Proﬁling of 149 Salivary Duct Carcinomas, Carcinoma Ex ... · gland cancer histologies, such as mucoepidermoid carcinoma, adenoid cystic carcinoma (ACC), acinic cell carcinomas

Personalized Medicine and Imaging

Profiling of 149 Salivary Duct Carcinomas,Carcinoma Ex Pleomorphic Adenomas, andAdenocarcinomas, Not Otherwise SpecifiedReveals Actionable Genomic AlterationsKai 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,RomanYelensky1, Doron Lipson1,VincentA.Miller1, Philip J. Stephens1, Hilary S. Serracino6,Jeffrey S. Ross1,6, and Daniel W. Bowles4

Abstract

Purpose: We sought to identify genomic alterations (GA) insalivary gland adenocarcinomas, not otherwise specified (NOS),salivary duct carcinomas (SDC), carcinoma ex pleomorphic ade-noma (ca ex PA), and salivary carcinoma, NOS.

Experimental Design: DNA was extracted from 149 tumors.Comprehensive genomic profiling (CGP) was performed onhybridization-captured adaptor ligation-based libraries of 182or 315 cancer-related genes plus introns from 14 or 28 genesfrequently rearranged for cancer and evaluated for all classes ofGAs.

Results: A total of 590 GAs were found in 157 unique genes(mean 3.9/tumor). GAs in the PI3K/AKT/mTOR pathway weremore common in SDC (53.6%) than other histologies (P ¼0.019) Cyclin-dependent kinase GAs varied among all histotypes:adenocarcinoma, NOS (34.6%); SDC (12.2%); ca ex PA (16.7%);

carcinoma, NOS (31.2%; P ¼ 0.043). RAS GAs were observed:adenocarcinoma, NOS (17.3%); SDC (26.8%); ca ex PA (4.2%);and carcinoma, NOS (9.4%; P ¼ 0.054). ERBB2 GAs, includingamplifications and mutations, were common: adenocarcinoma,NOS (13.5%); SDC (26.8%); ca ex PA (29.2%); carcinoma, NOS(18.8; P ¼ 0.249). Other notable GAs include TP53 in >45% ofeachhistotype;NOTCH1: adenocarcinoma,NOS (7.7%), ca ex PA(8.3%), carcinoma, NOS (21.6%); NF1: adenocarcinoma, NOS(9.6%), SDC (17.1%), carcinoma, NOS (18.8%). RET fusionswere identified in one adenocarcinoma, NOS (CCDC6-RET) andtwo SDCs (NCOA4-RET). Clinical responses were observed inpatients treated with anti-HER2 and anti-RET–targeted therapies.

Conclusions: CGP of salivary adenocarcinoma, NOS, SDCs, caex PA, and carcinoma, NOS revealed diverse GAs that may lead tonovel treatment options. Clin Cancer Res; 22(24); 6061–8.�2016 AACR.

IntroductionMalignant salivary gland carcinomas (SGC) are rare cancers

affecting less than 2,500 adults in the United States peryear (1). Moreover, SGCs are heterogeneous tumors, with 24distinct malignant histotypes recognized in the most recentWorld Health Organization (WHO) classification of salivarygland tumors (2). Although there are several unique salivarygland cancer histologies, such as mucoepidermoid carcinoma,adenoid cystic carcinoma (ACC), acinic cell carcinomas(AciCC), salivary duct carcinoma (SDC), and carcinoma expleomorphic adenoma (ca ex PA), a proportion of cases with-out specific histologic or immunophenotypic features are bestassigned to the category adenocarcinoma, not otherwise spec-ified (NOS; refs. 2, 3). Conventional and molecular cytogeneticanalysis has identified recurring translocations in a variety ofSGCs, including ACC (MYB-NFIB), mucoepidermoid carcino-ma (MECT1-MAML2), mammary analogue–secretory carcino-ma (MASC; ETV6-NTRK3), and hyalinizing clear cell carcinoma(HCCC; EWSR1-ATF1; refs. 4–7). However, little is knownabout the molecular features of SDC, adenocarcinomas, NOS,and ca ex PA. In general, conventional chemotherapy andradiation for SGCs has limited efficacy (8). Similarly, to date,

1Foundation Medicine, Inc, Cambridge, Massachusetts. 2Zhejiang Cancer Hos-pital, Hangzhou, China. 3Moffitt Cancer Center, University of South Florida,Tampa, Florida. 4Division of Medical Oncology, University of Colorado School ofMedicine, Aurora, Colorado. 5Department of Pathology and Laboratory Medi-cine, Albany Medical College, Albany, New York. 6Department of Pathology,University of Colorado School of Medicine, Aurora, Colorado. 7Centra Hema-tology Oncology Clinic, Alan B. Pearson Regional Cancer Center, Lynchburg,Virginia. 8Division of Hematology Oncology, Department of Medicine, MedicalCollege of Wisconsin, Milwaukee, Wisconsin.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Prior presentation: Preliminary account of this study has been presented in partat the American Society of Clinical Oncology Annual Meeting in Chicago 2015(abstract 6040) and the College of American Pathologists Annual Meeting inSan Diego, 2014.

Corresponding Authors: Daniel W. Bowles, University of Colorado School ofMedicine, 12801 E. 17th Ave, MS 8117, Aurora, CO 80045. Phone: 303-724-3801;Fax: 303-724-3889; E-mail: [email protected]; and Jeffery S. Ross,Department of Pathology and Laboratory Medicine, Albany Medical College, 47New Scotland Ave, MC8 81, Albany, NY 12208. E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-2568

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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targeted therapies in nonmolecularly preselected SGCs havebeen disappointing (8). HER2 (ERBB2) and the androgenreceptor (AR) have been recognized as important drivers ofSDC and adenocarcinomas, NOS (9, 10). Multiplex mutationor limited next-generation sequencing (NGS) analysis hasidentified TP53, activating PIK3CA, and RAS mutations asleading genetic alterations in SDC (11, 12). However, thesestudies have been limited by small mutation profiles (<50genomic alterations), insensitive assays for low purity tumorsamples, and/or small sample size. The genetic underpinningsof adenocarcinoma, NOS are largely unknown. Both adeno-carcinoma, NOS and SDC are associated with a worse prognosisthan other SGCs, heightening the need for novel, precisetherapies (13).

In the following study, we used comprehensive genomic pro-filing (CGP) to survey a large group of clinically advanced salivaryadenocarcinoma, NOS, SDC, ca ex PA, and unclassifiable salivarycarcinoma, NOS to search for novel therapy targets and demon-strate the impact of biomarker-selected targeted therapy in select-ed cases.

Materials and MethodsLocal site permissions were utilized for this study, and a full

description of the methods can be found in the SupplementaryMethods. SGC samples submitted for commercial CGP as salivaryadenocarcinoma or SDC underwent pathologic assessment usinghematoxylin and eosin (H&E) and review of prior IHC profiling.Images from each submitted tumor specimen were individuallyreviewed. Cases of other WHO SGC categories, including ACC,mucoepidermoid carcinoma, AciCC, epithelial-myoepithelialcarcinoma, myoepithelial carcinoma, MASC, basal cell adenocar-cinoma, HCCC, low-grade polymorphous adenocarcinoma, andsquamous cell carcinomas were excluded. The resulting sampleswere then classified into the WHO categories SDC, adenocarci-noma,NOS, and ca exPA, SDC.Carcinoma sampleswith a clinicalhistory as an SGC that did not fall into a clearWHOgroupingwereclassified as carcinoma, NOS. DNA was extracted from formalin-fixed and paraffin-embedded samples. Captured libraries weresequenced to amedian exon coverage depthof 600� for up to 315genes, and resultant sequences were analyzed for base substitu-tions, short insertions/deletions, copy number alterations (focal

amplifications and homozygous deletions), and gene fusions/rearrangements, as described previously (14).

Clinically relevant genomic alterations were defined as thoseidentifying anticancer drugs on themarket or in registered clinicaltrials. Statistical analysis was performed with the Fisher exact test,with the level of significance set at P � 0.05 using JMP (SAS).

ResultsOf the 149 patient samples included in this study 52 (34.9%)

were salivary adenocarcinoma, NOS, 41 (27.5%) were SDC, 24(16.1%) were ca ex PA, and 32 (21.5%) were carcinoma, NOS(Table 1). The male-to-female ratio differed by tumor type, withrelatively more men diagnosed with SDC (87.8%) and adeno-carcinoma, NOS (78.8%) (P < 0.001). The tumor source fromwhich the sample was obtained did not differ significantlybetween 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/regional (94/149, 63%) versus metastatic sites (55/149, 36.9%).

A total of 590 GAs were found in 157 unique genes (Figs. 1and 2; Table 2). One hundred and ninety-eight (33.6%) werebase substitutions or short indels, 163 (27.6%) were amplifica-tions, 44 (7.4%) were homozygous deletions, 23 (3.9%) wererearrangements or fusions, and 168 (28.5%) were gene trunca-tions. The mean number � SD of GAs per sample was 3.9 (2.8),with a difference across histotypes: adenocarcinoma, NOS (3.8� 3.1); SDC (3.6 � 1.9); ca ex PA (3 � 2), carcinoma, NOS(5.2 � 4.1; P ¼ 0.033). The majority of patients (142/149,95.3%) had at least one GA identified. Clinically relevant GAswere identified in 117 (78.5%) patient samples.

There were many similarities in GA profiles among histotypes.All had GAs in the PI3K/AKT/mTOR signaling pathway, cyclin-dependent kinases (CKD), and RAS (Figs. 1 and 2; Table 3 andSupplementary Table S1). In the PI3K/AKT/mTOR pathway, GAsoccurred most commonly in SDC [22 (53.6%)], followedby adenocarcinoma, NOS [19 (36.5%)], carcinoma, NOS[10 (31.2%)], and ca ex PA [4 (16.7%); P ¼ 0.019]. There wasa difference in CDK GAs among adenocarcinoma, NOS[18 (34.6%)], SDC [5 (12.2%)], ca ex PA [4 (16.7%)] andcarcinoma, NOS [10 (31.2%); P ¼ 0.043]. RAS family GAsappeared more frequently in SDC [11 (26.8%)] and adenocarci-noma, NOS [9 (17.3%)] than other histologies (<10%) but didnot reach statistical significance (P¼0.054). SimultaneousGAs inthe PI3K/mTOR pathway and RAS were more common in SDC[11 (26.8%)] than other histologies (P¼ 0.006). In fact each RASmutation in an SDC was accompanied by PI3K/mTOR pathwayactivation. There was no difference in concurrent GAs in PI3Kpathway and CDKs between histologies (P ¼ 0.473).

Other notableGAswere identified. Themost commonGA in alltumor types involved TP53: adenocarcinoma, NOS [29 (55.7%)];SDC [22 (53.7%)]; ca ex PA [11 (45.8%)]; carcinoma, NOS [19(59.4%); P ¼ 0.783]. ERBB2 GAs, including both amplificationsand activating mutations, were also seen across histologies:adenocarcinoma, NOS [7 (13.5%)]; SDC [11 (26.8%)]; ca ex PA[7 (29.2%)]; carcinoma, NOS [6 (18.8%); P ¼ 0.249]. NOTCH1GAs were not seen in SDC but were observed in adenocarcinoma,NOS [4 (7.7%)], ca ex PA [2 (8.3%)], and carcinoma, NOS[7 (21.9%); P ¼ 0.006. ARID1A GAs were observed in adenocar-cinoma, NOS [4 (7.7%)], SDC [1 (2.4%)], ca ex PA [3 (12.5%)],

Translational Relevance

Salivary gland adenocarcinomas, not otherwise specified(NOS), salivary duct carcinomas, carcinoma ex pleomorphicadenomas, and salivary carcinomas, NOS are rare and difficultto treat tumors, with little known about their genomic under-pinnings. Here, we describe the genomic alterations seen in149 such tumors using a commercially available comprehen-sive genomic profiling platform.We identified frequent altera-tions in key cancer genes and pathways, such as TP53, PIK3CA,RAS, ERBB2, and RET. There was preliminary evidence ofanticancer efficacy with targeted therapies. Two patients withRET translocations experienced tumor regressionwhen treatedwith RET-targeted therapies. This studymay provide an insightfor possible therapeutic targets in these rare head and neckcancers.

Wang et al.

Clin Cancer Res; 22(24) December 15, 2016 Clinical Cancer Research6062




and carcinoma, NOS [5 (15.6%); P¼ 0.182].NF1 GAs were seenmore common in SDC (7 [17.1%]) and carcinoma, NOS [6(18.8)] than ca ex PA [0 (0%)] or adenocarcinoma, NOS [5(9.6%); P ¼ 0.036]. BRAF mutations were found in both adeno-carcinoma, NOS [3 (5.8%)] and SDC [2 (4.9%)], but not otherhistologies. ETV6–NTRK3 translocations were seen only in 2(3.8%) adenocarcinomas, NOS. Neither of these tumors wasclearly suggestive of MASC tumors by light microscopy (Supple-mentary Fig. S1). One CCDC6–RET fusion was observed in anadenocarcinoma, NOS (1.9%), and two NCOA4–RET fusionswere found in SDC (4.9%).

Despite their rarity and the retrospective nature of this study,examples of successful use of precisions therapies were identified.A 62-year-oldmanwith a stage IVA (T4aN2bM0)HER2-positive,AR-positive parotid SDC who refused local therapy had a briskpartial response following 2 cycles of carboplatin, docetaxel, andtrastuzumab, an anti-HER2 antibody (Fig. 3A). Two patients withNCOA4–RET translocations derived benefit from RET-targetedtherapy. A 68-year-old man with a stage IVC (T4a M2c M1) AR-positive parotid SDC who had progressed following concurrentchemoradiation, combination chemotherapy, dual androgendeprivation, cetuximab, and everolimus had a chest wall lesion

biopsied that harbored an NCOA–RET fusion. He was givencabozantinib, a tyrosine kinase inhibitor targeting RET and otherkinases and had a dramatic improvement in his chest wall lesionsand mediastinal adenopathy after approximately 10 weeks(Fig. 3). A 79-year-old man with a stage IVA (T2 N2b Mx) AR-positive parotid SDC had a neck lesion biopsied after progressionfollowing surgery, radiation, and dual androgen deprivationtherapy. A NCOA4–RET fusion was identified and patient hadimprovement in his palpable neck relapse when treated withcabozantinib.

DiscussionSGCs are uncommon, diverse malignancies with limited ben-

efit from nontargeted systemic therapies. In this study, we iden-tified that many patients with salivary gland adenocarcinomas,NOS, salivary duct carcinomas, ca ex PA, and carcinoma, NOShave genetic alterations that may allow for precision therapyselection. To our knowledge, this is the most comprehensivestudy examining the genetic profiles of these tumors.

This study identified unique GAs that may be exploited ther-apeutically in SGC. Consistent with the great heterogeneity in

TP53RETRB1

PTENPIK3CA

NOTCH1NF1

MYCMDM2MCL1

KDM6AHRASFGF4FGF3

ERBB2CDKN2BCDKN2A

CDK6CDH1

CCND1BRAFBAP1

ARID1AAKT1

Gen

e

Percentage of cases with GA per gene

0% 10% 20% 30%Percentage of GAs

40% 50% 60% 70%

Disease type

Adenocarcinoma, NOSSDCca ex PACarcinoma, NOS

Figure 1.

Comparison of genes with >3 GAs insalivary adenocarcinoma, NOS, SDC, caex PA, or carcinoma, NOS.

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, NOSCharacteristic n ¼149 (%) n ¼ 52 (%) n ¼ 41 (%) n ¼ 24 (%) n ¼ 32 (%) P

Gender <0.001Male 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 0.4418Parotid 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)

Genomic Alterations in Salivary Adenocarcinoma

www.aacrjournals.org Clin Cancer Res; 22(24) December 15, 2016 6063




SGCs, the SGCs in this series had more actionable and nonac-tionable GAs than ACC, another SGC histotype (15). RET fusionswere observed in 1 adenocarcinoma, NOS (CCDC6-RET) and twoSDCs (NCOA4-RET). NCOA4-RET has been reported in 20% to

30% of papillary thyroid carcinoma cases and 1% of non–smallcell lung carcinoma (NSCLC; refs. 16–20). RET fusions have notbeen reported in SGCs to our knowledge. NSCLC patients withKIF5B–RET fusion have benefited from the RET inhibitor

TP53PIK3CAERBB2HRAS

CDKN2ANF1

CDKN2BPTEN

NOTCH1ARID1A

MYCBAP1CDH1

RB1MDM2MCL1SPEN

RETMYST3KDM6AFGFR1

FGF4FGF3

FBXW7CCND1

BRAFASXL1

RICTORHGF

FGF19EGFRCDK6CDK4BRIP1

AURKAAKT1

ZNF703TET2

SMARCB1ROS1

PTPRDPIK3R1NCOR1

MLL2MAP2K4

LRP1BKRAS

KDM5AFGFR2CDK12CCNE1CCND3CASP8BRCA1

BCORL1ATM

TSC2SPTA1SETD2PTCH1

PDGFRAPDCD1LG2

PALB2NOTCH2MUTYH

MAP3K1MAP2K1

KDRJAK2

FGF10FAT1

FANCAEZH2ETV6

ERBB3EMSYCHUKCHD2

CD274C17orf39

BRCA2BCORARID2AKT3

Adenocarcinoma, NOS

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

SDC ca ex PA Carcinoma, NOS

Figure 2.

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

Table 2. GAs in 149 cases salivary adenocarcinoma, NOS, SDC, ca ex PA, and carcinoma, NOS

All Adenocarcinoma, NOS SDC ca ex PA Carcinoma, NOSParameter 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) 0.033GAs per sample, mean 3.96 3.98 3.50 3.04 5.19Base substitutions/short indels, no. (%) 198 (33.6) 75 (36.2) 61 (42.4) 16 (21.9) 46 (27.7) 0.051Amplifications, no. (%) 163 (27.6) 48 (23.2) 40 (27.8) 26 (35.6) 49 (29.5) 0.408Homozygous deletions, no. (%) 44 (7.4) 20 (9.7) 4 (2.8) 6 (8.2) 14 (8.4) 0.111Rearrangements/fusions, no. (%) 23 (3.9) 9 (4.3) 5 (3.5) 3 (4.1) 6 (3.6) 0.866Gene truncations, no. (%) 168 (28.5) 57 (27.5) 36 (25.0) 22 (30.1) 53 (31.9) 0.137aP value corresponding to ANOVA of mean GAs per gene in each tumor type.

Wang et al.





cabozantinib, as have NSCLC patients with CCDC6–RET fusionstreated with vandetanib, another RET tyrosine kinase inhibitor(21, 22). In an ongoing phase II study of cabozantinib, 5responses have been achieved by NSCLC patients with RETfusions in their tumors (21). Similar to these other solid tumors,we found evidence of anticancer activity targeting RET in RET-activated tumors, as two patients with RET fusions in our retro-spective series had tumor shrinkage with cabozantinib.

The PI3K/AKT/mTOR was frequently altered in both histo-types, particularly SDCs. PIK3CA encodes the catalytically activesubunit of PI3K that is involved in cell growth, proliferation,differentiation, and survival (23). There are numerous PI3Kinhibitors under investigation, and prior studies have shown thatactivationof thePI3K/AKT/mTORpathway across different tumortypes may predict responses to PI3K/AKT/mTOR pathway inhi-bitors (24–27). In SDC, a small series of patients with PI3K/AKT/mTOR–activating GAs demonstrated clinical improvement whenthey were treated with temsirolimus, an mTOR inhibitor (28).Prior studies demonstrated PIK3CAmutations occurred in 19%ofSDC (11, 12), and they were reported in 7% of SGCs in COSMIC(29). In our datasets, we identified PIK3CAmutations in 19.2%ofadenocarcinoma, NOS and 36.6% in SDC, a higher number thanpreviously reported. Interestingly, although PIK3CA GAs were

also identified in carcinoma, NOS, they were not identified inductal ca ex PA. PTEN GAs were reported in 2% of salivary glandcarcinomas in COSMIC (29), whereas we found them in highernumbers in adenocarcinoma, NOS (7.7%), SDC (12.2%), andductal ca ex PA (12.5%). This study expands upon prior studies byidentifying frequent GAs not just in PIK3CA, but AKT, PTEN, andRICTOR that may make patients candidates for PI3K, AKT, ormTOR inhibitors.

Frequent simultaneous GAs in the PI3K/AKT/mTOR pathwayand the RAS family of genes were observed. In a series of multiplesolid tumorhistologies, pairedmutations inKRASwere commonlyobserved in PIK3CA-mutated tumors (30). A similar pattern withPIK3CA and HRAS has been noted in SDC (12). Targeting bothpathways may have a therapeutic advantage. For instance, coloncancer cells harboring both KRAS and PIK3CA mutations wereresistant to PI3K/mTOR inhibition alone, although solitary PI3Kinhibition could be overcome with combined MEK and PI3Kblockade (31, 32). In NRAS-mutated melanoma, combined MEKand PI3K inhibition is more effective than blockade of eitherpathway alone (33). Thus, dual pathway inhibition may be worthevaluating in PIK3CA and HRAS-mutated tumors.

A novel finding in this study was the frequent alteration of avariety of CDKs in all histologies. CDKs are a diverse set of critical

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

(9.6%)PTEN þ CDKN2Aþ CDKN2B – 1 (1.9%)

PTEN – 4 (7.7%) CDKN2B – 6 (11.5%) KRAS – 2 (3.8%) AKT1 þ HRAS – 2(3.8%)

PIK3CA þ CDKN2Aþ CDKN2B – 1 (1.9%)

RICTOR – 4 (7.7%) CCND1 – 3 (5.7%) AKT1 þ KRAS – 1(1.9%)

PIK3CA þ CDKN2A,CDKN2B, CDK6, CCND3– 1 (1.9%)

AKT1 – 3 (5.8%) CCND3 – 2 (3.7%) PIK3CAþ 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%) PIK3R1þCDKN1B – 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%) PIK3CAþHRAS – 10

(24.3%)RICTOR þ CDK4– 1 (2.4%)

PTEN – 5 (12.2%) CCNE1 – 1 (2.4%) PIK3CA þ AKT1 þHRAS – 1 (2.4%)

PTEN þ CDKN2A– 1 (2.4%)

RICTOR – 2 (4.9%) CDK4 – 1 (2.4%) PIK3CA þ CDKN2Aþ 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

(9.4%)PTEN þ CDKN2Aþ 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 (P¼ 0.019); CDK (P¼ 0.043); RAS (P¼ 0.052); PI3Kþ RAS (P¼ 0.006), PI3Kþ cyclin (P¼ 0.473).






regulatory proteins responsible for cell-cycle transitions (34, 35).Recently, CDKGAswere reported in awide variety of solid tumorsat varying frequencies (0%–81%), where they were associatedwithpoorer survival (36). In this study, CDKswere altered inmorethan 30% of adenocarcinoma, NOS and carcinoma, NOS.Because of diversity and complexity of CDK signaling, it has beendifficult to develop CDK-targeted therapies (37). However, pal-bociclib, a CDK4/6 inhibitor, was recently shown to improveprogression-free survival when combined with endocrine therapyin estrogen receptor–positive, HER2-negative breast cancer (38).SGCs harboring CDK GAs may be good candidates for trialstargeting CDKs or their partners.

Other common and rare GAs were identified that may havetherapeutic and/or diagnostic relevance. Consistent with priorstudies, amplification of ERBB2 was commonly seen in SDC butless so in adenocarcinomas,NOS (12, 39, 40). In addition,we alsoidentified a high number of ERBB2GAs in ductal ca ex PA. New tothis study was the identification of activating ERBB2mutations inadenocarcinoma, NOS, SDC, and ductal ca ex PA. HER2-targetedtherapies, such as trastuzumab, have previously demonstratedefficacy in ERBB2-amplified salivary gland cancers and contrib-uted a partial response in our series (41, 42). Select GAs in geneswith precision drugs currently commercially available includedROS1, MET, and BRAF, although BRAF GAs were seen less fre-quently than previously reported (11). BRAF inhibition wasrecently shown to be of benefit in one patient with a BRAF-mutated SDC (43). Interestingly, this study identified character-istic GAs in unexpected histologies. For instance, ETV6–NTRK3translocations, a translocation characteristic of MASCs, wereobserved in 2 (3.8%) of tumors submitted as adenocarcinoma,NOS (5). Thefinding of this translocation in the adenocarcinoma,NOS may speak to the lack of consensus for the IHC profile forMASC tumors and suggest thatCGPprofilingmaybeof diagnosticbenefit in histologically difficult cases (44). Finally, the carcino-

ma, NOS group in this study did not clearly cluster with any of thethree other histotypes, yet they harbored frequent actionableGAs. These findings suggest CGP may help clarify diagnosesand identify GAs for therapeutic exploitation in difficult to diag-nose cases.

This study has limitations. The greatest limitation is the lackof clinical correlations between identified GAs and diseasecharacteristics or patients outcomes. As this was a retrospectiveevaluation of samples submitted for clinical care, data abouttumor grade, cancer stage, response to therapies, and patientsurvival are not available. Another limitation is that, althoughthe CGP platform used in this study covers a wide range of genes,there may be clinically relevant GAs that were not assessed. Forinstance, although the assay routinely identifies the MYB–NFIBtranslocation commonly seen in ACC, it does not assess for theMECT1–MAML2 translocation commonly identified in mucoe-pidermoid carcinoma (4, 6). Designing a CGP panel for salivarygland cancers, including MECT-MAML2, may be more clinicallyapplicable to a broad range of tumors. Similarly, although thepathology reports and prior immunohistochemical staining pat-terns were reviewed, we could not perform additional/supple-mentary IHC characterization because specimens were submittedstrictly for CGP analysis. For SDC, AR staining aids in the diag-nosis and can be exploited therapeutically (9, 39, 45). Anotherlimitation is that the tumors tested in this study may not fullyrepresent the genetic spectrum of these diagnoses, as commercialCGP tends to be performed or more aggressive, advanced/met-astatic tumors, and many salivary gland cancers are somewhatindolent (46). Moreover, CGP was performed on both locore-gional disease and metastatic sites. There are no data in SGCs todemonstrate whether the GA profile of locoregional disease isrepresentative of metastatic sites, or vice versa, although thereappears to be high concordance in other tumors (47, 48). Finally,although this study demonstrates frequent actionableGAs, it does

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 withcarboplatin, 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.

Wang et al.





not address intratumoral heterogeneity and the relative impor-tance of each GA in patient outcomes. Recent studies in renal celland lung cancers have demonstrated that different clones havevarying driver mutations whose importance can fluctuate overtime (49, 50). Determining the inciting GA and relative impor-tance of subsequent GAs in SGC is worth exploring in futurestudies. Despite these limitations, this study greatly contributes tothe knowledge of the genetic drivers of these rare tumors.

In summary, deep genomic profiling with a comprehensivegenomic profiling assay of salivary adenocarcinomas, NOS, sal-ivary duct carcinomas, ca ex PA, and salivary carcinoma, NOSidentified a high percentage of potentially actionableGAs that caninfluence therapy selection and direct patients to enter clinicaltrials using precision therapies. TheGAs identified in this study arean important step to opening pathways for new therapeuticapproaches in these notoriously difficult to treat cancers.

Disclosure of Potential Conflicts of InterestK. Wang, J.A. Elvin, D. Khaira, A. Johnson, S.M. Ali, M. Murray, J. Chmieck-

ecki, R. Yelensky, D. Lipson, V.A. Miller, P.J. Stephens, and J.S. Ross haveownership interests (including patents) in Foundation Medicine. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K. Wang, J.S. Russell, J.A. Elvin, D. Khaira, S.J. Wong,V.A. Miller, J.S. Ross, D.W. Bowles

Development of methodology: K. Wang, R. Yelensky, D. Lipson, J.S. Ross,D.W. BowlesAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.S. Russell, J.A. Elvin, D. Khaira, T.A. Jennings,S.M. Ali, J.S. Ross, D.W. BowlesAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):K.Wang, J.D.McDermott,D. Khaira, A. Johnson, S.M.Ali, J. Chmielecki, R. Yelensky, P.J. Stephens, J.S. Ross, D.W. BowlesWriting, review, and/or revision of the manuscript: K. Wang, J.S. Russell, J.D.McDermott, J.A. Elvin, D. Khaira, S.M. Ali, M. Murray, C. Marshall, D. Wash-burn, S.J. Wong, V.A.Miller, P.J. Stephens, H.S. Serracino, J.S. Ross, D.W. BowlesAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.D. McDermott, M. Murray, J.S. Ross, D.W.BowlesStudy supervision: K. Wang, J.S. Ross, D.W. BowlesOther (case report contribution to the content of themanuscript):D.OldhamOther (minor contribution by detail writing and editing content by physi-cian): D. Washburn

AcknowledgmentsWe would like to thank Dr. Marino Leon fromMoffitt Cancer Center for the

H&E and androgen receptor photomicrographs of the RET fusion patientdescribed in this article.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 22, 2015; revised May 10, 2016; accepted May 23, 2016;published OnlineFirst June 22, 2016.

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Wang et al.




2016;22:6061-6068. Published OnlineFirst June 22, 2016.Clin Cancer Res Kai Wang, Jeffery S. Russell, Jessica D. McDermott, et al. Specified Reveals Actionable Genomic AlterationsPleomorphic Adenomas, and Adenocarcinomas, Not Otherwise Profiling of 149 Salivary Duct Carcinomas, Carcinoma Ex

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Proﬁling of 149 Salivary Duct Carcinomas, Carcinoma Ex ... · gland cancer histologies, such as mucoepidermoid carcinoma, adenoid cystic carcinoma (ACC), acinic cell carcinomas