Key Survival Pathways and Chemotherapeutic Sensitivity of Breast Implant Associated Anaplastic Large Cell Lymphomas,

Abstract 3739

Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) is of B-cell origin. Recently, a series of anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide in patients with saline and silicone breast implants (Lechner et al. Cancer 2010, Lazzeri D et al. Clin Breast Cancer 2011, Carty et al. Plast Reconstr Surg 2011, Popplewell et al. Leuk Lymphoma 2011, Roden et al. Mod Pathol 2008, de Jong et al. JAMA 2008, and others). These cases are striking for their homogeneous clinical presentation and pathology. Histological and cytogenetic analyses of the primary tumor biopsy specimens consistently demonstrates CD30⁺ EMA⁽⁺⁾ PAX5⁻ lymphoma cells with significant chromosomal atypia that lack the NPM-ALK (2;5) translocation, express pan T-cell markers, and show monoclonal TCR gene rearrangements. Increasing evidence suggests that these seroma-associated ALK⁻ ALCLs are a distinct clinicopathologic entity from either systemic ALK⁻ ALCL or cutaneous ALCL, and while the majority of cases are indolent, several patients have died from this disease.

In order to understand the nature of this newly emerging clinical entity, we have established three new cell lines, (TLBR-1, TLBR-2, TLBR-3,) from the primary tumor biopsy specimens of three patients diagnosed with breast implant-associated ALCL. Characterization of these pre-clinical models confirmed fidelity to the original tumor biopsy specimens and highlighted unique features that may aid the development of effective treatments for these cancers. Cytogenetics and FISH performed on TLBR-1, -2, and -3 revealed nuclear atypia with partial or complete trisomy (modal chromosome number 47, 76, and 81, respectively) and absence of the NPM-ALK (2;5) translocation. Phenotypic characterization of TLBR cell lines by flow cytometry and immunocytochemistry showed strong positivity for CD30, CD71, pan T cell CD2/5/7, natural killer (CD56), and antigen presentation (HLA-DR, CD80, CD86) markers, and IL-2 receptors (CD25, CD122). Multiplex or consensus sequence PCR confirmed monoclonal TCR gene rearrangements and showed no onco-virus incorporation (EBV, HTLV1/2), respectively, for TLBR-1, -2, and -3.

To identify effective therapies for patients with breast-implant associated ALCL, in vitro studies were used to evaluate the mechanisms by which TLBR evades immune detection and promotes cell cycle dysregulation. Gene expression analysis for proto-oncogenes, tumor suppressor genes, and regulators of apoptosis demonstrated significant up-regulation of survivin and down-regulation of pro-apoptotic genes (BID, BAK, BBC3) by all TLBR cell lines relative to healthy donor T cells. Interestingly, TLBR cell lines shared many features with human regulatory T cells, including phenotype (CD4⁺/8⁺, CD25⁺), high FoxP3 expression, up-regulation of immunosuppressive cytokines IL-10 and TGFβ, and suppression of T cell responses to stimuli. Inhibitor studies were used to identify critical signaling pathways in survival and proliferation of breast implant-associated ALCL models TLBR-1, -2, and -3. Data from these studies revealed that inhibition of the JAK/STAT, PI3K/mTOR, and WNT/beta-catenin signaling pathways resulted in a substantial increase in cell death in the TLBR cell lines. Significantly, TLBR-2, which was derived from a fatal breast implant associated ALCL, showed increased resistance to all inhibitors compared with TLBR-1 and TLBR-3, which were derived from more indolent cases. Analysis of protein lysates showed elevated cleaved Notch1 in TLBR-2, compared with cell lines TLBR-1 and -3, which may drive increased cell proliferation and resistance to apoptotic regulators. Since the treatment of these lymphomas has not been studied, we evaluated the sensitivity of the TLBR cell lines to mainstay chemotherapy regimens (CHOP, methotrexate). These studies found that doxorubicin and vinblastine exert potent cytotoxic effects against all three TLBR cell lines, whereas cyclophosphamide was largely ineffective. In summary, TLBR-1, -2, and -3 closely resemble the primary breast implant-associated lymphomas from which they were derived, and as such provide valuable preclinical models to study the cell of origin and biology of this newly emerging clinical entity.