Breast Implant-Associated, ALK-Negative, T-Cell, Anaplastic, Large-Cell Lymphoma: Establishment and Characterization of a Model Cell Line (TLBR-1) for This Newly Emerging Clinical Entity

Abstract

BACKGROUND

Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide.

METHODS

A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods.

RESULTS

Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)⁺ and perforin⁺. Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR⁺CD80⁺CD86⁺), IL-2 signaling (CD25⁺CD122⁺), and NK (CD56⁺) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor.

CONCLUSIONS

TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized disease entity.

Primary breast lymphoma after breast prostheses placement has previously been reported, and a survey of the literature from the past 30 years has identified sporadic reports of 5–11 cases of primary breast non-Hodgkin lymphoma (NHL) arising in patients with saline or silicone breast implants.^1–6 Several previous meta-analyses evaluating the risk of neoplasia associated with breast prostheses provided conflicting results or did not exclude patients with a previous breast malignancy, and, thus, the question remains to be answered definitively.^7,8 Brody et al⁹ recently identified 40 cases of breast implant-associated primary breast NHL of T-cell type occurring in patients with a specific type of textured breast prostheses. This case series is remarkable for the large number of patients identified and the similarity in patient presentation and disease. Primary non-Hodgkin lymphoma (NHL) of the breast is rare (900 incident cases reported annually; 0.5% of all breast malignancies), and the vast majority have a B-cell phenotype.^1,9

In the series reported by Brody et al, ⁹ all cases have been classified as T-cell, anaplastic, primary, breast non-Hodgkin lymphomas, an exceedingly rare diagnosis. The reported cases also share a similar presentation, clinical course, and implant style.⁹ The initial presentation for these patients (average age 44.7 years; range, 33–87 years) was late peri-implant seroma, severe capsular contracture, or pericapsular tumor mass, with an average time from implant of 5.8 years (range, 1–20 years).⁹ The clinical course of the malignancy was typically benign. Patients received surgical treatment, chemotherapy, and/or radio-therapy, and except for 1 patient, remain disease free.⁹ Implant information was available for 25 of 40 patients, and 23 of 25 shared a common lost-salt method of the textured shell.⁹ In this series, both silicone-gel and saline-filled implants were involved.⁹ Although previous reports have not conclusively shown an increased risk of primary breast NHL with silicone-gel or saline implants,^7,8 this recent series arising in textured salt-withdrawal breast implants may represent a subset of patients with an increased risk of malignancy.

Anaplastic large-cell lymphoma (ALCL) was first described by Stein et al¹⁰ in 1985 as a rare T- or null-cell NHL characterized by large, anaplastic, lymphoid cells with strong uniform CD30 expression. ALCL accounts for 3% of adult NHL and may involve nodal or extranodal sites.⁵ Several subclassifications of ALCL exist: systemic, secondary, and primary cutaneous (pc-) ALCL. Roden et al⁶ have also suggested seroma-associated (sa-) ALCL as a clinical entity related to pc-ALCL and occurring adjacent to breast implants. These 4 lymphomas are histologically indistinguishable, consisting of pleomorphic epithelioid tumor cells with blast-like appearance, severe cellular and nuclear atypia, and large nuclei and nucleoli.^11,12 However, these diagnoses represent distinct clinical entities.^12–13 Systemic and secondary ALCL are characterized by an aggressive clinical course and frequently express anaplastic large-cell kinase (ALK) subsequent to a reciprocal t(2;5) translocation fusing the nucleophosmin (NPM1) and ALK genes.^4,11–13 In contrast, pc- and sa-ALCL are indolent malignancies that rarely carry the t(2;5) translocation and are usually ALK-negative.¹⁴ Most cases of CD30⁺ ALK-negative, sa-, or pc-ALCL present as solitary or regional nodules and/or tumors showing ulceration, with extracutaneous or regional lymph node involvement seen in only 10% of patients.¹⁴ The preferred treatment for pc- or sa-ALCL is localized radiation or surgical excision, with systemic chemotherapy reserved for cases with large tumor burden and extracutaneous involvement.¹²

Other features reported in ALK-negative ALCL include T-cell markers, cytotoxic phenotype (perforin⁺, granzyme B⁺, TIA⁺), activation and antigen-presentation antigens (eg, CD25, HLA-DR, CD80, CD86), CD56, and transferrin receptor CD71.^15–17 T-cell neoplasms usually demonstrate clonal TCRγ gene rearrangement, but up to 10% of ALCL neoplasms also show rearrangement of the immunoglobulin heavy-chain (IgH) gene.¹⁸ Aberrant expression of cell-cycle genes and embryonic transcription factor Notch1 can contribute to malignant transformation in lymphoma, as well as overexpression of T-cell specific genes TAL1, HOX11, LYL1, and LMO1/2.^19,20

This article describes the case presentation of a primary, breast, T-cell, ALK-negative ALCL in a patient with cosmetic textured (lost-salt) breast implants from the series reported by Brody et al⁹ A model cell line, T-cell lymphoma breast-1 (TLBR-1), was established from the primary tumor tissue and recapitulates the phenotype and cytogenetics of the original tumor. This cell line has been made available for others in the scientific community through the American Tissue-Type Cell Collection (ATCC; www.atcc.org) and represents an important model for further studies of this disease.

MATERIALS AND METHODS

Cell Lines and Cells

The Karpas 299, Raji, HUT102, and Siha cell lines were obtained from ATCC. All cell lines were maintained in complete medium (Roswell Park Memorial Institute (RPMI) medium -1640 with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin) in a humidified 5% CO₂, 37°C incubator. Institutional review board approval from the University of Southern California Keck School of Medicine (HS-0600579) was obtained for the collection and use of human peripheral blood mononuclear cells (PBMC). PBMC were collected by routine venipuncture followed by density gradient centrifugation (Ficoll-Hypaque; Sigma, St. Louis, Mo).

Establishment of TLBR-1

A tumor biopsy on wet ice was obtained from the Peter MacCallum Cancer Center, Australia. Tumor cells were mechanically dissociated from stromal tissue and pooled with tumor cells obtained from the serous fluid contained in the biopsy material. Viable cells were then isolated by Ficoll-Hypaque differential gradient centrifugation and either frozen in liquid nitrogen for storage in RPMI-1640 medium containing 30% FCS and 10% dimethyl sulfoxide (DMSO) or placed in culture over a human fibroblast feeder layer with and without 50 IU/mL of recombinant IL-2 (R&D Systems, Minneapolis, Minn). This feeder layer has been used in the past by our laboratory to establish several human lymphoma cells lines.²¹ Under the microscope, the lymphoma cells were seen lying lightly on top of the feeder cells and began to grow after approximately 3 weeks in culture. At 2-week intervals, the unattached lymphoma cells were decanted, spun, and added to fresh feeder cells. Parallel cultures without IL-2 did not thrive. After approximately 8 weeks, the cells were successfully weaned from the feeder cell monolayer and grown in suspension cultures in the presence of IL-2. Cell doubling time was determined for TLBR-1 by cell count measurements at 24-hour intervals for 1 week. The minimum IL-2 concentration needed for viability in culture was determined by a 2-week culture of TLBR-1 cells in 10% FCS RPMI-1640 medium containing with 0–10,000 IU/mL rIL-2.

Heterotransplantation into SCID Mice

Six-week-old female severe combined immune deficiency (SCID) mice (n = 5; Harlan Sprague Dawley, Indianapolis, Ind) were injected subcutaneously in the flank with 5×10⁶ viable TLBR-1 cells. Institutional animal care and use committee-approved protocols and institutional guidelines for the proper humane care and use of animals in research were followed.

Immunohistochemistry

Cytospins of TLBR-1 cells from culture and tissue sections of TLBR-1 heterotransplanted tumors were used for immunohistochemistry (IHC) studies. Wright-Giemsa staining (Protocol Hema 3; Fisher, Kalamazoo, Mich) of TLBR-1 cells cytospin preparations was performed to assess morphology. Excised heterotransplanted TLBR-1 tumors from SCID mice were fixed overnight in 10% neutral buffered formalin and embedded in paraffin blocks, then stained with hematoxylin and eosin. Both TLBR-1 cytospin and paraffin tissue slides were stained for specific antigens with monoclonal antibodies. Observation, evaluation, and image acquisition were made using a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany) connected to an automated, digital SPOT RTke camera and SPOT Advanced Software (SPOT Imaging Solutions, a division of Diagnostic Instruments, Sterling Heights, Mich). Images were further resized and brightened for publication using Adobe Photoshop software (Adobe, San Jose, Calif).

Flow cytometry

Single cell suspensions (10⁶ cells in 100 μL) in fluorescence-activated cell-sorting (FACS) buffer (1% FCS in phosphate buffered saline [PBS]) were stained with fluorescence-conjugated antibodies. For intracellular staining, surface staining was performed first, followed by buffer fixation/permeabilization (eBioscience, San Diego, Calif) and intracellular staining. Antibodies and isotype controls were from BD Biosciences (San Jose, Calif) and eBioscience. Samples were run in duplicate on a FACS Calibur flow cytometer (BD Biosciences) and data acquisition and analysis were performed using Cell Quest Pro software (BD Biosciences) at the University of Southern California flow cytometry core facility.

Cytogenetics

Karyotype analysis was performed by the Division of Anatomic Pathology, City of Hope (Duarte, Calif) by using cultured TLBR-1 cells. Analysis included Giemsa banding of metaphase spreads and fluorescence in situ hybridization (FISH) procedures performed routinely by this laboratory.

Polymerase Chain Reaction for TCR and IgH Gene Rearrangements and Viral Screen

Genomic DNA was isolated from TLBR-1, Raji, HUT102, and PBMC using TRIreagent (Sigma) per manufacturer instructions. For PCR, 100 ng of DNA was amplified with specified primers using REDTaq Ready-Mix PCR Master Mix (Sigma, St. Louis, Mo) and run on an iCycler (BioRad, Hercules, Calif). TCRγ and IgH gene rearrangements were assessed by multiplex semi-nested polymerase chain reaction (PCR) as described previously.^15,18 Consensus primers for Epstein-Barr virus (EBV) EBNA and human T-cell leukemia virus (HTLV)-1/2 polymerase genes were used to screen for the presence of these respective viruses, as previously reported.^22,23

T-Cell Oncogene Analysis by Quantitative RT-PCR (qRT-PCR)

RNA was isolated from cultured cells or heterotransplant tissue by RNeasy Mini Kit (Qiagen, Valencia, Calif) and DNase treated using Turbo DNase (Applied Biosystems, Foster City, Calif) according to manufacturer instructions. For qRT-PCR, 100 ng of DNase-treated RNA was amplified with Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems) using primer sequences from the National Institutes of Health (NIH) qRT-PCR database (http://primerdepot.nci.nih.gov) synthesized by the University of Southern California core facility.²⁴ Amplification was performed on a Stratagene Mx3000P cycler with MxPro software (Strategene, an Agilent Technologies division, La Jolla, Calif). Gene-specific amplification was normalized to GAPDH and fold change in gene expression calculated relative to universal human reference RNA (Stratagene). For statistical analysis, the Student t test for independent samples was used with a significance level α = .05 by GraphPad Prism software (La Jolla, Calif).

Western Blot for Activated Notch1

Sonicated whole-cell lysates (15 μg protein) were fractionated on 10% Tris-glycine polyacrylamide gels, electro-transferred to PDVF membrane, and probed overnight for activated Notch1 (clone Val1744) (Cell Signaling, Danvers, Mass). Horseradish peroxidase-conjugated secondary antibodies (Caltag, Burlingame, Calif) were then applied followed by signal detection with Immobilon HRP Substrate (Millipore, Billerica, Mass). Blots were stripped and reprobed for GAPDH (clone FL-335) (Santa Cruz Biotech, Santa Cruz, Calif) to normalize the amount of sample loaded.

RESULTS

Case Report of a Patient Diagnosed With Breast Implant-Associated ALK-negative T-ALCL

The patient is a 42-year-old female with a history of celiac disease and gastroesophageal reflux. In February, 2005, aged 38 years, she underwent elective bilateral breast augmentation using saline filled Nagor SFX-HP250 (Nagor, a GC Aesthetics company, Glascow, Scotland, UK) implants with silicone shells and had an unremarkable postoperative course. In November 2008, she presented with a right chest wall rash, slight right arm swelling, and significant enlargement of the right breast reflecting a large seroma surrounding the implant. The seroma was drained, yielding 300 mL of fluid shown to be sterile by microbiologic tests. A dermal biopsy of the skin rash showed minor changes but no evidence of cutaneous lymphoma by histology or PCR testing for T-cell receptor (TCR) gamma and beta gene rearrangements. In March 2009, computed tomography (CT) and magnetic resonance imaging (MRI) scans showed reaccumulation of seroma fluid but no soft tissue masses or lymphadenopathy. In April 2009, an additional 350 mL of seroma fluid was drained, and cytological analysis of the fluid revealed the presence of a T-cell lymphoma.

In June 2009, the patient underwent surgery for the removal of the right breast implant and seroma-containing pseudocapsule (removed intact). The left breast implant and pseudocapsule were also removed but were unremarkable. The right axillary lymph nodes were not palpable and were not sampled. The right seroma fluid and the fibrous tissue on the surface of the pseudocapsule demonstrated malignant lymphoma cells. There was no mass lesion or lymphomatous infiltration of soft tissues. Immunoperoxidase stains of the implant-associated cell aggregates demonstrated a CD4, CD8, CD30, TIA-1, EMA, perforin positive, and ALK-1 and keratin negative population of anaplastic lymphoid cells (Fig. 1). Staining for CD2, CD5, CD7, ALK-1, CD20, PAX-5, CD56, TCRαβ, TCRγδ, HHV-8, and BF-1 were negative on flow cytometry. In situ hybridization staining for EBV-RNA was negative and PCR for TCRγ gene rearrangement demonstrated monoclonality. Cytogenetics performed on the fluid sample showed non-specific structural abnormalities involving 2q, 5p, 10p, and 16p, trisomies of 2 and 21, monosomy of chromosome 20, an isodicentric 21, and 2 additional marker chromosomes. Final pathologic diagnosis of the right breast implant seroma biopsy indicated an ALK-negative T-cell anaplastic large-cell lymphoma.

Figure 1.

Morphology and phenotype of original tumor biopsy is shown. (A) Low magnification view of pseudocapsule with fibrin covering; (B) Higher power view of clot section showing entrapped lymphoma cells; (C) IHC staining of biopsy cells for keratin (negative); (D) CD30 antigen (strong membrane positivity); (E) epithelial membrane antigen (strong cytoplasmic and peri-nuclear [golgi] positivity); and (F) ALK-1 (negative).

Subsequent staging with CT and ¹⁸F-FDG-PET scans and bone marrow biopsy confirmed disease localized to the breast. The patient was treated with local radiotherapy to the right breast and chest wall (40 Gy delivered in 20 fractions) with a good response, and she remained in remission 7 months postradiotherapy.

Establishment of the Cell Line

A cell line was established from the primary tumor biopsy specimen shipped from Australia to the University of Southern California Department of Pathology and designated TLBR-1. To establish the cell line, the lymphoma cells initially required both a stromal feeder layer and recombinant IL-2. Cultures without both of these requirements did not thrive. After about 8 weeks in culture, the lymphoma cells were able to be weaned from the feeder layer but retained their requirement for IL-2 supplementation (range, 5–50 IU/mL). The established cell line was found to have a relatively slow doubling time of 55 hours. In culture, TLBR-1 cells demonstrate a polymorphic cell shape ranging from spherical to dendritic (Fig. 2A). Wright-Giemsa staining of cytospin preparations showed cells with abundant cytoplasm, 1 to 4 large cytoplasmic vacuoles, enlarged nuclei, and prominent nucleoli (Fig. 2B) characteristic of other anaplastic large cell lymphomas.¹⁷ Ultrastructural analysis by transmission electron microscopy demonstrated markedly indented nuclei with prominent nucleoli and abundant cytoplasm (Fig. 2C, D).

Figure 2.

Novel human cell line TLBR-1 was established from a sample of the patient’s primary tumor. (A) Phase-contrast microphotograph of growing TLBR-1 cell line showing polymorphic cell shapes. (B) Photomicrograph of cytospin of TLBR-1 cells demonstrates abundant cytoplasm, 1–4 large cytoplasmic granules, and enlarged nuclei with frequent mitotic figures (Wright-Giemsa stain, ×100 original magnification). (C, D) Transmission electron micrograph of TLBR-1 cells shows markedly indented nuclei with prominent nucleoli and abundant cytoplasm (×3500 and ×7500 original magnification).

Heterotransplantation in SCID Mice

Cultured TLBR-1 cells were heterotransplanted subcutaneously in SCID mice to produce expanding tumors in 5 of 5 mice that recapitulated the original tumor morphology (Fig. 3). At 3 weeks, the tumors were excised and showed the presence of pleomorphic cells with highly atypical nuclei and prominent nucleoli. Mitotic figures were frequently seen, and the tumors had very little necrosis. These results demonstrate that TLBR-1 is highly transplantable in this mouse model and can be used by other investigators to explore experimental treatments or other in vivo studies.

Figure 3.

Heterotransplantation of TLBR-1 cell line. (A) Appearance of subcutaneous TLBR-1 tumor in SCID mouse. (B and C) Low and high magnification of TLBR-1 SCID tumor demonstrating similar morphological features to the original biopsy (hematoxylin and eosin [H&E] stain ×200 and ×400 original magnification).

Immunophenotype of TLBR-1 in Cell Culture and In Situ

Immunophenotypic characterization of TLBR-1 cells in culture and from tumors grown in SCID mice demonstrated similarity to the original tumor and confirmed an ALCL (Figs. 1 and 4). Neither the original tumor nor TLBR-1 cell line expressed the ALK protein or the associated t(2;5) translocation as determined by IHC and FISH analysis (Figs. 1 and 4, data not shown). TLBR-1 cells demonstrate strong uniform CD30 positivity and no keratin or nuclear PAX-5 expression (Fig. 4), similar to the original tumor. Epithelial membrane antigen (EMA) staining was decreased in the cytospin and heterotransplant in comparison with the original tumor biopsy.

Figure 4.

Immunoperoxidase staining of TLBR-1 cells in SCID mouse heterotransplant and in cytospin preparation for ALCL classification markers. Photomicrograph of immunoperoxidase staining of formalin-fixed, paraffin-embedded, tissue sections of TLBR-1 SCID mouse heterotransplant (left) and TLBR-1 cytospin preparation (right) for ALK-1, CD30, epithelial membrane antigen, pankeratins or PAX5 (×200 original magnification, inserts ×400). As seen in the higher magnification inserts, the CD30 stained preparation appears different because the cells in the cytospins still have intact membranes causing them to have an apparent cytoplasmic staining pattern. By contrast, the heterotranplanted tumor preparations show more of a classical ring pattern due to sectioning of the tissue. Both preparations, however, also show prominent golgi staining often seen with anti-CD30 antibodies.

Immunophenotype of the TLBR-1 cell line

The phenotype of TLBR-1 cell line was further characterized for expression of common ALCL and lymphoid markers by flow cytometry (Table 1). Compared with isotype controls, the TLBR-1 cell line was strongly positive for CD30 and CD26, T-cell coreceptors CD4 and CD8, and pan T-cell antigen CD2. Staining for other T-cell–lineage markers (CD3, CD5, CD7, TdT) and surface TCRαβ and γδ was negative. As expected from its reliance upon IL-2 in culture, TLBR-1 is strongly positive for CD25 (IL-2Rα) and shows moderate expression of CD122 (IL-2Rβ). Other T-cell activation markers expressed included granzyme B, FoxP3, and 41BB (CD137). TLBR-1 demonstrates strong expression of antigen presentation-associated markers (HLA-DR⁺CD80⁺CD86⁺), NK cell antigen CD56⁺, and CD71, the transferrin receptor. The cell line has variable expression of adhesion (CD11c⁺CD11b) and myeloid (CD13⁺CD14⁻CD15⁺CD68⁻) markers, and lacks surface expression of most B cell (CD10⁻CD19⁻CD20⁻ CD21⁻CD23⁺), dendritic cell (DC) (CD1a⁻), or stem cell (c-kit⁻CD133⁻) markers.

Table 1.

Immunophenotypic Analysis of TLBR-1 by Flow Cytometry

	% Positive		Mean Fluorescence Intensity
	Isotype Control	Antibody	Isotype Control	Antibody
T cell
CD2a	0.6	65.6	104.2	339.1
CD3	0.9	1.0	110.9	91.3
CD4a	3.0	81.4	180.4	404.2
CD5	3.0	5.9	180.4	141.3
CD7	13.6	4.2	307.0	222.5
CD8a	3.0	98.0	180.4	709.9
TdT	8.2	14.6	241.0	262.3
TCR αβ	13.6	6.7	307.0	246.2
TCR γδ	3.0	5.6	180.4	194.7
CD25a	0.6	95.2	104.2	547.1
CD122b	3.0	23.1	180.4	276.9
FoxP3b	8.2	55.4	241.0	328.1
GranzymeBa	43.5	100.0	306.7	668.5
CD28	13.6	13.6	307.0	272.6
GITR	0.6	0.9	104.2	90.5
CD40L	3.0	1.9	180.4	130.6
41BBb	0.6	7.9	104.2	197.0
B cell
CD24	0.9	1.2	110.9	109.6
CD22	0.6	0.8	104.2	89.0
CD21	57.5	4.0	351.7	224.6
CD20	4.1	1.5	192.0	106.2
CD19	13.6	6.7	307.0	248.0
CD10	0.1	0.8	109.3	86.5
CD23a	3.0	89.2	180.4	507.9
CD27b	3.0	11.2	180.4	247.5
Macrophage/myeloid/dendritic cell
CD11b		19.9		149.0
CD11ca	3.0	96.4	180.4	615.7
CD33b	0.6	5.9	104.2	170.6
CD13	13.6	16.8	307.0	282.3
CD14	4.1	1.6	192.0	134.4
CD15a	0.6	47.3	104.2	314.3
CD68	0.1	1.2	109.3	91.6
CD1a	0.6	1.2	104.2	104.9
HLA-DRa	4.1	99.2	192.0	624.2
CD80a	3.0	99.8	180.4	681.3
CD86a	0.6	98.1	104.2	588.2
Miscellaneous
CD30a	3.0	99.5	180.4	751.0
CD45a	0.6	99.0	104.2	517.6
CD56a	0.9	61.6	110.9	337.3
CD26a	0.6	99.4	104.2	653.3
CD71a	3.0	55.1	180.4	341.2
Stem cell
CD133	13.6	5.4	307.0	236.0
c-kit	8.2	14.2	241.0	260.8

Phenotype of TLBR-1 cells in culture was characterized by using monoclonal antibodies (left) and analyzed by flow cytometry. Percentage of positive staining of cells (middle) and mean fluorescence intensity (right) is shown for each antibody target and isotype control.

^aMean fluorescence intensity 100–500 above isotype control.

^bMean fluorescence intensity 50–100 above isotype control.

Cytogenetics

Cytogenetic analysis of the TLBR-1 cell line was performed in collaboration with the City of Hope (Duarte, Calif) (Fig. 5). All mitotic cells analyzed from the TLBR-1 cell line were clonally abnormal with a modal number of 47 chromosomes. The stemline clone showed partial trisomy 2, addition involving 5p, deletion involving 10p, gain of a suspected derivative chromosome 12, monosomy 16 and 20, and gain of 2 marker chromosomes. In addition to the aberrations detected in the stemline clone, 3 minor clonal populations were described. Sidelines 1, 2, and 3 were characterized by additional material of unknown origin on the short arms of chromosomes 16, 15, and 13, respectively. Based upon these karyotypes, TLBR-1 cells have significant chromosomal atypia, although the observed abnormalities are not specific for a particular tumor type. TLBR-1 cells did not show the t(2;5) translocation associated with ALK-NPM fusion and ALK expression, the t(7;9) translocation reported in T-cell lymphoblastic leukemia, or rearrangements involving the T-cell receptor gene loci on chromosomes 7 and 14. TLBR-1 cells also lacked all of those 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).^17,25 These cytogenetic findings were further confirmed by FISH analysis (data not shown).

Figure 5.

Karyotype of TLBR-1. The TLBR-1 stemline population demonstrated partial trisomy 2, deletion of 10p, an unbalanced translocation between chromosomes 12 and 17, and monosomy 16 and 20. Three clonal subpopulations showed additional abnormalities in the form of the addition of unknown genetic material to the short arms of chromosomes 16, 15, and 13 (data not shown).

TCR and IgH Gene Rearrangements Demonstrate Monoclonality

Clinical studies performed on the original tumor in Australia and multiplex PCR analysis of TLBR-1 cells demonstrated TCRγ monoclonality by PCR, suggesting T-cell origin and leading to a diagnosis of T-cell ALCL (Fig. 6A). Seminested PCR for IgH frameworks also revealed TLBR-1 to have IgH rearrangement (Fig. 6B). Reference T-cell lymphoma HUT102 and B-cell lymphoma Raji cell lines were run in parallel as negative and positive controls, respectively.

Figure 6.

PCR studies for TCRγ and IgH gene rearrangements and oncogenic viruses and Western blot analysis of activated Notch1 levels. TLBR-1 demonstrates TCRγ and IgH gene rearrangements by PCR but is negative for HTLV-1 and EBV oncogenic viruses. (A) Multiplex PCR analysis of TCRγ gene rearrangements (product 230 bp). (B) Seminested PCR analysis of IgH gene rearrangement using Fr3a/J_H primers (product 330–350 bp). Lane 1–6: Raji (B-cell lymphoma), HUT102 (T-cell lymphoma), PBMC, TLBR-1, water. (C and D) PCR studies for detection of EBV EBNA (C, product 596 bp) or HTLV-1 of 2 (D, product 150bp) with consensus primers. Lane 1–5: water, Raji (EBV+), negative control cell line, TLBR-1, HUT102 (HTLV+). (E) Notch1 is overexpressed in TLBR-1. Western blot studies on cell lysates demonstrate increased levels of activated (cleaved) Notch1 protein in T-ALCL cell lines TLBR-1 and Karpas 299 (overexpressed) relative to Siha cells (low expression).

Viral screen by PCR for HTLV1 of 2 and EBV

TLBR-1 cells were screened for oncogenic viruses EBV and HTLV-1/2 by PCR but did not show specific amplification for HTLV1/2 polymerase or EBV EBNA2 gene sequences (Fig. 6C and D). Reference T-cell lymphoma HUT102 (known HTLV positive), B-cell lymphoma Raji (known EBV positive), and known negative cell lines were run as controls.

Proto-oncogene expression

The cell line was also tested by qRT-PCR for expression levels of pertinent oncogenes and tumor-suppressor genes. Run in parallel with RNA extracted from the human ALCL Karpas 299 cell line and compared with normal human T-cell and Universal Human Reference RNAs, the overall expression levels of TLBR-1 closely paralleled that of Karpas 299, a documented T-ALCL cell line (data not shown). Statistical analysis revealed no significant differences in expression of p53, Rb, c-myc, or c-kit for TLBR-1 and Karpas 299. Both TLBR-1 and Karpas 299 showed a statistically significant decrease in mean expression of tumor-suppressor genes p53 and Rb relative to normal T cells (P < .05, data not shown). T-ALL associated oncogenes TAL1, HOX11, LYL1 and LMO1/2 were not upregulated in TLBR-1 or Karpas 299 cell lines (data not shown).

Activated Notch1 analysis

Activated Notch1 protein levels were determined by Western blot for TLBR-1 cells relative to over-expressing cell line Karpas 299 and low-expressing cell line Siha. Based upon these studies, TLBR-1 demonstrated an increased level of cleaved, activated, Notch1 protein (Fig. 6E).

DISCUSSION

Although it remains rare, primary breast T-cell ALCL has previously been reported in association with breast implants, with approximately 900 incident cases reported annually.^1–6 Although previous reports have not conclusively shown a positive correlation between the occurrence of primary breast NHL with a specific implant material (silicone-gel or saline), the most recent case series reported by Brody et al⁹ suggests that textured implants using the lost-salt method may pose an increased risk for this malignancy.^7–9 This report describes the clinical presentation of a primary breast lymphoma from this most recent case series and the establishment of a novel CD30⁺ ALK-negative IL-2 dependent T-cell ALCL cell line from the patient’s primary tumor biopsy.

The TLBR-1 cell line was established in culture from tumor-dissociated cells 5 days after removal of the primary lesion from the patient and exhibits similar phenotypic and genotypic characteristics to the primary tumor biopsy specimen. The morphology of TLBR-1 cells was consistent with ALCL by light and transmission electron microscopy studies.^13,26 Of note, the biopsy cells were observed to grow immediately without a latent period when placed on the stromal feeder layer and supplemented with rIL-2. Because of its rapid development in culture, the TLBR-1 cell line was found to be very similar to the primary tumor biopsy and showed both a CD30, CD4/8 coexpressing T-cell population and a cytotoxic phenotype without ALK expression or the associated t(2;5) translocation typically seen in systemic ALCL. Expression of pan-T cell, DC, and activation markers was similar between TLBR-1 and the primary tumor biopsy specimen. Notable differences between the patient tumor and TLBR-1 cells included stronger EMA expression in the primary tumor than in TLBR-1 cells or TLBR-1 SCID heterotransplants and positive CD56 expression on TLBR-1 cells but not in the tumor biopsy specimen. These differences are likely the result of expansion and outgrowth of a strongly neoplastic subclone from the total tumor cell population during establishment of the cell line. Karyotypes of the TLBR-1 cell line and the patient tumor biopsy specimen showed similar features, including trisomy 2, monosomy 20, nonspecific structural changes involving 10p and 5p, and gain of 2 marker chromosomes. Both karyotypes were notable for nuclear atypia as expected in ALCL.^17,25 Oncogene and tumor-suppressor gene analysis of TLBR-1 cells by quantitative RT-PCR demonstrated a profile typical of ALCL, notable for significant overexpression of Notch1.²⁵

The differential diagnosis for ALK-negative ALCL in this patient included primary sa-, pc-, primary systemic, and secondary ALCL.^6,11–13 The primary tumor specimen from the patient was most consistent with a diagnosis of sa-ALCL by tissue histology and immunostaining, although pc-ALCL is a closely related disease with many shared features. pc-ALCL is less likely because this patient did not have evidence of skin involvement to indicate that the implant-associated neoplastic cells were a local spread of pc-ALCL. The indolent clinical course of the disease and the absence of ALK expression favors classification of sa-ALCL over systemic ALCL, and a diagnosis of secondary ALCL was excluded because of the absence of a history of prior malignancy. Table 2 compares the tumor specimen from the patient and the derived cell line TLBR-1 to the characteristic features described in systemic, sa-ALCL, and pc-ALCL.^6,17,25

Table 2.

Comparison of Patient Tumor and TLBR-1 Cell Line to Primary Systemic, Primary Cutaneous, and Seroma-Associated ALCLs

	Primary Systemic ALCL	Primary Cutaneous ALCL	Seroma- Associated ALCL	Primary Tumor Specimen	TLBR-1 Cell Line & SCID Heterotransplant
CD30	+	+	+	+	+
TCRγ monoclonality	+	+	+	+	+
ALK	+ (60–80%)	− (Rarely +)	− (Rarely +)	−	−
t(2;5)	+ (60–80%)	−	−	−	−
EMA	+	−	+	+	Weak
Clinical course	Aggressive	Indolent	Indolent	Indolent	NA

The TLBR-1 cell line appears to be unique as a model of sa-ALCL and will provide investigators with a new tool to study the pathogenesis of this newly characterized implant-associated neoplasm. TLBR-1 cells express several antigens that are reported with moderate frequency in T-cell ALCL tumors but are rarely present in established ALCL cell lines, including high expression of IL-2 receptor chains CD25 and CD122, NK cell marker CD56, and surface antigens HLA-DR, CD80, and CD86.^17,25 PCR analysis of TCRγ and IgH gene rearrangement demonstrate that TLBR-1 cells show monoclonal rearrangement at these loci, which is found in only 10% of ALCL cases.¹⁸ TLBR-1 is also unique in being an ALK-negative T-cell ALCL cell line derived directly from primary tumor tissue. Although ALCL cell lines have been established and characterized, they have been obtained mainly from effusions or solid biopsy materials heterotransplanted into mice rather than from direct cultivation of disaggregated solid tumor biopsy material. ¹⁷ Cell lines derived from heterotransplanted tumors often acquire xenotropic viruses that can change the cellular characteristics of the cell line. A review of ALCL cell lines in the literature suggests that TLBR-1 represents a novel phenotype distinct from other existing cell lines.^17,25 In addition, very few ALCL cell lines are representative of ALK-negative ALCL, Mac-2A being the only reported cell line.^17,25 Furthermore, the observation of activated Notch1 overexpression by TLBR-1 cells highlights a potential therapeutic target for these primary breast lymphomas and other T-cell ALCL.²⁷ This case report is 1 in a larger series of cases reported by Brody et al⁹ suggesting an association between textured breast implants and ALK-negative T-cell ALCL. TLBR-1 represents an important model of this emerging clinical entity and should aid investigations studying its pathogenesis and treatment.