenocarcinoma (n ¼ 134) 1 (0.7) 0 1 (0.7) 0 0 0 0 Breast carcinoma (n ¼ 506) 3 (0.6) 0 1 (0.2) 0 2 (0.4) 0 0 Abbreviation: HNSCC, head and neck squamous cell carcinoma. aThe term fusion was used when RET was rearranged with known fusion partner (e.g., KIF5B-RET). On the other hand, the term rearrangement was used when there was no specific identified fusion partner (e.g., RET rearrangement, exon 11).

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Duplicaon (1.1%) Rearrangement (3.4%) Loss (1.1%) Figure 1. Overview of RET aberrations among diverse cancers (N ¼ 4,871). Among diverse cancers (N ¼ 4,871), 88 cases (1.8%) harbored RET aberrations. Among 88 cases with RET aberrations, 38.6% (34/88) were mutations, 30.7% Amplificaon Mutaon (38.6%) (27/88) were fusions (defined as RET RET Aberraons (25%) rearrangement with known fusion (1.8%) partner, e.g., KIF5B-RET), 25% (22/88) were amplifications, 3.4% (3/88) were rearrangements (defined as RET No RET aberraons (98.2%) Fusion (30.7%) rearrangement without specific identified fusion partner, e.g., RET rearrangement, exon 11), 1.1% (1/88) were duplication, and 1.1% (1/88) were loss. N = 4,871 N = 88

[5.7% (2/35)] and glioblastoma [0.7% (2/281); Supplementary 292 coaberrations were identified. Among 292 coaberrations, Table S5; Supplementary Fig. S1]. 80.8% (236/292) were potentially targetable with FDA-approved agents as off-label use, and an additional 8.2% (24/292) were Overview of cancer diagnosis and specific RET aberrations theoretically targetable with therapies that are currently in clinical As mentioned, the most common type of RET aberrations were trials. Altogether, among all coaberrations, 89.0% (260/292) were mutations [38.6% (34/88)], followed by fusions [30.7% (27/88)] potentially actionable either with therapies that are approved by and amplifications [25.0% (22/88); Fig. 1]. This observation was the FDA (albeit off label) or with therapies that are in clinical trials similar to cBioPortal data in relative frequencies, although the (Supplementary Tables S3 and S7). actual percentages for cBioPortal mutations, fusions, and ampli- Among 292 coexisting aberrations, 200 were molecularly dis- fications differed a bit from our data [60.2% (109/181), 15.5% tinct alterations, occurring either in separate genes or distinct (28/181), and 12.7% (23/181), respectively; Supplementary alterations within the same gene. However, there were 16 cases of Table S5; Supplementary Fig. S1]. In the current report, RET CDKN2A/B loss, and those were considered as single aberration. mutations were most commonly seen in patients with medullary Among these molecularly distinct aberrations, 80.0% (160/200) thyroid carcinoma [80% (4/5)], followed by paraganglioma [25% were targetable by an FDA-approved drug, and an additional 7.5% (1/4)], anaplastic thyroid carcinoma [16.7% (2/12)], and ureter (15/200) were targetable by drugs that are under investigation in urothelial carcinoma [16.7% (1/6); Table 1; Fig. 2]. RET fusions clinical trials (Supplementary Tables S3 and S7). were seen in patients with lung carcinosarcoma [16.7% (1/6)], Among 88 patients with RET aberrations, the median number followed by papillary thyroid carcinoma [8.7% (2/23)] and lung of coaberrations per patient was three (range, 0–16; excluding RET adenocarcinoma [3.9% (16/412)]. RET amplifications were alterations). The median number of coaberrations was similar detected in patients with fallopian tube adenocarcinoma [8.3% among patients who were tested with the 182-gene panel (n ¼ 16 (1/12)], uterine carcinosarcoma [5.3% (1/19)], and duodenal patients, median of 3 coaberrations, range 0–6) and 236-gene adenocarcinoma [5.0% (1/20); Table 1; Fig. 2]. panel (n ¼ 72 patients, median of 3 coaberrations, range 0–16; Supplementary Table S4 for list of genes). The median number of Coaberrant oncogenic pathways associated with RET potentially targetable coaberrations per patient was two (range, aberrations 0–16; Fig. 4). Among all 88 patients with RET aberrations, 78.4% Among 88 patients with RET aberrations, 72 also harbored (69/88) of patients had theoretically actionable coaberrations coaberrations (Supplementary Table S3). Those include coaber- by an FDA-approved agent, and an additional 2.3% (2/88) rations with TP53-associated genes [59.1% (52/88)], cell cycle– patients had coaberrations targetable with investigational agents associated genes [39.8% (35/88)], aberrations in the PI3K sig- in clinical trial. Altogether, 80.6% (71/88) patients had action- naling pathway [30.7% (27/88)], MAPK effectors [22.7% able coaberrations either with FDA-approved or with investiga- (20/88)], and other tyrosine kinases families [21.6% (19/88); tional agents. However, if we only focus on 72 patients who Fig. 3; Supplementary Table S5]. Coaberrant oncogenic pathways had coaberrations along with RET aberrations, almost all pati- were all readily observed (greater than 20%) among RET muta- ents had actionable coaberrations [98.6% (71/72)] either with tions, amplifications, and fusions, except the coaberrations with FDA-approved or with investigational agents (Fig. 4; Supplemen- tyrosine kinase families and MAPK signaling pathway were tary Tables S3 and S7). both infrequently associated with RET fusions [7.4% (2/27) and 0% (0/27), respectively; Supplementary Table S5]. Distinctness of genomic aberrations among 88 patients with RET aberrations Number of cogenetic aberrations associated with RET Among 88 patients with RET aberrations, 12 patients had aberrations and possible cognate targeted therapies identical genomic portfolios [RET C634R (ID #1 and #2), RET As mentioned, among 88 cases with RET aberrations, 72 M918T (patient ID #6, #8, #9, #11, and #12), RET-NCOA4 fusion patients also had cogenetic aberrations along with RET aberra- (ID #37 and #38), and RET-CCDC6 fusion (ID #57, #58, and tions (Supplementary Table S3). Among these 72 cases, a total of #60); Supplementary Table S3]. However, among 72 patients

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All

Medullary thyroid carcinoma ≈

Anaplastic thyroid carcinoma

Lung carcinosarcoma

Ureter urothelial carcinoma

Uterine carcinosarcoma

Papillary thyroid carcinoma

Basal cell carcinoma

Merkel cell carcinoma

Atypical lung carcinoid

Fallopian tube adenocarcinoma

Ovarian epithelial carcinoma

Salivary gland adenocarcinoma

Lung adenocarcinoma

Meningioma

Duodenal adenocarcinoma

20% ≈ 75% 0%0% 5%5% 1010%% 115%5% 20% 25% 80%30% 85%35% Fusion Mutation Rearrangement Amplification Duplication Loss

Figure 2. Frequencies and distributions of RET aberrations. RET aberrations were frequently seen in patients with medullary thyroid carcinoma [80% (4/5)], all being mutations. This was followed by anaplastic thyroid carcinoma [16.7% (2/12)], lung carcinosarcoma [16.7% (1/6)], and ureter urothelial carcinoma [16.7% (1/6)]. Included when RET was aberrant in 5% of cases and if at least 5 cancer diagnoses were tested for the aberration. Please see Table 1 for a complete list of cancer diagnoses found to be positive for RET aberrations. The term fusion was used when RET was rearranged with known fusion partner (e.g., KIF5B- RET). On the other hand, the term rearrangement was used when there was no speciﬁcidentiﬁed fusion partner (e.g., RET rearrangement, exon 11).

harboring coaberrations along with RET aberrations, there were to a major response (Fig. 5A); the second patient was a 35-year-old no two patients with identical genomic portfolios (Supplemen- man with sporadic medullary thyroid carcinoma and a RET tary Table S3). If we consider the genetic alterations at the level of M918T mutation as well as ATM L804fs4 and ATM S978fs12 the gene (and not the specific molecular aberration), then five alterations. Additional tumor evaluation with an IHC panel also patients had coaberrations identical to at least one other patient. showed strong positivity of phospho-AKT. The patient was ini- Those include patient ID #32 and #87 with KRAS and TP53 and ID tially treated with single-agent vandetanib with prolonged stable #39, #41, and #45 with RB1, STK11, and TP53 coaberrations disease; however, the addition of everolimus led to significant (Supplementary Table S3). tumor shrinkage (Fig. 5B).

Clinical impact of multikinase inhibitors with anti-RET activity in patients with RET aberrations Discussion To demonstrate the impact of therapies with anti-RET activity in We report a comprehensive landscape of RET aberrations cancer patients harboring RET aberrations, we report two patients among 4,871 patients with diverse cancers. RET aberrations were with RET alterations who were treated with multikinase inhibitors identified in 1.8% (88/4,871) of tumors, with mutations being that possess anti-RET activity. The first is a 43-year-old woman the most frequent aberration [38.6% (34/88)], followed by with adenocarcinoma of the lung and a KIF5B-RET fusion, refrac- fusions [e.g., KIF5B-RET; 30.7% (27/88)], amplifications tory to multiple lines of therapies including the multikinase RET/ [25.0% (22/88)], rearrangements without specific identified MET/VEGFR2 inhibitor cabozantinib. Treatment with another fusion partner [e.g., RET rearrangement, exon 11; 3.4% (3/88)] multikinase inhibitor, vandetanib (RET/EGFR/VEGFR2 inhibi- and n ¼ 1 each of duplication and loss (Fig. 1). The overall tor) in combination with everolimus (an mTOR inhibitor), led frequency of RET aberrations in this current report is similar to the

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RET Aberrations in Cancer

Cell-cycle–associated genes (39.8%) Tyrosine kinases families (21.6%)

CDKN2A/B (18.2%) FGFR1 (3.4%) EGFR (2.3%) DDR2 (1.1%) FLT3 (3.4%) CDKN2A (8.0%) FGFR2 (2.3%) ERBB2 (2.3%) FLT4 (1.1%) CDKN2C Figure 3. (1.1%) FGFR3 (3.4%) KDR (2.3%) ALK JAK2 Coaberrant oncogenic pathways FGFR4 (1.1%) (1.1%) (2.3%) PDGFRA (2.3%) associated with RET aberrations. KIT (2.3%) PDGFRB (1.1%) SRC (1.1%) Among 88 patients with RET CCND1 (3.4%) CDK6 (2.3%) aberrations, some patients also CCND2 (3.4%) harbored coaberrations that can lead to tumorigenesis. Those coaberrations MAPK Signaling (22.7%) PI3K Signaling (30.7%) include TP53-associated genes [e.g., CCNE1 (6.8%) RB1 (6.8%) KRAS (10.2%) PIK3CA (10.2%) MDM2, ATM,orTP53; 59.1% (52/88)], NF1 PTEN (5.7%) (6.8%) NRAS (3.4%) PIK3R1 (1.1%) cell-cycle–associated genes [e.g., CDKN2A/B, CDK6,orRB1; 39.8% Cell-cycle progression (35/88)], PI3K signaling pathway [e.g., AKT2 (1.1%) BRAF (4.5%) PIK3CA, PTEN, AKT,orMTOR; 30.7% (27/88)], MAPK effectors [e.g., KRAS, NF1,orBRAF; 22.7% (20/88)], and TP53-associated genes (59.1%) TSC1 (2.3%) other tyrosine kinase families [e.g., STK11 (4.5%) TSC2 (2.3%) MDM2 (5.7%) ATM (4.5%) FGFR, EGFR, ERBB2, ALK,orKIT; 21.6% (19/88)]. Please see Supplementary Tables S3 and S5 for a complete list of RPTOR (1.1%) co-occurring aberrations associated TP53 (52.3%) MTOR (1.1%) NF2 (2.3%) with RET aberrations. RICTOR (3.4%) Cell survival and proliferaon Cell survival and proliferaon frequency reported in the cBioPortal dataset 3.0% (181/6,011; 43% to 71% of sporadic medullary thyroid carcinomas (5–8). Supplementary Table S5; Supplementary Fig. S1). RET M918T is the most common mutation reported in RET mutations are a hallmark of medullary thyroid can- sporadic disease (5, 6, 8), which is consistent with the current cer (both sporadic and familial cases); they are reported in report, wherein four of ﬁve medullary thyroid cancers

16 ≈

11 10

Figure 4. 8 Number of all reported coaberrations and possibly actionable coaberrations 7 per patient. Among 88 patients with RET aberrations, there was a median 6 of 3 coaberrations per patient (range, 0–16) and a median of 2 (range, 0–16) 5 possibly actionable coaberrations per

patient. Please see Supplementary aberrations Number of 4 Table S6 for a complete list of co-occurring aberrations and rationale 3 for possible targeted therapies. 2

1 0

0 2 4 6 8 1012141618 Number of patients All reported coaberrations Possibly actionable coaberrations

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A Pretreatment Posttreatment

B Pretreatment Posttreatment 100 mm 100 mm

Figure 5. Case reports of patients with RET aberrations treated with anti-RET multikinase inhibitors. A, Never-smoker lung adenocarcinoma with KIF5B-RET fusion. Posttreatment PET scan was performed at 8 weeks and showed improvement in the right lung hypermetabolic tumor. A 43-year-old female never-smoker was diagnosed with stage IV poorly differentiated adenocarcinoma of the lung with focal signet features. Comprehensive genomic profiling revealed a KIF5B-RET fusion with CDK4 and MDM2 amplification. She was started on treatment with a multikinase RET/MET/VEGFR2 inhibitor, cabozantinib. She responded initially to treatment and derived clinical benefit for 12 months. On progression, she was then enrolled on a trial with carboplatin/paclitaxel plus a heat shock protein 90 inhibitor. She progressed after 7 months and was started on pemetrexed plus bevacizumab. Because of progression, she was enrolled on vandetanib, a multikinase inhibitor of RET/EGFR/VEGFR2 inhibitor, in combination with everolimus (an mTOR inhibitor; ClinicalTrials.gov; NCT01582191). Her performance status and pain improved while on therapy. After two cycles, FDG PET scans showed interval improvement in the right lung hypermetabolic tumor (A) and improvement in metastases to the bones and liver. She had a 76% decrease per RECIST version 1.1. However, she developed new sites of disease after four cycles and was taken off the trial and is now enrolled on another RET inhibitor trial. B, Recurrent medullary thyroid carcinoma sporadic type with RET M918T mutation. Posttreatment CT scan was performed at 8 weeks, which showed improvement of lymphadenopathy. A 45-year-old man without known family history of endocrine malignancy initially underwent thyroidectomy for localized medullary thyroid carcinoma. The patient subsequently developed multiple areas of lymphadenopathy and liver metastasis that was biopsy proven to be recurrent disease. Comprehensive genomic profiling revealed RET M918T as well as ATM L804fs4andATM S978fs12. The patient was treated with vandetanib for 12 months with overall stable disease with eventual slow increase in size. Meanwhile, the patient had further evaluation of the tumor sample with an IHC panel, including phospho-AKT, which showed positive intensity (3þ) in 100% of cells, suggesting coactivation of the PI3K pathway. He was subsequently enrolled on a trial with vandetanib, a multikinase inhibitor of RET/EGFR/VEGFR inhibitor, in combination with everolimus (an mTOR inhibitor; ClinicalTrials.gov; NCT01582191) with improvement in lymphadenopathy (B). His tumor demonstrated a 25% decrease per RECIST version 1.1 that lasted eight cycles.

harbored RET mutations [M918T (n ¼ 3) and C634R (N ¼ Various FDA-approved multikinase inhibitors that possess 1); Table 1 and Supplementary Table S3; Fig. 2]. Activating anti-RET activity have recently become available: vandetanib, RET mutations lead to enhanced downstream signaling with cabozantinib, lenvatinib, ponatinib, sunitinib, regorafenib, and multiple effectors, including those in the MAPK and PI3K sorafenib (19, 20). Among these agents, one of the earliest studies pathways, resulting in increased cell proliferation and that demonstrated clinical activity against RET-mutated tumors anchorage-independent cell growth (33–35). Clinically, RET was a phase I trial with cabozantinib (RET/MET/VEGFR2 inhib- mutations are associated with poor clinical outcomes, includ- itor), which enrolled 37 patients with medullary thyroid carci- ing larger tumor size, metastasis, and poorer survival, when noma (8). In that study, 81% (25/31) of analyzed tumors har- compared with RET wild-type cases among patients with bored activating RET mutations (including 3 patients with germ- medullary thyroid carcinoma (7). line RET mutations). Stable disease of at least 6 months or a partial

1994 Clin Cancer Res; 23(8) April 15, 2017 Clinical Cancer Research

RET Aberrations in Cancer

response (PR) was observed in 68% (25/37) of patients [PR, tanib (RET/EGFR/VEGFR2 inhibitor)-based therapy (Fig. 5A). 25.9% (17/37)]. Tumor regression was also observed among Along with these promising reports (15, 21, 24), there are medullary thyroid carcinomas without identified RET mutations, multiple ongoing phase I and II trials targeting RET fusions in which could be due to anti-VEGFR2 and anti-MET activities patients with NSCLC and other advanced solid tumors (Sup- (2 patients had MET amplification) of cabozantinib or because plementary Table S1). of other unknown aberrations in the RET pathway (8). Cabozan- Ofnote,wehaveidentified a patient (papillary thyroid; case tinib was further studied in a double-blind, phase III trial in #61, Supplementary Table S3) with a sequestosome 1 patients with advanced medullary thyroid carcinoma and dem- (SQSTM1)-RET fusion,which,toourknowledge,hasnotbeen onstrated statistically significant progression-free survival (PFS) previously reported (2, 12–18, 20). The functional and clinical when compared with placebo (PFS, 11.2 months versus 4.0 relevance of SQSTM1-RET is worth investigating as the SQSTM1 months; HR, 0.28; P < 0.001), with a subgroup analysis demon- fusion with other partners, such as ALK (37), showed trans- strating statistically significant HR only seen among the RET forming activity in vitro, and there is a case report of a patient mutation–positive group (22). On the basis of these studies, with NSCLC and a SQSTM1-NTRK1 fusion demonstrating a cabozantinib is currently approved for patients with advanced durable response to entrectinib (a multikinase inhibitor with medullary thyroid carcinoma. In this current report, RET-activat- targets including TrkA, B, and C; ref. 38). ing mutations were also found in diverse cancers other than We have also evaluated aberrations that cooccurred with medullary thyroid carcinoma with variable frequencies: anaplas- RET. Indeed, 81.8% (72/88) of cases harboring RET aberra- tic thyroid [16.7% (2/12)], Merkel cell [10% (1/10)], GIST [3.3% tions had additional alterations. The most common pathways (1/30)], hepatocellular [2.3% (1/44)], endometrial [1.3% altered involved TP53-associated genes [59.1% (52/88)], fol- (1/79)], colorectal [0.7% (2/300)], and breast [0.2% (1/506)] lowed by cell cycle–associated genes [39.8% (35/88)], the carcinomas (Table 1 and Supplementary Table S3; Fig. 2). Addi- PI3K signaling pathway [30.7% (27/88)], MAPK effectors tional clinical trials targeting diverse cancer types with RET muta- [22.7% (20/88)], and other tyrosine kinase families [21.6% tions in so-called basket trials may yield insights as to the (19/88); Fig. 3]. In fact, among 292 coexisting aberrations relevance of histology in the presence of RET mutations (Supple- detected in this report, 80.8% (236/292) were potentially mentary Table S1). It is important to also note that medullary targetable with FDA-approved agents (albeit off label) and thyroid carcinoma patients being targeted with multikinase an additional 8.2% (24/292) with experimental agents that inhibitors can have frequent initial declines in tumor measure- are under investigation in clinical trials (Supplementary ments and markers (calcitonin and carcinoembryonic antigen), Tables S3 and S7). Moreover, almost all patients [98.6% followed by short-lived cycling patterns that include an increase (71/72)] harboring coaberrations with RET had potentially in tumor measurements more than 20% above nadir values actionable coaberrations with either FDA-approved or with (which is defined as progressive disease per RECIST version investigational agents (Supplementary Tables S3 and S7). 1.1) with or without increases in tumor markers (36). However, Although targeting RET aberrations has been reported to be we have previously demonstrated that these fluctuations can be successful, especially when RET was the only identifiable transient, and continued RET inhibition was associated with target (15, 21), the current report suggests that therapeutic durable tumor regression (36). combination approaches may be necessary to move beyond RET fusions were the second most common RET aberrations PRs with limited durability. Several such trials have been identified [30.7% (27/88); Table 1 and Supplementary Table suggested or described previously (39–42). Consistent with S3; Fig. 1]. Most fusion partners contain coiled–coil or leucine this notion, we show a patient with sporadic medullary zipper domains that drive the dimerization or oligomerization thyroid carcinoma harboring a RET M918T alteration, who of the fusion kinase and lead to ligand-independent RET acti- had prolonged stable disease on vandetanib (12 months) vation (20). RET fusions have been described in approximately and had strong positivity for phospho-AKT by IHC. The 20% to 40% of papillary thyroid cancers (2, 11), with even patient's tumor demonstrated 25% reduction with the addi- higher frequency when associated with radioiodine exposure tion of everolimus (an mTOR inhibitor; Fig. 5B). (60%; ref. 12). More than 10 RET fusion partners have been Interestingly, RET fusions were mutually exclusive with reported (2). In our study, 8.7% of papillary thyroid carcinomas MAPK signaling pathway (which includes NF1, KRAS, NRAS, (2/23) harbored RET fusions (Table 1; Fig. 2). The differences in and BRAF that were seen with RET mutations/amplifications) frequency between previous reports and the current study may and infrequently associated with aberrations in other tyrosine be due to small sample size and/or differing detection methods. kinase families [7.4% (2/27); Fig. 3; Supplementary Table S6], Recent studies also demonstrated RET fusions in about 1% to which is consistent with a previous report (15). However, 2% of patients with NSCLC, with several of the fusion partners coaberrations in effectors of MAPK signaling or other tyrosine overlapping with those identified in papillary thyroid carcinoma kinases were readily (greater than 20%) seen among tumors (e.g., CCDC6, KIF5B, NCOA4, and TRIM33; refs. 13–18, 24), with either RET mutations or amplifications (Fig. 3; Supple- which is consistent with findings in this current report (Table 1 mentary Table S6). The extent to which this holds true in larger and Supplementary Table S3; Fig. 2). Studies analyzing case groups of patients merits investigation. series have reported that individual patients with NSCLC har- There are several limitations to the current data. First, corre- boring a CCDC6-RET fusion treated with vandetanib (n ¼ 1; lations with overall disease outcome were not feasible, as the ref. 21) as well as NSCLC bearing TRIM33-RET (n ¼ 1) or KIF5B- data were not clinically annotated. Second, the impact of RET (n ¼ 1) treated with cabozantinib have achieved PRs (15). germline mutations [especially important for medullary thy- Herein, we also show a patient with lung adenocarcinoma and roid carcinoma and pheochromocytoma that can be associated a KIF5B-RET fusion, refractory to multiple lines of therapy, with MEN (9, 10)] was not evaluable. Third, the possibility of who attained a major response (76% regression) with vande- sample size bias exists as the number of cases in each

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Kato et al.

malignancy relied on the number of specimens submitted by cine, Genentech, Guardant, Merck, Pfizer, and Squenom. No potential physicians for NGS. Finally, the diagnosis was determined on conflicts of interest were disclosed by the other authors. the basis of the submitting physician's designation. However, despite these limitations, the current report provides a large and Authors' Contributions comprehensive analysis of RET aberrations in diverse cancers. Conception and design: S. Kato, E. Marchlik, S.K. Elkin, J.L. Carter, R. Kurzrock In conclusion, we have evaluated 4,871 patients with diverse Development of methodology: S. Kato, E. Marchlik, S.K. Elkin cancers and shown that aberrations in RET are found in 1.8% (88/ Acquisition of data (provided animals, acquired and managed patients, 4,871) of cases. We have also identified a novel SQSTM1-RET provided facilities, etc.): V. Subbiah, E. Marchlik, S.K. Elkin fusion (n ¼ 1) in a papillary thyroid tumor. Most patients [81.8% Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.Kato,V.Subbiah,E.Marchlik,S.K.Elkin, (72/88)] had coexisting genomic aberrations that accompanied R. Kurzrock their RET alterations. In the vast majority of individuals [98.6% Writing, review, and/or revision of the manuscript: S. Kato, V. Subbiah, (71/72)], at least one of the coalterations was pharmacologically E. Marchlik, S.K. Elkin, J.L. Carter, R. Kurzrock tractable. Although various trials targeting RET aberrations are Administrative, technical, or material support (i.e., reporting or organizing ongoing (Supplementary Table S1), the current report suggests data, constructing databases): V. Subbiah that individualized cotargeting of multiple aberrant genes along Study supervision: R. Kurzrock with RET inhibition may be required for optimal clinical outcome. Grant Support This study was funded in part by the Joan and Irwin Jacobs fund. Disclosure of Potential Conflicts of Interest The costs of publication of this article were defrayed in part by the V. Subbiah reports receiving commercial research grants from Novartis. payment of page charges. This article must therefore be hereby marked advertisement S.K. Elkin and J.L. Carter have ownership interest (including patents) in in accordance with 18 U.S.C. Section 1734 solely to indicate N-of-One. R. Kurzrock is an employee of and has ownership interests this fact. (including patents) in Novena Inc. and Curematch, Inc.; is a consultant/ advisory board member for Actuate Therapeutics, Squenom, and Xbiotech; Received July 7, 2016; revised August 22, 2016; accepted September 4, 2016; and reports receiving commercial research grants from Foundation Medi- published OnlineFirst September 28, 2016.

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www.aacrjournals.org Clin Cancer Res; 23(8) April 15, 2017 1997

RET Aberrations in Diverse Cancers: Next-Generation Sequencing of 4,871 Patients

Shumei Kato, Vivek Subbiah, Erica Marchlik, et al.

Clin Cancer Res 2017;23:1988-1997. Published OnlineFirst September 28, 2016.

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