Profiling of 149 Salivary Duct Carcinomas, Carcinoma Ex Pleomorphic Adenomas, and Adenocarcinomas, Not Otherwise Specified Reveals Actionable Genomic Alterations

Published OnlineFirst June 22, 2016; DOI: 10.1158/1078-0432.CCR-15-2568

Personalized Medicine and Imaging Clinical Cancer Research Proﬁling of 149 Salivary Duct Carcinomas, Carcinoma Ex Pleomorphic Adenomas, and Adenocarcinomas, Not Otherwise Speciﬁed Reveals Actionable Genomic Alterations Kai Wang1,2, Jeffery S. Russell3, Jessica D. McDermott4, Julia A. Elvin1, Depinder Khaira1, Adrienne Johnson1, Timothy A. Jennings5, Siraj M. Ali1, Molly Murray1, Carrie Marshall6, Dwight S. Oldham7, Donna Washburn7, Stuart J. Wong8, Juliann Chmielecki1, Roman Yelensky1, Doron Lipson1,Vincent A. Miller1, Philip J. Stephens1, Hilary S. Serracino6, Jeffrey S. Ross1,6, and Daniel W. Bowles4

Abstract

Purpose: We sought to identify genomic alterations (GA) in carcinoma, NOS (31.2%; P ¼ 0.043). RAS GAs were observed: salivary gland adenocarcinomas, not otherwise specified (NOS), adenocarcinoma, NOS (17.3%); SDC (26.8%); ca ex PA (4.2%); salivary duct carcinomas (SDC), carcinoma ex pleomorphic ade- and carcinoma, NOS (9.4%; P ¼ 0.054). ERBB2 GAs, including noma (ca ex PA), and salivary carcinoma, NOS. amplifications and mutations, were common: adenocarcinoma, Experimental Design: DNA was extracted from 149 tumors. NOS (13.5%); SDC (26.8%); ca ex PA (29.2%); carcinoma, NOS Comprehensive genomic profiling (CGP) was performed on (18.8; P ¼ 0.249). Other notable GAs include TP53 in >45% of hybridization-captured adaptor ligation-based libraries of 182 each histotype; NOTCH1: adenocarcinoma, NOS (7.7%), ca ex PA or 315 cancer-related genes plus introns from 14 or 28 genes (8.3%), carcinoma, NOS (21.6%); NF1: adenocarcinoma, NOS frequently rearranged for cancer and evaluated for all classes of (9.6%), SDC (17.1%), carcinoma, NOS (18.8%). RET fusions GAs. were identified in one adenocarcinoma, NOS (CCDC6-RET) and Results: A total of 590 GAs were found in 157 unique genes two SDCs (NCOA4-RET). Clinical responses were observed in (mean 3.9/tumor). GAs in the PI3K/AKT/mTOR pathway were patients treated with anti-HER2 and anti-RET–targeted therapies. more common in SDC (53.6%) than other histologies (P ¼ Conclusions: CGP of salivary adenocarcinoma, NOS, SDCs, ca 0.019) Cyclin-dependent kinase GAs varied among all histotypes: ex PA, and carcinoma, NOS revealed diverse GAs that may lead to adenocarcinoma, NOS (34.6%); SDC (12.2%); ca ex PA (16.7%); novel treatment options. Clin Cancer Res; 1–8. 2016 AACR.

Introduction 1Foundation Medicine, Inc, Cambridge, Massachusetts. 2Zhejiang Cancer Hos- pital, Hangzhou, China. 3Moffitt Cancer Center, University of South Florida, Malignant salivary gland carcinomas (SGC) are rare cancers Tampa, Florida. 4Division of Medical Oncology, University of Colorado School of affecting less than 2,500 adults in the United States per 5 Medicine, Aurora, Colorado. Department of Pathology and Laboratory Medi- year (1). Moreover, SGCs are heterogeneous tumors, with 24 cine, Albany Medical College, Albany, New York. 6Department of Pathology, 7 distinct malignant histotypes recognized in the most recent University of Colorado School of Medicine, Aurora, Colorado. Centra Hema- fi tology Oncology Clinic, Alan B. Pearson Regional Cancer Center, Lynchburg, World Health Organization (WHO) classi cation of salivary Virginia. 8Division of Hematology Oncology, Department of Medicine, Medical gland tumors (2). Although there are several unique salivary College of Wisconsin, Milwaukee, Wisconsin. gland cancer histologies, such as mucoepidermoid carcinoma, Note: Supplementary data for this article are available at Clinical Cancer adenoid cystic carcinoma (ACC), acinic cell carcinomas Research Online (http://clincancerres.aacrjournals.org/). (AciCC), salivary duct carcinoma (SDC), and carcinoma ex pleomorphic adenoma (ca ex PA), a proportion of cases with- Prior presentation: Preliminary account of this study has been presented in part fi at the American Society of Clinical Oncology Annual Meeting in Chicago 2015 out speci c histologic or immunophenotypic features are best (abstract 6040) and the College of American Pathologists Annual Meeting in assigned to the category adenocarcinoma, not otherwise spec- San Diego, 2014. ified (NOS; refs. 2, 3). Conventional and molecular cytogenetic fi Corresponding Authors: Daniel W. Bowles, University of Colorado School of analysis has identi ed recurring translocations in a variety of Medicine, 12801 E. 17th Ave, MS 8117, Aurora, CO 80045. Phone: 303-724-3801; SGCs, including ACC (MYB-NFIB), mucoepidermoid carcino- Fax: 303-724-3889; E-mail: [email protected]; and Jeffery S. Ross, ma (MECT1-MAML2), mammary analogue–secretory carcino- Department of Pathology and Laboratory Medicine, Albany Medical College, 47 ma (MASC; ETV6-NTRK3), and hyalinizing clear cell carcinoma New Scotland Ave, MC8 81, Albany, NY 12208. E-mail: (HCCC; EWSR1-ATF1;refs.4–7).However,littleisknown [email protected] about the molecular features of SDC, adenocarcinomas, NOS, doi: 10.1158/1078-0432.CCR-15-2568 and ca ex PA. In general, conventional chemotherapy and 2016 American Association for Cancer Research. radiation for SGCs has limited efficacy (8). Similarly, to date,

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amplifications and homozygous deletions), and gene fusions/ Translational Relevance rearrangements, as described previously (14). Salivary gland adenocarcinomas, not otherwise specified Clinically relevant genomic alterations were defined as those (NOS), salivary duct carcinomas, carcinoma ex pleomorphic identifying anticancer drugs on the market or in registered clinical adenomas, and salivary carcinomas, NOS are rare and difficult trials. Statistical analysis was performed with the Fisher exact test, to treat tumors, with little known about their genomic under- with the level of significance set at P 0.05 using JMP (SAS). pinnings. Here, we describe the genomic alterations seen in 149 such tumors using a commercially available comprehensive genomic profiling platform. We identified frequent altera- Results tions in key cancer genes and pathways, such as TP53, PIK3CA, Of the 149 patient samples included in this study 52 (34.9%) RAS, ERBB2, and RET. There was preliminary evidence of were salivary adenocarcinoma, NOS, 41 (27.5%) were SDC, 24 anticancer efficacy with targeted therapies. Two patients with (16.1%) were ca ex PA, and 32 (21.5%) were carcinoma, NOS RET translocations experienced tumor regression when treated (Table 1). The male-to-female ratio differed by tumor type, with with RET-targeted therapies. This study may provide an insight relatively more men diagnosed with SDC (87.8%) and adeno- for possible therapeutic targets in these rare head and neck carcinoma, NOS (78.8%) (P < 0.001). The tumor source from cancers. which the sample was obtained did not differ significantly between groups with most samples taken from the parotid gland (32.2%), followed by a head and neck nonspecified site (12.1%), lung (12.8%), lymph node (10.1%), and salivary gland (8.7%; P ¼ 0.442). The majority of tissue samples came from local/ targeted therapies in nonmolecularly preselected SGCs have regional (94/149, 63%) versus metastatic sites (55/149, 36.9%). been disappointing (8). HER2 (ERBB2) and the androgen A total of 590 GAs were found in 157 unique genes (Figs. 1 receptor (AR) have been recognized as important drivers of and 2; Table 2). One hundred and ninety-eight (33.6%) were SDC and adenocarcinomas, NOS (9, 10). Multiplex mutation base substitutions or short indels, 163 (27.6%) were amplifica- or limited next-generation sequencing (NGS) analysis has tions, 44 (7.4%) were homozygous deletions, 23 (3.9%) were identified TP53, activating PIK3CA,andRAS mutations as rearrangements or fusions, and 168 (28.5%) were gene trunca- leading genetic alterations in SDC (11, 12). However, these tions. The mean number SD of GAs per sample was 3.9 (2.8), studies have been limited by small mutation profiles (<50 with a difference across histotypes: adenocarcinoma, NOS (3.8 genomic alterations), insensitive assays for low purity tumor 3.1); SDC (3.6 1.9); ca ex PA (3 2),carcinoma,NOS samples, and/or small sample size. The genetic underpinnings (5.2 4.1; P ¼ 0.033). The majority of patients (142/149, of adenocarcinoma, NOS are largely unknown. Both adeno- 95.3%) had at least one GA identified. Clinically relevant GAs carcinoma, NOS and SDC are associated with a worse prognosis were identified in 117 (78.5%) patient samples. than other SGCs, heightening the need for novel, precise There were many similarities in GA profiles among histotypes. therapies (13). All had GAs in the PI3K/AKT/mTOR signaling pathway, cyclin- In the following study, we used comprehensive genomic pro- dependent kinases (CKD), and RAS (Figs. 1 and 2; Table 3 and filing (CGP) to survey a large group of clinically advanced salivary Supplementary Table S1). In the PI3K/AKT/mTOR pathway, GAs adenocarcinoma, NOS, SDC, ca ex PA, and unclassifiable salivary occurred most commonly in SDC [22 (53.6%)], followed carcinoma, NOS to search for novel therapy targets and demon- by adenocarcinoma, NOS [19 (36.5%)], carcinoma, NOS strate the impact of biomarker-selected targeted therapy in select- [10 (31.2%)], and ca ex PA [4 (16.7%); P ¼ 0.019]. There was ed cases. a difference in CDK GAs among adenocarcinoma, NOS [18 (34.6%)], SDC [5 (12.2%)], ca ex PA [4 (16.7%)] and Materials and Methods carcinoma, NOS [10 (31.2%); P ¼ 0.043]. RAS family GAs Local site permissions were utilized for this study, and a full appeared more frequently in SDC [11 (26.8%)] and adenocarci- description of the methods can be found in the Supplementary noma, NOS [9 (17.3%)] than other histologies (<10%) but did Methods. SGC samples submitted for commercial CGP as salivary not reach statistical significance (P ¼ 0.054). Simultaneous GAs in adenocarcinoma or SDC underwent pathologic assessment using the PI3K/mTOR pathway and RAS were more common in SDC hematoxylin and eosin (H&E) and review of prior IHC profiling. [11 (26.8%)] than other histologies (P ¼ 0.006). In fact each RAS Images from each submitted tumor specimen were individually mutation in an SDC was accompanied by PI3K/mTOR pathway reviewed. Cases of other WHO SGC categories, including ACC, activation. There was no difference in concurrent GAs in PI3K mucoepidermoid carcinoma, AciCC, epithelial-myoepithelial pathway and CDKs between histologies (P ¼ 0.473). carcinoma, myoepithelial carcinoma, MASC, basal cell adenocar- Other notable GAs were identified. The most common GA in all cinoma, HCCC, low-grade polymorphous adenocarcinoma, and tumor types involved TP53 : adenocarcinoma, NOS [29 (55.7%)]; squamous cell carcinomas were excluded. The resulting samples SDC [22 (53.7%)]; ca ex PA [11 (45.8%)]; carcinoma, NOS [19 were then classified into the WHO categories SDC, adenocarci- (59.4%); P ¼ 0.783]. ERBB2 GAs, including both amplifications noma, NOS, and ca ex PA, SDC. Carcinoma samples with a clinical and activating mutations, were also seen across histologies: history as an SGC that did not fall into a clear WHO grouping were adenocarcinoma, NOS [7 (13.5%)]; SDC [11 (26.8%)]; ca ex PA classified as carcinoma, NOS. DNA was extracted from formalin- [7 (29.2%)]; carcinoma, NOS [6 (18.8%); P ¼ 0.249]. NOTCH1 fixed and paraffin-embedded samples. Captured libraries were GAs were not seen in SDC but were observed in adenocarcinoma, sequenced to a median exon coverage depth of 600 for up to 315 NOS [4 (7.7%)], ca ex PA [2 (8.3%)], and carcinoma, NOS genes, and resultant sequences were analyzed for base substitu- [7 (21.9%); P ¼ 0.006. ARID1A GAs were observed in adenocar- tions, short insertions/deletions, copy number alterations (focal cinoma, NOS [4 (7.7%)], SDC [1 (2.4%)], ca ex PA [3 (12.5%)],

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Table 1. Clinical characteristics of patients and tissue samples of 149 cases salivary adenocarcinoma, NOS, SDC, ca ex PA, and carcinoma, NOS All Adenocarcinoma, NOS SDC ca ex PA Carcinoma, NOS Characteristic n ¼149 (%) n ¼ 52 (%) n ¼ 41 (%) n ¼ 24 (%) n ¼ 32 (%) P Gender <0.001 Male 105 (70.5) 41 (78.8) 36 (87.8) 11 (45.8) 17 (53.1) Female 44 (29.5) 11 (21.2) 5 (12.2) 13 (54.2) 15 (46.9) Tissue source 0.4418 Parotid 48 (32.2) 15 (28.8) 17 (41.4) 9 (37.5) 7 (21.9) Salivary gland 13 (8.7) 2 (3.8) 6 (14.6) 2 (8.3) 3 (9.4) Lymph node 15 (10.1) 7 (13.9) 3 (7.3) 1 (4.1) 4 (12.5) Head/neck 18 (12.1) 8 (15.4) 2 (4.8) 2 (8.3) 6 (25) Lung 17 (12.8) 5 (9.6) 5 (12.2) 2 (8.3) 5 (15.6) Bone 8 (5.5) 4 (7.7) 2 (4.8) 1 (4.1) 1 (3.1) Brain 5 (3.4) 3 (5.8) 0 (0) 2 (8.3) 0 (0) Other 25 (16.8) 8 (15.4) 6 (14.6) 5 (20.8) 6 (18.8) and carcinoma, NOS [5 (15.6%); P ¼ 0.182]. NF1 GAs were seen biopsied that harbored an NCOA–RET fusion. He was given more common in SDC (7 [17.1%]) and carcinoma, NOS [6 cabozantinib, a tyrosine kinase inhibitor targeting RET and other (18.8)] than ca ex PA [0 (0%)] or adenocarcinoma, NOS [5 kinases and had a dramatic improvement in his chest wall lesions (9.6%); P ¼ 0.036]. BRAF mutations were found in both adeno- and mediastinal adenopathy after approximately 10 weeks carcinoma, NOS [3 (5.8%)] and SDC [2 (4.9%)], but not other (Fig. 3). A 79-year-old man with a stage IVA (T2 N2b Mx) AR- histologies. ETV6–NTRK3 translocations were seen only in 2 positive parotid SDC had a neck lesion biopsied after progression (3.8%) adenocarcinomas, NOS. Neither of these tumors was following surgery, radiation, and dual androgen deprivation clearly suggestive of MASC tumors by light microscopy (Supple- therapy. A NCOA4–RET fusion was identified and patient had mentary Fig. S1). One CCDC6–RET fusion was observed in an improvement in his palpable neck relapse when treated with adenocarcinoma, NOS (1.9%), and two NCOA4–RET fusions cabozantinib. were found in SDC (4.9%). Despite their rarity and the retrospective nature of this study, examples of successful use of precisions therapies were identified. Discussion A 62-year-old man with a stage IVA (T4a N2b M0) HER2-positive, SGCs are uncommon, diverse malignancies with limited ben- AR-positive parotid SDC who refused local therapy had a brisk efit from nontargeted systemic therapies. In this study, we iden- partial response following 2 cycles of carboplatin, docetaxel, and tified that many patients with salivary gland adenocarcinomas, trastuzumab, an anti-HER2 antibody (Fig. 3A). Two patients with NOS, salivary duct carcinomas, ca ex PA, and carcinoma, NOS NCOA4–RET translocations derived benefit from RET-targeted have genetic alterations that may allow for precision therapy therapy. A 68-year-old man with a stage IVC (T4a M2c M1) AR- selection. To our knowledge, this is the most comprehensive positive parotid SDC who had progressed following concurrent study examining the genetic profiles of these tumors. chemoradiation, combination chemotherapy, dual androgen This study identified unique GAs that may be exploited ther- deprivation, cetuximab, and everolimus had a chest wall lesion apeutically in SGC. Consistent with the great heterogeneity in

Percentage of cases with GA per gene Disease type TP53 Adenocarcinoma, NOS RET SDC RB1 ca ex PA PTEN PIK3CA Carcinoma, NOS NOTCH1 NF1 MYC MDM2 MCL1 KDM6A Figure 1. HRAS Comparison of genes with >3 GAs in FGF4 Gene salivary adenocarcinoma, NOS, SDC, ca FGF3 ex PA, or carcinoma, NOS. ERBB2 CDKN2B CDKN2A CDK6 CDH1 CCND1 BRAF BAP1 ARID1A AKT1 0% 10% 20% 30% 40% 50% 60% 70% Percentage of GAs

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Adenocarcinoma, NOS SDC ca ex PA Carcinoma, NOS

TP53 PIK3CA ERBB2 HRAS CDKN2A NF1 CDKN2B PTEN NOTCH1 ARID1A MYC BAP1 CDH1 RB1 MDM2 MCL1 SPEN RET MYST3 KDM6A FGFR1 FGF4 FGF3 FBXW7 CCND1 BRAF ASXL1 RICTOR HGF FGF19 EGFR CDK6 CDK4 BRIP1 AURKA AKT1 ZNF703 TET2 SMARCB1 ROS1 PTPRD PIK3R1 NCOR1 MLL2 MAP2K4 LRP1B KRAS KDM5A FGFR2 CDK12 CCNE1 CCND3 CASP8 BRCA1 BCORL1 ATM TSC2 SPTA1 SETD2 PTCH1 PDGFRA PDCD1LG2 PALB2 NOTCH2 MUTYH MAP3K1 MAP2K1 KDR JAK2 FGF10 FAT1 FANCA EZH2 ETV6 ERBB3 EMSY CHUK CHD2 CD274 C17orf39 BRCA2 BCOR ARID2 AKT3

Substitution/indel Gene amplification Gene homozygous deletion Truncation Gene fusion/rearr.

Figure 2. Tile plot of 149 salivary adenocarcinomas, NOS, SDC, ca ex PA, or carcinoma, NOS.

SGCs, the SGCs in this series had more actionable and nonac- 30% of papillary thyroid carcinoma cases and 1% of non–small tionable GAs than ACC, another SGC histotype (15). RET fusions cell lung carcinoma (NSCLC; refs. 16–20). RET fusions have not were observed in 1 adenocarcinoma, NOS (CCDC6-RET) and two been reported in SGCs to our knowledge. NSCLC patients with SDCs (NCOA4-RET). NCOA4-RET has been reported in 20% to KIF5B–RET fusion have beneﬁted from the RET inhibitor

Table 2. GAs in 149 cases salivary adenocarcinoma, NOS, SDC, ca ex PA, and carcinoma, NOS All Adenocarcinoma, NOS SDC ca ex PA Carcinoma, NOS Parameter n ¼ 149 (%) n ¼ 52 (%) n ¼ 41 (%) n ¼ 24 (%) n ¼ 32 (%) Pa Total GAs, no. 590 207 (34.9) 144 (24.4) 73 (12.4) 166 (28.1) 0.033 GAs per sample, mean 3.96 3.98 3.50 3.04 5.19 Base substitutions/short indels, no. (%) 198 (33.6) 75 (36.2) 61 (42.4) 16 (21.9) 46 (27.7) 0.051 Ampliﬁcations, no. (%) 163 (27.6) 48 (23.2) 40 (27.8) 26 (35.6) 49 (29.5) 0.408 Homozygous deletions, no. (%) 44 (7.4) 20 (9.7) 4 (2.8) 6 (8.2) 14 (8.4) 0.111 Rearrangements/fusions, no. (%) 23 (3.9) 9 (4.3) 5 (3.5) 3 (4.1) 6 (3.6) 0.866 Gene truncations, no. (%) 168 (28.5) 57 (27.5) 36 (25.0) 22 (30.1) 53 (31.9) 0.137 aP value corresponding to ANOVA of mean GAs per gene in each tumor type.

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Table 3. GAs by pathway in 149 cases salivary adenocarcinoma, NOS, salivary duct carcinoma, ca ex PA, and carcinoma, NOS Tumor type PI3K pathway CDKs RAS PI3K þ RAS PI3K þ Cyclin Adenocarcinoma, NOS All – 19 (36/5%) All – 18 (34.6%) All – 9 (17.3%) All – 8 (15.3%) All – 6 (11.5%) n ¼ 52 PIK3CA – 10 (19.2%) CDKN2A – 9 (17.3%) HRAS – 7 (13.5%) PIK3CA þ HRAS – 5 PTEN þ CDKN2A (9.6%) þ CDKN2B – 1 (1.9%) PTEN – 4 (7.7%) CDKN2B – 6 (11.5%) KRAS – 2 (3.8%) AKT1 þ HRAS – 2 PIK3CA þ CDKN2A (3.8%) þ CDKN2B – 1 (1.9%) RICTOR – 4 (7.7%) CCND1 – 3 (5.7%) AKT1 þ KRAS – 1 PIK3CA þ CDKN2A, (1.9%) CDKN2B, CDK6, CCND3 – 1 (1.9%) AKT1 – 3 (5.8%) CCND3 – 2 (3.7%) PIK3CA þ CCND3 – 1 (1.9%) AKT3 – 1 (1.9%) CCNE1 – 2 (3.8%) AKT3 þ CCNE1 – 1 (1.9%) PIK3R1 – 1 (1.9%) CDK12 – 2 (3.78%) PIK3R1 þ CDKN1B – 1 (1.9%) CDK6 – 2 (3.8%) CDKN1B – 1 (1.9%) CCND2 – 1 (1.9%) SDC All – 22 (53.6%) All – 5 (12.2%) All – 11 (26.8%) All – 11 (26.8%) All – 4 (9.8%) n ¼ 41 PIK3CA – 15 (36.6%) CDKN2A – 3 (7.3%) HRAS – 11 (26.8%) PIK3CA þ HRAS – 10 RICTOR þ CDK4 (24.3%) – 1 (2.4%) PTEN – 5 (12.2%) CCNE1 – 1 (2.4%) PIK3CA þ AKT1 þ PTEN þ CDKN2A HRAS – 1 (2.4%) – 1 (2.4%) RICTOR – 2 (4.9%) CDK4 – 1 (2.4%) PIK3CA þ CDKN2A þ CDKN2B – 1 (2.4%) AKT3 – 1 (2.4%) CDKN2B – 1 (2.4%) PIK3CA þ CDKN2A – 1 (2.4%) AKT1 – 1 (2.4%) PIK3R1 – 1 (2.4%) ca ex PA All – 4 (16.7%) All – 4 (16.7%) All – 1 (4.2%) All – 0 (0.0%) All – 1 (4.2%) n ¼ 24 PTEN – 3 (12.5%) CDKN2A – 2 (8.3%) KRAS – 1 (4.2%) PTEN þ CCND3 – 1 (4.2%) RICTOR – 1 (4.2%) CDKN2B – 2 (8.3%) CCND3 – 1 (4.2%) CDK4 – 1 Carcinoma, NOS All – 10 (31.2%) All – 10 (31.2%) All – 3 (9.4%) All – 3 (9.4%) All – 1 (3.1%) n ¼ 32 PIK3CA – 7 (21.9%) CDKN2A – 7 (21.9%) HRAS – 3 (9.4%) PIK3CA þ HRAS – 3 PTEN þ CDKN2A (9.4%) þ CDKN2B – 1 (3.1%) AKT – 1 (3.1%) CDKN2B – 6 (18.7%) PIK3CB – 1 (3.1%) CDK4 – 2 (6.2%) PIK3R1 – 1 (3.1%) CCND1 – 2 (6.2%) PTEN – 1 (3.1%) CDK12 – 1 (3.1%) RICTOR – 1 (3.1%) CDK6 – 1 (3.1%) NOTE: P values for comparisons among groups: PI3K pathway (P ¼ 0.019); CDK (P ¼ 0.043); RAS (P ¼ 0.052); PI3K þ RAS (P ¼ 0.006), PI3K þ cyclin (P ¼ 0.473).

cabozantinib, as have NSCLC patients with CCDC6–RET fusions also identified in carcinoma, NOS, they were not identified in treated with vandetanib, another RET tyrosine kinase inhibitor ductal ca ex PA. PTEN GAs were reported in 2% of salivary gland (21, 22). In an ongoing phase II study of cabozantinib, 5 carcinomas in COSMIC (29), whereas we found them in higher responses have been achieved by NSCLC patients with RET numbers in adenocarcinoma, NOS (7.7%), SDC (12.2%), and fusions in their tumors (21). Similar to these other solid tumors, ductal ca ex PA (12.5%). This study expands upon prior studies by we found evidence of anticancer activity targeting RET in RET- identifying frequent GAs not just in PIK3CA, but AKT, PTEN, and activated tumors, as two patients with RET fusions in our retro- RICTOR that may make patients candidates for PI3K, AKT, or spective series had tumor shrinkage with cabozantinib. mTOR inhibitors. The PI3K/AKT/mTOR was frequently altered in both histo- Frequent simultaneous GAs in the PI3K/AKT/mTOR pathway types, particularly SDCs. PIK3CA encodes the catalytically active and the RAS family of genes were observed. In a series of multiple subunit of PI3K that is involved in cell growth, proliferation, solid tumor histologies, paired mutations in KRAS were commonly differentiation, and survival (23). There are numerous PI3K observed in PIK3CA-mutated tumors (30). A similar pattern with inhibitors under investigation, and prior studies have shown that PIK3CA and HRAS has been noted in SDC (12). Targeting both activation of the PI3K/AKT/mTOR pathway across different tumor pathways may have a therapeutic advantage. For instance, colon types may predict responses to PI3K/AKT/mTOR pathway inhi- cancer cells harboring both KRAS and PIK3CA mutations were bitors (24–27). In SDC, a small series of patients with PI3K/AKT/ resistant to PI3K/mTOR inhibition alone, although solitary PI3K mTOR–activating GAs demonstrated clinical improvement when inhibition could be overcome with combined MEK and PI3K they were treated with temsirolimus, an mTOR inhibitor (28). blockade (31, 32). In NRAS-mutated melanoma, combined MEK Prior studies demonstrated PIK3CA mutations occurred in 19% of and PI3K inhibition is more effective than blockade of either SDC (11, 12), and they were reported in 7% of SGCs in COSMIC pathway alone (33). Thus, dual pathway inhibition may be worth (29). In our datasets, we identified PIK3CA mutations in 19.2% of evaluating in PIK3CA and HRAS-mutated tumors. adenocarcinoma, NOS and 36.6% in SDC, a higher number than A novel finding in this study was the frequent alteration of a previously reported. Interestingly, although PIK3CA GAs were variety of CDKs in all histologies. CDKs are a diverse set of critical

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Figure 3. A, H&E photomicrograph of an HER2þ SDC (left), HER2 immunostaining (middle left), MRI face with contrast before (middle right) and after (right) treatment with carboplatin, docetaxel, and trastuzumab. B, H&E photomicrograph of a salivary duct carcinoma harboring an NCOA–RET fusion (left), AR staining (middle left), CT chest before (middle right) and after (right) treatment with cabozantinib.

regulatory proteins responsible for cell-cycle transitions (34, 35). ma, NOS group in this study did not clearly cluster with any of the Recently, CDK GAs were reported in a wide variety of solid tumors three other histotypes, yet they harbored frequent actionable at varying frequencies (0%–81%), where they were associated GAs. These findings suggest CGP may help clarify diagnoses with poorer survival (36). In this study, CDKs were altered in more and identify GAs for therapeutic exploitation in difficult to diag- than 30% of adenocarcinoma, NOS and carcinoma, NOS. nose cases. Because of diversity and complexity of CDK signaling, it has been This study has limitations. The greatest limitation is the lack difficult to develop CDK-targeted therapies (37). However, pal- of clinical correlations between identified GAs and disease bociclib, a CDK4/6 inhibitor, was recently shown to improve characteristics or patients outcomes. As this was a retrospective progression-free survival when combined with endocrine therapy evaluation of samples submitted for clinical care, data about in estrogen receptor–positive, HER2-negative breast cancer (38). tumor grade, cancer stage, response to therapies, and patient SGCs harboring CDK GAs may be good candidates for trials survival are not available. Another limitation is that, although targeting CDKs or their partners. the CGP platform used in this study covers a wide range of genes, Other common and rare GAs were identified that may have there may be clinically relevant GAs that were not assessed. For therapeutic and/or diagnostic relevance. Consistent with prior instance, although the assay routinely identifies the MYB–NFIB studies, amplification of ERBB2 was commonly seen in SDC but translocation commonly seen in ACC, it does not assess for the less so in adenocarcinomas, NOS (12, 39, 40). In addition, we also MECT1–MAML2 translocation commonly identified in mucoe- identified a high number of ERBB2 GAs in ductal ca ex PA. New to pidermoid carcinoma (4, 6). Designing a CGP panel for salivary this study was the identification of activating ERBB2 mutations in gland cancers, including MECT-MAML2, may be more clinically adenocarcinoma, NOS, SDC, and ductal ca ex PA. HER2-targeted applicable to a broad range of tumors. Similarly, although the therapies, such as trastuzumab, have previously demonstrated pathology reports and prior immunohistochemical staining pat- efficacy in ERBB2-amplified salivary gland cancers and contrib- terns were reviewed, we could not perform additional/supple- uted a partial response in our series (41, 42). Select GAs in genes mentary IHC characterization because specimens were submitted with precision drugs currently commercially available included strictly for CGP analysis. For SDC, AR staining aids in the diag- ROS1, MET, and BRAF, although BRAF GAs were seen less fre- nosis and can be exploited therapeutically (9, 39, 45). Another quently than previously reported (11). BRAF inhibition was limitation is that the tumors tested in this study may not fully recently shown to be of benefit in one patient with a BRAF- represent the genetic spectrum of these diagnoses, as commercial mutated SDC (43). Interestingly, this study identified character- CGP tends to be performed or more aggressive, advanced/met- istic GAs in unexpected histologies. For instance, ETV6–NTRK3 astatic tumors, and many salivary gland cancers are somewhat translocations, a translocation characteristic of MASCs, were indolent (46). Moreover, CGP was performed on both locore- observed in 2 (3.8%) of tumors submitted as adenocarcinoma, gional disease and metastatic sites. There are no data in SGCs to NOS (5). The finding of this translocation in the adenocarcinoma, demonstrate whether the GA profile of locoregional disease is NOS may speak to the lack of consensus for the IHC profile for representative of metastatic sites, or vice versa, although there MASC tumors and suggest that CGP profiling may be of diagnostic appears to be high concordance in other tumors (47, 48). Finally, benefit in histologically difficult cases (44). Finally, the carcino- although this study demonstrates frequent actionable GAs, it does

OF6 Clin Cancer Res; 2016 Clinical Cancer Research

Genomic Alterations in Salivary Adenocarcinoma

not address intratumoral heterogeneity and the relative impor- Development of methodology: K. Wang, R. Yelensky, D. Lipson, J.S. Ross, tance of each GA in patient outcomes. Recent studies in renal cell D.W. Bowles and lung cancers have demonstrated that different clones have Acquisition of data (provided animals, acquired and managed patients, fl provided facilities, etc.): J.S. Russell, J.A. Elvin, D. Khaira, T.A. Jennings, varying driver mutations whose importance can uctuate over S.M. Ali, J.S. Ross, D.W. Bowles time (49, 50). Determining the inciting GA and relative impor- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tance of subsequent GAs in SGC is worth exploring in future computational analysis): K. Wang, J.D. McDermott, D. Khaira, A. Johnson, S.M. studies. Despite these limitations, this study greatly contributes to Ali, J. Chmielecki, R. Yelensky, P.J. Stephens, J.S. Ross, D.W. Bowles the knowledge of the genetic drivers of these rare tumors. Writing, review, and/or revision of the manuscript: K. Wang, J.S. Russell, J.D. In summary, deep genomic profiling with a comprehensive McDermott, J.A. Elvin, D. Khaira, S.M. Ali, M. Murray, C. Marshall, D. Wash- fi burn, S.J. Wong, V.A. Miller, P.J. Stephens, H.S. Serracino, J.S. Ross, D.W. Bowles genomic pro ling assay of salivary adenocarcinomas, NOS, sal- Administrative, technical, or material support (i.e., reporting or organizing ivary duct carcinomas, ca ex PA, and salivary carcinoma, NOS data, constructing databases): J.D. McDermott, M. Murray, J.S. Ross, D.W. identified a high percentage of potentially actionable GAs that can Bowles influence therapy selection and direct patients to enter clinical Study supervision: K. Wang, J.S. Ross, D.W. Bowles trials using precision therapies. The GAs identified in this study are Other (case report contribution to the content of the manuscript): D. Oldham an important step to opening pathways for new therapeutic Other (minor contribution by detail writing and editing content by physi- cian): D. Washburn approaches in these notoriously difficult to treat cancers. Acknowledgments fl Disclosure of Potential Con icts of Interest We would like to thank Dr. Marino Leon from Moffitt Cancer Center for the K. Wang, J.A. Elvin, D. Khaira, A. Johnson, S.M. Ali, M. Murray, J. Chmieck- H&E and androgen receptor photomicrographs of the RET fusion patient ecki, R. Yelensky, D. Lipson, V.A. Miller, P.J. Stephens, and J.S. Ross have described in this article. ownership interests (including patents) in Foundation Medicine. No potential The costs of publication of this article were defrayed in part by the payment of conflicts of interest were disclosed by the other authors. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: K. Wang, J.S. Russell, J.A. Elvin, D. Khaira, S.J. Wong, Received October 22, 2015; revised May 10, 2016; accepted May 23, 2016; V.A. Miller, J.S. Ross, D.W. Bowles published OnlineFirst June 22, 2016.

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OF8 Clin Cancer Res; 2016 Clinical Cancer Research

Profiling of 149 Salivary Duct Carcinomas, Carcinoma Ex Pleomorphic Adenomas, and Adenocarcinomas, Not Otherwise Specified Reveals Actionable Genomic Alterations

Kai Wang, Jeffery S. Russell, Jessica D. McDermott, et al.

Clin Cancer Res Published OnlineFirst June 22, 2016.

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