Next-Gen Sequencing Exposes Frequent MED12Mutations and Actionable Therapeutic Targetsin Phyllodes TumorsAndi K. Cani1, Daniel H. Hovelson2, Andrew S. McDaniel1, Seth Sadis3, Michaela J. Haller1,Venkata Yadati1, Anmol M. Amin1, Jarred Bratley1, Santhoshi Bandla3, Paul D.Williams3,Kate Rhodes4, Chia-Jen Liu1, Michael J. Quist1,5, Daniel R. Rhodes3, Catherine S. Grasso1,5,Celina G. Kleer1, and Scott A. Tomlins1,6,7

Abstract

Phyllodes tumors are rare fibroepithelial tumors with variableclinical behavior accounting for a small subset of all breastneoplasms, yet little is known about the genetic alterations thatdrive tumor initiation and/or progression. Here, targeted next-generation sequencing (NGS) was used to identify somatic altera-tions in formalin-fixed paraffin-embedded (FFPE) patient speci-mens from malignant, borderline, and benign cases. NGSrevealedmutations inmediator complex subunit 12 (MED12) affect-ing the G44 hotspot residue in the majority (67%) of casesspanning all three histologic grades. In addition, loss-of-functionmutations in p53 (TP53) as well as deleterious mutations in thetumor suppressors retinoblastoma (RB1) and neurofibromin 1 (NF1)were identified exclusively in malignant tumors. High-level copy-number alterations (CNA) were nearly exclusively confined tomalignant tumors, including potentially clinically actionable

gene amplifications in IGF1R and EGFR. Taken together, thisstudy defines the genomic landscape underlying phyllodes tumordevelopment, suggests potential molecular correlates to histolog-ic grade, expands the spectrum of human tumors with frequentrecurrent MED12 mutations, and identifies IGF1R and EGFR aspotential therapeutic targets in malignant cases.

Implications: Integrated genomic sequencing and mutationalprofiling provides insight into the molecular origin of phyllodestumors and indicates potential druggable targets in malignantdisease.

Visual Overview: http://mcr.aacrjournals.org/content/early/2015/04/02/1541-7786.MCR-14-0578/F1.large.jpg.Mol Cancer Res; 13(4); 613–9. �2015 AACR.

IntroductionPhyllodes tumors of the breast are relatively rare fibroepithelial

tumors that account for approximately 1% of all breast neo-plasms. Like benign breast fibroadenomas, they are characterizedby proliferation of both stromal and epithelial components, but,in contrast, they have considerable malignant potential. Phyl-lodes tumors are classified as benign (�65%), borderline(�25%), and malignant (�10%) based on histologic features,

including cellular atypia, mitotic activity, stromal overgrowth,stromal cellularity, and tumor margins (1). However, this histo-pathologic classification often fails to predict which phyllodestumors will recur or metastasize after treatment and does notaccurately inform on treatment options. Although local recur-rence after resection is most prevalent in histologically malignantcases (approximately 30%, depending on width of excised mar-gins), borderline, and benign tumors can also recur locally inabout 15% and 10% of cases, respectively, demonstrating thelimitations of current prognostic approaches (2). Likewise,although approximately 10% of all phyllodes tumors progressto distant metastases, only approximately 20% of histologicallymalignant cases do so (3, 4), leaving a substantial number ofborderline and even histologically benign cases that have meta-static potential. Conversely, although most histologically benigncases will behave as such, there are a proportion of phyllodestumors classified asmalignant andborderline thatwill behave in abenign manner. Current treatment guidelines for phyllodestumors require wide surgical resection margins, but efficacioustreatment options for the 10% of all phyllodes tumors thatprogress to metastatic disease are lacking and survival rates aredismal (3).

The key genetic alterations driving phyllodes tumor deve-lopment and molecular correlates with histologic grade andmalignant behavior are poorly characterized. Comparativegenomic hybridization (CGH) and array CGH (aCGH) studies

1Department of Pathology, Michigan Center for Translational Pathol-ogy, Ann Arbor, Michigan. 2Department of Computational Medicineand Bioinformatics University of MichiganMedical School, Ann Arbor,Michigan. 3Life Sciences Solutions, ThermoFisher Scientific, AnnArbor, Michigan. 4Life Sciences Solutions, ThermoFisher Scientific,Carlsbad, California. 5Department of Pathology, Oregon Health andSciences University, Portland, Oregon. 6Department of Urology, Uni-versity of Michigan Medical School, Ann Arbor, Michigan. 7Compre-hensive Cancer Center, University of Michigan Medical School, AnnArbor, Michigan.

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

CorrespondingAuthor: Scott A. Tomlins, University of MichiganMedical School,1524 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109. Phone: 734-764-1549;Fax: 734-647-7950; E-mail: tomlinss@umich.edu

doi: 10.1158/1541-7786.MCR-14-0578

�2015 American Association for Cancer Research.

MolecularCancerResearch

www.aacrjournals.org 613

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578

have shown multiple recurrent, broad somatic chromosomalcopy-number alterations (CNA) in phyllodes tumors, includinggains of chromosome 1q and losses in 13q, 6q, 9p; however,their prognostic utility is unclear (5–9). Several genes havebeen implicated in phyllodes tumor development by virtue ofbeing localized to areas of CNA, including EGFR, which wasrecently shown by FISH to be amplified in 2% to 16% of cases(10, 11). In addition, gene expression and IHC studies haveimplicated various signaling pathways, including insulin-likegrowth factor (IGF) and Wnt/b-catenin, as being activated inphyllodes tumors (1). To more comprehensively assesssomatic molecular alterations in phyllodes tumors and identifypotential opportunities for personalized medicine, we per-formed next-generation sequencing (NGS) of 15 formalin-fixedparaffin-embedded (FFPE) phyllodes tumors representing thehistologic grade spectrum.

Materials and MethodsCase selection

We identified a cohort of 15 archived, routine clinical FFPEphyllodes tumor specimens from the University of Michigan,Department of Pathology Tissue Archive. Clinicopathologic infor-mation for each case was obtained from the clinical archive.Hematoxylin and eosin (H&E)–stained slides for all cases werereviewed by a board-certified Anatomic Pathologist (S.A. Tomlins)to ensure sufficient tumor content and confirm histologic grade.

Targeted next-generation sequencingTargeted NGS of tumor tissue was performed with IRB approv-

al. For each specimen, 4 to 10 � 10-mm FFPE sections were cutfrom a single representative block per case, usingmacrodissectionwith a scalpel as needed to enrich for at least 50% tumor content(as defined by areas of stromal overgrowth). DNA was isolatedusing theQiagenAllprep FFPEDNA/RNAKit (Qiagen), accordingto the manufacturer's instructions except for adding a 2 minuteroom temperature incubation and extending centrifugation timeto 5 minutes during the xylene deparaffinization (step 1) andethanol washing of xylene (step 2). DNAwas quantified using theQubit 2.0 fluorometer (Life Technologies).

Targeted, multiplexed PCR-based NGS was performed on eachcomponent using a custom panel comprised of 2,462 ampliconstargeting 130 genes and Ion Torrent–based sequencing. Genesincluded in this panelwere selected based on pan-cancerNGS andcopy-number profiling data analysis that prioritized somatic,recurrently altered oncogenes, tumors suppressors, genes presentin high-level copy gains/losses, and known/investigational ther-apeutic targets. Barcoded libraries were generated from 20 ng ofDNA per sample using the Ion Ampliseq library Kit 2.0 (LifeTechnologies) according to the manufacturer's instructions withbarcode incorporation. Templates were prepared using the IonPGM Template OT2 200 Kit (Life Technologies) on the Ion OneTouch 2 according to themanufacturer's instructions. Sequencingof multiplexed templates was performed using the Ion TorrentPersonal GenomeMachine (PGM) on Ion 318 chips using the IonPGM Sequencing 200 Kit v2 (Life Technologies) according to themanufacturer's instructions.

Data analysis was performed essentially as described previ-ously (12) in Torrent Suite 4.0.2, with alignment by TMAP usingdefault parameters, and variant calling using the Torrent VariantCaller plugin (version 4.0-r76860) using default low-stringency

somatic variant settings. Variants were annotated using Annovar(13). Called variants were filtered to remove synonymous ornoncoding variants, those with flow corrected read depths(FDP) less than 20, flow corrected variant allele containingreads (FAO) less than 6, variant allele frequencies (FAO/FDP)less than 0.10, extreme skewing of forward/reverse flow cor-rected reads calling the variant (FSAF/FSAR <0.2 or >5, or FSAFor FSAR <3), or indels within homopolymer runs >4. Variantsoccurring exclusively in reads containing other variants (single-nucleotide variants or indels) or those occurring in the lastmapped base of a read were excluded. Variants with allelefrequencies >0.5% in ESP6500 or 1,000 genomes or thosereported in ESP6500 or 1,000 genomes with observed variantallele frequencies between 0.40 and 0.60 or >0.9 were consid-ered germ line variants. High-confidence somatic variants pass-ing the above criteria were then visualized in IGV. We havepreviously confirmed that these filtering criteria identify variantsthat pass PCR validation with >95% accuracy (12). To prioritizepotential driving alterations, we used Oncomine softwaretools (powertools.oncomine.com) to annotate called variants,which uses pan-cancer NGS data to identify genes as oncogenesor tumor suppressors, based on overrepresentation of hotspotor deleterious mutations, respectively. Variants in oncogenesare then considered gain-of-function if at a hotspot and variantsin tumor suppressors are considered loss-of-function if delete-rious or at a hotspot (S.A. Tomlins; unpublished data).

Copy-number analysisTo identify CNAs, normalized, GC-content corrected read

counts per amplicon for each sample were divided by those froma pool of normal male genomic DNA samples (FFPE and frozentissue, individual, and pooled samples), yielding a copy-numberratio for each amplicon. Gene-level copy-number estimates weredetermined by taking the coverage-weighted mean of the per-probe ratios, with expected error determined by the probe-to-probe variance (12); a detailed article describing this technique isin submission (C.S. Grasso; submitted for publication). Geneswith a log2 copy-number estimate of <�1 or >0.6were consideredto have high-level loss or gain, respectively.

Sanger sequencing to validate called somatic variantsBidirectional Sanger sequencing was performed over the

observedMED12mutation hotspot (G44) on all tumor samples.Genomic DNA (10 ng) was used as template in PCR amplifica-tions with Invitrogen Platinum PCR Supermix Hi-Fi (Life Tech-nologies) with the suggested initial denaturation and cyclingconditions. Primer sequences were as previously reported(14, 15) with the addition of universal M13 adaptors (M13forward: TGTAAAACGACGGCCAGT and M13 reverse: CAG-GAAACAGCTATGACC). PCR products were subjected to bidirec-tional Sanger sequencing for both primer pairs by the Universityof Michigan DNA Sequencing Core after treatment with ExoSAP-IT (GE Healthcare) and sequences were analyzed using SeqManPro software (DNASTAR).

qPCR to validate copy-number variationsEGFR, IGF1R, and CDKN2A copy-number changes were sub-

jected to validation through quantitative real-time PCR (qPCR)for 12 samples with sufficient DNA. PH13, 14 and 30 hadinsufficient DNA (no CNAs in these genes were identified byNGS) for qPCR and PH5 had sufficient DNA only for assessing

Cani et al.

Mol Cancer Res; 13(4) April 2015 Molecular Cancer Research614

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578

EGFR and CDKN2A (no IGF1R CNAs were identified by NGS).Primers and probes (50 FAM; ZEN/Iowa Black FQ dual quench-ers) were designed using PrimerQuest (http://www.idtdna.com/Primerquest/Home/Index, hg 19 genome assembly) andobtained from IDT. Assay specificity was confirmed usingBLAST and BLAT and primers/probes in areas of SNPs wereexcluded. Primer/probe sequences are available upon request.qPCR reactions (15 mL) were performed in triplicate usingTaqMan Genotyping Master Mix (Applied Biosystems), 5 nggenomic DNA per reaction and a final concentration of0.9 mmol/L each primer and 0.25 mmol/L probe in 384-wellplates on the QuantStudio 12K Flex (Applied Biosystems).Automatic baseline and Ct thresholds were set using Quant-Studio 12K Flex Real-Time PCR System Software. Log2 copynumber of EGFR, CDKN2A, and IGF1R were determined by theDDCt method using the average Ct of DNMT3A, FBXW7, andMYO18A as the reference (copy-number neutral by NGS in all

PH samples) and PH 22 (copy-number neutral by NGS) as thecalibrator sample.

Statistical analysisComparisons of the number of mutations or CNAs per

sample by tumor grade were performed using the Kruskal–Wallis test with post-hoc pairwise comparison of subgroupsusing MedCalc 13.1.2.0. Comparison of the frequency ofMED12 mutations by tumor grade was performed by the Fisherexact test using R 3.1.0.

ResultsWe performed targeted NGS on a cohort of 15 FFPE phyllodes

tumors comprised of 5 cases each of benign, borderline, andmalignant histologic grade; representative photomicrographs andclinical characteristics of all patients are presented in Fig. 1A.

PH-08PH-19 PH-06PH-05

PH-08PH-19 PH-06PH-05

Size (cm)ProcedureAgeTypeGradeCase1.1Ex./Lump. 47PrimaryBenignPH-18

PH-19 1.5Ex./Lump. 32PrimaryBenign3.0Ex./Lump. 42PrimaryBenignPH-20

15.0Ex./Lump. 24PrimaryBenignPH-223.2Ex./Lump. 33PrimaryBenignPH-304.4Ex./Lump. 37PrimaryBorderlinePH-04

PH-08 3.4Ex./Lump. 21RecurrenceBorderline4.0Ex./Lump. 30PrimaryBorderlinePH-113.0Ex./Lump. 35PrimaryBorderlinePH-139.1Ex./Lump. 13PrimaryBorderlinePH-178.1Ex./Lump. 30PrimaryMalignantPH-03

PH-05 7.0Mast. 67RecurrenceMalignantPH-06 1.4Ex./Lump. 26PrimaryMalignant

N/ACore Bx. 60Lung MetMalignantPH-1410.0Mast. 39PrimaryMalignantPH-16

BenignBorderlineMalignant

PrimaryRecurrenceMetastasis

≤30 y31–49 y≥50 y

Copy gainCopy lossNonsyn. SNVFs. indelFp. indelSplice site

Grade Type

Patient age

Alteration

PH

-30

PH

-18

PH

-19

PH

-22

PH

-20

PH

-11

PH

-17

PH

-04

PH

-13

PH

-08

PH

-03

PH

-05

PH

-16

PH

-14

PH

-06

GradeTypeAgeProcedure

MED12TP53TERTIGF1RMCL1EGFRCCNE1MYCZNF217NF1CDKN2APDGFRACCND1CD44RB1BCL9

Core biopsyExc. / Lump.Mastectomy

Procedure

Nonsense

A

B

Figure 1.Histology and clinicopathologicinformation for FFPE phyllodestumors assessed by targeted NGS andintegrative molecular heatmap ofdriving molecular alterations inphyllodes tumors. A, H&E-stainedsections of representative benign(PH-19), borderline (PH-08), andmalignant (PH-05 and PH-06)phyllodes tumors sequenced areshown. Top, �4 originalmagnification; bottom �20magnification. Clinicopathologicinformation, including histologicgrade, specimen type, patient age,procedure type, and tumor size, for allcases is given (Ex./Lump, excisionalbiopsy or lumpectomy; Mast,mastectomy; Core bx, Core biopsy).B, targeted NGS of 15 FFPE phyllodestumors was performed to identifypotentially driving/actionablemolecular alterations. All high-confidence, gain- or loss-of-functionsomatic mutations in oncogenes andtumor suppressors, in addition tohigh-level CNA are indicated for eachcase. Specific alteration types areindicated according to the legend(Nonsyn SNV, nonsynonymous SNV;Fs and Fp indel, frame-shifting andframe-preserving indels,respectively). Slashed boxes indicatetwo alterations. Clinicopathologicinformation is shown above theheatmap according to the legend andas in A.

Next-Generation Sequencing of Phyllodes Tumors

www.aacrjournals.org Mol Cancer Res; 13(4) April 2015 615

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578

We isolated an average of 0.65 mg DNA per case from 4 to 10 �10-mm sections using macrodissection to enrich tumor contentas needed. NGS was performed using a multiplexed PCR-basedcustom Ion Torrent Ampliseq panel comprised of 2,462 ampli-cons targeting 130 genes and Ion Torrent–based sequencing onthe PGM. Targeted genes were selected on the basis of pan-cancer NGS and copy-number profiling data analysis to prior-itize somatic, recurrently altered oncogenes, tumors suppres-sors and genes present in high-level CNAs. Detailed character-ization of this panel will be reported separately (S.A. Tomlins;unpublished data).

NGS of multiplexed templates on the Ion Torrent PGM gen-erated an average of 1,011,571 mapped reads yielding �409targeted base coverage across the 15 samples (SupplementaryTable S1). We identified a total of 26 high-confidence, likelysomatic nonsynonymous or splice site altering point mutationsand short insertion/deletions (indels) across the 15 samples(median 2; range, 0–4) as shown in Supplementary Tables S2and S3. The number of high-confidence somatic nonsynony-mous mutations were not significantly different between thehistologic grades (Kruskal–Wallis test, P ¼ 0.09), as shown inSupplementary Fig. S1A. Copy-number analysis of NGS datayielded a total of 16 high-level CNAs (median 0; range, 0–6).The number of high level CNAs differed significantly betweenhistologic grades (Kruskal-Wallis test, P¼ 0.002), withmalignanttumors harboring significantly more high-level CNAs persample (median 2; range, 2–6) than borderline (median 0; range,0–0) or benign (median 0; range, 0–2) tumors (Kruskal–Wallistest, post-hoc analysis, both P < 0.05), as shown in SupplementaryFig. S1B. Prioritized likely gain- or loss-of-function somaticmutations in oncogenes and tumor suppressors (see below)and high-level CNAs for each case are shown in an integrativeheatmap (Fig. 1B).

By NGS, we found that MED12, which encodes subunit 12 ofthe Mediator complex (the multiprotein assembly that serves as ageneral coactivator of transcription by RNA polymerase II) wasmutated in 10 of 15 samples (67%; one sample with biallelicmutations) by automated variant calling and visual read inspec-tion in IGV (as some called variants were filtered due to skewedread support). All mutations were localized to the exon 2 hotspotregion near residue G44 (Fig. 2A and Supplementary Tables S2and S3), which has recently been reported to be recurrentlymutated at high frequency in uterine leiomyomas (14–16) andbenign breast fibroadenomas (17), and more rarely in uterineleiomyosarcomas (15, 16, 18, 19). Five of 11 total MED12mutations were point mutations at G44 (3 p.G44S, 1 p.G44C,and 1 p.G44R) whereas three mutations were frame-preservingdeletions adjacent to or including G44 (p.38_43, p.41_49, andp.42_51). Two mutations were intronic point mutations justupstream of exon 2, at a previously reported splice acceptor sitecausing retentionof an additional 6bases in the transcript (c.IVS-8p.E33_D34insPQ; refs. 14, 17). PH-11, which harbored a c.IVS-8mutation, also harbored an additional intronic mutation furtherupstream (c.IVS-15), consistent with biallelic intronic MED12mutations in this sample. There was no significant difference inthe presence of MED12 mutations between tumors of differenthistologic grade (benign 4/5, borderline 4/5, malignant 2/5,Fisher exact test, P ¼ 0.5). All MED12 mutations were confirmedby bidirectional Sanger sequencing (Fig. 2B).

To prioritize potential driving alterations from the remainingnon-MED12 point mutations/indels, we used the Oncomine

Plugin in Ion Reporter for assessing gain- or loss-of-function.This analysis identified five loss-of-function alterations, includingthree mutations in TP53 (F270L in PH-14, Q192X in PH-16,and C242Y in PH3), and one mutation each in RB1 (E533X inPH16) and NF1 (p.1152_1153del in PH-05), as shown in theintegrative heatmap of driving alterations (Fig. 1B and Supple-mentary Tables S2 and S3). Intriguingly, these loss-of-functionalterations occurred exclusively in malignant tumors.

Copy-number analysis of NGS data demonstrated recurrentlow-level CNAs, including gain of chromosome 1q and loss ofchromosome 13q, consistent with previous reports (5–9). Thesewere more prevalent in malignant tumors (5/5), but were alsopresent in two borderline and one benign case (Fig. 3A andSupplementary Fig. S2). High-level CNAs were nearly exclusivelypresent in malignant specimens 14 of 16 alterations, as shownin Fig. 3A and B. Of note, PH-03 showed high-level EGFR (copy-number ratio > 6) and IGF1R amplifications, whereas PH-16also showed ahigh-level IGF1R amplification (copy-number ratio> 32, Fig. 3B). TERT amplifications were also observed in threemalignant tumors, whereas PH-05 harbored a focal high-levelCDKN2A (p16INK4A) loss. We confirmed EGFR, IGF1R, andCDKN2A CNAs by qPCR as shown in Fig. 3C.

DiscussionWe performed targeted NGS of 15 FFPE phyllodes tumors

representing all three histologic grades to identify somaticalterations associated with tumor development and potentialtargetable alterations. Mutations in MED12 were present in 10of 15 cases (67%) and affected the known exon 2 G44 residuehotspot through multiple mechanisms. No significant differencein MED12 mutation frequency was observed across histologicgrades, although our cohort size is limited. Our IRB approvedprotocol does not allow NGS of matched normal tissue; how-ever, the observed MED12 variant allele frequencies are consis-tent with somatic events as seen in other tumors. SimilarMED12somatic mutations are frequent (50%–70%) in benign uterineleiomyomas (14–16), but less common in malignant uterineleiomyosarcomas (7%–30%; refs. 15, 16, 18, 19). Recently, Limand colleagues identified similar MED12 mutations in 59% ofbenign breast fibroadenomas through exome sequencing (17).Given the morphologic similarity of breast fibroadenomas andbenign phyllodes tumors, frequent MED12 mutations in bothentities support a closely related molecular origin. In addition,although our findings will need to be replicated in largercohorts, the similar frequency of MED12 mutations across thehistologic spectrum of phyllodes tumors (in addition to benignfibroadenomas) suggests that MED12 mutations in the breastare early events that may be unrelated to malignant behavior, incontrast with uterine leiomyomas and leiomyosarcomas, whichshow notable differences in MED12 mutation frequencies asjust described. Our results also support the evolution of malig-nant phyllodes tumors from less aggressive fibroadenomas orphyllodes tumors (possibly through loss of key tumor suppres-sors). AlthoughMED12 hotspot mutations have been identifiedinfrequently in extrauterine or extramammary tumors (18, 20),functional studies support a role for MED12 mutations affect-ing the G44 hotspot in dysregulation of estrogen signaling inestrogen responsive cells (17), and the Mediator complex isknown to interact with the estrogen receptor (21).

Cani et al.

Mol Cancer Res; 13(4) April 2015 Molecular Cancer Research616

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578

Although surgical resection of phyllodes tumors may becurative, local recurrence is not uncommon and distant metas-tasis is associated with poor survival. Furthermore, the histo-logic features do not accurately predict clinical behavior ofphyllodes tumors. Hence, targetable alterations, particularly inmalignant phyllodes tumors, may be useful for personalizedmedicine strategies. Through copy-number analysis of NGSdata (and confirmed by qPCR), we identified potentially clin-ically actionable high-level, focal amplifications of EGFR andIGF1R in 7% and 13% of cases, respectively (1/5 and 2/5

malignant cases). EGFR has been shown to be highly amplifiedin phyllodes tumors by FISH in up to 16% of cases (10, 11),consistent with our findings. Dysregulation of the IGF pathwayhas been implicated in phyllodes tumors by IHC (1); however,IGF1R amplification has not been reported.

Direct comparison of additional CNAs identified in ourstudy and previous studies is challenging due to platformdifferences. However, broad, low-level gains in genes on 1qand losses on 13q were observed in malignant as well asborderline and benign tumors, consistent with previous

MED12

Intron 1

c.IVS-15

c.IVS-8 (2)

Exon 2

p. G44S (3)

p. G44C

p. G44R

p. 38_43delInsE

p. 41_49delinsA

p. 42_51delinsI

Nonsyn. SNV

Fp. indel

Splice site

Alteration

c. 113_127del15; p. 38_43delInsEPH-03

c. 130G>A; p. G44SPH-05

c. 122_145del24; p. 41_49delinsAPH-04

PH-11c. IVS-8, cIVS-15 ; p. E33_D34insPQ?

c. 130G>T; p. G44CPH-13 PH-17 c. IVS-8; p. E33_D34insPQ

PH-18 c. 130G>A; p. G44S

PH-19 c. 130G>A; p. G44S

PH-22 c130G>C; p. G44R PH-30 c. 124_151del27; p. 42_51delinsI

A

B

Figure 2.Identification of recurrent MED12mutations in phyllodes tumors. NGSand Sanger sequencing identified 11MED12 mutations in 10 of 15 phyllodestumors subjected to NGS. A, schematicrepresentation of MED12 intron 1 andexon 2 junction with locations of allobserved mutations shown. Mutationtype is indicated in the legendand the frequency of observedmutations is indicated in parentheses.B, bidirectional Sanger sequencingwasperformed on all specimens. Traces ofcases withMED12mutations are shown(only one trace direction shown) withthe indicated nucleotide and aminoacid changes noted. The mutation(s)position is indicated by the arrow.

Next-Generation Sequencing of Phyllodes Tumors

www.aacrjournals.org Mol Cancer Res; 13(4) April 2015 617

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578

reports. On the other hand, we did not observe gains in MDM2orMDM4, which have been reported in previous aCGH studies,and were targeted herein; we hypothesize this may be due to thehigh TP53 alteration rate in our phyllodes tumors with highnumbers of CNAs. Finally, our panel did not target genes insome previously reported regions of CNA (such as 6q), pre-cluding comparisons of these alterations.

Besides MED12 hotspot mutations, other potential drivingsomatic point mutations/indels, which included loss-of-functionalterations in TP53, RB1, and NF1, occurred exclusively in malig-nant tumors. In addition, high-level, focal CNAs (such as those inEGFR and IGF1R) were only observed inmalignant cases. Togeth-er, these findings supportmolecular correlates to histologic grade.Whether such molecular alterations may be useful in cases withchallenging histology or show prognostic potential can be inves-tigated in additional cohorts.

Taken together, our results demonstrate frequentMED12muta-tions in phyllodes tumors, supporting a shared originwith benignbreast fibroadenomas. In addition, our results suggest potentialtherapeutic targets in malignant tumors, including EGFR andIGF1R. Finally, as both driving somatic mutations/indels otherthan MED12 and high-level, focal CNAs occurred exclusively inmalignant tumors in our cohort, such alterations may be usefulfor classification or prognostication in borderline tumors if con-firmed in other cohorts.

Disclosure of Potential Conflicts of InterestS.A. Tomlins has a separate sponsored research agreement with Com-

pendia Bioscience/Life Technologies/ThermoFisher Scientific that providesaccess to the sequencing panel used herein. No other aspect of the studydescribed herein was supported by Compendia Bioscience/Life Technolo-gies/ThermoFisher Scientific. S. Sadis, S. Bandla, P.D. Williams, K. Rhodes,

−5.0

−2.5

0.0

2.5

5.0

MTO

RM

YC

L1B

CL9

MC

L1M

DM

4M

YC

ND

NM

T3A

MS

H2

SF

3B1

VH

LP

PAR

GB

AP

1P

IK3C

AS

OX

2AT

P11

BD

CU

N1D

1F

GF

R3

PD

GF

RA

KIT

KD

RT

ET

2F

BX

W7

TE

RT

PIK

3R1

AP

CIL

6E

GF

RC

DK

6M

ET

FG

FR

1M

YC

JAK

2C

D27

4P

DC

D1L

G2

CD

KN

2AP

TC

H1

AB

L1T

SC

1N

OT

CH

1G

ATA

3P

TE

NF

GF

R2

WT

1C

D44

CC

ND

1B

IRC

3B

IRC

2AT

MK

RA

SA

CV

RL1

CD

K4

MD

M2

FLT

3B

RC

A2

RB

1G

AS

6A

PE

X1

PN

PN

KX

2−1

AK

T1

IGF

1RT

SC

2C

DH

1T

P53

MYO

18A

TIA

F1

NF

1E

RB

B2

BR

CA

1R

PS

6KB

1S

TK

11C

CN

E1

CS

NK

2A1

BC

L2L1

ZN

F21

7S

MA

RC

B1

NF

2A

R

Log 2 c

opy

num

ber r

atio

Log 2 c

opy

num

ber r

atio

Log 2 c

opy

num

ber r

atio

Log 2 c

opy

num

ber r

atio

TERTEGFRCDKN2AIGF1ROther

−5.0

−2.5

0.0

2.5

5.0

Chr 1 2 3 4 5 7 8 9 10 11 12 13 14 16 17 19 20 22 X15−3.0

−1.5

0.0

1.5

3.0

−3.0

−1.5

0.0

1.5

3.0

IGF1R

IGF1R

CDKN2A

EGFR

TERT

-3

-1

0

1

3

6IGF1REGFRCDKN2A

PH-03

PH-05

PH-16

PH

-18

PH

-19

PH

-20

PH

-11

PH

-17

PH

-04

PH

-08

PH

-06

PH

-03

PH

-05

PH

-16 Benign

BorderlineMalignant

Tumor grade

Log 2

cop

y nu

mbe

r ra

tio

Chr 1 Chr X

A

B

C

Figure 3.Copy-number analysis of phyllodestumor identifies potential therapeutictargets in malignant samples. Copy-number analysis was performed fromNGS data. For each sequencedphyllodes tumor, GC contentcorrected, normalized read counts peramplicon were divided by those froma composite normal sample, yielding acopy-number ratio for each amplicon.Gene-level copy-number estimateswere determined by taking theweighted mean of the per-probecopy-number ratios. A, summary ofgene-level copy-number ratios (log2)for all profiled samples. Selectedgenes of interestwith high-level CNAsare colored according to the legend.B, copy-number profiles for threemalignant phyllodes tumors withhigh-level CNAs. Log2 copy-numberratios per amplicon are plotted (witheach individual amplicon representedby a single dot, and each individualgene indicated by different colors),with gene-level copy-numberestimates (black bars) determined bytaking the weighted mean of the per-probe copy-number ratios. Selectedhigh-level CNAs are indicated. C,qPCR confirmation of high-level CNAsin EGFR, IGF1R, andCDKN2A. qPCR ongenomic DNA from indicated sampleswas performed using DNMT3A,FBXW7, andMYO18A as the referencegenes. Normalizedmean IGF1R (blue),EGFR (red), andCDKN2A (green) log2copy-number ratios [using PH22 (noCNAs by NGS) as the calibrator] fromtriplicate qPCR � SD are plotted.

Cani et al.

Mol Cancer Res; 13(4) April 2015 Molecular Cancer Research618

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578

and D.R. Rhodes are employees of ThermoFisher Scientific. The otherauthors have no competing interests to declare.

Authors' ContributionsConception and design: A.K. Cani, A.S. McDaniel, K. Rhodes, D.R. Rhodes,S.A. TomlinsDevelopment of methodology: A.K. Cani, S. Sadis, S. Bandla, K. Rhodes,D.R. RhodesAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.K. Cani, A.S. McDaniel, V. Yadati, A.M. Amin,J. Bratley, K. RhodesAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.K. Cani, D.H. Hovelson, M.J. Quist, C.S. Grasso,C.G. Kleer, S.A. TomlinsWriting, review, and/or revision of the manuscript: A.K. Cani, D.H.Hovelson, A.S. McDaniel, S. Sadis, M.J. Haller, A.M. Amin, C.S. Grasso,C.G. Kleer, S.A. Tomlins

Administrative, technical, or material support (i.e., reporting ororganizing data, constructing databases): D.H. Hovelson, A.M. Amin,P.D. Williams, C.-J. LiuStudy supervision: A.K. Cani, P.D. Williams, K. Rhodes, S.A. Tomlins

AcknowledgmentsThe authors thank Javed Siddiqui and Mandy Davis for technical

assistance.

Grant SupportS.A. Tomlins is supported by the A. Alfred Taubman Medical Research

Institute.

Received October 27, 2014; revised December 9, 2014; accepted December15, 2014; published OnlineFirst March 30, 2015.

References1. Yang X, Kandil D, Cosar EF, Khan A. Fibroepithelial tumors of the breast:

pathologic and immunohistochemical features and molecular mechan-isms. Arch Pathol Lab Med 2014;138:25–36.

2. Tan PH, Thike AA, Tan WJ, Thu MM, Busmanis I, Li H, et al. Predictingclinical behaviour of breast phyllodes tumours: a nomogram based onhistological criteria and surgical margins. J Clin Pathol 2012;65:69–76.

3. Parker SJ, Harries SA. Phyllodes tumors. Postgrad Med J 2001;77:428–35.4. Moffat CJ, Pinder SE, DixonAR, ElstonCW, Blamey RW, Ellis IO. Phyllodes

tumours of the breast: a clinicopathological review of thirty-two cases.Histopathology 1995;27:205–18.

5. Jones AM, Mitter R, Springall R, Graham T, Winter E, Gillett C, et al. Acomprehensive genetic profile of phyllodes tumours of the breast detectsimportant mutations, intra-tumoral genetic heterogeneity and new geneticchanges on recurrence. J Pathol 2008;214:533–44.

6. Kuijper A, Snijders AM, Berns EM, Kuenen-Boumeester V, van der Wall E,Albertson DG, et al. Genomic profiling by array comparative genomichybridization reveals novel DNA copy number changes in breast phyllodestumours. Cell Oncol 2009;31:31–9.

7. Lae M, Vincent-Salomon A, Savignoni A, Huon I, Freneaux P, Sigal-ZafraniB, et al. Phyllodes tumors of the breast segregate in two groups according togenetic criteria. Mod Pathol 2007;20:435–44.

8. Lu YJ, Birdsall S, Osin P, Gusterson B, Shipley J. Phyllodes tumors of thebreast analyzed by comparative genomic hybridization and association ofincreased 1q copy number with stromal overgrowth and recurrence. GenesChromosomes Cancer 1997;20:275–81.

9. Lv S, Niu Y, Wei L, Liu Q, Wang X, Chen Y. Chromosomal aberrations andgenetic relations in benign, borderline andmalignant phyllodes tumors ofthe breast: a comparative genomic hybridization study. Breast Cancer ResTreat 2008;112:411–8.

10. Kersting C, Kuijper A, Schmidt H, Packeisen J, Liedtke C, Tidow N, et al.Amplifications of the epidermal growth factor receptor gene (egfr) arecommon in phyllodes tumors of the breast and are associated with tumorprogression. Lab Invest 2006;86:54–61.

11. Tse GM, Lui PC, Vong JS, Lau KM, Putti TC, Karim R, et al. Increasedepidermal growth factor receptor (EGFR) expression in malignant mam-mary phyllodes tumors. Breast Cancer Res Treat 2009;114:441–8.

12. McDaniel AS, Zhai Y, Cho KR, Dhanasekaran SM, Montgomery JS, Pala-pattu G, et al. HRAS mutations are frequent in inverted urothelial neo-plasms. Hum Pathol 2014;45:1957–65.

13. Chang X, Wang K. wANNOVAR: annotating genetic variants for personalgenomes via the web. J Med Genet 2012;49:433–6.

14. Makinen N, Mehine M, Tolvanen J, Kaasinen E, Li Y, Lehtonen HJ, et al.MED12, the mediator complex subunit 12 gene, is mutated at highfrequency in uterine leiomyomas. Science 2011;334:252–5.

15. de Graaff MA, Cleton-Jansen AM, Szuhai K, Bovee JV. Mediator complexsubunit 12 exon 2 mutation analysis in different subtypes of smoothmuscle tumors confirms genetic heterogeneity. Hum Pathol 2013;44:1597–604.

16. Schwetye KE, Pfeifer JD,Duncavage EJ.MED12 exon2mutations in uterineand extrauterine smooth muscle tumors. Hum Pathol 2014;45:65–70.

17. Lim WK, Ong CK, Tan J, Thike AA, Ng CC, Rajasegaran V, et al. Exomesequencing identifies highly recurrent MED12 somatic mutations in breastfibroadenoma. Nat Genet 2014;46:877–80.

18. KampjarviK,MakinenN,KilpivaaraO,Arola J,HeinonenHR, Bohm J, et al.Somatic MED12 mutations in uterine leiomyosarcoma and colorectalcancer. Br J Cancer 2012;107:1761–5.

19. Ravegnini G, Marino-Enriquez A, Slater J, Eilers G, Wang Y, Zhu M, et al.MED12 mutations in leiomyosarcoma and extrauterine leiomyoma. ModPathol 2013;26:743–9.

20. Barbieri CE, Baca SC, LawrenceMS, Demichelis F, Blattner M, Theurillat JP,et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12mutations in prostate cancer. Nat Genet 2012;44:685–9.

21. Kang YK, Guermah M, Yuan CX, Roeder RG. The TRAP/Mediator coacti-vator complex interacts directly with estrogen receptors alpha and betathrough the TRAP220 subunit and directly enhances estrogen receptorfunction in vitro. Proc Natl Acad Sci U S A 2002;99:2642–7.

www.aacrjournals.org Mol Cancer Res; 13(4) April 2015 619

Next-Generation Sequencing of Phyllodes Tumors

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578

2015;13:613-619. Published OnlineFirst January 15, 2015.Mol Cancer Res   Andi K. Cani, Daniel H. Hovelson, Andrew S. McDaniel, et al.   Actionable Therapeutic Targets in Phyllodes Tumors

Mutations andMED12Next-Gen Sequencing Exposes Frequent

  Updated version

  10.1158/1541-7786.MCR-14-0578doi:

Access the most recent version of this article at:

  Material

Supplementary

  http://mcr.aacrjournals.org/content/suppl/2015/01/14/1541-7786.MCR-14-0578.DC1

Access the most recent supplemental material at:

   

  Overview

Visual 

http://mcr.aacrjournals.org/content/13/4/613/F1.large.jpgA diagrammatic summary of the major findings and biological implications:

   

  Cited articles

  http://mcr.aacrjournals.org/content/13/4/613.full#ref-list-1

This article cites 21 articles, 5 of which you can access for free at:

  Citing articles

  http://mcr.aacrjournals.org/content/13/4/613.full#related-urls

This article has been cited by 13 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.org

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/13/4/613To request permission to re-use all or part of this article, use this link

on June 11, 2020. © 2015 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 15, 2015; DOI: 10.1158/1541-7786.MCR-14-0578