Clin Cancer Res-2012-Lechner-4549-59

Human Cancer Biology
Survival Signals and Targets for Therapy in Breast Implant–
Associated ALKÀ
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
Abstract
Purpose: 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 TLBR models in vitro and may 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
from which they were derived and as such provide valuable preclinical models to study their unique biology.
Clin Cancer Res; 18(17); 4549–59. Ó2012 AACR.
Introduction
Breast implant–associated (BIA) T-cell anaplastic large
cell lymphoma (ALCL) is a recently recognized clinical
entity, with 80 cases identified worldwide to date and four
disease-specific fatalities (1–15). BIA-ALCL presents com-
monly as a late seroma and/or tumor mass attached to the
scar capsule containing malignant cells an average of 5.8
years after implant placement (range, 0.4–20 years;
ref. 13). While most cases are indolent and respond well
to capsulectomy with local adjuvant radiation therapy,
10% of cases present with metastasis and 5% of cases are
fatal (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 and nuclear atypia, and large nuclei and
nucleoli (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 and
kinase domain of the ALK (16–19). Disease is subcategor-
ized as ALKþ
systemic, ALKÀ
systemic, or primary cutaneous
(pc-) ALCL, and each group exhibits distinct clinical behav-
ior (16, 18). ALKÀ
systemic ALCL is aggressive, with a 5-year
overall survival (OS) rate of only 49%, compared with ALKþ
ALCL (70% 5-year OS rate) and pc-ALCL (90% 5-year OS
rate; ref. 20). Seroma-associated ALCL was proposed by
Roden and colleagues (5) in 2008 to address BIA-ALCL,
which shares morphologic features of both primary system-
ic ALKÀ
ALCL and pc-ALCL but is distinct in its presentation
with malignant seroma fluid and varied clinical progres-
sion (indolent to aggressive). T-ALCLs express a range of
immune markers, including T-cell antigens, cytotoxic gran-
ules, and antigen presentation molecules, and, like other
T-cell neoplasms, show clonal T-cell receptor (TCR) gene
rearrangement (21–23).
As more cases of BIA-ALCLs are recognized, questions
about tumor etiology have emerged and the identification
of 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 1
Pathology, 2
Medicine, and 3
Plastic
Surgery, Keck School of Medicine, University of Southern California, Los
Angeles, California
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Alan L. Epstein, Department of Pathology, USC
Keck School of Medicine, 2011 Zonal Ave, HMR 205, Los Angeles, CA
90033. Phone: 323-442-1172; Fax: 323-442-3049; E-mail:
aepstein@usc.edu
doi: 10.1158/1078-0432.CCR-12-0101
Ó2012 American Association for Cancer Research.
Clinical
Cancer
Research
www.aacrjournals.org 4549
on August 18, 2015. © 2012 American Association for Cancer Research.clincancerres.aacrjournals.orgDownloaded from
Published OnlineFirst July 12, 2012; DOI: 10.1158/1078-0432.CCR-12-0101

initial report, we have established and characterized 2 new
cell lines from patients with BIA-ALCL, including 1 of the 4
fatal cases, designated TLBR-2 and -3. Using these models of
BIA-ALCLs, we now describe fully the phenotypic and
functional features of this newly emerging clinical entity,
including identification of aberrancies in cell signaling and
apoptosis regulators that seem to be excellent molecular
targets for therapy.
Materials and Methods
Cell lines and cells
Karpas299 (Karpas), Raji, HUT102, and Jurkat cell lines
were obtained from American Type Culture Collection
(authentication by short tandem repeat) and maintained
in 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 Keck
School of Medicine (University of Southern California, Los
Angeles, CA; HS-0600579) was obtained for the collection
of peripheral blood mononuclear cells from healthy
donors.
Cytogenetics
Karyotype analysis was conducted by the Division of
Anatomic Pathology, City of Hope (Duarte, CA) using early
passages of each cell line. Patient cytogenetic information
was reported by the treating physician.
Heterotransplantation
Xenografts of the TLBR cell lines were attempted in 6- to 8-
week-old female nude, severe combined immunodeficient
(SCID; Harlan), nonobese diabetic (NOD)/SCID, and
NOD/SCID-g mice (Jackson Labs) using 106
cells in a
0.2-mL subcutaneous inoculum.
Morphology
Formalin-fixed, paraffin-embedded (FFPE) xenograft
tumors or cultured cells were sectioned and stained using
hematoxylin–eosin (HE), Wright–Giemsa (W–G), or
monoclonal antibodies (Supplementary Table S1) using
immunocytochemical techniques, as described previously.
Observation and image acquisition were made using a Leica
DM2500 microscope (Leica Microsystems, www.leica-
microsystems.com), digital SPOT RTke camera, and SPOT
Advanced Software (SPOT Diagnostic Instrument Inc.,
www.diaginc.com). Images were resized and brightened
for publication using Adobe Photoshop software (Adobe,
www.adobe.com).
Flow cytometry
Single-cell suspensions were stained with fluorescence-
conjugated antibodies as described previously (24). Anti-
bodies and isotype controls were from BD Biosciences and
eBiosciences (Supplementary Table S1). Samples were run
in duplicate on a BD FACSCalibur flow cytometer using
CellQuestPro software. Mean fluorescence intensity (MFI)
and percentage of positive staining cells (difference between
MFI of sample and isotype 50) were determined for 15,000
events.
PCR and quantitative reverse transcriptase PCR
PCR to assess TCRg gene rearrangement and screen for
oncogene incorporation was carried out as described pre-
viously (1, 21, 25–27). Quantitative reverse transcriptase
PCR (qRT-PCR) to measure gene expression was carried out
as described previously (24). Gene-specific amplification
was normalized to glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) and fold change calculated relative to
healthy donor peripheral blood T cells. Differences in mean
expression of tumor suppressor, proto-oncogenes, and apo-
ptosis-related genes among tumor cell lines and normal
donor T cells were evaluated for statistical significance by
ANOVA followed by Dunnett test.
Immunoblotting
Whole-cell lysates in radioimmunoprecipitation (RIPA)
buffer were fractionated on 10% Tris-glycine PAGE, electro-
transferred to nitrocellulose, and probed overnight for
target proteins with primary antibodies (Cell Signaling and
Santa Cruz Biotech; Supplementary Table S1), as described
previously (1). Protein concentration was determined by
the bicinchoninic acid (BCA) assay.
Measurement of lymphoma-derived cytokines
Levels of cytokines in supernatants from 24-hour cultures
of TLBR-1, -2, -3, Karpas299, or Jurkat cells were measured
by cytometric bead array and analyzed on a BD LSRII flow
cytometer using FACSDiva software for acquisition and
compensation. Differences in mean levels of cytokine pro-
duction were tested for statistical significance by ANOVA
followed by Dunnett test with comparison to medium
alone.
Drug studies
TLBR-1, -2, -3, and Karpas299 cells were cultured (106
cells/mL) in vitro in the presence or absence of cell signaling
Translational Relevance
Numerous cases of rare T-cell ALKÀ
anaplastic large
cell lymphoma have recently been identified in women
with textured silicone and saline breast implants. In
2011, the U.S. Food and Drug Administration issued a
warning for these implants out of concern for this newly
emerging clinical entity. In this study, we identify
increased STAT3 activation related to dysregulation of
the SHP-1 phosphatase and autocrine production of
interleukins as a driver of cell survival in breast
implant–associated anaplastic large cell lymphomas.
Improved understanding of the biology of these tumors
will facilitate changes to implant design to prevent new
cancer cases and development of effective therapies for
this disease.
Lechner et al.
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 30
minutes, then washed twice with cold complete medium,
and cultured in the absence of drug for 48 hours. Cell
viability was measured by staining with Annexin V/propi-
dium iodide (PI; Invitrogen) and analyzed on a BD LSRII
flow cytometer using FACSDiva software for acquisition
and compensation (!15,000 events per sample). Reagents
evaluated 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 treated with
inhibitors or vehicle alone were evaluated by ANOVA and
Dunnett post-test or pairwise comparisons by the Student t
test with Bonferroni correction.
Results
Clinical presentation
The TLBR cell lines were established from the primary
tumor specimens of women with BIA-ALCL, as summarized
in Table 1, under IRB-approved protocol HS-10-00254 and
reported previously for TLBR-1 (1). These cases were typical
of BIA-ALCLs in that the women presented with unilateral
seroma fluid accumulation 3 to 15 years after elective breast
augmentation with textured saline implants (Fig. 1A). The
seromas uniformly recurred within months of initial drain-
age and were found to contain malignant cells consistent
with ALKÀ
ALCLs (Fig. 1B). All patients underwent bilateral
implant removal and capsulectomy and had no evidence of
contralateral breast involvement, skin manifestations, or
spread to regional lymph nodes at that time. Patients 1 and
3 received local radiotherapy to the affected breast and chest
wall 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 ALKÀ
seroma–associated
T-ALCL, absent t(2;5) 42-y-old
female
ALKÀ
seroma–associated T-
ALCL, absent t(2;5) 43-y-old
female
ALKÀ
seroma–associated
T-ALCL, absent t(2;5) 45-y-old
female
Implant type Textured saline Nagor SFX-HP
250cc
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 to
the capsule seen by imaging
Tumor specimen
cytology
Large mononuclear CD30þ
ALKÀ
CD4þ
CD8þ
TIA-1þ
Perforinþ
KeratinÀ
PAX5À
CD20À
anaplastic lymphoma cells
ALKÀ
CD45þ
CD20À
CD15À
CD43À
Cytokeratins (Cam 5.2)À
anaplastic lymphoma cells
ALKÀ
CD45þ
CD4þ
CD43þ
TIA-1þ
CD8À
CD15À
CD20À
CD68À
PAX5À
anaplastic lymphoma
cells
Patient treatment
and outcome
Surgery (bilateral implant removal
and capsulectomy) and
radiation therapy (40 Gy
delivered in 20 fractions)
and capsulectomy)
and capsulectomy) and
radiation therapy (36 Gy
delivered in 20 fractions)
Patient in remission at time of
publication (3 y after initial
presentation)
Recurrent disease involving
axillary lymph nodes,
supraclavicular fossa,
mediastinum, and bilateral lung
infiltrates
Patient in remission at time of
publication (14 mo after initial
presentation)
Chemotherapy and radiation
therapy, unsuccessful
Patient died of disease 9 mo after
initial 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 h
Viral status EBVÀ
, HTLV1/2À
, HPVÀ
EBVÀ
, HTLV1/2À
, HPVÀ
EBVÀ
, HTLV1/2À
, HPVÀ
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 2
months after surgery with involvement of the axillary and
supraclavicular lymph nodes and bilateral pleural effusions.
She received radiation therapy to which she showed a
dramatic response with significant tumor shrinkage. How-
ever, computed tomographic imaging of the chest 2 months
later showed spread of her disease into the mediastinum
with airway compression and bilateral lung infiltrates. Her
status progressively worsened and she died of ALCL-related
complications 9 months after initial presentation.
Establishment of TLBR cell lines from patient tumor
specimens
All 3 TLBR cell lines grow in suspension cultures and
exhibit interleukin (IL)-2–dependent growth (Table 1).
Wright–Giemsa staining of cytospin preparations of the
TLBR cell lines showed cells with abundant cytoplasm, 1
to 4 large cytoplasmic vacuoles, enlarged nuclei, and prom-
inent nucleoli characteristic of other ALCLs and similar to
the original specimens (Fig. 1C; refs. 23, 28). Multiplex PCR
analysis of TLBR-1, -2, and -3 cells showed TCRg mono-
clonality, confirming a neoplastic T-cell origin of the cell
lines and their derivation from the T-ALCL patient speci-
mens (Table 1).
Chromosomal atypia
Conventional cytogenetic and spectral karyotyping anal-
ysis of mitotically active TLBR-2 and -3 cells showed clonally
abnormal, hypertriploid, and complex karyotypes (Supple-
mentary Fig. S1). TLBR-2 cells had a modal number of
chromosomes of 76 and showed gains of chromosomes
1, 2, 5, 6, 10, 11, 14, 17, as well as clonal loss of one copy of
chromosome 18, relative to a triploid genome. TLBR-3 cells
showed a modal number of chromosomes of 81, gains of
chromosomes X, 2, 5, 7, 8, 10, 11, 12, 14, 19, 20, 21, and 22,
and clonal losses of one copy of chromosomes 9, 16, and
17, relative to a triploid genome. Cytogenetic analysis of the
TLBR-1 cell line was reported previously (1). None of the
TLBR cell lines show the NPM-ALK t(2;5) (consistent with
the primary tumor specimens), the (7;9) translocation
reported in T-cell lymphoblastic leukemia or rearrange-
ments involving the TCR gene loci on chromosomes 7 and
Figure 1. Establishment of model
cell lines for BIA-ALCLs from
primary tumor specimens. A, BIA-
ALCL in patient 3 presented as
seroma fluid accumulation (arrow)
around the right breast implant
(left). Intra-operative photographs
of the breast implants and
surrounding scar capsule tissue
removed from patient 3 (middle,
right). Anatomic pathology
identified the focal adhesions on
the right capsule as focal, foreign-
body type granulomatous
inflammation with refractile
nonpolarizable material, likely
silicone from the textured implant
surface. Wright–Giemsa–stained
cytospins of (B) tumor cells in
seroma fluid isolated from patient 2
and (C) early passages of TLBR-1,
-2, and -3 cells in culture show
features typical of ALCL, including
enlarged nuclei with frequent
mitotic figures, multiple prominent
nucleoli, pale cytoplasm with
vesiculation, and occasional
multinucleated giant cells (original
magnification, Â400). D,
immunohistochemistry for
lymphoma markers of FFPE tissue
sections of TLBR -1, -2, and -3
xenotransplant tumors (original
magnification, Â200 for HE and
Â400 for all others).
Lechner et al.

14. All 3 TLBR cell lines also lacked other translocations
frequently found in germinal center cell, mantle cell, diffuse
large 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 ALKÀ
ALCL and fidelity to
original tumors
Immunophenotypic 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 3
TLBR models showed strong CD30 positivity, weak expres-
sion of epithelial membrane antigen (EMA), and absent
ALK-1 [t(2;5) product], keratins (squamous tissue antigen)
or nuclear PAX-5 (Hodgkin lymphoma antigen; ref. 28).
Comparison to normal T-cell lineages
To understand better the BIA-ALCL cell biology, the TLBR
cell lines were characterized for expression of normal T-cell
lineage markers and transcription factors. Expression of
immune-related proteins by TLBR cell lines was examined
by flow cytometry (Supplementary Table S2). The TLBR cell
lines varied in their expression of T-cell lineage markers,
CD4, CD8, and TCRab/gd, suggesting arrest at different
stages 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 cell
lines showed positivity for cytotoxicity protein Granzyme
B and strong expression of antigen presentation–associated
markers (HLA-DRþ
CD80þ
CD86þ
) and CD71, the trans-
ferrin receptor. Expression of adhesion (CD11cþ
CD11bÀ
)
and myeloid (CD13þ
CD14À
CD15þ
CD68À
) markers was
variable, and TLBR cells generally lacked B-cell
(CD10À
CD19À
CD20À
CD21À
CD23þ
), dendritic cell (DC;
CD1aÀ
), and stem cell (c-kitÀ
CD133À
) markers. In regard
to normal T-cell lineages, analysis of T-cell transcription
factors [Th1 (T-bet), Th2 (GATA-3), Th17 (RORg), and
T-regulatory (FoxP3)] showed strongest positivity for T-bet
and FoxP3 and weak positivity for RORg.
Dysregulation of cell-cycle and apoptosis controls
Aberrant expression of cell-cycle control genes and escape
from homeostatic programmed cell death pathways can
facilitate 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 -3
and established PTCL cell lines Karpas299 and Jurkat using
qRT-PCR techniques. As shown in Fig. 2A, ALCL cell lines
Figure 2. Survival regulators. A,
expression of antiapoptotic genes in
TLBR cell lines relative to normal
donor T cells by qRT-PCR
techniques and confirmation of
elevated survivin by immunoblotting.
Karpas299, known to overexpress
survivin, was run in parallel for
comparison. B, expression of
proapoptotic and tumor suppressor
genes in the TLBR cell lines. A and B,
for all graphs, gene expression
measured by qRT-PCR techniques
was normalized to GAPDH and mean
fold change relative to normal donor
T cells is shown with SEM. Significant
differences in mean gene expression
from normal donor T cells are
indicated by an asterisk. All samples
within the brackets had significant
differences in expression relative to
normal donor T cells. ALK
þ
ALCL
Karpas299 and T-ALL Jurkat cell
lines were run in parallel for
comparison. C, increased
expression and phosphorylation of
STAT3 in TLBR cell lines were
evaluated by immunoblotting
techniques. Karpas299 is known to
have aberrant STAT3
overexpression and activation. D,
detection of pSTAT3 in FFPE tissue
sections of TLBR -1, -2, and -3
xenotransplant tumors (original
magnification, Â400).
Breast Implant ALCL

(TLBR and Karpas299) showed significant upregulation of
antiapoptotic genes survivin (P 0.05) and BCL2L2 (P
0.05). Strong expression of survivin by ALCL cell lines was
confirmed by immunoblotting and showed similar levels of
expression among the TLBR cell lines. Proapoptotic genes
and 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 acute
lymphocytic leukemia (T-ALL)-associated oncogenes TAL1,
HOX11, LYL1 and LMO1/2, and the results of these studies
were negative (data not shown).
Activation of STAT3 signaling
Activation of STAT3 can upregulate survival signals and
downregulate proapoptotic mediators in lymphoid cells
(29–31). Immunoblotting confirmed increased translation
and activation of STAT3 proteins in these models (Fig. 2C),
with the greatest activity in the cell line derived from the
most aggressive case of BIA-ALCL (TLBR-2), and at levels
comparable with STAT3-overexpressing Karpas299 cells
(33, 34). High levels of pSTAT3 were also seen in xenograft
tumors of TLBR-1, -2, and -3 (Fig. 2D).
Cytokine expression and functional studies
ALK 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 cell
lines 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-A
was measured for the TLBR cell lines in culture (Fig. 3A).
The TLBR cell lines showed strong secretion of cytokines
associated with multiple T-cell subsets, most notably IL-6
and IL-10, compared with other PTCL models (Jurkat,
Karpas299). We hypothesized that survival signals in
these cells may be driven, in part, by autocrine responses
to cytokines, many of which act through JAK/STAT sig-
naling. TLBR-1, -2, and -3 were uniformly positive for the
IL-6 receptor (Fig. 3B), and TLBR-2 and -3 showed weak
positivity for the IL-10 receptor (data not shown), sug-
gesting that these cells are capable of responding to these
factors. Neutralization experiments for IL-6 showed a
modest but insignificant decrease in TLBR cell prolifera-
tion (data not shown), likely related to the very high
levels of IL-6 produced by these models. Regulatory T cell
(Treg)-like suppressive function was also suggested for
TLBR cell lines by FoxP3þ
and IL-10 and TGFb secretion
(TLBR-2 and -3). To evaluate suppressive ability, TLBR
cell lines were co-cultured with naive normal donor T
cells in the presence of CD3/CD28 stimulation, and T-cell
proliferation was measured by carboxyfluorescein succi-
nimidyl ester (CFSE) dilution after 3 days, as carried out
routinely by our laboratory (24). Surprisingly, all 3 TLBR
cell lines were found to augment T-cell proliferation (data
not shown), perhaps as a result of their strong production
of T-cell–activating cytokines (e.g., IFNg, IL-2). The TLBR
cell 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 rapid
growth when cultured at higher density.
Sensitivity to STAT3 inhibition
To determine the influence of JAK/STAT3 signaling on
TLBR cell survival, cells were cultured in the presence of
STAT3-specific inhibitors WP1066 and S3I-201 or JAK/
STAT3-targeted tyrosine kinase inhibitor sunitinib, and cell
viability was assessed by Annexin V/PI staining. As shown
in Fig. 3C, STAT3-specific inhibition by WP1066 produced
significant cell death in all 3 TLBR cell lines in a dose-
dependent manner. Similar effects on cell viability were
seen with S3I-201 (data not shown). Furthermore, sunitinib
produced striking cell death in all TLBR cell lines across
a range of doses (Fig. 3D). The ALKþ
ALCL cell line
Karpas299 was run in parallel as a positive control in these
experiments.
Downregulation of STAT3-negative regulator SHP-1
STAT3 activation can also result from decreased levels
of negative regulating phosphatase SHP-1 (33, 35). TLBR
cells 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-1
expression relative to STAT3, even relative to Karpas299,
an ALCL model previously reported to have significant
SHP-1 loss (37). SHP-1 activation by honokiol produced
significant cell death in the TLBR cell lines, with loss of
pSTAT3 confirmed in cell lysates by immunoblotting (Fig.
4C and D). In addition, the chemotherapeutic agent 5-
aza-20
-deoxycytidine (AZA), which was previously shown
to 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 aggressive
TLBR-2
Evaluation of TLBR and established PTCL cell lines
showed strong expression of Notch1 and Notch2 receptors
and unique expression of a major Notch ligand, Jagged 2,on
the 3 TLBR cell lines (Supplementary Fig. S4). Aberrant
expression and activation of the embryonic transcription
factor Notch1 can contribute to malignant transformation
in some adult PTCLs (38). Levels of cleaved, activated
Notch1 protein were previously found to be elevated in
TLBR-1 and Karpas299 cells (1). TLBR-2 and -3 also have
significant cleaved Notch1 and Notch1 levels (Supplemen-
tary Fig. S4). The much higher levels of cleaved Notch1 in
TLBR-2 cells may drive the faster division and more aggres-
sive behavior of this model and the tumor from which it was
established. However, modulation of Notch1 signaling
using g-secretase inhibitors (FBHA, DAPT) or activators
(SBHA, valproate) failed to produce any significant change
Lechner et al.

in TLBR-1, -2, -3, or Karpas299 cell viability (Supplementary
Fig. S4).
Evaluating cytotoxic therapies
In 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 TLBR
model cells lines were exposed to CHOP constituent drugs
[vinblastine (vincristine analogue with in vitro activity),
doxorubicin, 4-hydroperoxycyclophosphamide (active
metabolite of cyclophosphamide)] briefly and cell viability
was then assessed. As shown in Fig. 5A, TLBR-1, -2, and -3
cells 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 17.5
mmol/L dose, P 0.001). The TLBR cell lines showed
moderate sensitivity to vinblastine (0.9 and 9 mmol/L) and
to a very high dose of 4-hydroperoxycyclophosphamide
(100 mmol/L; Fig. 5B and C).
Discussion
As recently reported, breast implant–associated T-cell
anaplastic large cell lymphomas are an emerging clinical
entity (2–15). Three model cell lines, designated TLBR-1, -2,
and -3, have been established from the primary tumor
specimens from patients with a spectrum of indolent to
aggressive BIA-ALCLs to facilitate studies of the etiology and
potential therapy for these cancers. Morphologic and cyto-
genetic studies confirmed the ALKÀ
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. Mean
cytokine levels in cell culture supernatants with SEM are shown; asterisk indicates levels significantly increased from media controls (P 0.05). For all
3 TLBR cell lines, production of IL-6 and IL-10 was very high (boxed bars on graphs). B, surface expression of IL-6R measured by flow cytometry for TLBR cell
lines (open, sample; shaded, isotype control; representative histograms shown from 2 independent experiments). C and D, TLBR-1, -2, and -3 and
Karpas299 cells were treated in vitro with STAT3-specific inhibitor WP1066 (C) or tyrosine kinase inhibitor sunitinib (D), and cell viability was assessed by
Annexin V/PI staining and analysis by flow cytometry after 48 hours. Graphs show mean with SEM; significant differences in cell viability with drug treatment
compared with vehicle alone are indicated by an asterisk, P 0.001.
Breast Implant ALCL

the BIA-ALCL TLBR cell lines and their similarity to the
original tumor biopsy specimens. The molecular features of
ALKÀ
ALCLs and other ALCL subsets are largely unknown, a
fact that is mirrored by the 30% to 50% of ALCL cases
designated as not otherwise specified (ALCL-NOS) by his-
topathology (40). Functional studies of the TLBR cell lines
identified high production of T-cell–associated cytokines
IL-6 and IL-10, activation of JAK/STAT3 signaling pathways,
and strongest expression of transcription factors associated
with the T-helper cell (TH)1 and Treg cell lineages. This
molecular profile may be compared with that recently
reported for ALKþ
systemic ALCLs, namely upregulation of
TH17-related genes [e.g., IL-17A, IL-22, retinoic acid–related
orphan receptor (ROR)], JAK/STAT3 signaling, and cyto-
toxic molecules (32).
Compared with naive, normal donor T cells, the TLBR cell
lines showed dysregulation of cell-cycle and apoptotic
regulators, namely survivin, and activation of JAK/STAT3
pathways. Functional characterization and in vitro studies of
the TLBR cell lines yielded a working model of BIA-ALCL
tumor cell biology (Fig. 5D), with an emphasis on autocrine
cytokine signaling that promotes tumor cell survival. A
milieu rich in immune stimulatory cytokines, like IL-6 and
IL-2, which promotes rapid division of host lymphocytes
may cause the initial tumorigenic changes that lead to
BIA-ALCL in some patients. Chronic inflammation is well
recognized as a promoter of cancer (41). Autocrine IL-6
production has been identified as a driver of tumorigenesis
in some diffuse large B-cell lymphomas, as well as solid
tumors including breast, lung, and ovarian carcinomas (42–
44). The cytokine profile of BIA-ALCL cell lines, specifically
IL-6, TGFb, and IL-10, has also been shown to induce
immune suppressor cell populations (Tregs and myeloid-
derived suppressor cells) that could inhibit host antitumor
immunity and facilitate cancer development (45, 46). Pre-
vious studies of women with saline and silicone breast
implants found no increased risk of primary lymphoma or
breast cancer compared with women without implants
(15), suggesting that the present case series is directly linked
to newer device features. Texturing of the implant silicone
shell, an aesthetic advance introduced in the late 1980s to
reduce contractures and one recurring feature in these
cancer cases, results in greater silicone particle shedding in
the surrounding scar capsule. Indeed, histologic analysis of
mass lesions in cases of BIA-ALCLs, including patient 3 (Fig.
1A), shows nonrefractive particles consistent with shed
Figure 4. Downregulation of
STAT3-negative regulator SHP-1.
A and B, expression of
phosphatase SHP-1 and the ratio
of SHP-1 to STAT3 expression
were significantly decreased in all 3
TLBR cell lines relative to healthy
donor T cells, as measured by qRT-
PCR techniques. Graph shows
mean (n ¼ 3) gene expression as a
percentage of GAPDH, with SEM;
all cell lines were significantly
different from normal T cells
(ÃÃÃ
, P 0.0001). ALK
þ
ALCL
Karpas299 and T-ALL Jurkat cell
lines were run in parallel for
comparison. C, TLBR cell lines
were treated with the SHP-1
activator honokiol and viability
assessed by Annexin V/PI staining
after 48 hours. Mean is shown with
SEM; significant differences in cell
viability with drug treatment
compared with vehicle (V) alone are
indicated by an asterisk, P 0.001.
D, honokiol treatment effects on
STAT3 phosphorylation were
measured by immunoblotting of
cell lysates at 24 hours, with
GAPDH.
Lechner et al.

silicone among granulomatous inflammation and tumor
cells. Whether this inflammatory stimulus is increased in
textured implants and may play a role in the development of
BIA-ALCLs are questions that will require future investiga-
tion, but the prominent role of IL-6 found in the TLBR cell
lines suggests that immune reactions are important to the
progression of this disease.
It is important also to acknowledge that IL-2 signaling
almost certainly contributes to BIA-ALCL cell survival, as
the TLBR cell lines all die in the absence of this cytokine
and have strong expression of IL-2R proteins. In experi-
mental systems, IL-2 overexpression has been shown to
produce 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) to
CHOP chemotherapy drugs: doxorubicin (DOX), vinblastine [(VIN), vincristine in vitro analogue], or 4-hydroperoxycyclophosphamide [(4HC), active metabolite
of cyclophosphamide], and cell viability was measured 48 hours later by Annexin V/PI staining. For all graphs, mean is shown with SEM; significant
differences in cell death with drug treatment compared with vehicle alone are indicated with an asterisk, P 0.001. D, schematic of BIA-ALCL biology and
potential therapies. TLBR cell lines have significant production of T-cell–associated cytokines, including IL-6, IL-10, IFNg, and IL-2, and express the
cognate receptors for these cytokines. Autocrine cytokine signals and aberrantly low levels of SHP-1 phosphatase contribute to increased JAK/STAT
signaling. In the nucleus, phosphorylated STAT dimers lead to transcription of more T-cell cytokines and promote cell survival by increasing expression of
antiapoptotic genes (e.g., survivin, BCL2L2). This understanding of TLBR cell function helped to identify effective therapies to induce cell death, namely
STAT3 inhibitors and SHP-1 activators, in addition to existing chemotherapy drugs for lymphoma. ER, endoplasmic reticulum.
Breast Implant ALCL

produce only low levels of IL-2, insufficient to sustain
their own survival in culture, it is likely that other
immune cells in the implant microenvironment are pres-
ent and actively secreting this factor. This would also
be consistent with the development of BIA-ALCLs in the
setting of ongoing host inflammatory responses at the
implant scar capsule.
Notch1 activation in the TLBR cell lines was interesting
because the highest levels were observed in TLBR-2, the cell
line derived from a treatment-resistant, fatal case of BIA-
ALCLs. Notch1 activation therefore might be a marker of
more aggressive diseases, and studies to evaluate cleaved
Notch1 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, suggesting
that the cells have sufficiently strong survival signals pro-
vided by other factors or pathways to overcome inhibition
of Notch1. Future studies evaluating Notch inhibition in
combination with cytokine signaling interruption may
identify highly effective therapies for aggressive cases of
BIA-ALCLs.
Using newly established models of BIA-ALCLs, we have
made significant improvements in the understanding of
the biology of these tumors and identified potential
targets for therapy that are readily translatable to the
clinic. The identification of a successful xenotransplanta-
tion model for the TLBR cell lines should facilitate future
evaluation 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 been
deposited with the American Tissue Culture Collection
(www.atcc.org).
Disclosure of Potential Conflicts of Interest
A.L. Epstein has a commercial research grant from Mentor Corporation
and Allergan, Inc. No potential conflicts of interest were disclosed by the
other authors.
Authors' Contributions
Conception and design: M.G. Lechner, C.H. Church, R.B. Sevell, A.L.
Epstein
Development of methodology: M.G. Lechner, C. Megiel, C.H. Church,
S.M. Russell, R.B. Sevell, A.L. Epstein
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): C. Megiel, C.H. Church, T.E. Angell, S.M. Russell,
J.K. Jang, G.S. Brody
Analysis 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. Jang
Writing, 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.Angell
Administrative, technical, or material support (i.e., reporting or orga-
nizing data, constructing databases): R.B. Sevell, A.L. Epstein
Study supervision: A.L. Epstein
Execution of experiments: M.G. Lechner
Acknowledgments
The authors thank the expert work of Victoria Bedell and the City of Hope
Cytogenetic Core Facility (Duarte, CA) in conducting the cytogenetic studies;
and Michael F. Bohley (Aesthetic Breast Care Center, Portland, OR), Thomas
W. Martin (Puget Sound Institute of Pathology, Seattle, WA), and James H.
Blackburn (Plastic Surgery Bellingham, Bellingham, WA) in the clinical care
of the patients and the collection of specimens and clinical information for
these studies.
Grant Support
This 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 marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received January 13, 2012; revised May 31, 2012; accepted June 29, 2012;
published OnlineFirst July 12, 2012.
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Breast Implant ALCL

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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