Julie K. Jang

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

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. ©2012 AACR.

MHC Class I Loss is a Frequent Mechanism of Immune Escape in Papillary Thyroid Cancer that is Reversed by Interferon and Selumetinib Treatment in vitro

Purpose: To evaluate MHC class I expression on papillary thyroid cancer (PTC) and analyze changes in MHC expression and associated immune activation with current and experimental treatments for thyroid cancer using in vitro PTC cell lines. Experimental Design: MHC class I expression and assessment of tumor-infiltrating leukocyte populations were evaluated by immunohistochemistry. PTC cell lines were analyzed for HLA-ABC expression by flow cytometry following tyrosine kinase inhibitor, IFNα or IFNγ, or radiation treatment. Functional changes in antigenicity were assessed by coculture of allogeneic donor peripheral blood leukocytes (PBL) with pretreated or untreated PTC cell lines and measurement of T-cell activation and cytokine production. Results: Both MHC class I and β2-microglobulin expression was reduced or absent in 76% of PTC specimens and was associated with reduced tumor-infiltrating immune cells, including effector (CD3+, CD8+, CD16+) and suppressor (FoxP3+) populations. Treatment of PTC cell lines with the MEK1/2 inhibitor selumetinib or IFN increased HLA-ABC expression. This phenotypic change was associated with increased T-cell activation (%CD25+ of CD3+) and IL2 production by PBL cocultured with treated PTC cell lines. Additive effects were seen with combination selumetinib and IFN treatment. Conclusions: MHC class I expression loss is frequent in human PTC specimens and represents a significant mechanism of immune escape. Increased antigenicity following selumetinib and IFN treatment warrants further study for immunotherapy of progressive PTC. Clin Cancer Res; 20(23); 6034–44. ©2014 AACR.

BRAFV600E in Papillary Thyroid Carcinoma Is Associated with Increased Programmed Death Ligand 1 Expression and Suppressive Immune Cell Infiltration

BACKGROUND There remain a small number of patients with papillary thyroid cancer (PTC) who suffer recurrence, metastases, or death. While mutation of the BRAF gene, corresponding to the constitutively active BRAF(V600E) protein, has been associated with worse clinical outcomes in thyroid cancer, the reasons underlying this observation are presently unknown. Disruption of endogenous host immune surveillance and promotion of tumor immune escape is one mechanism by which BRAF(V600E) tumors may achieve more aggressive behavior. This study evaluated the relationship between BRAF(V600E) status and known strategies of tumor-mediated immune suppression. METHODS Tissue sections of PTC tumors from 33 patients were evaluated by immunohistochemistry for tumor-expressed suppressive ligands and enzymes and effector and suppressor populations of tumor-infiltrating immune cells. Presence of BRAF(V600E) was evaluated by direct DNA sequencing of PTC specimens and the results correlated with tumor-expressed molecules and tumor-infiltrating immune cell populations, as well as patient characteristics and pathologic findings. RESULTS BRAF(V600E) tumors more often express high levels of immunosuppressive ligands programmed death ligand 1 (53% vs. 12.5%) and human leukocyte antigen G (41% vs. 12.5%) compared to BRAF wild-type tumors. There was no association between indoleamine 2,3-dioxygenase 1 expression and BRAF(V600E) status. Furthermore, BRAF(V600E) tumors demonstrate both lower CD8(+) effector to FoxP3(+) regulatory T cell, and CD68(+) pan-macrophage to CD163(+) M2 macrophage ratios, indicating relative increases in suppressive T cell and macrophage components, respectively. CONCLUSIONS Overall, BRAF(V600E) PTC tumors display a broadly immunosuppressive profile and evidence of disturbed host tumor immune surveillance that may contribute to the poorer outcomes observed in this subset of patients with thyroid cancer.

Generation of tumor-targeted antibody-CpG conjugates.

A number of monoclonal antibodies against tumor-associated antigens have been developed for the treatment of cancer. The anti-tumor effects of such antibodies can be enhanced by conjugation to immune stimulatory ligands, such as the toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG). The present study describes methods for the conjugation of CpG to two clinically approved monoclonal antibodies (rituximab and trastuzumab) via a Sulfo-EMCS maleimide linker. This conjugation method yielded stable joining of CpG and antibody (molar range 2.2-4.3:1). Immunofluorescence studies showed intact antigen-specific antibody binding of the immunoconjugates, that were comparable to unmodified antibody. Furthermore, antibody-CpG conjugates demonstrated improved (rituximab) or equivalent (trastuzumab) immune stimulatory activity compared to free CpG in vitro. These studies demonstrate the feasibility of antibody-CpG immunoconjugates and provide the foundation for future in vivo immunotherapy evaluation.

Phase 1 Dose-Escalation Study with LEC/Chtnt-3 and Toceranib Phosphate (Palladia) in Dogs with Spontaneous Malignancies

OBJECTIVES LEC chemokine promotes TH1 responses and recruits immune cells to inflammatory sites. By linking LEC to an antibody targeting tumor necrosis, LEC/chTNT-3 can be used for the immunotherapeutic treatment of tumors. The primary objective of this study was to determine the safety profile of LEC/chTNT-3 and toceranib phosphate (Palladia®) combination therapy in dogs with spontaneous malignancies. Secondary purpose was to determine objective responses to treatment. METHODS Twenty-three dogs with cancer were enrolled, covering nine different malignancies. In this dose escalation study, dogs received LEC/chTNT-3 for five days, and toceranib every 48 hours for the remainder of the study. Dogs received physical exams, chemistry panel, urinalysis, and complete blood counts on days 0, 10, 28 of the study, and every 6-8 weeks thereafter. RESULTS Lethargy was noted in 13% dogs. There were no statistical differences in the prevalence of anorexia, diarrhea, thrombocytopenia, renal toxicity, or hepatic toxicity before or during the study. There were trends in increases in the prevalence of vomiting, lymphopenia, and neutropenia (all grade 2 or lower, p=0.07) over the initial 28 days of the study. By day 28, 10% of dogs had partial responses, 58% had stable disease, and 32% had progressive disease. CONCLUSIONS LEC/chTNT-3 and toceranib were well tolerated. This combination therapy showed some biological activity against a variety of cancers at a low dose and short duration of LEC/chTNT-3 administration.

Abstract 1539: Antibody targeted CpG for the immunotherapy of solid tumors

A method is described to improve the therapeutic effects of unmethylated CpG oligodeoxynucleotides (ODNs) analogues, which can be used to stimulate the innate immune system for the treatment of cancer. The study is based on the generation of novel reagents that can be used to target tumors after linkage of CpG ODNs analogues to tumor-targeting monoclonal antibodies. This approach is novel in concept and may yield important information regarding the effectiveness of innate immunity in the treatment of cancer. CpG has been used primarily as an adjuvant injected locally at the site of interest or mixed with tumor vaccines. For systemic diseases such as cancer, however, the local administration of CpG would limit its potential to the site of injection. To address this, we have chemically linked active CpG motif analogues to chTNT-3, a monoclonal antibody which targets tumor necrosis, a site rich in tumor antigens. Our findings validate the usefulness of this novel approach and show that, compared to the parental antibody, the CpG immunoconjugate is able to target tumor and produce a 50% greater reduction in tumor growth. It is anticipated that the availability of antibody targeted CpG will enable research to optimize the effectiveness of this approach, especially when combined with other forms of immunotherapy, for the successful treatment of solid tumors. Additional studies using CpG motif analogues conjugated to rituximab (anti-CD20) and trastuzumab (anti-HER2/neu) are underway to demonstrate the full potential of this approach in tumors positive for these common biomarkers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1539. doi:1538-7445.AM2012-1539