clin cancer res-2012-lechner-4549-59

Human Cancer Biology

Survival Signals and Targets for Therapy in Breast Implant–Associated ALK� Anaplastic Large Cell Lymphoma

Melissa G. Lechner1, Carolina Megiel1, Connor H. Church1, Trevor E. Angell2, Sarah M. Russell1,Rikki B. Sevell1, Julie K. Jang1, Garry S. Brody3, and Alan L. Epstein1

AbstractPurpose: Anaplastic lymphoma kinase (ALK)–negative, T-cell, anaplastic, non–Hodgkin lymphoma

(T-ALCL) in patients with textured saline and silicone breast implants is a recently recognized clinical entity

for which the etiology and optimal treatment remain unknown.

Experimental Design: Using three newly established model cell lines from patient biopsy specimens,

designated T-cell breast lymphoma (TLBR)-1 to -3, we characterized the phenotype and function of these

tumors to identify mechanisms of cell survival and potential therapeutic targets.

Results: Cytogenetics revealed chromosomal atypia with partial or complete trisomy and absence of the

NPM-ALK (2;5) translocation. Phenotypic characterization showed strong positivity for CD30, CD71, T-cell

CD2/5/7, and antigen presentation (HLA-DR, CD80, CD86) markers, and interleukin (IL)-2 (CD25,

CD122) and IL-6 receptors. Studies of these model cell lines showed strong activation of STAT3 signaling,

likely related to autocrine production of IL-6 and decreased SHP-1. STAT3 inhibition, directly or by recovery

of SHP-1, and cyclophosphamide–Adriamycin–vincristine–prednisone (CHOP) chemotherapy reagents,

effectively kill cells of all three TLBRmodels in vitro andmay be pursued as therapies for patients with breast

implant–associated T-ALCLs.

Conclusions: The TLBR cell lines closely resemble the primary breast implant–associated lymphomas

fromwhich theywere derived and as such provide valuable preclinicalmodels to study their unique biology.

Clin Cancer Res; 18(17); 4549–59. �2012 AACR.

IntroductionBreast implant–associated (BIA) T-cell anaplastic large

cell lymphoma (ALCL) is a recently recognized clinicalentity, with 80 cases identified worldwide to date and fourdisease-specific fatalities (1–15). BIA-ALCL presents com-monly as a late seroma and/or tumor mass attached to thescar capsule containing malignant cells an average of 5.8years after implant placement (range, 0.4–20 years;ref. 13). While most cases are indolent and respond wellto capsulectomy with local adjuvant radiation therapy,10% of cases present with metastasis and 5% of cases arefatal (12, 13).T-ALCL is a subset of adult peripheral T-cell lymphomas

(PTCL) with strong CD30 positivity and consisting of

pleomorphic epitheliod tumor cells with blast-like appear-ance, severe cellular andnuclear atypia, and large nuclei andnucleoli (16–18). A subset expresses the anaplastic lym-phoma kinase (ALK) as a result of reciprocal (2;5) translo-cation between the nucleophosmin (NPM1) gene andkinase domain of the ALK (16–19). Disease is subcategor-ized as ALKþ systemic, ALK� systemic, or primary cutaneous(pc-) ALCL, and each group exhibits distinct clinical behav-ior (16, 18). ALK� systemic ALCL is aggressive, with a 5-yearoverall survival (OS) rate of only 49%, comparedwithALKþ

ALCL (70% 5-year OS rate) and pc-ALCL (90% 5-year OSrate; ref. 20). Seroma-associated ALCL was proposed byRoden and colleagues (5) in 2008 to address BIA-ALCL,which sharesmorphologic features of both primary system-ic ALK�ALCL and pc-ALCL but is distinct in its presentationwith malignant seroma fluid and varied clinical progres-sion (indolent to aggressive). T-ALCLs express a range ofimmune markers, including T-cell antigens, cytotoxic gran-ules, and antigen presentation molecules, and, like otherT-cell neoplasms, show clonal T-cell receptor (TCR) generearrangement (21–23).

As more cases of BIA-ALCLs are recognized, questionsabout tumor etiology have emerged and the identificationof effective treatments becomes more important. Previous-ly, we established the first model cell line for BIA-ALCL,designated TLBR-1, for studies of this disease (1). Since that

Authors' Affiliations: Departments of 1Pathology, 2Medicine, and 3PlasticSurgery, Keck School of Medicine, University of Southern California, LosAngeles, California

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

Corresponding Author: Alan L. Epstein, Department of Pathology, USCKeck School of Medicine, 2011 Zonal Ave, HMR 205, Los Angeles, CA90033. Phone: 323-442-1172; Fax: 323-442-3049; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-0101

�2012 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 4549

on August 18, 2015. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 12, 2012; DOI: 10.1158/1078-0432.CCR-12-0101

http://clincancerres.aacrjournals.org/

initial report, we have established and characterized 2 newcell lines from patients with BIA-ALCL, including 1 of the 4fatal cases, designated TLBR-2 and -3. Using thesemodels ofBIA-ALCLs, we now describe fully the phenotypic andfunctional features of this newly emerging clinical entity,including identification of aberrancies in cell signaling andapoptosis regulators that seem to be excellent moleculartargets for therapy.

Materials and MethodsCell lines and cells

Karpas299 (Karpas), Raji, HUT102, and Jurkat cell lineswere obtained from American Type Culture Collection(authentication by short tandem repeat) and maintainedin RPMI-1640 with 10% fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL strepto-mycin in a humidified 5% CO2, 37

�C incubator. Institu-tional Review Board (IRB) approval from the USC KeckSchool of Medicine (University of Southern California, LosAngeles, CA; HS-0600579) was obtained for the collectionof peripheral blood mononuclear cells from healthydonors.

CytogeneticsKaryotype analysis was conducted by the Division of

Anatomic Pathology, City of Hope (Duarte, CA) using earlypassages of each cell line. Patient cytogenetic informationwas reported by the treating physician.

HeterotransplantationXenografts of the TLBR cell lineswere attempted in6- to8-

week-old female nude, severe combined immunodeficient(SCID; Harlan), nonobese diabetic (NOD)/SCID, andNOD/SCID-g mice (Jackson Labs) using 106 cells in a0.2-mL subcutaneous inoculum.

MorphologyFormalin-fixed, paraffin-embedded (FFPE) xenograft

tumors or cultured cells were sectioned and stained using

hematoxylin–eosin (H&E), Wright–Giemsa (W–G), ormonoclonal antibodies (Supplementary Table S1) usingimmunocytochemical techniques, as described previously.Observation and image acquisitionweremade using a LeicaDM2500 microscope (Leica Microsystems, www.leica-microsystems.com), digital SPOT RTke camera, and SPOTAdvanced Software (SPOT Diagnostic Instrument Inc.,www.diaginc.com). Images were resized and brightenedfor publication using Adobe Photoshop software (Adobe,www.adobe.com).

Flow cytometrySingle-cell suspensions were stained with fluorescence-

conjugated antibodies as described previously (24). Anti-bodies and isotype controls were from BD Biosciences andeBiosciences (Supplementary Table S1). Samples were runin duplicate on a BD FACSCalibur flow cytometer usingCellQuestPro software. Mean fluorescence intensity (MFI)andpercentage of positive staining cells (difference betweenMFI of sample and isotype>50)were determined for 15,000events.

PCR and quantitative reverse transcriptase PCRPCR to assess TCRg gene rearrangement and screen for

oncogene incorporation was carried out as described pre-viously (1, 21, 25–27). Quantitative reverse transcriptasePCR (qRT-PCR) tomeasure gene expression was carried outas described previously (24). Gene-specific amplificationwas normalized to glyceraldehyde-3-phosphate dehydro-genase (GAPDH) and fold change calculated relative tohealthy donor peripheral blood T cells. Differences inmeanexpression of tumor suppressor, proto-oncogenes, and apo-ptosis-related genes among tumor cell lines and normaldonor T cells were evaluated for statistical significance byANOVA followed by Dunnett test.

ImmunoblottingWhole-cell lysates in radioimmunoprecipitation (RIPA)

buffer were fractionated on 10% Tris-glycine PAGE, electro-transferred to nitrocellulose, and probed overnight fortarget proteins with primary antibodies (Cell Signaling andSanta Cruz Biotech; Supplementary Table S1), as describedpreviously (1). Protein concentration was determined bythe bicinchoninic acid (BCA) assay.

Measurement of lymphoma-derived cytokinesLevels of cytokines in supernatants from24-hour cultures

of TLBR-1, -2, -3, Karpas299, or Jurkat cells were measuredby cytometric bead array and analyzed on a BD LSRII flowcytometer using FACSDiva software for acquisition andcompensation. Differences in mean levels of cytokine pro-duction were tested for statistical significance by ANOVAfollowed by Dunnett test with comparison to mediumalone.

Drug studiesTLBR-1, -2, -3, and Karpas299 cells were cultured (106

cells/mL) in vitro in the presence or absence of cell signaling

Translational RelevanceNumerous cases of rare T-cell ALK� anaplastic large

cell lymphoma have recently been identified in womenwith textured silicone and saline breast implants. In2011, the U.S. Food and Drug Administration issued awarning for these implants out of concern for this newlyemerging clinical entity. In this study, we identifyincreased STAT3 activation related to dysregulation ofthe SHP-1 phosphatase and autocrine production ofinterleukins as a driver of cell survival in breastimplant–associated anaplastic large cell lymphomas.Improved understanding of the biology of these tumorswill facilitate changes to implant design to prevent newcancer cases and development of effective therapies forthis disease.

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Clin Cancer Res; 18(17) September 1, 2012 Clinical Cancer Research4550




inhibitors or chemotherapeutic drugs. For the chemother-apy studies, cells were exposed to drug or vehicle for 30minutes, then washed twice with cold complete medium,and cultured in the absence of drug for 48 hours. Cellviability was measured by staining with Annexin V/propi-dium iodide (PI; Invitrogen) and analyzed on a BD LSRIIflow cytometer using FACSDiva software for acquisitionand compensation (�15,000 events per sample). Reagentsevaluated included WP1066, sunitinib malate, honokiol,and 4-hydroperoxycyclophosphamide (4HC; Santa Cruz);S3I-201 (EMD Chemicals); 5-aza-20-deoxycytidine (AZA),vinblastine, doxorubicin, FBHA,DAPT, suberoyl bis-hydro-xamic acid (SBHA), and valproate (Sigma). Mean percen-tages of positive staining cells for groups of cells treatedwithinhibitors or vehicle alone were evaluated by ANOVA andDunnett post-test or pairwise comparisons by the Student ttest with Bonferroni correction.

ResultsClinical presentation

The TLBR cell lines were established from the primarytumor specimens of womenwith BIA-ALCL, as summarizedin Table 1, under IRB-approved protocol HS-10-00254 andreported previously for TLBR-1 (1). These cases were typicalof BIA-ALCLs in that the women presented with unilateralseroma fluid accumulation 3 to 15 years after elective breastaugmentation with textured saline implants (Fig. 1A). Theseromas uniformly recurred within months of initial drain-age and were found to contain malignant cells consistentwith ALK� ALCLs (Fig. 1B). All patients underwent bilateralimplant removal and capsulectomy and had no evidence ofcontralateral breast involvement, skin manifestations, orspread to regional lymph nodes at that time. Patients 1 and3 received local radiotherapy to the affected breast and chestwall following surgery and remain disease free at the time of

Table 1. Disease diagnosis, patient characteristics, growth characteristics, and viral status of the TLBR-1,-2, and -3 cell lines

Patient 1: TLBR-1 Patient 2: TLBR-2 Patient 3: TLBR-3

Patient diagnosis ALK� seroma–associatedT-ALCL, absent t(2;5) 42-y-oldfemale

ALK� seroma–associated T-ALCL, absent t(2;5) 43-y-oldfemale

ALK� seroma–associatedT-ALCL, absent t(2;5) 45-y-oldfemale

Implant type Textured saline Nagor SFX-HP250cc

Textured saline McGhan/Inamed/Allergan 410cc

Textured saline McGhan/Inamed/Allergan 480cc

Clinical presentation Unilateral malignant seroma,recurrent after initial drainage

Unilateral malignant seroma,recurrent after initial drainage

Unilateral malignant seroma,recurrent after initial drainage,and mass lesions attached tothe capsule seen by imaging

Tumor specimencytology

Large mononuclear CD30þ ALK�

CD4þ CD8þ TIA-1þ Perforinþ

Keratin� PAX5� CD20�

anaplastic lymphoma cells


CD45þ CD20� CD15� CD43�

Cytokeratins (Cam 5.2)�

anaplastic lymphoma cells


CD45þ CD4þ CD43þ TIA-1þ

CD8� CD15� CD20� CD68�

PAX5� anaplastic lymphomacells

Patient treatmentand outcome

Surgery (bilateral implant removaland capsulectomy) andradiation therapy (40 Gydelivered in 20 fractions)

Surgery (bilateral implant removaland capsulectomy)

Surgery (bilateral implant removaland capsulectomy) andradiation therapy (36 Gydelivered in 20 fractions)

Patient in remission at time ofpublication (3 y after initialpresentation)

Recurrent disease involvingaxillary lymph nodes,supraclavicular fossa,mediastinum, and bilateral lunginfiltrates

Patient in remission at time ofpublication (14 mo after initialpresentation)

Chemotherapy and radiationtherapy, unsuccessful

Patient died of disease 9 mo afterinitial presentation

Cell line culture Suspension culture, IL-2–dependent

Suspension culture, IL-2–dependent

Suspension culture, IL-2–dependent

Doubling time 55 h 37 h 77 hViral status EBV�, HTLV1/2�, HPV� EBV�, HTLV1/2�, HPV� EBV�, HTLV1/2�, HPV�

Malignant origin Karyotype, TCRg rearrangement,heterotransplantation

Karyotype, TCRg rearrangement Karyotype, TCRg rearrangement

Year established 2009 2011 2011

Breast Implant ALCL

www.aacrjournals.org Clin Cancer Res; 18(17) September 1, 2012 4551




publication. Patient 2 developed a local recurrence 2months after surgery with involvement of the axillary andsupraclavicular lymphnodes andbilateral pleural effusions.She received radiation therapy to which she showed adramatic response with significant tumor shrinkage. How-ever, computed tomographic imaging of the chest 2monthslater showed spread of her disease into the mediastinumwith airway compression and bilateral lung infiltrates. Herstatus progressively worsened and she died of ALCL-relatedcomplications 9 months after initial presentation.

Establishment of TLBR cell lines from patient tumorspecimens

All 3 TLBR cell lines grow in suspension cultures andexhibit interleukin (IL)-2–dependent growth (Table 1).Wright–Giemsa staining of cytospin preparations of theTLBR cell lines showed cells with abundant cytoplasm, 1to 4 large cytoplasmic vacuoles, enlarged nuclei, and prom-inent nucleoli characteristic of other ALCLs and similar tothe original specimens (Fig. 1C; refs. 23, 28).Multiplex PCRanalysis of TLBR-1, -2, and -3 cells showed TCRg mono-

clonality, confirming a neoplastic T-cell origin of the celllines and their derivation from the T-ALCL patient speci-mens (Table 1).

Chromosomal atypiaConventional cytogenetic and spectral karyotyping anal-

ysis ofmitotically active TLBR-2 and -3 cells showed clonallyabnormal, hypertriploid, and complex karyotypes (Supple-mentary Fig. S1). TLBR-2 cells had a modal number ofchromosomes of 76 and showed gains of chromosomes1, 2, 5, 6, 10, 11, 14, 17, as well as clonal loss of one copy ofchromosome 18, relative to a triploid genome. TLBR-3 cellsshowed a modal number of chromosomes of 81, gains ofchromosomes X, 2, 5, 7, 8, 10, 11, 12, 14, 19, 20, 21, and 22,and clonal losses of one copy of chromosomes 9, 16, and17, relative to a triploid genome. Cytogenetic analysis of theTLBR-1 cell line was reported previously (1). None of theTLBR cell lines show the NPM-ALK t(2;5) (consistent withthe primary tumor specimens), the (7;9) translocationreported in T-cell lymphoblastic leukemia or rearrange-ments involving the TCR gene loci on chromosomes 7 and

Figure 1. Establishment of modelcell lines for BIA-ALCLs fromprimary tumor specimens. A, BIA-ALCL in patient 3 presented asseroma fluid accumulation (arrow)around the right breast implant(left). Intra-operative photographsof the breast implants andsurrounding scar capsule tissueremoved from patient 3 (middle,right). Anatomic pathologyidentified the focal adhesions onthe right capsule as focal, foreign-body type granulomatousinflammation with refractilenonpolarizable material, likelysilicone from the textured implantsurface. Wright–Giemsa–stainedcytospins of (B) tumor cells inseroma fluid isolated from patient 2and (C) early passages of TLBR-1,-2, and -3 cells in culture showfeatures typical of ALCL, includingenlarged nuclei with frequentmitotic figures, multiple prominentnucleoli, pale cytoplasm withvesiculation, and occasionalmultinucleated giant cells (originalmagnification, �400). D,immunohistochemistry forlymphoma markers of FFPE tissuesections of TLBR -1, -2, and -3xenotransplant tumors (originalmagnification, �200 for H&E and�400 for all others).

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14. All 3 TLBR cell lines also lacked other translocationsfrequently found in germinal center cell,mantle cell, diffuselarge B-cell, and Burkitt lymphomas: t(14;18), t(11;14), t(3;14), t(3;22), t(8;2), t(8;14), and t(8;22) (23, 28).

Immunophenotype confirms ALK� ALCL and fidelity tooriginal tumorsImmunophenotypic characterization of TLBR-1, -2, and

-3 xenograft tumors (Fig. 1D) and cells in culture[Supplementary Fig. S2 or previously shown (ref. 1)]showed similarity to the original tumor specimens. All 3TLBR models showed strong CD30 positivity, weak expres-sion of epithelial membrane antigen (EMA), and absentALK-1 [t(2;5) product], keratins (squamous tissue antigen)or nuclear PAX-5 (Hodgkin lymphoma antigen; ref. 28).

Comparison to normal T-cell lineagesTo understand better the BIA-ALCL cell biology, the TLBR

cell lines were characterized for expression of normal T-celllineage markers and transcription factors. Expression ofimmune-related proteins by TLBR cell lines was examinedby flow cytometry (Supplementary Table S2). The TLBR celllines varied in their expression of T-cell lineage markers,CD4, CD8, and TCRab/gd, suggesting arrest at differentstages of maturation. Consistent with their T-cell origin and

IL-2 dependence, TLBR cell lines were positive for CD25(IL-2Ra) and CD122 (IL-2Rb). TLBR-1, -2, and -3 celllines showed positivity for cytotoxicity protein GranzymeB and strong expression of antigen presentation–associatedmarkers (HLA-DRþCD80þCD86þ) and CD71, the trans-ferrin receptor. Expression of adhesion (CD11cþCD11b�)and myeloid (CD13þCD14�CD15þCD68�) markers wasvariable, and TLBR cells generally lacked B-cell(CD10�CD19�CD20�CD21�CD23þ), dendritic cell (DC;CD1a�), and stem cell (c-kit�CD133�) markers. In regardto normal T-cell lineages, analysis of T-cell transcriptionfactors [Th1 (T-bet), Th2 (GATA-3), Th17 (RORg), andT-regulatory (FoxP3)] showed strongest positivity for T-betand FoxP3 and weak positivity for RORg .

Dysregulation of cell-cycle and apoptosis controlsAberrant expression of cell-cycle control genes and escape

from homeostatic programmed cell death pathways canfacilitate lymphoma tumorigenesis and progression (29–32). In this study, expression of tumor suppressor, (proto-)oncogenes, and apoptosis regulators [antiapoptotic: survi-vin,BCL2L2,MCL-1 (short transcript),BCL-2; proapoptotic:BID, BAX, BBC3, BAK] was evaluated in TLBR-1, -2, and -3and established PTCL cell lines Karpas299 and Jurkat usingqRT-PCR techniques. As shown in Fig. 2A, ALCL cell lines

Figure 2. Survival regulators. A,expression of antiapoptotic genes inTLBR cell lines relative to normaldonor T cells by qRT-PCRtechniques and confirmation ofelevated survivin by immunoblotting.Karpas299, known to overexpresssurvivin, was run in parallel forcomparison. B, expression ofproapoptotic and tumor suppressorgenes in the TLBR cell lines. A and B,for all graphs, gene expressionmeasured by qRT-PCR techniqueswas normalized toGAPDH andmeanfold change relative to normal donorTcells is shownwithSEM.Significantdifferences in mean gene expressionfrom normal donor T cells areindicated by an asterisk. All sampleswithin the brackets had significantdifferences in expression relative tonormal donor T cells. ALKþ ALCLKarpas299 and T-ALL Jurkat celllines were run in parallel forcomparison. C, increasedexpression and phosphorylation ofSTAT3 in TLBR cell lines wereevaluated by immunoblottingtechniques. Karpas299 is known tohave aberrant STAT3overexpression and activation. D,detection of pSTAT3 in FFPE tissuesections of TLBR -1, -2, and -3xenotransplant tumors (originalmagnification, �400).

Breast Implant ALCL





(TLBR and Karpas299) showed significant upregulation ofantiapoptotic genes survivin (P < 0.05) and BCL2L2 (P <0.05). Strong expression of survivin by ALCL cell lines wasconfirmed by immunoblotting and showed similar levels ofexpression among the TLBR cell lines. Proapoptotic genesand tumor suppressor genes were significantly downregu-lated relative to normal donor T cells, most notably for BID,BAK, and BBC3, with some variance among cell lines (Fig.2B). The TLBR cell lines were also evaluated for incorpo-ration of oncogenic viruses human T-cell leukemia virus(HTLV)-1/2 and Epstein-Barr virus (EBV), and T-cell acutelymphocytic leukemia (T-ALL)-associated oncogenes TAL1,HOX11, LYL1 and LMO1/2, and the results of these studieswere negative (data not shown).

Activation of STAT3 signalingActivation of STAT3 can upregulate survival signals and

downregulate proapoptotic mediators in lymphoid cells(29–31). Immunoblotting confirmed increased translationand activation of STAT3 proteins in these models (Fig. 2C),with the greatest activity in the cell line derived from themost aggressive case of BIA-ALCL (TLBR-2), and at levelscomparable with STAT3-overexpressing Karpas299 cells(33, 34). High levels of pSTAT3 were also seen in xenografttumors of TLBR-1, -2, and -3 (Fig. 2D).

Cytokine expression and functional studiesALK expression drives STAT3 activation and survival in

ALKþ ALCLs (34, 35), but in the absence of this translo-cation or activating point mutations (data not shown;ref. 36), the driver of high pSTAT3 in the BIA-ALCL celllines was unclear. Expression of T-cell cytokines (IL-2,IFNg , TNFa, IL-10, IL-4, IL-6, and IL-17A), immunosup-pressive cytokine TGFb, and angiogenic factor VEGF-Awas measured for the TLBR cell lines in culture (Fig. 3A).The TLBR cell lines showed strong secretion of cytokinesassociated with multiple T-cell subsets, most notably IL-6and IL-10, compared with other PTCL models (Jurkat,Karpas299). We hypothesized that survival signals inthese cells may be driven, in part, by autocrine responsesto cytokines, many of which act through JAK/STAT sig-naling. TLBR-1, -2, and -3 were uniformly positive for theIL-6 receptor (Fig. 3B), and TLBR-2 and -3 showed weakpositivity for the IL-10 receptor (data not shown), sug-gesting that these cells are capable of responding to thesefactors. Neutralization experiments for IL-6 showed amodest but insignificant decrease in TLBR cell prolifera-tion (data not shown), likely related to the very highlevels of IL-6 produced by these models. Regulatory T cell(Treg)-like suppressive function was also suggested forTLBR cell lines by FoxP3þ and IL-10 and TGFb secretion(TLBR-2 and -3). To evaluate suppressive ability, TLBRcell lines were co-cultured with naive normal donor Tcells in the presence of CD3/CD28 stimulation, and T-cellproliferation was measured by carboxyfluorescein succi-nimidyl ester (CFSE) dilution after 3 days, as carried outroutinely by our laboratory (24). Surprisingly, all 3 TLBRcell lines were found to augment T-cell proliferation (data

not shown), perhaps as a result of their strong productionof T-cell–activating cytokines (e.g., IFNg , IL-2). The TLBRcell lines are strongly positive for IL-2Ra and IL-2Rb,make detectable amounts of IL-2 in culture, exhibit IL-2–dependent growth in vitro, and show more rapidgrowth when cultured at higher density.

Sensitivity to STAT3 inhibitionTo determine the influence of JAK/STAT3 signaling on

TLBR cell survival, cells were cultured in the presence ofSTAT3-specific inhibitors WP1066 and S3I-201 or JAK/STAT3-targeted tyrosine kinase inhibitor sunitinib, and cellviability was assessed by Annexin V/PI staining. As shownin Fig. 3C, STAT3-specific inhibition by WP1066 producedsignificant cell death in all 3 TLBR cell lines in a dose-dependent manner. Similar effects on cell viability wereseenwith S3I-201 (data not shown). Furthermore, sunitinibproduced striking cell death in all TLBR cell lines acrossa range of doses (Fig. 3D). The ALKþ ALCL cell lineKarpas299 was run in parallel as a positive control in theseexperiments.

Downregulation of STAT3-negative regulator SHP-1STAT3 activation can also result from decreased levels

of negative regulating phosphatase SHP-1 (33, 35). TLBRcells had significantly downregulated SHP-1 expression(P < 0.05) and decreased SHP-1/STAT3 ratios (P < 0.05)compared with normal donor T cells (Fig. 4A and B).TLBR-2 and -3 had the most dramatic loss of SHP-1expression relative to STAT3, even relative to Karpas299,an ALCL model previously reported to have significantSHP-1 loss (37). SHP-1 activation by honokiol producedsignificant cell death in the TLBR cell lines, with loss ofpSTAT3 confirmed in cell lysates by immunoblotting (Fig.4C and D). In addition, the chemotherapeutic agent 5-aza-20-deoxycytidine (AZA), which was previously shownto increase levels of SHP-1 protein in PTCL cell lines (37),produced dose-related cell death in TLBR cells (Supple-mentary Fig. S3).

Increased levels of activated Notch1 in aggressiveTLBR-2

Evaluation of TLBR and established PTCL cell linesshowed strong expression of Notch1 and Notch2 receptorsandunique expressionof amajorNotch ligand, Jagged 2, onthe 3 TLBR cell lines (Supplementary Fig. S4). Aberrantexpression and activation of the embryonic transcriptionfactor Notch1 can contribute to malignant transformationin some adult PTCLs (38). Levels of cleaved, activatedNotch1 protein were previously found to be elevated inTLBR-1 and Karpas299 cells (1). TLBR-2 and -3 also havesignificant cleaved Notch1 and Notch1 levels (Supplemen-tary Fig. S4). The much higher levels of cleaved Notch1 inTLBR-2 cells may drive the faster division and more aggres-sive behavior of thismodel and the tumor fromwhich itwasestablished. However, modulation of Notch1 signalingusing g-secretase inhibitors (FBHA, DAPT) or activators(SBHA, valproate) failed to produce any significant change

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in TLBR-1, -2, -3, or Karpas299 cell viability (SupplementaryFig. S4).

Evaluating cytotoxic therapiesIn cases of BIA-ALCLs requiring adjuvant therapy after

capsulectomy, cyclophosphamide–Adriamycin–vincris-tine–prednisone (CHOP) chemotherapy may be beneficial(39). To estimate BIA-ALCL sensitivity to CHOP, the TLBRmodel cells lines were exposed to CHOP constituent drugs[vinblastine (vincristine analogue with in vitro activity),doxorubicin, 4-hydroperoxycyclophosphamide (activemetabolite of cyclophosphamide)] briefly and cell viabilitywas then assessed. As shown in Fig. 5A, TLBR-1, -2, and -3cells were highly sensitive to doxorubicin treatment (>80%cell death after 30-minute exposure to lower dose of 1.75

mmol/L, P < 0.001, and near-complete cell death at 17.5mmol/L dose, P < 0.001). The TLBR cell lines showedmoderate sensitivity to vinblastine (0.9 and 9 mmol/L) andto a very high dose of 4-hydroperoxycyclophosphamide(100 mmol/L; Fig. 5B and C).

DiscussionAs recently reported, breast implant–associated T-cell

anaplastic large cell lymphomas are an emerging clinicalentity (2–15). Threemodel cell lines, designated TLBR-1, -2,and -3, have been established from the primary tumorspecimens from patients with a spectrum of indolent toaggressive BIA-ALCLs to facilitate studies of the etiology andpotential therapy for these cancers. Morphologic and cyto-genetic studies confirmed the ALK� T-ALCL classification of

Figure 3. Cytokine signaling and sensitivity to STAT3 inhibition. A, production of TH1/TH2/TH17 and immunosuppressive cytokines by TLBR cell lines. Meancytokine levels in cell culture supernatants with SEM are shown; asterisk indicates levels significantly increased from media controls (P < 0.05). For all3 TLBR cell lines, production of IL-6 and IL-10was very high (boxed bars on graphs). B, surface expression of IL-6Rmeasured by flow cytometry for TLBR celllines (open, sample; shaded, isotype control; representative histograms shown from 2 independent experiments). C and D, TLBR-1, -2, and -3 andKarpas299 cells were treated in vitro with STAT3-specific inhibitor WP1066 (C) or tyrosine kinase inhibitor sunitinib (D), and cell viability was assessed byAnnexin V/PI staining and analysis by flow cytometry after 48 hours. Graphs showmean with SEM; significant differences in cell viability with drug treatmentcompared with vehicle alone are indicated by an asterisk, P < 0.001.

Breast Implant ALCL





the BIA-ALCL TLBR cell lines and their similarity to theoriginal tumor biopsy specimens. Themolecular features ofALK� ALCLs and other ALCL subsets are largely unknown, afact that is mirrored by the 30% to 50% of ALCL casesdesignated as not otherwise specified (ALCL-NOS) by his-topathology (40). Functional studies of the TLBR cell linesidentified high production of T-cell–associated cytokinesIL-6 and IL-10, activation of JAK/STAT3 signaling pathways,and strongest expression of transcription factors associatedwith the T-helper cell (TH)1 and Treg cell lineages. Thismolecular profile may be compared with that recentlyreported for ALKþ systemic ALCLs, namely upregulation ofTH17-related genes [e.g., IL-17A, IL-22, retinoic acid–relatedorphan receptor (ROR)], JAK/STAT3 signaling, and cyto-toxic molecules (32).

Comparedwith naive, normal donor T cells, the TLBR celllines showed dysregulation of cell-cycle and apoptoticregulators, namely survivin, and activation of JAK/STAT3pathways. Functional characterization and in vitro studies ofthe TLBR cell lines yielded a working model of BIA-ALCLtumor cell biology (Fig. 5D),with an emphasis on autocrinecytokine signaling that promotes tumor cell survival. Amilieu rich in immune stimulatory cytokines, like IL-6 and

IL-2, which promotes rapid division of host lymphocytesmay cause the initial tumorigenic changes that lead toBIA-ALCL in some patients. Chronic inflammation is wellrecognized as a promoter of cancer (41). Autocrine IL-6production has been identified as a driver of tumorigenesisin some diffuse large B-cell lymphomas, as well as solidtumors includingbreast, lung, andovarian carcinomas (42–44). The cytokine profile of BIA-ALCL cell lines, specificallyIL-6, TGFb, and IL-10, has also been shown to induceimmune suppressor cell populations (Tregs and myeloid-derived suppressor cells) that could inhibit host antitumorimmunity and facilitate cancer development (45, 46). Pre-vious studies of women with saline and silicone breastimplants found no increased risk of primary lymphoma orbreast cancer compared with women without implants(15), suggesting that the present case series is directly linkedto newer device features. Texturing of the implant siliconeshell, an aesthetic advance introduced in the late 1980s toreduce contractures and one recurring feature in thesecancer cases, results in greater silicone particle shedding inthe surrounding scar capsule. Indeed, histologic analysis ofmass lesions in cases of BIA-ALCLs, including patient 3 (Fig.1A), shows nonrefractive particles consistent with shed

Figure 4. Downregulation ofSTAT3-negative regulator SHP-1.A and B, expression ofphosphatase SHP-1 and the ratioof SHP-1 to STAT3 expressionwere significantly decreased in all 3TLBR cell lines relative to healthydonor T cells, asmeasuredby qRT-PCR techniques. Graph showsmean (n ¼ 3) gene expression as apercentage of GAPDH, with SEM;all cell lines were significantlydifferent from normal T cells(��, P < 0.0001). ALKþ ALCLKarpas299 and T-ALL Jurkat celllines were run in parallel forcomparison. C, TLBR cell lineswere treated with the SHP-1activator honokiol and viabilityassessed by Annexin V/PI stainingafter 48 hours. Mean is shown withSEM; significant differences in cellviability with drug treatmentcomparedwith vehicle (V) alone areindicated by an asterisk, P < 0.001.D, honokiol treatment effects onSTAT3 phosphorylation weremeasured by immunoblotting ofcell lysates at 24 hours, withGAPDH.

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silicone among granulomatous inflammation and tumorcells. Whether this inflammatory stimulus is increased intextured implants andmayplay a role in the development ofBIA-ALCLs are questions that will require future investiga-tion, but the prominent role of IL-6 found in the TLBR celllines suggests that immune reactions are important to theprogression of this disease.

It is important also to acknowledge that IL-2 signalingalmost certainly contributes to BIA-ALCL cell survival, asthe TLBR cell lines all die in the absence of this cytokineand have strong expression of IL-2R proteins. In experi-mental systems, IL-2 overexpression has been shown toproduce autonomous cell growth and malignant trans-formation in T cells (47, 48). Because the TLBR cell lines

Figure 5. Chemotherapy sensitivity and therapeutic targets in BIA-ALCL. A–C, TLBR-1, -2, -3 cells and Karpas299 cells were exposed briefly (30 minutes) toCHOPchemotherapydrugs: doxorubicin (DOX), vinblastine [(VIN), vincristine in vitro analogue], or 4-hydroperoxycyclophosphamide [(4HC), activemetaboliteof cyclophosphamide], and cell viability was measured 48 hours later by Annexin V/PI staining. For all graphs, mean is shown with SEM; significantdifferences in cell death with drug treatment compared with vehicle alone are indicated with an asterisk, P < 0.001. D, schematic of BIA-ALCL biology andpotential therapies. TLBR cell lines have significant production of T-cell–associated cytokines, including IL-6, IL-10, IFNg , and IL-2, and express thecognate receptors for these cytokines. Autocrine cytokine signals and aberrantly low levels of SHP-1 phosphatase contribute to increased JAK/STATsignaling. In the nucleus, phosphorylated STAT dimers lead to transcription of more T-cell cytokines and promote cell survival by increasing expression ofantiapoptotic genes (e.g., survivin, BCL2L2). This understanding of TLBR cell function helped to identify effective therapies to induce cell death, namelySTAT3 inhibitors and SHP-1 activators, in addition to existing chemotherapy drugs for lymphoma. ER, endoplasmic reticulum.

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produce only low levels of IL-2, insufficient to sustaintheir own survival in culture, it is likely that otherimmune cells in the implant microenvironment are pres-ent and actively secreting this factor. This would alsobe consistent with the development of BIA-ALCLs in thesetting of ongoing host inflammatory responses at theimplant scar capsule.

Notch1 activation in the TLBR cell lines was interestingbecause the highest levels were observed in TLBR-2, the cellline derived from a treatment-resistant, fatal case of BIA-ALCLs. Notch1 activation therefore might be a marker ofmore aggressive diseases, and studies to evaluate cleavedNotch1 levels in tumor specimens from patients with BIA-ALCL may provide useful prognostic information. g-Secre-tase inhibitors failed to affect cell viability in vitro, suggestingthat the cells have sufficiently strong survival signals pro-vided by other factors or pathways to overcome inhibitionof Notch1. Future studies evaluating Notch inhibition incombination with cytokine signaling interruption mayidentify highly effective therapies for aggressive cases ofBIA-ALCLs.

Using newly established models of BIA-ALCLs, we havemade significant improvements in the understanding ofthe biology of these tumors and identified potentialtargets for therapy that are readily translatable to theclinic. The identification of a successful xenotransplanta-tion model for the TLBR cell lines should facilitate futureevaluation of targeted therapies against cytokine path-ways (e.g., IL-6, IL-2) and cell survival molecules (e.g.,survivin), as well as confirmation of chemotherapy sen-sitivity, in the in vivo setting. The TLBR cell lines have beendeposited with the American Tissue Culture Collection(www.atcc.org).

Disclosure of Potential Conflicts of InterestA.L. Epstein has a commercial research grant from Mentor Corporation

and Allergan, Inc. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: M.G. Lechner, C.H. Church, R.B. Sevell, A.L.EpsteinDevelopment of methodology: M.G. Lechner, C. Megiel, C.H. Church,S.M. Russell, R.B. Sevell, A.L. EpsteinAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): C. Megiel, C.H. Church, T.E. Angell, S.M. Russell,J.K. Jang, G.S. BrodyAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M.G. Lechner, C. Megiel, C.H. Church,T.E. Angell, S.M. Russell, J.K. JangWriting, review, and/or revision of the manuscript: M.G. Lechner, C.Megiel, C.H.Church, S.M.Russell, R.B. Sevell, J.K. Jang,A.L. Epstein, T.E.AngellAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R.B. Sevell, A.L. EpsteinStudy supervision: A.L. EpsteinExecution of experiments: M.G. Lechner

AcknowledgmentsThe authors thank the expert work of Victoria Bedell and the City of Hope

Cytogenetic Core Facility (Duarte, CA) in conducting the cytogenetic studies;andMichael F. Bohley (Aesthetic Breast Care Center, Portland, OR), ThomasW. Martin (Puget Sound Institute of Pathology, Seattle, WA), and James H.Blackburn (Plastic Surgery Bellingham, Bellingham, WA) in the clinical careof the patients and the collection of specimens and clinical information forthese studies.

Grant SupportThis work was supported by Mentor Corporation, Allergan, Inc., and

Cancer Therapeutics Laboratories, Inc., of which A.L. Epstein is a co-founder.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 13, 2012; revisedMay 31, 2012; accepted June 29, 2012;published OnlineFirst July 12, 2012.

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2012;18:4549-4559. Published OnlineFirst July 12, 2012.Clin Cancer Res Melissa G. Lechner, Carolina Megiel, Connor H. Church, et al.

Anaplastic Large Cell Lymphoma−Associated ALK−Survival Signals and Targets for Therapy in Breast Implant

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