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Cancer Therapy: Preclinical Ligand-Dependent Activation of EGFR in Follicular Dendritic Cells Sarcoma is Sustained by Local Production of Cognate Ligands William Vermi 1,7 , Emanuele Giurisato 2 , Silvia Lonardi 1 , Piera Balzarini 1 , Elisa Rossi 2 , Daniela Medicina 1 , Daniela Bosisio 1 , Silvano Sozzani 1,8 , Wilma Pellegrini 1 , Claudio Doglioni 3 , Antonio Marchetti 4 , Giulio Rossi 5 , Stefano Pileri 6 , and Fabio Facchetti 1 Abstract Purpose: The aim of this study was to investigate the biological and clinical significance of epidermal growth factor receptor (EGFR) signaling pathway in follicular dendritic cell sarcoma (FDC-S). Experimental Design: Expression of EGFR and cognate ligands as well as activation of EGFR signaling components was assessed in clinical samples and in a primary FDC-S short-term culture (referred as FDC- AM09). Biological effects of the EGFR antagonists cetuximab and panitumumab and the MEK inhibitor UO126 on FDC-S cells were determined in vitro on FDC-AM09. Direct sequencing of KRAS, BRAF, and PI3KCA was conducted on tumor DNA. Results: We found a strong EGFR expression on dysplastic and neoplastic FDCs. On FDC-AM09, we could show that engagement of surface EGFR by cognate ligands drives the survival and proliferation of FDC-S cells, by signaling to the nucleus mainly via MAPK and STAT pathways. Among EGFR ligands, heparin-binding EGF-like growth factor, TGF-a and Betacellulin (BTC) are produced in the tumor micro- environment of FDC-S at RNA level. By extending this finding at protein level we found that BTC is abundantly produced by FDC-S cells and surrounding stromal cells. Finally, direct sequencing of tumor- derived genomic DNA showed that mutations in KRAS, NRAS, BRAF, and PI3KCA, which predicts resistance to anti-EGFR MoAb in other cancer models, are not observed in FDC-S. Conclusion: Activation of EGFR by cognate ligands produced in the tumor microenvironment sustain viability and proliferation of FDC-S indicating that the receptor blockade might be clinically relevant in this neoplasm. Clin Cancer Res; 19(18); 1–12. Ó2013 AACR. Introduction EGFR, a well-characterized member of the ErbB receptor family is normally expressed by epithelial and mesenchy- mal cells and plays a major role in their proliferation and differentiation by binding a large set of endogenous ligands (1). The intracellular signal induced through EGFR activa- tion is transmitted by 3 main synergic cascades including Ras/Raf which activate mitogen-activated protein kinases (MAPK), PI3K/AKT, and JAK/STAT (2). These intracellular pathways ultimately translate extracellular stimuli into a nuclear message to sustain a large set of biological activities, including cell differentiation, survival, and proliferation. As largely showed by numerous preclinical models, unchecked increase of EGFR signaling might also support the transfor- mation of different cell types, as well as progression of carcinomas and gliomas (3–5). In these neoplasms, aber- rant EGFR signaling is perpetuated in a ligand-independent fashion by genetic variants inherent to transformed cells (e.g., gene amplification or activating mutations) of the EGFR allele (6, 7). Alternatively, activation of the receptor might also occur in EGFR overexpressing transformed cells with a wild-type configuration of the EGFR allele via binding to endogenous ligands abundantly produced in the tumor microenvironment, as recently showed in soft tissue sarcomas (8). In the hematopoietic system EGFR is functional only on subsets of monocyte-derived cells, namely osteoblasts and osteoclasts (9). In contrast, all the remaining lineages are Authors' Afliations: 1 Department of Molecular and Translational Med- icine, Section of Anatomic Pathology, Oncology and Experimental immu- nology, University of Brescia, Brescia; 2 Department of Physiopathology, Experimental Medicine and Public Health, University of Siena, Siena; 3 Department of Pathology, San Raffaele Scientic Institute, Milan; 4 Center of Predictive Molecular Medicine, Center of Excellence on Aging Univer- sity-Foundation, Chieti; 5 Unita' Operativa di Anatomia Patologica, Azienda Arcispedale S. Maria Nuova/IRCCS, Reggio Emilia; 6 Hematopathology Section, Policlinico S. Orsola, University of Bologna, Bologna; 7 Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, Missouri; and 8 Humanitas Clinical and Research Center Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Authors: William Vermi, Department of Molecular and Translational Medicine, Section of Pathology, University of Brescia, 25123, Brescia, Italy. Phone: 39-030-3998425; Fax: 39-030-2995377; E-mail: [email protected]; and Fabio Facchetti, [email protected] doi: 10.1158/1078-0432.CCR-13-1275 Ó2013 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org OF1 Research. on August 4, 2018. © 2013 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst July 25, 2013; DOI: 10.1158/1078-0432.CCR-13-1275

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Cancer Therapy: Preclinical

Ligand-Dependent Activation of EGFR in Follicular DendriticCells Sarcoma is Sustained by Local Production of CognateLigands

William Vermi1,7, Emanuele Giurisato2, Silvia Lonardi1, Piera Balzarini1, Elisa Rossi2, Daniela Medicina1,Daniela Bosisio1, Silvano Sozzani1,8, Wilma Pellegrini1, Claudio Doglioni3, Antonio Marchetti4, Giulio Rossi5,Stefano Pileri6, and Fabio Facchetti1

AbstractPurpose: The aim of this study was to investigate the biological and clinical significance of epidermal

growth factor receptor (EGFR) signaling pathway in follicular dendritic cell sarcoma (FDC-S).

Experimental Design: Expression of EGFR and cognate ligands as well as activation of EGFR signaling

components was assessed in clinical samples and in a primary FDC-S short-term culture (referred as FDC-

AM09). Biological effects of the EGFR antagonists cetuximab and panitumumab and the MEK inhibitor

UO126 on FDC-S cells were determined in vitro on FDC-AM09. Direct sequencing of KRAS, BRAF, and

PI3KCA was conducted on tumor DNA.

Results: We found a strong EGFR expression on dysplastic and neoplastic FDCs. On FDC-AM09, we

could show that engagement of surface EGFR by cognate ligands drives the survival and proliferation of

FDC-S cells, by signaling to the nucleus mainly via MAPK and STAT pathways. Among EGFR ligands,

heparin-binding EGF-like growth factor, TGF-a and Betacellulin (BTC) are produced in the tumor micro-

environment of FDC-S at RNA level. By extending this finding at protein level we found that BTC is

abundantly produced by FDC-S cells and surrounding stromal cells. Finally, direct sequencing of tumor-

derived genomicDNA showed thatmutations inKRAS,NRAS,BRAF, and PI3KCA, which predicts resistance

to anti-EGFR MoAb in other cancer models, are not observed in FDC-S.

Conclusion: Activation of EGFR by cognate ligands produced in the tumor microenvironment sustain

viability and proliferation of FDC-S indicating that the receptor blockademight be clinically relevant in this

neoplasm. Clin Cancer Res; 19(18); 1–12. �2013 AACR.

IntroductionEGFR, a well-characterized member of the ErbB receptor

family is normally expressed by epithelial and mesenchy-mal cells and plays a major role in their proliferation anddifferentiation by binding a large set of endogenous ligands

(1). The intracellular signal induced through EGFR activa-tion is transmitted by 3 main synergic cascades includingRas/Raf which activate mitogen-activated protein kinases(MAPK), PI3K/AKT, and JAK/STAT (2). These intracellularpathways ultimately translate extracellular stimuli into anuclearmessage to sustain a large set of biological activities,including cell differentiation, survival, and proliferation. Aslargely showed bynumerous preclinicalmodels, uncheckedincrease of EGFR signaling might also support the transfor-mation of different cell types, as well as progression ofcarcinomas and gliomas (3–5). In these neoplasms, aber-rant EGFR signaling is perpetuated in a ligand-independentfashion by genetic variants inherent to transformed cells(e.g., gene amplification or activating mutations) of theEGFR allele (6, 7). Alternatively, activation of the receptormight also occur in EGFR overexpressing transformedcells with a wild-type configuration of the EGFR allele viabinding to endogenous ligands abundantly produced in thetumor microenvironment, as recently showed in soft tissuesarcomas (8).

In the hematopoietic system EGFR is functional only onsubsets of monocyte-derived cells, namely osteoblasts andosteoclasts (9). In contrast, all the remaining lineages are

Authors' Affiliations: 1Department of Molecular and Translational Med-icine, Section of Anatomic Pathology, Oncology and Experimental immu-nology, University of Brescia, Brescia; 2Department of Physiopathology,Experimental Medicine and Public Health, University of Siena, Siena;3Department of Pathology, San Raffaele Scientific Institute, Milan; 4Centerof Predictive Molecular Medicine, Center of Excellence on Aging Univer-sity-Foundation, Chieti; 5Unita' Operativa di Anatomia Patologica, AziendaArcispedale S. Maria Nuova/IRCCS, Reggio Emilia; 6HematopathologySection, PoliclinicoS.Orsola,University ofBologna,Bologna; 7Departmentof Pathology and Immunology, Washington University School of Medicine,Saint Louis, Missouri; and 8Humanitas Clinical and Research Center

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

Corresponding Authors: William Vermi, Department of Molecular andTranslational Medicine, Section of Pathology, University of Brescia,25123, Brescia, Italy. Phone: 39-030-3998425; Fax: 39-030-2995377;E-mail: [email protected]; and Fabio Facchetti, [email protected]

doi: 10.1158/1078-0432.CCR-13-1275

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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independent from this receptor for their biological activities.Recently, we and others reported that EGFR expressionmarkfollicular dendritic cells sarcomas (FDC-S; refs. 10, 11), aneoplasmderived fromFDCs. FDCare stromal cells typicallyfound in the B-cell compartment of secondary lymphoidorgans. Landmark studies have supported the notion thatmost FDCs are ofmesenchymal origin andplay a critical rolein the germinal center dynamics by presenting antigen to Bcells through antigen–antibody complexes (12, 13). How-ever, the extreme difficulty to purify and grow human FDChas represented a significant limitation in the understandingof their biology in normal and disease states (14).

FDC-S, the neoplastic counterpart of the B-cell follicleFDC, represents a rare spindle cell malignancy that canoccur at nodal and extra nodal sites (15–17). PrimaryFDC-S are treated with surgery, however a consistent frac-tion of them relapse. Unfortunately, studies on the biologyof FDC-S have been rarely translated into a meaningful andclinically actionable knowledge. In this study,we report thatEGFR overexpression by immunohistochemistry marks theentire spectrum of lymphoid organs stromal cells includingFDCs and derived neoplasms. By signaling to the nucleusmainly viaMAPK and STAT pathways, we show that engage-ment of surface EGFR by cognate ligands drives the survivaland proliferation of FDC-S cells. Among the latter, heparin-binding EGF-like growth factor (HBEGF), TGF-a, and Beta-cellulin (BTC) are produced in the tumor microenviron-ment at RNA level. By extending this finding at protein level,we found that BTC is abundantly produced by FDC-S cellsand surrounding stromal cells. Remarkably, mutations inKRAS,NRAS, BRAF, and PI3KCA, which predicts resistanceto anti-EGFR MoAb in other cancer models, are notobserved in FDC-S thus indicating that the receptor block-ade might be clinically relevant.

Materials and MethodsPatients and tissues

Samples included formalin-fixed paraffin-embedded(FFPE) tissue blocks. Clinical data were obtained frompatients’ records. Reactive human lymph nodes and tonsilsas well as normal spleen and pancreas were used as controltissues. Pathologic cases were represented by 16 FDC-Scases and 5 cases of hyaline vascular Castleman’ disease(HV-CD). The study has been conducted in compliancewith the Helsinki Declaration and with policies approvedby the local Ethics Board (Spedali Civili di Brescia, Italy).

Immunostaining proceduresFour-micrometer tissue sections from FFPE blocks were

used for immunohistochemical staining. Primary antibodiesare listed in the Supplementary Material. Proper isotypecontrol or irrelevant antibodies have been included in theprotocol. Double immunohistochemistry were conductedas previously reported (18). Immunohistochemistry wasinterpreted byWV, SL, and FF. For phosphorylated proteins,cases were scored positive when showing main reactivity inthenucleus (forpSTAT3,pAKT, andpERK)or cellmembrane(for pEGFR) in>5%of tumor cells. Positive caseswere scoredbasedon thepercentageof positive cells as follow:þ¼ 5% to20% of neoplastic cells;þþ ¼ 20% to 50%;þþþ ¼ >50%.

Culture procedure and cytogenetic analysisPart of lesion from an FDC-S case was removed under

sterile conditions and transferred in RPMI 1640 mediumwithout serum kept at 4�C until transfer to the cell culturefacility. For culture, the tissue was finely minced anddigested for 24 hours at 37�C in RPMI 1640 containing0.2% of Collagenase type II. The cells suspension waswashed with PBS, plated on T25 flask with complete RPMI1640 supplemented with 10% FCS. Cells were maintainedat 37�C in a humidified atmosphere with 5% CO2. Themediumwas changed twice a week and after trypsinization,the cells were passed in a new flask for cells expansion andcytogenetic analysis. The cytogenetic analysis was con-ducted on total of 20 cells in G-banded metaphases cellsobtained from 5-day culture. Cell line authentication bymorphology and phenotype is described in detail in theresult section.

FISHFISH for EGFR/CEP7 was applied as previously reported

(19), by using LSI EGFR Spectrum Orange/CEP7 SpectrumGreen probes (Abbott Molecular). Tumor samples wereexamined with an epifluorescent microscope (Nikon,Eclipse 90I). In each tumor sample, signals were countedin a total of 100 nonoverlapping tumor-cell nuclei. Themean signal number of the EGFR gene as well as CEP7 wascalculated and the EGFR/CEP7 ratio determined. Geneamplification was considered for EGFR/CEP7 ratio � 2.

qPCRRNAwas extracted using TRIzol reagent (Cat. No. 15 596;

Invitrogen) according to the manufacturer’s instructions.

Translational RelevanceFollicular dendritic cell sarcoma (FDC-S) is treatedwith

surgery however a consistent fraction of them relapse.Adjuvant radiation and/or chemotherapy provides nosignificant benefit in term of disease-free survival. Theextreme difficulty to purify human FDC and the rarity ofFDC-Shave represented a limitation in theunderstandingof the FDC-S biology. Here, we report that EGF receptor(EGFR) overexpression marks the entire spectrum ofFDC-derived neoplastic lesions. By using blocking experi-ments on a newly developed patient-derived cellularsystem, we show that the engagement of surface EGFRby EGF drives the survival and proliferation of FDC-S viaMAPK and STAT pathways. Among EGFR ligands, hepa-rin-binding EGF-like growth factor, TGF-a, and Betacel-lulin are produced in the tumormicroenvironment. Lackof somatic mutations ofKRAS, NRAS, BRAF, and PI3KCAin FDC-S tumor samples predicts sensitivity to anti-EGFRmonoclonal antibody and suggests possible candidatesfor EGFR blockade in this neoplasm.

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After RNA purification, samples were treated with DNase toremove contaminating genomic DNA (DNaseI amplifica-tion grade, Cat. No. 18 068; Invitrogen). Reverse transcrip-tion was conducted using random hexamers (Cat. No. 48190; Invitrogen) and Superscript II reverse transcriptase(Cat. No. 18 964; Invitrogen). Primers are listed in theSupplementaryMaterial. iQTM SYBRGreen Supermix (Cat.No. 170-8882; Bio-Rad) was used to run relative quantita-tive real-time PCR of the samples according to the manu-facturer’s instructions. Reactionswere run in triplicate on aniCyclerTM (Bio-Rad Laboratories Inc.) and generated pro-ducts analyzed with the iCyclerTM iQ Optical System Soft-ware (Version 3.0a; Bio-Rad). Gene expression was normal-ized based on both GAPDHmRNA and 18S rRNA contents.Data are displayed as 2-dCt values.

Flow cytometry analysis of EGFRThe cell surface expression of EGFRwas determined using

flow cytometry analysis using anti-EGFR mAb (BD Trans-duction Laboratories). Details are reported in the Supple-mentary Material.

Cell stimulation and immunoblottingA total of 15�104 tumor cellswere seeded in 6-well plate

containing RPMI/10% FBS. After 24 hours cells werestarved for 48 hours in RPMI/0.1% FBS and then stimu-lated with indicated concentration of EGF (Sigma) for 10minutes at 37�C. Where indicated, FDC-S were incubatedin starvation medium with and without anti-EGFR mAbcetuximab (20 mg/mL) for 4 hours at 37�C, before stimu-lation with 40 ng/mL of EGF. In some experiments, FDC-Swere pretreated with the MEK inhibitor UO126 (1 and 10mmol/L; Calbiochem) at 37�C for 30 minutes. After wash-ing cells were lysed and analyzed by immunoblotting asreported in the Supplementary Material.

FDC-S cell-proliferation assayCellswere assayed for cell-cycle progressionbymonitoring

the incorporation of 5-ethynyl-20-deoxyuridine (EdU) andits subsequent detection by a fluorescent azide through aCu(I)-catalyzed [3þ2] cycloaddition reaction ("click" chem-istry; Invitrogen) as described in Supplementary Material.

Cell growth analysisFDC-S cells (5 � 103/well into of a 24-wells plate) were

incubated with cetuximab (0.1, 1, 10, 20, 40, and 100mg/mL) or left untreated in RPMI/0,1% FBS. Where indi-cated, FDC-S were pretreated with the MEK inhibitorUO126 (1 and 10 mmol/L) at 37�C for 30 minutes. After48 hours, cells were stimulated without (-EGF) or withindicated concentration of EGF. After 5 days, FDC-Sgrowth was analyzed by microscopy as described in theSupplementary Material.

Double staining with acridine orange (AO) andethidium bromide (EB) for apoptosis detectionFDC-S cells (4 � 103/well of a 24-well plate) were seeded

on gelatin-covered glasses in RPMI/10% FBS for 24 hours.

Cells were incubated without (controls) or with anti-EGFRmAb cetuximab (20 mg/mL) in RPMI/0.1% FBS. At day 4,cells were stimulatedwith 40 ng/mLof EGFwithout removethe medium. At day 7, plates were centrifuged and cellapoptosis was detected by EB/AO staining procedures asreported in the Supplementary Material.

Direct sequencingFormalin-fixed paraffin-embedded blocks were reviewed

for quality control and tumor content. A representativeblock, containing from 30% to 80% of malignant cells wasselected for each case. Genomic DNA was extracted usingQIAamp DNA FFPE Tissue kit (Qiagen). Direct sequencingof EGFR (exons 18, 19, and 21), KRAS (exon 1), NRAS(exons 1 and 2), BRAF (exon 15), and PI3KCA (exons 9 and20) was conducted on all samples as previously reported(20, 21). All samples were subjected to automated sequenc-ing by ABI PRISM 3130 (Applied Biosystems). Controlswere represented by wild-type and mutated cases obtainedfrom colon and lung carcinoma cases as well as frommelanoma.

ResultsEGFR is strongly expressed by FDCs and theirneoplastic counterpart

By characterizing a cohort of FDC-S on the basis of theirphenotype, we andothers have recently identified EGFR as ahighly sensitive marker of neoplastic FDC (10, 17, 22, 23).To further confirm this finding, we applied immunohis-tochemistry to a more extended cohort of clinical samplesincluding a total of 16 FDC-S as well as reactive lymphnodes and tonsils by using 2different anti-EGFR antibodies.All FDC-S cases (16/16; 100%) showed strong and diffuseEGFR reactivity in the cytoplasm and on the cell membrane(Fig. 1A and B). The level of tumor heterogeneity of EGFRexpressionwas very limited andmost FDC-S cases expressedthis marker on more than 75% of neoplastic cells. Remark-ably, only a faint reactivity on occasional neoplastic cellswas observed for HER2 (not shown), whereas HER4 wasweakly expressed on 8 of 15 FDC-S cases (53.3%; Fig. 1C).We tested whether EGFR expression is acquired duringneoplastic transformation by extending this characteriza-tion to normal and dysplastic FDC. In reactive tonsils,lymph nodes, and spleen, anti-EGFR antibodies specificallystain the 2 major stromal cells populations, namely FDCand fibroblastic reticulum cells (FRC; Fig. 1D and E), as alsoconfirmed by staining of serial sections of reactive lymphnode with the FDCmarker CD21 and the FRCmarker anti-smoothmuscle actin (SMA; Supplementary Fig. S1); expect-edly, a strong signal was also observed on surface and cryptepithelium in the tonsils (not shown). These data indicatedthat EGFR can be expressed by nontransformed lymphoidorgan stromal cells. Dysplastic FDCs are commonly obs-erved in the HV-CD, the putative precursor of FDC-S (24–26). We found diffuse expression of EGFR on FDCs, includ-ing those characterized by evident dysplastic features, in allHV-CD–tested cases (5 of 5; Fig. 1F). Overall, these data

EGFR in FDC Sarcoma

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confirm that in lymphoid organs EGFR expression isrestricted to the stromal cell compartment including FDCand FRC and is maintained during neoplastic transforma-tion of FDC.

High expression of EGFR in FDC-S is not associated tonumerical alterationof chromosome7or amplificationof the EGFR alleles

Oncogenic addition to EGFR is observed in a large varietyof humanmalignancies (27). In these cases, overexpressionof the protein is frequently associated to genetic variantsintrinsic to cancer cells. IncreasedEGFRexpression in cancercells at the protein level might depend on the occurrence ofgene amplification. Molecular studies on FDC-S are limitedto sporadic cytogenetic analysis reporting multiple chro-mosomal aberrations. Among the latter, abnormalitiesinvolving the chromosome 7 containing the EGFR locushave been reported in form of translocations (28) or tet-rasomy (29). By using FISH, we could show that all casesshowed eusomy of the chromosome 7 with normal gene

copy number of EGFR as measured by EGFR/CEP7 ratio(Fig. 2A–D).

FDC-S cells proliferation and survival is induced by theactivation of EGFR-mediated signaling

To our knowledge, stable FDC-S cell lines are notavailable for in vitro or in vivo studies. To characterizethe biological effects associated to EGFR signaling, wegenerated a short-term culture from a FDC-S case (fromhere referred as FDC-AM09). Culture cells showed anFDC-S-like morphology (Fig. 3A and B) and expressedthe FDC-S marker CD21 (Fig. 3C). By cytogenetic analysisconducted on G-banded metaphases cells, numerical andstructural abnormalities were observed (Fig. 3E–H),including gain of chromosome 21, chromosome ring,chromosome marker, derivative of chromosome 1, gainof chromosome 11, and chromosome marker, but notincluding the chromosome 7. On flow cytometry analysis,FDC-AM09 expressed EGFR on the surface (Fig. 3D).Significantly, EGF-induced FDC-AM09 cell growth is dose

A B

C D

E F

Figure 1. EGFR expression in FDCand FDC-S cells. Sections areobtained from FDC-S cases (A–C),reactive tonsils (D) and lymph node(E), and an HV-CD case (F) stainedfor EGFR (A, B, D–F) and HER4 (C).EGFR expression is diffuse in thecytoplasm and on the cellmembrane on FDC-S cells (A andB), reactive FDC (D), FRC (E), anddysplastic FDC (F). InC, diffuse andstrong expression of HER4 isobserved in the cytoplasm ofFDC-S cells. Sections arecounterstained with Meyer'shematoxylin. Magnification/scalebar: 200�/100 mm (A–D and F) and400�/50 mm (E).

Vermi et al.

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dependent and ERK phosphorylation is already detect-able at low concentration of EGF (e.g., 1 ng/mL; Supple-mentary Fig. S2). To test whether EGFR engagementinduced FDC-S survival and proliferation, cells weretreated with EGF (40 ng/mL) for 48 hours and S-phaseentry was measured by nucleoside analog EdU incorpo-ration into DNA. EGF significantly promoted S-phaseentry in at least 40% of FDC-S cells (Fig. 4A) associatedto ERK1/2 activation and phosphorylation of AKT andSTAT3 (Fig. 4B). To further confirm that this effect wasdependent on EGFR, FDC-S cells were preincubated withthe blocking anti-EGFR monoclonal antibody cetuximabat different concentrations for 48 hours followed bytreatment with 40 ng/mL EGF for 5 days. As shown inFig. 4C, preincubation with cetuximab inhibited FDC-Scell growth in a dose-dependent manner. Because treat-ment with anti-EGFR monoclonal antibody not onlyaffects cell proliferation but also results in a strong induc-tion of cell death in several tumor cells lines, we inves-tigated whether cetuximab induced FDC-S cell death.Using fluorescent DNA binding dyes, we found thatpretreatment with 20 mg/mL of cetuximab induced apo-ptosis in more than 40% of FDC-S cells (Fig. 4D–F). Todetermine whether cetuximab inhibits EGF-induced sig-naling pathways, subconfluent cells were serum starved,treated with cetuximab or control IgG2, before EGF inc-ubation. As shown in Fig. 4B, in contrast to EGF stimu-lation, pretreatment with cetuximab inhibited ligand-induced ERK1/2 activation, AKT phosphorylation, andreduced STAT3 phosphorylation, indicating that Cetux-imab inhibits EGFR-induced signaling cascades. Blocking

of EGF-dependent signaling and biological effects wassimilar when using an alternative anti-EGFR monoclonalantibody Panitumumab (not shown). The role of EGFR-MAPK signaling on FDC-S growth was also investigatedafter inhibition of ERK activation. Preincubation with thespecific MEK inhibitor UO126 resulted in a significantreduction of EGF-dependent FDC-AM09 cell growth(Supplementary Fig. S3), indicating that EGFR-inducedERK activation is required for FDC-S tumor growth.

All together, these results indicate that in vitro, FDC-S cellsproliferation and survival can be induced by the activationof EGFR-mediated signaling cascades via cognate ligands.

Local production of EGFR ligands in FDC-SBased on the results from the previous set of experiments,

wehypothesized that local productionof endogenous EGFRligands could sustain viability of reactive and neoplasticFDC-S. In 4 cases of FDC-S, frozenmaterialwas available fornucleic acid extraction. By qPCR we found messenger RNAfor a set of EGFR ligands includingHBEGF, TGF-a, andBTC,whereas no or very low signal was observed for EGF,amphiregulin (AREG), epiregulin (EREG), and epithelialmitogen homolog (EPGN; Fig. 5A). To validate this findingat protein level and identify the cellular source of theseligands, we took advantage on an anti-BTC antibody whichspecifically recognizes the protein on FFPE tissues. BTCbinds different combinations of ErbB receptors includingEGFR (30, 31) andwas identified on endocrinea cells in thepancreatic islets where it promotes proliferation and differ-entiation of pancreatic b cells (32, 33). Accordingly, in FFPEsections of human pancreas, anti-BTC antibody recognizes

A B

C D

Figure 2. FISH analysis of EGFRgene in FDC-S cases. Comparativeanalysis of EGFRexpression (AandB) and EGFR/CEP7 FISH (C and D)conducted on 2 FDC-S case. Byimmunohistochemistry, FDC-Scells are strongly positive for EGFR(A and B), whereas no geneamplification or chromosome 7numerical abnormalities aredetectable (C and D). Sections arecounterstained with Meyer'shematoxylin (A and B) and DAPI (Cand D). FISH is conducted with LSIEGFR SpectrumOrange and CEP7Spectrum Green probes.Magnification/Scale bar: 400x/50micron (A and B), 1000x (C and D).

EGFR in FDC Sarcoma

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normal and neoplastic a cells as also confirmed by doublestain for BTC coupled with insulin and glucagon (Supple-mentary Fig. S4). In reactive lymph nodes and tonsils, BTCstain germinal center macrophages and FRC, whereas nosignal is observed in lymphoid cells (Fig. 5B and C).Remarkably, in the large majority of FDC-S–tested cases,expression of BTC was observed in FDC-S cells (13 of 16;81.25%) and in microenvironment stromal cells (16 of 16;

100%), the lattermore likely representing FRC surroundingneoplastic FDC (Fig. 5D and E). Interestingly, in HV-CDcases, BTC signal was observed in CD11cþ germinal centermacrophages and also on dysplastic FDC (Fig. 5F–H). Alltogether, these data indicate that the EGFR ligands arelocally produced in the tumor microenvironment ofFDC-S and the major cellular sources are represented byneoplastic cells or tumor-associated stromal cells.

A B

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E F

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Figure 3. Characterization of FDC-AM09. On cytospin preparation,FDC-AM09 cells from passage 1(A) and passage 3 (B) maintain aspindle cell morphology with largenuclei containing multiple nucleoliwith expression of CD21 (C).Magnification/scale bar: 400�/50mm (A–C). By flow cytometry, FDC-AM09 cells show surfaceexpression of EGFR (D). FDC-AM09 are stained with PE-conjugated anti-EGFR (black line)or with PE-conjugated IgG isotypecontrol antibody (shaded graycurve). As positive control, A431cell linewasused (gray line). In E–H,representative metaphases shownumerical and structuralchromosome aberration found inFDC-AM09, as indicated bycolored arrows, including gain ofchromosome 21 (E, blue),chromosome ring (E–F, red),chromosome marker (E, green),derivative of chromosome 1 (G,gray), gain of chromosome 11 (G,pink), and chromosome marker (H,violet).

Vermi et al.

Clin Cancer Res; 19(18) September 15, 2013 Clinical Cancer ResearchOF6

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EGFR and downstream pathways are activated inprimary FDC-STo substantiate the activation of the EGFR pathway in vivo

in FDC-S, sections were stained for antibodies directedagainst phosphorylated forms of EGFR, AKT[Ser473], STAT3[Tyr705], and ERK [Thr202/Tyr204]. Detection of phos-phorylated proteins in archival FFPE sections by immu-nohistochemistry is characterized by a significant level ofheterogeneity (34–36). We validated the expression ofpEGFR[Tyr1068] and pEGFR[Tyr1173] on EGF-treated and-untreated fixed cells as well as in FFPE sections obtainedfrom cases of lung adenocarcinomawith L858R and exon 19delE746 activating mutations of the tyrosine kinase domainof EGFR. This set of positive controls gave a recognizablecytoplasmic and membrane stain (Supplementary Fig. S5).Summary of the data on FDC-S are reported in a Supple-mentary Table S6. The combinationof pEGFR[Tyr1173] andpEGFR[Tyr1068] allowed to identify anti-pEGFR signal in alltested FDC-S cases (15 of 15; 100%), indicating ongoingactivation of the receptor in vivo (Fig. 6A and B). In addition,pSTAT3[Tyr705] expression was clearly detected in thenuclei in 9 of 16 cases (56.25%), whereas pERK [Thr202/Tyr204] was detected in the nuclei and cytoplasmof 12 of 16cases (75%; Fig. 6C and D). By double immunohistochem-

istry, we could detect coexpression of pEGFR[Tyr1173] withpSTAT3[Tyr705] and pERK [Thr202/Tyr204] (Fig. 6E andinsert). pAKT[Ser473] showed a faint-to-moderate stain in 7of 16 FDC-S (43.75%; Fig. 6F).

These findings suggest that EGFR activation might occurin vivo in FDC-S cells. Persistent activation of EGFR down-stream signaling pathways in other cancers might dependon mutations of EGFR or other intracellular components.By direct sequencing of genomic DNA purified from allFDC-S cases, we tested for the occurrence of the mostcommon genetic variants of EGFR (substitutions in exons18 and 21 and deletion in exon 19), KRAS (exon 1), NRAS(exons 1 and 2), BRAF (exon 15), and PI3KCA (exons 9 and20). No mutations of these genes were found in all casetested (16 of 16; 100%). Overall these data indicate thatsustained activationof EGFR-downstreampathways is inde-pendent from somatic events inmajor oncogenes and likelyresult from ligand-dependent activation of the receptor.

DiscussionAmong pathways involved in cell transformation and

tumor progression, the most extensively characterized arethose associatedwith receptors tyrosine kinase, in particular

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Figure 4. Biological effects of EGFR signaling on FDC-AM09. EGF-induced proliferation of FDC-AM09 cells is shown in A. Quantitative analysis is from 3independent experiments (� denotes a P value of 0.05). Effect of EGF and cetuximab on ERK1/2, AKT, and STAT3 activation in FDC-AM09 is shown in B.PMA and ionomycin (P/I) were used as positive control for ERK-MAPK activation. Cells lysates were analyzed by Western blotting. Cetuximab (Cetx) blocksof FDC-AM09cell growth in adose-dependentmanner is shown inC.Quantitative analysis of cells proliferation are from2 independent experiments (� denotesa P value of 0.01. �� denotes a P value of 0.05). Cetuximab induced apoptosis of FDC-AM09 is shown in D–F. Representative images showing apoptosis ofFDC-AM09 cells under AO/EB staining. FDC-S cells were treated without (D) or with (E) cetuximab, stimulated with EGF and stained with AO/EB. AOþapoptotic cells are stained in orange.Magnification: 400�. In F, quantitative analysis of live and apoptotic cells are from2 independent experiments (� denotesa P value of 0.01. �� denotes a P value of 0.05).

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those belonging to the ErbB family. Abnormalities in keycomponents of the ErbB receptors, including EGFR, areparticularly relevant to epithelial cells and derived carci-nomas. However, EGFR signaling has also been implicat-ed in the transformation and progression of nonepithelialcancer including soft tissue sarcomas (8, 37). Here, weprovide evidences for a role of the EGFR pathway in thebiology of human FDC and their neoplastic counterpart.This finding uncovers a new key molecule in the organi-

zation of secondary lymphoid organs and suggests thatEGFR activation might sustains survival and proliferationof neoplastic FDC (FDC-S). In particular, EGFR is stronglyexpressed on FDC-S cells and signal through downstreampathways in vitro and in vivo. Receptor activation in FDC-Sis dependent on the availability of cognate endogenousligands in the tumor microenvironment, particularlyBTC, that is produced by tumor cells and by the surround-ing stroma.

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Figure 5. Production of EGFRligands in primary FDC-S andHV-CD. Message RNA for theEGFR-ligands BTC, HBEGF, andTGF-a is detected in 4 FDC-Scases by qPCR (A). Sections arefrom reactive lymph nodes (B andC), 2 FDC-S cases (D and E) and 2HV-CDcases (F–H) stained forBTC(brown in B–H) and CD11c (blue, Hand insert in B). In reactive lymphnodes, BTC is expressed byCD11cþ germinal centermacrophages (B) and fibroblasticreticulum cells (C). In FDC-S, BTCis expressed by neoplastic (D) andsurrounding FRC (E). In HV-CD,BTC reactivity is found indysplastic FDC (F) and in CD11cþgerminal center macrophages (G)as confirmed by doubleimmunohistochemistry (H).Sections are counterstained withMeyer's hematoxylin.Magnification/scale bar: 200�/100mm (B, C, F, and G) and 400�/50mm (D, E, and H).

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The biology of normal and transformed human FDC isstill poorly understood. This limitation ismainly due to therarity of this neoplasm and to difficulties in establishingcell purification and culture systems strategies (14). Thishas severely hampered the development of molecularlybased therapeutic strategies for FDC-S. Previous studieshave suggested that the transformation process of FDC canbe paralleled by the overexpression of EGFR (11). In thisstudy, we have found that EGFR expression trace thedevelopment of all major subsets of human lymphoidorgan stromal cells including FDC and FRC and is main-tained during neoplastic transformation of FDC. Based onFISH analysis of FDC-S cases, we could exclude that over-expression of EGFR is associated to an increased copynumber of the gene or numerical abnormalities of chro-mosome 7. As alternative, EGFR protein overexpressionmight derive from increased transcription of the wild-typeallele or impairment of receptor homeostasis, the latterlikely due to somatic abnormalities in genes that regulatereceptor endocytosis and degradation.

By deriving the short-term culture FDC-AM09 from anFDC-S case, we could show that the engagement of EGFRprovides a sufficient amount of intracellular signal thatsupport survival and proliferation of FDC-S and activatesdownstream pathways. Although this finding requires val-idation on a larger set of cells lines, it was further confirmedin vivo in a set of clinical samples by immunohistochem-istry, showing frequent coexpression of the phosphorylatedforms of EGFR, ERK, and STAT3 by FDC-S. This activation isnot dependent on the occurrence of somatic mutations inmajormolecular drivers downstream to the EGFR pathwayssuch as KRAS, BRAF, and PI3KCA as showed by directsequencing of tumor-derived genomic DNA. The latterresults point toward a ligand-dependent activation of EGFRin FDC-S. Accordingly, message RNA for HBEGF, TGF-a,andBTCwas found in FDC-S samples. The latter findingwaslimited to 4 FDC-S cases with available frozen material.However, we could extend this observation at the proteinlevel showing that BTC expression, by tumor cells and bysurrounding stromal cell, is found in themajority of FDC-S.

A B

C D

E F

Figure 6. Expression ofphosphorylated EGFR, AKT,STAT3, and ERK in primary FDC-S.Sections are from a FDC-S caseand stained for pEGFR[Tyr1068](brown in A and F),pEGFR[Tyr1173] (brown in B),pSTAT3 [Tyr705] (brown in C; bluein F), pERK [Thr202/Tyr204] (brownin D), and pAKT[Ser473] (brown inE). Membrane expression of the 2pEGFR clones is diffuse in FDC-Scells (A and B); whereas pSTAT3,pERK, and pAKT can be observedin the cytoplasm and in the nucleus(C, D, and F). Coexpression ofpEGFR with pSTAT3p (E) andpERK (insert in E) is observed in aFDC-S case. Sections arecounterstained with Meyer'shematoxylin. Magnification/scalebar: 400�/50 mm (A–F).

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BTC is synthesized indifferent tissues andby a large numberof mesenchymal and epithelial cells in culture (38, 39). Itsbiological activity in various cellular systems ismediated bybinding to many different combinations of ErbB receptors(31, 40). Significantly, we could document that BTC is alsoproduced in reactive lymph nodes and HV-CD by germinalcenter macrophages. The latter population might thus sup-port viability and proliferation of normal EGFR-expressingFDCduring the life cycle of a B-cell follicles, including thosede novo generated in inflamed peripheral tissues. In addi-tion, germinal center macrophages-derived BTC might alsoprovide a sufficient amount of signal to initially trans-formed FDC before their outgrowth outside the germinalcenter. Although not investigated in this study, how FDCacquire the capability of producing high level of BTC upontransformation is of relevance and might represent also toother cancer models.

In different form of carcinomas, a meaningful clinicalbenefit has been showed with the addition of therapiestargeting EGFR to standard treatments (27). Four anti-EGFRcompounds have been approved for the clinical use in thelast decade. Two small molecule inhibitors, gefitinib anderlotinib, have shown anunprecedented clinical benefit in asubset of lung adenocarcinoma with activating somaticmutation of EGFR (41, 42). In addition, 2 monoclonalantibodies to extracellular portion of EGFR (EGFR-MoAbs),cetuximab and Panitumumab, have proven to be effectivein combination with chemotherapy or as single agents fortreatment of colorectal carcinomas (43–45); whereas noEGFR mutation are detected in FDC-S, addition of Cetux-imab to FDC-AM09 resulted in a significant blockade of theproliferation and induction of cell death. Although preclin-ical models in soft tissue sarcomas have indicated a poten-tial clinical benefit for EGFR-MoAbs, a recently conductedphase II trial has concluded that cetuximab is not an activecompound in EGFR-expressing advanced sarcoma, indicat-ing that additional biomarkers of response have to beunequivocally identified (46). It is now well establishedthatKRASmutated cases have a scarce probability to achievean objective response to EGFR-MoAbs, as well as to obtain abenefit in terms survival. Additional negative predictors ofresponse to EGFR-MoAbs such as somatic mutations ofNRAS, BRAF, and PI3KCA, has recently proposed (47–49). By direct sequencing of genomic DNA obtained fromFDC-S, this study shows that FDC-S lackmutations inKRAS,NRAS, BRAF, and PI3KCA indicating integrity of the intra-cellular pathways downstream to EGFR. Increased produc-tion of endogenous EGFR ligands in the tumor microenvi-ronment positively correlate with disease control in colo-rectal cancer patients treated with EGFR-MoAbs (50). Byimmunohistochemistry on routine biopsies, we could showthat EGFRanddownstreampathways are activated in FDC-S

and EGFR ligands, particularly BTC, are locally produced. Itis conceivable that a combination of these biomarkersmight represent a useful strategy to identify FDC-S patientseventually responding to EGFR-MoAbs.

The large majority of FDC-S is treated with surgery.However, a consistent fraction of them relapse with limitedcurative options. Findings from this study suggest that EGFRsignaling induced by locally produced cognate ligandssustain viability and proliferation of FDC-S cells by activat-ing intracellular pathways. To this end, gene-targetingexperiments of the recently proposed FDC-precursorsmightsignificantly contribute to the generation of valuable pre-clinical models mimicking FDC transformation (12). Asproved for other EGFR-dependent neoplasms, functionalinhibition of this receptor might provide an additionalclinical benefit to FDC-S patients.

Disclosure of Potential Conflicts of InterestS.A. Pileri is a consultant/advisory board member in Takeda, CelGene,

and TopoTarget. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: W. Vermi, E. Giurisato, F. FacchettiDevelopment of methodology: E. Giurisato, S. Lonardi, P. Balzarini,D. Medicina, D. BosisioAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): P. Balzarini, E. Rossi, D. Medicina, W. Pellegrini,C. Doglioni, G. Rossi, S.A. PileriAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): W. Vermi, E. Giurisato, S. Lonardi,P. Balzarini, E. Rossi, D. Medicina, D. Bosisio, S. Sozzani, W. Pellegrini,A. Marchetti, F. FacchettiWriting, review, and/or revision of the manuscript: W. Vermi, E. Giur-isato, S. Lonardi, E. Rossi, C. Doglioni, G. Rossi, S.A. Pileri, F. FacchettiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Lonardi, E. Rossi, W. Pellegrini,G. RossiStudy supervision: W. Vermi, F. Facchetti, S. Sozzani

AcknowledgmentsThe authors are grateful to P. Bossini and L. Fappani for their technical

assistance and to S.R. Chan (Washington University, Saint Louis, MI, USA)and A. Zaniboni (Department of Oncology, Fondazione Poliambulanza,Brescia, Italy) for criticisms and comments.

Grant SupportThis study is supported by PRIN (Ministero dell’Istruzione

dell’Universit�a e della Ricerca) to W. Vermi (2009CKARAL), S. Sozzani(2010–2011) and F. Facchetti (20104HBZ8E) and AIRC (AssociazioneItaliana per la ricerca sul cancro) to W. Vermi (IG 11924) and S. Sozzani(IG12064). This work has received financial support from the IstitutoToscano Tumori (ITT, grant 2008 to E. Giurisato).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 9, 2013; revised July 11, 2013; accepted July 17, 2013;published OnlineFirst July 25, 2013.

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Published OnlineFirst July 25, 2013.Clin Cancer Res   William Vermi, Emanuele Giurisato, Silvia Lonardi, et al.   LigandsCells Sarcoma is Sustained by Local Production of Cognate Ligand-Dependent Activation of EGFR in Follicular Dendritic

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