Comprehensive Genomic Profiling of Patient ... · Comprehensive Genomic Profiling of...

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Genomics Comprehensive Genomic Proling of Patient- matched Head and Neck Cancer Cells: A Preclinical Pipeline for Metastatic and Recurrent Disease Lluís Nisa 1,2,3 , David Barras 4 , Michaela Medov a 1,2 , Daniel M. Aebersold 1,2 , Mat u s Medo 1,2 , Michaela Poliakov a 1,2 , Jonas Koch 1,2 , Beat Bojaxhiu 1 , Olgun Eli¸ cin 1 , Matthias S. Dettmer 5 , Paolo Angelino 4 , Roland Giger 3 , Urs Borner 3 , Marco D. Caversaccio 3 , Thomas E. Carey 6,7 , Liza Ho 8 , Thomas A. McKee 8 , Mauro Delorenzi 4,9,10 , and Yitzhak Zimmer 1,2 Abstract Metastases and tumor recurrence have a major prognostic impact in head and neck squamous cell carcinoma (HNSCC); however, cellular models that comprehensively characterize metastatic and recurrent HNSCC are lacking. To this end, we obtained genomic, transcriptomic, and copy number proles of the UM-SCC cell line panel, encompass- ing patient-matched metastatic and recurrent cells. UM-SCC cells recapitulate the most prevalent genomic alterations described in HNSCC, featuring common TP53, PI3K, NOTCH, and Hippo pathway mutations. This analysis identied a novel F977Y kinase domain PIK3CA mutation exclusively present in a recurrent cell line (UM-SCC14B), potentially conferring resistance to PI3K inhibitors. Small proline-rich protein 2A (SPRR2A), a protein involved in epithelial homeostasis and invasion, was one of the most consistently downregulated transcripts in metastatic and recurrent UM-SCC cells. Assessment of SPRR2A protein expression in a clinical cohort of patients with HNSCC conrmed common SPRR2A downregulation in primary tumors (61.9% of cases) and lymph node metastases (31.3%), but not in normal tissue. High expression of SPRR2A in lymph node metastases was, along with non- oropharyngeal location of the primary tumor, an indepen- dent prognostic factor for regional disease recurrence after surgery and radiotherapy (HR 2.81; 95% CI, 1.166.79; P ¼ 0.02). These results suggest that SPRR2A plays a dual role in invasion and therapeutic resistance in HNSCC, respectively through its downregulation and overexpression. Implications: The current study reveals translationally rele- vant mechanisms underlying metastasis and recurrence in HNSCC and represents an adjuvant tool for preclinical research in this disease setting. Underlining its discovery potential this approach identied a PIK3CA-resistant muta- tion as well as SPRR2A as possible theragnostic markers. Mol Cancer Res; 16(12); 191226. Ó2018 AACR. Introduction Development of regional or distant metastases and recurrence after denitive therapy are arguably the most outstanding issues in the management of patients with head and neck squamous cell carcinoma (HNSCC). Presence of lymph node metastases has a major prognostic impact, as it reduces survival of HNSCC patients roughly by half (1). Moreover, lymph node metastases do not necessarily respond to given therapeutic approaches in the same way as their matched primaries do, an observation likely under- lying different intrinsic biological mechanisms (2). Such a het- erogeneous behavior poses an obvious challenge to achieve simultaneous local (primary tumor) and regional (lymph node metastases) disease control. Indeed, while most early-stage (with the exception of oral squamous cell carcinoma) and some advanced-stage primaries can be usually managed by radiother- apy (with or without chemotherapy/cetuximab), presence of lymph node metastases often requires combined approaches, encompassing neck dissection and irradiation (3). Distant metas- tases develop in approximately 10%30% of HNSCC patients after denitive therapy and carries a meager prognosis (approx- imately 5% overall 5-year survival; ref. 4). 1 Department of Radiation Oncology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland. 2 Department for BioMedical Research, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland. 3 Department of Otorhinolaryngology Head and Neck Surgery, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland. 4 SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland. 5 Institute of Pathology, University of Bern, Bern, Switzerland. 6 Department of Otolaryngology - Head and Neck Surgery, University of Michigan School of Medicine, Ann Arbor, Michigan. 7 Comprehensive Cancer Center, University of Michigan School of Medicine, Ann Arbor, Michigan. 8 Division of Clinical Pathology, Geneva University Hospitals, Geneva, Switzerland. 9 Ludwig Center for Cancer Research, University of Lausanne, Lausanne, Switzerland. 10 Department of Oncology, University of Lausanne, Lausanne, Switzerland. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). L. Nisa and D. Barras are co-rst authors of this article. M. Delorenzi and Y. Zimmer are co-senior authors of this article. Corresponding Author: Yitzhak Zimmer, Department for BioMedical Research, Maurice E. Muller-Haus, E-807, Murtenstrasse 35, Bern 3008, Switzerland. Phone: 413-1632-2642; Fax: 413-1632-3297; E-mail: [email protected]. doi: 10.1158/1541-7786.MCR-18-0056 Ó2018 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 16(12) December 2018 1912 on June 19, 2020. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst August 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0056

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Genomics

Comprehensive Genomic Profiling of Patient-matchedHeadandNeckCancerCells:APreclinicalPipeline for Metastatic and Recurrent DiseaseLluís Nisa1,2,3, David Barras4, Michaela Medov�a1,2, Daniel M. Aebersold1,2,Mat�u�s Medo1,2, Michaela Poliakov�a1,2, Jonas Koch1,2, Beat Bojaxhiu1, Olgun Elicin1,Matthias S. Dettmer5, Paolo Angelino4, Roland Giger3, Urs Borner3,Marco D. Caversaccio3, Thomas E. Carey6,7, Liza Ho8, Thomas A. McKee8,Mauro Delorenzi4,9,10, and Yitzhak Zimmer1,2

Abstract

Metastases and tumor recurrence have a major prognosticimpact in head and neck squamous cell carcinoma(HNSCC); however, cellular models that comprehensivelycharacterize metastatic and recurrent HNSCC are lacking. Tothis end, we obtained genomic, transcriptomic, and copynumber profiles of the UM-SCC cell line panel, encompass-ing patient-matched metastatic and recurrent cells. UM-SCCcells recapitulate the most prevalent genomic alterationsdescribed in HNSCC, featuring common TP53, PI3K,NOTCH, and Hippo pathway mutations. This analysisidentified a novel F977Y kinase domain PIK3CA mutationexclusively present in a recurrent cell line (UM-SCC14B),potentially conferring resistance to PI3K inhibitors. Smallproline-rich protein 2A (SPRR2A), a protein involved inepithelial homeostasis and invasion, was one of the mostconsistently downregulated transcripts in metastatic andrecurrent UM-SCC cells. Assessment of SPRR2A proteinexpression in a clinical cohort of patients with HNSCC

confirmed common SPRR2A downregulation in primarytumors (61.9% of cases) and lymph node metastases(31.3%), but not in normal tissue. High expression ofSPRR2A in lymph node metastases was, along with non-oropharyngeal location of the primary tumor, an indepen-dent prognostic factor for regional disease recurrence aftersurgery and radiotherapy (HR 2.81; 95% CI, 1.16–6.79; P ¼0.02). These results suggest that SPRR2A plays a dual role ininvasion and therapeutic resistance in HNSCC, respectivelythrough its downregulation and overexpression.

Implications: The current study reveals translationally rele-vant mechanisms underlying metastasis and recurrence inHNSCC and represents an adjuvant tool for preclinicalresearch in this disease setting. Underlining its discoverypotential this approach identified a PIK3CA-resistant muta-tion as well as SPRR2A as possible theragnostic markers.Mol Cancer Res; 16(12); 1912–26. �2018 AACR.

IntroductionDevelopment of regional or distant metastases and recurrence

after definitive therapy are arguably themost outstanding issues inthe management of patients with head and neck squamous cellcarcinoma (HNSCC). Presence of lymph node metastases has amajor prognostic impact, as it reduces survival ofHNSCCpatientsroughly by half (1). Moreover, lymph node metastases do notnecessarily respond to given therapeutic approaches in the sameway as their matched primaries do, an observation likely under-lying different intrinsic biological mechanisms (2). Such a het-erogeneous behavior poses an obvious challenge to achievesimultaneous local (primary tumor) and regional (lymph nodemetastases) disease control. Indeed, while most early-stage (withthe exception of oral squamous cell carcinoma) and someadvanced-stage primaries can be usually managed by radiother-apy (with or without chemotherapy/cetuximab), presence oflymph node metastases often requires combined approaches,encompassing neck dissection and irradiation (3). Distant metas-tases develop in approximately 10%–30% of HNSCC patientsafter definitive therapy and carries a meager prognosis (approx-imately 5% overall 5-year survival; ref. 4).

1Department of Radiation Oncology, Inselspital, Bern University Hospital,University of Bern, Bern, Switzerland. 2Department for BioMedical Research,Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland.3Department of Otorhinolaryngology – Head and Neck Surgery, Inselspital,Bern University Hospital, University of Bern, Bern, Switzerland. 4SIB SwissInstitute of Bioinformatics, Lausanne, Switzerland. 5Institute of Pathology,University of Bern, Bern, Switzerland. 6Department of Otolaryngology - Headand Neck Surgery, University of Michigan School of Medicine, Ann Arbor,Michigan. 7Comprehensive Cancer Center, University of Michigan School ofMedicine, Ann Arbor, Michigan. 8Division of Clinical Pathology, GenevaUniversity Hospitals, Geneva, Switzerland. 9Ludwig Center for Cancer Research,University of Lausanne, Lausanne, Switzerland. 10Department of Oncology,University of Lausanne, Lausanne, Switzerland.

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

L. Nisa and D. Barras are co-first authors of this article.

M. Delorenzi and Y. Zimmer are co-senior authors of this article.

Corresponding Author: Yitzhak Zimmer, Department for BioMedical Research,Maurice E. M€uller-Haus, E-807, Murtenstrasse 35, Bern 3008, Switzerland.Phone: 413-1632-2642; Fax: 413-1632-3297; E-mail: [email protected].

doi: 10.1158/1541-7786.MCR-18-0056

�2018 American Association for Cancer Research.

MolecularCancerResearch

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With respect to therapeutic resistance, recurrences after defin-itive therapy in HNSCC occur in 30%–50% of the cases, depend-ing on primary tumor anatomic location, stage at presentation,andhumanpapillomavirus (HPV) status among other factors (5).Management options for treatment failures after multimodaltherapy, including extensive salvage surgery, reirradiation, orpalliation, are limited and often of limited efficacy. Moreover,such approaches carry almost systematically high rates of severetoxicity and/or functional impairment of breathing, swallowing,airway protection, and speech production (6, 7).

The inescapable corollary of these clinical considerations is thepressing need to develop and implement more effective thera-peutic strategies for metastatic and recurrent HNSCC.

The ability to tailor individual management approaches and toimplement them requires thorough characterization of themolec-ular alterations in HNSCC and extensive preclinical validation.In this sense, concerted next-generation sequencing (NGS)approaches in HNSCC have been published over the last fewyears, providing an unprecedented amount of information on themost frequent alterations and mechanisms underlying tumori-genesis (8–12). With respect to preclinical validation, cancer celllines and their related in vivo models represent major researchtools in preclinical oncology when it comes to addressing issuesrelated to tumor biology and target discovery. Comprehensivegenomic characterization is often unavailable for commonly usedcell lines, a limitation that hinders the generalization of resultsand accounts at least to a certain extent for the discordantresponses to novel therapies seen in preclinical versus clinicalsettings (13).

Recognizing the crucial nature of this issue, Barretina andcolleagues (14) established genomic profiles of a large numberof human cancer cell lines. In their study, the authors exploredcorrelations between genomic background and responsesto particular therapeutic agents, emphasizing the relevance ofgenetically driven management approaches. Several other stud-ies provide similar datasets for cell lines from different tumorentities, and databases such as the catalogue of somatic muta-tions in cancer (COSMIC) systematically gather results fromthese studies to facilitate access to the research community(www.cancer.sanger.ac.uk/cosmic). With respect to HNSCC,Martin and colleagues (15) obtained full exome and transcrip-tome profiles of 21 HNSCC cell lines, showing that most typicalgenomic alterations found in HNSCC patients were recapitu-lated in these cell lines. While this study and others cover thespectrum of alterations in primary tumors, patient-derivedmodels to address research questions pertaining to mechanismsof invasion and therapeutic resistance have so far not beencomprehensively characterized from a genomic perspective.

Paralleling the sequencing approaches performed in primaryHNSCCs, two recent studies investigated the genomic landscapeof metastatic and recurrent HNSCC (16, 17). Hedberg and col-leagues (16) performed whole-exome sequencing from 13HNSCC lymph node metastases and 10 recurrent tumors andtheir matched primaries, whileMorris and colleagues (17) carriedout a targeted mutational approach in 410 cancer-related genesand copy number profiles of several head and neck tumors,including 53 HNSCCs.

Here we provide comprehensive genomic profiles of a HNSCCcell line panel derived from primaries and their patient-matchedmetastases/recurrences (18). Our results show that the UM-SCCpanel is representative ofHNSCC fromamolecular perspective, as

themost common aberrations found inHNSCC are recapitulatedin the cell lines. Moreover, the current datasets underline thediscovery potential of the UM-SCC panel and represent a uniquetoolset for the preclinical study of invasion and treatment resis-tance mechanisms in HNSCC.

Materials and MethodsEthical statement

Patients' tissue and clinical datawere collected after approval bythe Bernese Cantonal Ethical Committee (Protocol 050/14). TheUM-SCC cell lines were obtained from surgically excised tissuesfrom patients treated at the University of Michigan (Ann Arbor,MI) who gave written informed consent for the use of their tissuesfor research studies, including the development of permanent celllines.

Cell lines and reagentsUM-SCC cell lines were provided by Professor T. Carey

(University of Michigan, Ann Arbor, MI). Culture, mainte-nance, and cell line identity confirmation were performed asdescribed previously (18). For group analysis, cell lines weredivided in invasive/metastatic (UM-SCC-10B, -14C, -17B and-22B) and recurrent (UM-SCC-11B, -14B, -74B and -81B), andcompared with their matched primaries ("A" series).

PI3K was inhibited with pictilisib (GDC-0941, AbMole Bio-Science). This drug was dissolved in DMSO and stored at�20�C.

KaryotypesFor chromosome analysis, cells were grown as monolayers on

coverslips (35 mmdish, 22� 22mmGlass, MatTek corporation)and harvested in situ. Cultures in exponential growth werearrested at metaphase by adding 10 mg/mL colcemid (KaryoMaxColcemid, Gibco) for 20 minutes at 37�C. Cells were thenharvested with hypotonic saline solution (60 mmol/L KCl), fixedwith fresh fixative (3:1methanol: acetic acid, Scharlau), dried in adrying chamber (25�C and 40% humidity, CDS-5, Thermotron),aged for 1 hour at 93�C on a hot plate (Pr€azitherm, Huber&CoAG), and stained using Giemsa (azur eosin methylene bluesolution, Scharlau) and trypsin (Difco Trypsin 250, Dr. GroggChemie). Capturing and karyotyping was performed with thecommercial software Genikon (Nikon).

Transcriptome and exome profilingNucleic acids were extracted following lysis of cell pellets in

600 mL RTL lysis buffer (AllPrep DNA/RNA/miRNAUniversal Kit,Qiagen). The lysates were homogenized in CK14 Precellyshomogenization tubes (Labgene Scientific) using the Minilyshomogenizer (Bertin Technologies). DNA and RNAwere purifiedfrom the homogenized lysates using a robotic workstation(Qiacube, Qiagen) following manufacturer's instructions andtheir quality assessed using the Fragment Analyzer (AdvancedAnalytical Technologies Inc.). RNA sequencing libraries wereprepared using the Illumina TruSeq Stranded Total RNA reagents(catalog number RS-122-2201; Illumina) according to the pro-tocol suppliedby themanufacturer andusing 750ngof total RNA.Exome sequencing libraries were produced as follows: 3 mg ofgenomic DNA was fragmented into 150–200 base pair fragmentsusing the Covaris Sample Preparation System (S-series). Sampleswere prepared using SureSelect library prep kit (Agilent).The resulting adaptor-tagged libraries were then hybridized with

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exon biotin-coated baits from the SureSelect V5 kit (Agilent) for24 hours at 65�C.Hybrids were capturedwith streptavidin-coatedbeads, PCR amplified and indexed.

Cluster generation was performed with the RNA and DNAlibraries using the Illumina HiSeq PE Cluster Kit v4 cBot reagents(catalog number PE-401-400) and sequenced on the IlluminaHiSeq 2500 using HiSeq SBS Kit V4 reagents (catalog number FC-401-4002). Sequencing data were processed using the IlluminaPipeline Software version 1.84. Exome sequencing initial numberof reads averaged 164 � 41 (SD) million per sample. The rate ofmapping to the hGRC37 genome was of 99.9% � 0.1% (SD).

RNA sequencing initial number of reads averaged 69� 13 (SD)million per sample. Reads were first trimmed to remove polyAand Illumina TruSeq adapter sequences using cutadapt (19), thenaligned to the human reference hGRC37 genome using the STARaligner (20). Reads that uniquelymapped to the reference genomeaveraged 92.6% � 1.6% (SD). The number of counts weresummarized at the gene level using featureCounts (21). Geneexpression values were computed from the RPKM (reads perkilobase per million) values produced by the rpkm function ofthe edgeR package (version 3.16.5) by adding a pseudocount of 1and log2-transforming the results.

Somatic mutation and copy number variation callingExome sequences were processed according to the Genome

Analysis Toolkit (GATK) best practices pipeline, which involvestrimming, alignment using BWA-MEM, marking of duplicates,local realignment around indels, and base recalibration (Supple-mentary Methods; ref. 22). Single-nucleotide polymorphisms(SNP) calling was performed against a panel of eight normaltissue samples taken from the salivary gland of independentpatients obtained within the frame of a different study, using thealgorithms of MuTect (23) and VarScan2 (24) with default set-tings. The variants identifiedwith bothMuTect and Varscan2wereannotated using ANNOVAR (25). To specifically find mutationsinvolved in recurrence and metastatic progression, the variantcalling was also performed comparing each recurrent or meta-static cell line directly against its primary tumor counterpart, byusing the same algorithms.

We determined known cancer-relevant genes contained inchromosomal regions that were amplified or lost, using thecurated cancer-driver gene database derived from Rubio-Perezand colleagues (https://www.intogen.org/downloads; ref. 26).The GISTIC2 software was used to find copy number alterationsrecurring in multiple samples.

Somatic copy number variation between primary cell linesand metastatic/recurrent counterparts was estimated using theVEGAWES R package (27) and represented in Circos plots (28).Use of VEGAWES for CNV (copy number variation) calling waspreviously validated by establishing karyotypes of two randomlyselected cell lines (UM-SCC-17A and UM-SCC-17B). Karyotypesand VEGAWES calling was performed and showed a strikinglyhigh concordance (Supplementary Fig. S1).

Transcriptome and pathway-level analysesPaired differential gene expression analyses were performed

with the generalized linear modeling functions glmFit andglmLRT of the edgeR package (version 3.16.5) using integer readcounts as input (29). Only genes whose expression was consis-tently up- or downregulated in all 4 cell lines of either group(metastases and/or recurrences)were kept. The false discovery rate

(FDR) values computed by edgeR were used to select the differ-entially expressed genes. Genes with an FDR < 0.25 were kept.Genes/cell lines biclustering and heatmaps were constructedusing correlation distance as metrics and the average linkageagglomeration algorithm and heatmaps (R package nclust version1.9.0, http://bcf.isb-sib.ch/Resources.html).

Pathway analyses were performedwith the single sample GSEA(ssGSEA) and the preranked GSEA methods available at theGenePattern web interface of the Broad Institute (http://software.broadinstitute.org/cancer/software/genepattern/). Genedirectional likelihood ratios resulting from differential geneexpression analyses were used as weights in the preranked GSEAmethod and pathways with an FDR < 0.25 were kept. The singlesamples GSEA signature score of these significant pathwaysare presented in heatmaps created with the pheatmap package.We used the Hallmark collection of the MSigDB portal (http://www.broadinstitute.org/gsea/msigdb). The network of 24 pro-teins SPRR2A interacts with was obtained from the STRINGdatabase (medium confidence, all interaction sources, interac-tions areweightedwith the combined score reported by STRING).

TCGA data analysisLevel 3 TCGA RNA sequencing gene expression data was

acquired using the RTCGA package. We used the RSEM normal-ized expression values and log2-transformed them adding apseudocount of 1. The gene classifier that allows categorizingTCGA patients into HNSCC molecular subtypes was kindly pro-vided by V. Walter, University of North Carolina at Chapel Hill,Chapel Hill, NC (30). HNSCC subtype scores are defined as thecorrelation of the sample gene expression with the subtypecentroids. TCGA patients and UM-SCC lines were then classifiedas subtypes with the highest correlation score. To analyze corre-lation of SPRR2A RNA levels and survival, we included patientsfor which both transcriptomic data (FPKM-UQ) of primarytumors and survival/follow-up information was available tocalculate overall survival and disease-free survival. A total of498 patients out of the 806 available (as of February 27, 2018)fulfilled these criteria.

Real-time PCR and genomic locus sequencingTotal RNAwas extracted from80% confluent cell cultures using

TRIzol lysis according to manufacturer's instructions (Roche).Reverse transcription of mRNA was performed using the Omnis-cript RT Kit (Qiagen). Quantitative PCR of SPRR2A was per-formed using a 7900HT fast real-time PCR systemwith a TaqManassay (Applied Biosystems). The mean Ct was determined fromtriplicate experiments and mRNA levels normalized to thoseobtained for 28S ribosomal protein. Changes in expression weredetermined by calculations of DDCt.

A 1 kbp region around the SNP of interest was amplified byPCR, purified on agarose gel, and sequenced by the Sangercapillarymethod. The resulting electropherogramsweremanuallyanalyzed to confirm the presence of the SNP of interest.

Cell proliferation, clonogenic assays, and cell migrationCell viability was determined using a resazurin sodium salt

reduction assay (Sigma). Briefly, at the indicated experimentalendpoints cells cultured in 96-well plates were supplementedwith medium containing 44 mmol/L resazurin. Resazurin reduc-tion was colorimetrically estimated 1 and 6 hours later (570/600 nm) using a Tecan Reader (Tecan Group Ltd.). The

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measurements obtained in every treatment condition were nor-malized to vehicle-treated controls. Results are representative ofthree independent experiments.

For clonogenic assays, cells were plated and left to attachovernight. Irradiation was delivered using a 137Cs research irra-diator (MDS Nordion) with a dose rate of 0.75 Gy/minute. Thesurviving fraction was normalized to the plating efficiency (PE ¼colonies formed/cells plated). Radiosensitization was evaluatedusing the radiation enhancement ratio (RER). A RER significantlysuperior to 1 according to one-sample t test was deemed toindicate radiosensitization (31).

Cell migration was assessed with the Oris Cell MigrationAssembly Kit (AMS Biotechnology) after 3 days of culture.Briefly, cells were plated in 96-well plates in presence of astopper which creates a 2-mm diameter cell-free circular area inthe middle of each well. After cells are attached, cell stoppers areremoved and cells may freely migrate. Pictures were captured atbaseline and after 72 hours using a Leica DC 300F microscope.The invaded area in each treatment condition was determinedwith the ImageJ software (imagej.nih.gov/ij/).

HNSCC patient cohortValidations in patient-derived tissues were based on a retro-

spective cohort includingHNSCC formalin-fixed paraffin-embed-ded (FFPE) specimens of patient-matched primary tumors, lymphnode metastases, and normal reference tissue. All tumors werestage IV-A and IV-B according to the UICC 7th edition (2009). Allpatients were treated with curative intent in the Departments ofRadiation Oncology and Otorhinolaryngology-Head and NeckSurgery, Inselspital Bern University Hospital (Bern, Switzerland)and received radiotherapy, either as primary therapeutic strategyor following surgery for primary tumors, with or without con-comitant systemic therapy (either cisplatin/5-fluorouracil orcetuximab). All patients received neck dissections (Supplemen-tary Table S1).

Construction of tissue microarrays and IHCTissue microarrays (TMAs) were constructed using a new-

generation tissue arrayer. Briefly, FFPE blocks were retrieved fromthe archives of the Institute of Pathology, University of Bern (Bern,Switzerland). Puncheswith adiameter of 0.6mmwere transferredto receptor TMA blocks. IHC staining was performed with anautomated systemBONDRX (Leica Biosystems). Sections of 4mmwere deparaffinized and rehydrated in dewax solution (LeicaBiosystems). Endogenous peroxide activity was blocked inH2O2 for 4 minutes. Samples were incubated with specific pri-mary antibodies for 30 minutes at room temperature: SPRR2A(1:500; Novus Biologicals). Slides were scanned using the Pan-noramic Midi digital slide scanner (3DHISTECH Ltd.). Stainingintensity was scored using the following scores: 0, no detectablestaining; 1, weak staining; 2, moderate to strong in up to 50% oftumor cells; and 3, strong staining in more than 50% of tumorcells. For subsequent ad hoc group comparisons, scores 0 and 1were considered as "low staining intensity" and 2 and 3 as "highstaining intensity."

Statistical analysisDescriptive and comparative analyses were performed using

GraphPad v5.03 (GraphPad Software, Inc.). Student t test wasperformed for intergroup comparison, association between stain-ings and clinicopathologic features were evaluated using x2 test

or Fisher exact test. Univariate survival analysis was plottedaccording to the Kaplan–Meier method and compared with thelog-rank test. Multivariate survival analysis was performed usingCox proportional hazards regression analysis, using establishedrisk factors for decreased disease control or survival. All P valueswere two-sided and statistical significance was set at 0.05.

ResultsGenomic profiles of UM-SCC cell lines recapitulate themolecular heterogeneity and most common alterations inHNSCC

We employed whole-exome sequencing, copy number varia-tion (CNV), and RNA sequencing to characterize the UM-SCCpanel. This panel, which includes 15 cell lines derived from 7different patients (3 females and 4 males), encompasses allanatomical HNSCC subsites (Fig. 1A and B; ref. 18).

We applied the molecular HNSCC subtype classifier initiallyreported by Chung and colleagues (32) and subsequentlyrefined and used by a number of studies, including the TCGA(9, 30). After validation of the subtype classifier in the TCGAcohort, UM-SCC cell lines were classified as atypical (n ¼ 9),mesenchymal (n ¼ 5), and classical (n ¼ 1; Fig. 1C; Supple-mentary Fig. S2A and S2B). Importantly, no cell line displayedhigh centroid correlation for any given molecular subtype,reflecting a high degree of molecular heterogeneity, which fullyparallels observations in HNSCC (30). Equally relevant interms of expression profiles, we found that most cell linescluster with their patient-matched counterparts, with the excep-tion of UM-SCC-11A/B (Supplementary Fig. S2C). UM-SCC-11B clustered better with UM-SCC-74A/B than with its ownmatched counterpart, an observation most likely because thesethree cell lines have a clearly higher mesenchymal centroidcorrelation and phenotype than UM-SCC-11A (Fig. 1B and C).In addition, gene expression correlation was not necessarilydetermined by primary anatomic location or specimen's origin(primary, metastatic, etc.), a finding congruent with previousstudies (Supplementary Fig. S2D; refs. 9, 16, 17, 30).

Also consistent with previous NGS studies, the mutationalprofiles of the UM-SCC panel featured common alterations ofTP53, members of the Notch, the PI3K and the Hippo pathway,and of the histone methyltransferases KMT2D and NSD1 (Fig.1D; Table 1; Fig. 2A; Supplementary Document S1; refs. 8, 9, 12,15, 33). Conversely, however, mutations in other frequentlyaltered genes such as CDKN2A, CASP8, and TGFBR2 were notfound in this panel (Fig. 2A). Alterations in TRAF3, commonlyassociated with HPV-positive HNSCC, were not encountered inthis panel of exclusively HPV-negative cell lines (9).

To explore recurrently mutated genes in an unbiased manner,we focused on genes whose mutation rate across cell lines washigh and whose length was short to middle, as long genes arestatistically more likely to accumulate passenger mutations(Fig. 1E). Among these were NOD2, a gene involved in immuneresponses whosemutations have been reported in several types ofcancers (34). Another interesting finding was presence ofADAMTSL4, member of a gene family involved in cellular adhe-sion and angiogenesis (35). In addition, our unbiased approachrevealed a number of genes known to be consistently false-positive findings in mutational studies (36).

Finally, we performed gene expression pathway analysis usingsingle sample gene set enrichment analysis (ssGSEA), with the

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Figure 1.

General characteristics of the 15 UM-SCC cell lines. A, Clinical, histologic, and therapeutic characteristics of UM-SCC cell lines. PT, primary tumors; LN, lymphnodemetastasis; PD, poorly differentiated; P-MD, poorly tomoderately differentiated; MD,moderately differentiated; M-WD,moderately towell differentiated; N/A,not available. B, Representative pictures of the 15 UM-SCC cell lines (20� magnification). C, HNSCC molecular subtype score for each of the UM-SCC celllines. The final HNSCC subtype call is defined as the one with the highest subtype centroid correlation score. D, Nonsynonymous mutations of the UM-SCCsamples detected in driver genes taken from the TCGA, known HNSCC driver pathways and in genes found recurrently mutated (see Supplementary Fig. S1).A genewas consideredmutated (black square) if at least one variant was found. The number of variants per gene and the percentage of sampleswithmutated genesare reported as side bars. A full list of variants appears in supplementary Fig. 2A. E, Scatterplot showing mutations summarized at the gene level accordingto their frequency in UM-SCC cell lines and to their gene length. Genes highlighted in green have a good mutation frequency to gene length ratio. Genes in red areknown drivers. F, Heatmap of single sample geneset enrichment analysis (ssGSEA) scores. The upper heatmap includes the gene sets of the MSigDB Hallmarkcollection that displayed low correlation of the gene profile across cell lines (see Materials and Methods section for details) and thus represent pathways withdifferent activation/enrichment patterns across cell lines. The lower heatmap displays ssGSEA results for pathways that are considered as drivers in HNSCC.

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hallmark gene signature collections available from the MSigDBmolecular signature database. We selected pathways whose cor-relationmatrices displayed at least one low correlation coefficientbetween two cell lines (r � 0.5), to identify variable activationpatterns across cell lines (Fig. 1F). Frequently altered HNSCCpathways were equally included. Mirroring previously publisheddata, gene expression pathway analysis displayed very heteroge-neous degrees of reliance on specific pathways (Fig. 1F). Consid-ering such patterns of expression is most relevant in preclinicalresearch when considering genomic correlates of responses togiven therapies and allows selection models to address specificresearch questions in a genomically guided manner (9, 14).Altogether, these observations show that the UM-SCC panel isgenomically representative of HNSCC.

Landscapes of UM-SCC cell lines display differentiallydistributed mutations with potential therapeutic implications

Two recent sequencing studies demonstrated that metastaticand recurrent HNSCC specimens aremutationally similar to theirpatient-matched primaries (16, 17). Mirroring such findings,variant allele frequency (VAF) in the UM-SCC panel displayedhigh concordance between cell lines derived from matched pairsof primaries and recurrences/metastases (Supplementary Fig. S3Aand S3B).

Even thoughmutations exclusively present inmetastatic/recur-rent tumors seem to be overall rare, such differential distributionis potentially relevant for implementation of therapeuticapproaches. For instance, Hedberg and colleagues (16) foundDDR2mutations in somemetastatic/recurrent specimens, absentin their patient-matched primaries. Ectopic expression of such

DDR2mutations in cell lines rendered them exquisitely sensitiveto dasatinib (16), underlying possible implementation of geno-mically guided personalized approaches in metastatic/recurrentHNSCC.

In spite of the small number of samples analyzed in this study,we identified a novel kinase domain PIK3CA mutation inUM-SCC-14B (recurrence), absent in UM-SCC-14A (primary)and UM-SCC-14C (metastasis; Fig. 2A). The allelic frequency ofthis mutated variant was 25% (Fig. 2B). Presence of the PIK3CAT2930A mutation (resulting in a F977Y protein change) wasconfirmed by direct sequencing of the region of interest (Fig.2C). Next, to assess the impact of this mutation on cell prolifer-ation, UM-SCC-14A, UM-SCC-14B, and UM-SCC-14C cells wereexposed for 72 hours to the pan-class I PI3K inhibitor pictilisib.Interestingly, pictilisib was less effective at impairing proliferationinUM-SCC-14B than in theother twomatched cell lines (Fig. 2D).

Given that the PI3K pathway is one of the most altered inHNSCC and PIK3CA among the top mutated oncogenes,PI3K signaling is considered a potential therapeutic target inHNSCC (9, 33). Our observation suggests existence of drug-resistant PIK3CA mutations and strongly stresses the importanceof mutationally guided pretherapeutic patient stratification. Weconsulted the TCGA datasets through the cBioPortal for CancerGenomics (www.cbioportal.org) to assess presence of this muta-tion. Although this specific mutation was not found, as UM-SCC-14Bwas derived froma recurrent tumor following radiotherapy, itis possible that the F977Y mutation be treatment-induced.

Differentially distributed mutations in more than one cell linewere only found in metastatic lines (Supplementary Table S2).More specifically, DCHS2 mutations were found in three meta-static cell lines. DCHS2 encodes for the dachsous homolog 2protein, also known as cadherin-27 or protocadherin-23, and isrelated to Hippo pathway signaling (37). In addition, threedifferent mutations in the dystrophin gene (DMD) were foundinUM-SCC-14C andUM-SCC-22B. Nevertheless, as bothDCHS2and DMD are very large genes, the likelihood that identifiedmutations are mere passenger events is high, especially in thecase of DMD, the largest human gene. Most relevant perhaps wasthe A1G variant of TLR8 (Toll-like receptor 8) in 2 metastatic celllines (Supplementary Table S2). This variant has been reported toactivate NFkB signaling, as well as modify cytokine secretionand metabolic patterns in a number of infectious diseases, result-ing in more efficient protection against disease. Along the samelines, TLR8 is thought to potentially confer enhanced tumorinvasiveness by stimulating an NFkB-mediated proinflammatoryresponse (38). Our results warrant further characterization of therole of TLR8 in metastatic HNSCC.

Comparative gene-copy number variation profiles in UM-SCCcell lines

Asnext step,we performed somatic CNV calling tofindwhetherspecific chromosomal aberrations were recurrently present inmetastatic or recurrent cell lines (Fig. 3A and B; SupplementaryDocument 2). Using GISTIC2, we found three chromosomalregions significantly amplified in metastatic cell lines (4q35.1,9q34.3, and 14q24.1), and only one significantly deleted inrecurrent cell lines (21q22.3; Supplementary Fig. S4). The ampli-fied regions in metastatic cell lines contained genes encoding forestablished drivers such as IRF2, FAT1, NOTCH1, MAX, andMLH3. With the exception of FAT1, all genes were overexpressedat least in 3 of 4metastatic cell lines (Fig. 3A). The third amplified

Table 1. Comparison of mutation rates in top mutated genes in UM-SCC cells

UM-SCCpanel

Public databases/Publications (%)

Gene %TCGA(9)

Stransky(12)

Agrawal(8)

Pickering(10)

KMT2D 66.7 16 11 0 10TP53 53.3 72 62 69 60DCHS2 46.7 6 1.4 0 0FAT1 46.7 23 12 0 30FAT4 46.7 8 11 6 2.5FAT2 46.7 7 11 3 2.5NOTCH1 40 18 12 13 10PIK3C2A 26.7 1.2 1.4 0 0NOTCH3 26.7 4 4 0 0NSD1 26.7 12 9 0 8PTEN 26.7 2.7 5 0 0FAT3 26.7 9 12 0 5NF1 26.7 2.7 4 0 0PIK3CG 20 4 5 3 0PIK3CA 20 18 8 9 15PIK3C2G 20 2.2 1.4 0 0AJUBA 20 6 0 0 0RICTOR 13.3 1.8 0 0 0WWC1 13.3 2 0 0 5PIK3CD 13.3 1.6 1.4 0 0DCHS1 13.3 4 8 0 0HRAS 13.3 6 5 9 13KRAS 13.3 0.2 1.4 0 0MAML3 6.7 0.6 0 0 2.5MAML1 6.7 1.2 0 0 0LATS1 6.7 1.4 0 0 0MST1 6.7 0.2 0 0 0TEAD4 6.7 0.8 1.4 0 0

NOTE: Data were retrieved from the cBioPortal for Cancer Genomics.

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Figure 2.

Variant detection and characterization of a novel PIK3CA kinase domain mutation in UM-SCC cell lines. A, Nonsynonymous mutations of the UM-SCC samplesdetected in driver genes taken from the TCGA, known HNSCC driver pathways and in genes found recurrently mutated (see B). Black squares represent mutationfound in both primary tumor and matched recurrent/metastasis counterpart while blue and red squares represent those found only in primary, respectivelyrecurrence/metastasis. B, Allelic frequency of the PIK3CA T2930A mutation according to exome- and RNA-sequencing data. C, Presence of the T2930A (F977Y)PIK3CAmutation in the UM-SCC-14 cell lineswas confirmed by Sanger sequencing.D,UM-SCC-14A/B/C cellswere treatedwith increasing concentrations of the PI3Kinhibitor pictilisib for 72 hours. After this period, cell viability was assessed (RFU, relative fluorescence units; P values: �� , P < 0.01; ��� , P < 0.001).

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Figure 3.

Somatic copy number variation in metastatic and recurrent cell lines. Circos representations showing somatic chromosomal copy number gains (in green) orlosses (in red) in metastatic (A) and recurrent (B) HNSSC cell lines. Chromosomal regions found to be significantly recurrently gained or lost by GISTIC2 arehighlighted. The potential driver genes found in these regions are highlighted and their expression in the respective cell lines is shown in inset panels.

Profiling of Recurrent and Metastatic HNSCC Cell Lines

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region (14q24.1) in metastatic cell lines contains, among others,MYC associated factor X gene (MAX) and MLH3, two geneswhose mutations have been involved in pheochromocytomaand colorectal cancer development, respectively (39, 40). Theonly significantly deleted region in recurrent cell lines(21q22.3) contains RUNX1, a tumor suppressor that poten-tially interacts with p53 (41).

Taken together, our findings suggest that, while differences inCNV between primary and matched metastatic/recurrent tumorsseem to be overall discrete, genes involved in progression andtherapeutic resistance may be often altered and most likely playrelevant roles in these processes.

Differentially regulated pathway analysis in metastatic andrecurrent cell lines highlights the relevance of epithelial-to-mesenchymal transition as a mechanism of therapeuticresistance

To characterize differentially regulated pathways in metastaticand recurrent cell lines, we performed pathway enrichment anal-ysis using the gene likelihood ratios obtained by differential geneanalysis as weight factors (Fig. 4A). Among the frequentlyenriched pathways in recurrent tumors, we identified severalproinflammatory signatures (IFNa/g response, IL6/JAK/STAT3and IL2/STAT5 signaling, and TNFa signaling via NFkB). Altera-tions of proinflammatory signaling are frequent in HNSCCtumors and cell lines, and have been linked to potential thera-peutic resistance (9, 15, 42). Another process involved in resis-tance to chemotherapeutic agents, xenobiotic metabolism, wasequally upregulated in 3 of 4 recurrent cell lines. Metabolicreprogramming is indeed thought to confer a selection advantageto tumor cells upon genotoxic therapy (43).

Most prominently, epithelial-to-mesenchymal transition(EMT) was upregulated in all recurrent cell lines, while noconsistently enriched pathways could be identified in metastaticcell lines (Fig. 4A). In linewith theobservedEMTupregulation,wefound that HNSCC subtype score progression in recurrent but notmetastatic cell lines is characterized by an increase in the mesen-chymal score (Fig. 4B). To further assess the differences in EMT'senrichment in metastases and recurrent cell lines, we performeddifferential expression analysis and compared the results for genesin the EMT group versus all genes in metastatic and recurrent celllines (Fig. 4C). In contrast to recurrent cell lines, EMT genes with Pvalues < 10�3 were absent in metastatic cell lines. Focusing on 14EMT genes with an FDR < 0.25 in the differential expressionanalysis, we found these genes to be well connected and consis-tently upregulated in recurrent cell lines (Fig. 4D).

The consistent upregulation of EMT in recurrent but not met-astatic cell lines is congruentwith recentfindings in pancreatic andmammary carcinomas, supporting the notion that EMTmay havea more important role in therapeutic resistance than in invasive-ness (44–46).

Comparative expression profiles of UM-SCC cell lines unveil apotential role of SPRR2A as effector of therapeutic resistanceand potential prognostic marker in regionally invasive HNSCC

The differential expression analysis results were further used toidentify individual genes involved inmetastasis and/or recurrencein the UM-SCC panel. The tenmost differentially expressed genesin metastatic and/or recurrent UM-SCC cell lines include ECMcomponents (SRGN), genes involved in extracellular matrix(ECM) remodeling (MMP2 and KLK5), or epidermal differenti-

ation and homeostasis (PRRX1, ALPK2, FLG, and IVL; Fig. 5A). Afull list of differentially regulated genes (FDR < 1) is provided inSupplementary Document S3.

Interestingly, small proline-rich protein 2A (SPRR2A) was theonly gene differentially expressed in both metastatic (FDR ¼0.089) and recurrent (FDR ¼ 0.004) cell lines (Fig. 5A). Down-regulation was confirmed in 3 of 4 cell lines tested by means ofqPCR (Fig. 5B). SPRR2A is one of the 14 SPRR-family of proteinsand acts as a keratinocyte cross-linking protein, ensuring tissueintegrity when facing injury and oxidative stress (47).

To gain further mechanistic insights into the potential roles ofSPRR2A in metastasis and recurrence, we used Cytoscape tovisualize the network of established SPRR2A interactors accordingto the STRING database and their fold-change results from thetranscriptomic analysis. Genes of the SPRR family were coordi-nately downregulated both in metastatic and recurrent cell lineswhen compared with their matched primaries (Fig. 5C and D).Other genes involved in epithelial homeostasis, such as POU2F2and CNFN, were similarly downregulated.

The oncogenic role of SPRR2A is complex. Specht and collea-gues (48) demonstrated that stable SPRR2A transfection of cho-langiocarcinoma cell lines injected intrasplenically led to inabilityto form liver metastases, however increasing local tumor inva-siveness. Conversely, cell lines with downregulated SPRR2A read-ily developed liver metastases. From a mechanistic perspective,SPRR2A binds SH3 domain–containing kinases, including Src,Yes1, and Abl (47). In our transcriptomic dataset, SPRR2A down-regulation did not seem to have an impact at the transcriptionallevel on Src-related kinases (Fig. 5C and D). However, given thatinteractions between SPRR2A and Src-related kinases occur at theprotein-protein level, it is anticipated that SPRR2A downregula-tion may have an impact on prosurvival signaling mediated bythese kinases.

While a limited number of studies have addressed the role ofSPRR2A in hepatobiliary malignancies, the relevance of SPRR2Ain HNSCC has not been explored. Thus, we proceeded to assessexpression of SPRR2A by qPCR in 51 patient-matched sets ofHNSCC primary tumors and lymph node metastases, demon-strating significant downregulation of SPRR2A mRNA levels inmetastatic specimens (Fig. 6A). Next, to explore the potentialclinical relevance of SPRR2A, protein expression levels wereassessed by IHC in 426 primary tumors from 181 patients, 530lymph node metastases from 236 patients, and 295 normaltissues (mainly salivary gland tissue) from 237 patients. SPRR2Awas highly expressed in 49.53% of primaries and 26.98% oflymph node metastases, while its expression was not detected innormal tissues (Fig. 6B).

Matched sets of primary tumor, lymph node metastasis, andnormal tissue, as well as full clinical and survival data wereavailable for 147 patients. Patients were classified according toSPRR2A expression (low: scores 0 and 1; vs. high: scores 2 and 3),in primary tumors and lymph node metastases (SupplementaryTable S3). In this set of matched samples, SPRR2A was expressedaccording to 4 different patterns (Fig. 6C). In 44.9% of the cases,SPRR2A was highly expressed in primary tumors only, in 14.29%in lymph node metastases only, and in 17.01% in both primaryandmatched lymphnodemetastases. SPRR2Awasnot detected in23.81% of the cases (Fig. 6C). SPRR2A expression was notassociated with tumoral stage, site, or development of distantmetastases. SPRR2A low expression in primary tumors was sig-nificantly associated with local recurrences (P < 0.01) in the

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Figure 4.

Pathway analysis comparisons in UM-SCC cell lines. A, Preranked GSEA using the likelihood ratio statistics of the differential expression testing as weight vector.The MSigDB hallmark geneset collection was used and ssGSEA score were used to draw the heatmap. Only pathways with a FDR below 0.25 in the prerankedGSEA analysis were kept for plotting. B, Progression of the HNSCC subtype score from primary tumor cell lines to their recurrent/metastatic counterparts.The progression score is computed as the difference between the recurrence/metastasis score and the primary tumor score for each cell line. C, Fraction of EMTversus all genes in metastatic and recurrent cell lines. D, Interaction networks of differentially expressed EMT genes with an FDR < 0.25 and their estimatedlogarithmic fold change values.

Profiling of Recurrent and Metastatic HNSCC Cell Lines

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Figure 5.

Differentially expressed genes and relevance of SPRR2A expression in metastatic and recurrent HNSCC cell lines. A, Expression of the ten most differentiallyexpressed genes (all with FDR < 0.25) separately in recurrent and metastatic cell lines versus their respective primaries. Only genes that were consistently up- ordownregulated in all four cell lines are shown. B, SPRR2A mRNA levels in metastatic cell lines. C and D, SPRR2A interaction networks based on transcriptomic datain metastatic and recurrent cell lines. Link width is proportional to the combined evidence score reported by STRING, and node color indicates the estimatedlogarithmic fold change in the primary-metastasis and primary-recurrent comparisons, respectively.

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Figure 6.

Patterns of SPRR2A expression and impact on survival in regionally metastatic HNSCC tissue samples. A, Comparison of SPRR2A mRNA levels in 51 patient-matched primary tumors versus lymph node metastases. B, SPRR2A IHC staining intensity in HNSCC samples. C, SPRR2A staining patterns in patient-matchedprimaries, lymph node (LN) metastases, and normal tissue. D, Kaplan–Meier plots illustrating regional disease-free survival as a function of SPRR2A expression.E, Migration assay with UM-SCC-10A/B and UM-SCC-22A/B cell lines. Cells were allowed to migrate for 72 hours, after which the invaded area wasmeasured. F, Colony-forming assays. RER, radiation enhancement ratio.

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Profiling of Recurrent and Metastatic HNSCC Cell Lines

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univariate time-independent analysis. SPRR2A low expression inlymph node metastases was significantly more prevalent inpatients older than 60 years old (P ¼ 0.02), while SPRR2A highexpression in lymph node metastases was more prevalent inpatients that experienced regional recurrences (P ¼ 0.04) andthose who got concomitant systemic therapy (P ¼ 0.04) (Sup-plementary Table S3).

In the univariate survival analysis, only high SPRR2A expres-sion in lymph node metastases was significantly associated withreduced regional recurrence-free survival (RRFS; HR ¼ 2.80, 95%CI: 1.16–6.79, P¼ 0.02; Fig. 6D; Supplementary Table S4). In themultivariate analysis, nonoropharyngeal location of the primarytumor and high SPRR2A expression in lymph node metastaseswere both independent predictors of poor regional recurrence-free survival (Supplementary Table S4). Our multivariate modelscould not identify significant predictors of overall survival or localrecurrence-free survival. Surgical resection of the primary tumorand oropharyngeal primary site predicted improved distantmetastasis-free survival (Supplementary Table S4).

We next performed similar analyses using the TCGA datasetsavailable for 498 patients and found no correlation betweenSPRR2A RNA levels and survival (Supplementary Fig. S5). Thisobservation is not surprising, as data is derived from primarytumors and protein levels are not available in the TCGA cohorts.

Because the clinical data seems to suggest that high expressionof SPRR2A may contribute to radioresistance and low expressionmay facilitate metastasis, we performed two supplementaryin vitro approaches. For this purpose, UM-SCC-10A/B and UM-SCC-22A/B cells were plated and allowed tomigrate for 72 hours.The UM-SCC-10 pair migrated faster than UM-SCC-22 cells.Nevertheless, for both pairs of cell lines, the SPRR2A low expres-sing 10B and 22B migrated significantly faster than 10A and 22A,respectively (Fig. 6E). To assess radiosensitivity, cells were irradi-ated with increasing doses (0, 2, and 4 Gy), and left to proliferatefor 7 days. The SPRR2A-high line UM-SCC-10A was significantlymore resistant to irradiation than its matched SPRR2A-low celllineUM-SCC-10B.Wedid notfind significant differences betweenUM-SCC-22A and 22B (Fig. 6F).

DiscussionIn this study, we provide the most comprehensive molecular

characterization of a panel of cell lines derived from metastaticand recurrent HNSCC specimens to datewith the aim to provide asuitable tool for preclinical research in the metastatic and recur-rent setting of HNSCC. Molecular portraits of HNSCC, includingcomparisons betweenHPV-positive andHPV-negative tumors, aswell as primaries versus lymph node metastases and recurrenttumors, have emerged in recent years (8, 9, 11, 12, 16, 17).Implementation of novel findings in clinical practice, however,requires thorough preclinical validation in well-characterizedmodels (14).

Our current results show that the UM-SCC panel is represen-tative of the most common and most characteristic molecularalterations found inHNSCC. Indeed,mutational profiling ofUM-SCC cells revealed typical alterations affecting TP53, PI3K, and theNOTCH pathways. Amajor aspect of interest was the observationofmutations exclusively present inmetastases or recurrences, andthe subsequent possibility to study differential sensitivities todiverse management approaches in primaries versus theirmatchedmetastases and/or recurrences based on theirmutational

background. For instance, it was recently demonstrated thatexclusive presence ofDDR2mutations in lymph node metastasesresulted in increased sensitization of HNSCC preclinical modelstodasatinib (16). In this study andhinting at a potential resistancemechanism, we identified a novel kinase domain PIK3CA muta-tion (F977Y) that impairs responses to PI3K inhibitors in cell lines(Fig. 2D). This mutation was found in a cell line derived from arecurrent HNSCC specimen and is possibly treatment induced.Thus, given the potential relevance of the PI3K pathway as atherapeutic target alongwith the high rate of alterations along thispathway, elucidating the implications of such alterations in termsof treatment responses is of utmost importance prior to imple-mentation of PI3K pathway inhibitors in recurrent HNSCC.

Mutations of components of theHippo pathway were found inall cell lines (Figs. 1Dand2A).AberrantHippopathway activationhas been reported in several types of human malignancies,including lung, colorectal, ovarian, and liver cancer (49). MostprevalentlymutatedwereDCHS2 and FAT isoforms 1 to 4. FAT1 isone of the most commonly mutated genes in HNSCC and isthought to act upstream of the Hippo pathway mediating cross-talk with the Wnt pathway, thus contributing to acquisition ofinvasive features (9, 49). Nevertheless, the oncogenic significanceof FAT1, DCHS1, andDCHS2mutations is not fully clear, as thesegenes are large and high prevalence of mutations in these genesmay be due, in part, to their size. Moreover, while FAT and DCHSare well established Hippo pathway modulators in Drosophilamelanogaster, their precise role in mammalians are not fully clear(49). Besides mutations, we found hemicentin 1 (HMCN1), anincreasingly recognized key upstream regulator ofHippopathwaycore kinases MST1/MST2 and LATS1/LATS2, to be consistentlyupregulated in metastatic cell lines (Fig. 5A). Given the markeddifferences in Hippo pathway members' mutations in primaryversus metastatic and recurrent HNSCCs, the impact of Hipposignaling in such settings warrant further investigation.

CNV profiles were highly preserved in metastatic and recurrentversus primary tumors, with only 3 significant peaks inmetastaticand 1 in recurrent cell lines (Fig. 3A and B). Such discretedifferences are consistent with previously findings (16). Never-theless, altered regions contained relevant drivers or tumor sup-pressors and underline potentially relevant mechanisms in devel-opment of metastases or recurrences.

Expression data revealed that transcriptomic differencesbetween primary tumors and theirmatchedmetastases weremoremarked than in the case of recurrences. Among the frequentlyupregulated pathways in recurrent cell lines were pathwaysinvolved in the inflammatory response and, most significantly,EMT (Fig. 4A). While EMT is considered a hallmark process ofmetastasizing tumors, emergent evidence links EMT to treatmentresistance (45). Indeed, recent studies in mammary and pancre-atic tumormodels convincingly show that impairment of EMT byblockade of either ZEB1 andZEB2 or Snail and Twist does not leadto decreased metastatic ability (44, 46). Importantly however,inability to undergo EMT resulted in increased sensitivity tocyclophosphamide and gemcitabine in the samemodels (44, 46).

When considering individual cell lines, EMT signatures fea-tured most prominently in UM-SCC-74A, UM-SCC-74B, andUM-SCC-11B, all derived from patients previously managedwith chemoradiotherapy and with phenotypical characteristicsof EMT (Fig. 1A and B; ref. 18).

The heterogeneity in terms of pathway analysis seen in the UM-SCC panel reflects the heterogeneity found in actual tumors and

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needs to be considered when exploring novel therapeuticapproaches (9, 14, 30, 32). Relevant to this point, the character-ization provided in UM-SCC panel allows investigation ofmechanisms and target validation taking into consideration themolecular background of such cell lines. An important implica-tion of such approach is the possibility to unveil heterogeneity intreatment responses and potentially identifying predictivebiomarkers.

Finally, the finding that most clearly illustrates the discoveryand validation potential of the UM-SCC panel concerns theidentification of SPRR2A as potential effector of therapeuticresistance and biomarker in HNSCC. In cholangiocarcinoma andliver cancer models SPRR2A is activated by STAT-3 both in biliaryepithelial cells and cholangiocarcinoma, coorchestrating EMTthrough interaction with ZEB-1 (47, 50). In addition, SPRR2Areduces p53 acetylation by impairing p300-p53 interactions,leading to inhibition of p53 transcriptional targets (50). More-over, SPRR2A acts as an SH3 domain ligand, hence promotingprotection against oxidative stress and DNA damage (47). In ourFFPE validation cohort, most primary tumors (61.91%) highlyexpressed SPRR2A, while only a minority of lymph node metas-tases did (31.30%), perhaps reflecting necessary SPRR2A down-regulation in certain clones within the primary tumor to give riseto metastases (48). Importantly, however, our results emphasizean additional aspect on the role of SPRR2A in therapeutic resis-tance. Indeed, as high SPRR2A expression in lymph node metas-tases is an independent risk factor for development of regionalrecurrence after therapy, it could be hypothesized that aftermetastatic colonization SPRR2A expression may be restored inneoplastic cells. In this context, SPRR2A would contribute to thestress response upon radiotherapy/chemoradiation by virtue of itsantioxidative stress ability and through protein–protein interac-tions with multiple prosurvival signaling molecules through itsSH3domain ligand activity (including Src, Yes1, and others) (47).In vitro migration and radiosensitization assays seem to suggestthat indeed high SPRR2A expression may contribute to radio-resistance, while low SPRR2A expression may stimulate invasivefeatures.

Factors that determine SPRR2A reestablished expression inmetastases are yet to be fully investigated and may provide novelmechanistic insights with potential therapeutic relevance, asSPRR2A cannot be directly targeted.

In addition, it would be of high interest to determine SPRR2Aexpression levels in recurrent lymph node metastases after radio-

therapy and performing prospective validation of SPRR2A as abiomarker for risk stratification.

Limitations of this study include the absence of HPV-positivecell lines and limited amount of cell lines included. However, itshould be noted that availability of metastatic/recurrent andprimary tumor patient-matched HNSCC cell lines, especiallyHPV-positive ones, remains limited.

Disclosure of Potential Conflicts of InterestO. Elicin is a consultant/advisory boardmember for AstraZeneca,Merck, and

Merck Serono. T.E. Carey has ownership interest (including stock, patents, etc.)in licensed cell lines to Millipore EMD. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: L. Nisa, D. Barras, M. Medov�a, D.M. Aebersold,R. Giger, M. Delorenzi, Y. ZimmerDevelopment ofmethodology: L. Nisa, D. Barras,M. S.Dettmer, R. Giger, L. HoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Nisa, M. Paliakov�a, J. Koch, B. Bojaxhiu, O. Elicin,M. S. Dettmer, R. Giger, M.D. Caversaccio, T.E. Carey, T.A. McKeeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Nisa, D. Barras, M. Medov�a, M. Medo,M. Paliakov�a, J. Koch, P. Angelino, R. Giger, M. Delorenzi , Y. ZimmerWriting, review, and/or revision of the manuscript: L. Nisa, D. Barras,M. Medov�a, D.M. Aebersold, O. Elicin, R. Giger, U. Borner, M.D. Caversaccio,T.E. Carey, T.A. McKee, M. Delorenzi, Y. ZimmerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Nisa, D. Barras, M. Medov�a, J. Koch,B. Bojaxhiu, O. Elicin, M. S. Dettmer, R. Giger, U. Borner, M.D. Caversaccio,M. DelorenziStudy supervision: L. Nisa, D. Barras, M. Medov�a, R. Giger, M. Delorenzi,Y. Zimmer

AcknowledgmentsThis work was supported by Swiss National Science Foundation

(grant no. 31003A_156816, to Y. Zimmer) and by a Bernische Krebsliga grant(to M. Medov�a). The authors gratefully acknowledge the help of Sabina Gallati,PhD andNijas Aliu, MSc, from the Division of HumanGenetics, Department ofPediatrics (Inselspital, Bern, Switzerland) for establishing karyotypes.

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 January 17, 2018; revised April 28, 2018; accepted August 7, 2018;published first August 14, 2018.

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2018;16:1912-1926. Published OnlineFirst August 14, 2018.Mol Cancer Res   Lluís Nisa, David Barras, Michaela Medová, et al.   Recurrent DiseaseNeck Cancer Cells: A Preclinical Pipeline for Metastatic and Comprehensive Genomic Profiling of Patient-matched Head and

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