SPATA2 and CYLD inhibit T cell infiltration into colorectal cancer via regulation of IFN-γ/STAT1 axis.

Introduction: Colorectal cancer (CRC) is largely refractory to currently available immunotherapies such as blockade of programmed cell death protein-1 (PD-1). Results: In this study, we identified SPATA2 and its protein partner CYLD as novel regulators of CXC-ligand 10 (CXCL10), a T-cell-attractant chemokine, in CRC. By specifically deleting SPATA2 and CYLD in human and mouse CRC cell lines, we showed that these two proteins inhibit STAT1 accumulation and activation and subsequently CXCL10 expression in tumor cells. At steady-state, STAT1 is highly ubiquitinated in a SPATA2/CYLD-dependent manner. Finally, we demonstrated that tumor-specific deletion of SPATA2 and CYLD enhances anti-PD-1 response in vivo. Discussion: Our data suggest that SPATA2 and CYLD represent two potential novel targets for treatment of immune-excluded, PD-1-resistant tumors.

Preclinical characterization and clinical translation of pharmacodynamic markers for MK-5890: a human CD27 activating antibody for cancer immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI therapy. CD27 is a member of the tumor necrosis factor receptor superfamily that plays a critical role in promoting responses of T cells, B cells and NK cells. METHODS: Anti-CD27 antibodies were generated and selected for agonist activity using NF-кB luciferase reporter assays. Antibodies were humanized and characterized for agonism using in vitro T-cell proliferation assays. The epitope recognized on CD27 by MK-5890 was established by X-ray crystallography. Anti-tumor activity was evaluated in a human CD27 knock-in mouse. Preclinical safety was tested in rhesus monkeys. Pharmacodynamic properties were examined in mouse, rhesus monkeys and a phase 1 dose escalation clinical study in patients with cancer. RESULTS: Humanized anti-CD27 antibody MK-5890 (hIgG1) was shown to bind human CD27 on the cell surface with sub-nanomolar potency and to partially block binding to its ligand, CD70. Crystallization studies revealed that MK-5890 binds to a unique epitope in the cysteine-rich domain 1 (CRD1). MK-5890 activated CD27 expressed on 293T NF-κB luciferase reporter cells and, conditional on CD3 stimulation, in purified CD8+ T cells without the requirement of crosslinking. Functional Fc-receptor interaction was required to activate CD8+ T cells in an ex vivo tumor explant system and to induce antitumor efficacy in syngeneic murine subcutaneous tumor models. MK-5890 had monotherapy efficacy in these models and enhanced efficacy of PD-1 blockade. MK-5890 reduced in an isotype-dependent and dose-dependent manner circulating, but not tumor-infiltrating T-cell numbers in these mouse models. In rhesus monkey and human patients, reduction in circulating T cells was transient and less pronounced than in mouse. MK-5890 induced transient elevation of chemokines MCP-1, MIP-1α, and MIP-1ß in the serum of mice, rhesus monkeys and patients with cancer. MK-5890 was well tolerated in rhesus monkeys and systemic exposure to MK-5890 was associated with CD27 occupancy at all doses. CONCLUSIONS: MK-5890 is a novel CD27 agonistic antibody with the potential to complement the activity of PD-1 checkpoint inhibition in cancer immunotherapy and is currently undergoing clinical evaluation.

Reverse Translating Molecular Determinants of Anti-Programmed Death 1 Immunotherapy Response in Mouse Syngeneic Tumor Models.

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFß biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Combination of EP4 antagonist MF-766 and anti-PD-1 promotes anti-tumor efficacy by modulating both lymphocytes and myeloid cells.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2-mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

Biophysical and Immunological Characterization and In Vivo Pharmacokinetics and Toxicology in Nonhuman Primates of the Anti-PD-1 Antibody Pembrolizumab.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

Characterization of murine CEACAM1 in vivo reveals low expression on CD8+ T cells and no tumor growth modulating activity by anti-CEACAM1 mAb CC1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been reported to mediate both tumorigenic and anti-tumor effects in vivo. Blockade of the CEACAM1 signaling pathway has recently been implicated as a novel mechanism for cancer immunotherapy. CC1, a mouse anti-CEACAM1 monoclonal antibody (mAb), has been widely used as a pharmacological tool in preclinical studies to inform on CEACAM1 pathway biology although limited data are available on its CEACAM1 blocking characteristics or pharmacodynamic-pharmacokinetic profiles. We sought to investigate CEACAM1 expression on mouse tumor and immune cells, characterize CC1 mAb binding, and evaluate CC1 in syngeneic mouse oncology models as a monotherapy and in combination with an anti-PD-1 mAb. CEACAM1 expression was observed at high levels on neutrophils, NK cells and myeloid-derived suppressor cells (MDSCs), while the expression on tumor-infiltrating CD8+ T cells was low. Unexpectedly, rather than blocking, CC1 facilitated binding of soluble CEACAM1 to CEACAM1 expressing cells. No anti-tumor effects were observed in CT26, MBT2 or A20 models when tested up to 30 mg/kg dose, a dose that was estimated to achieve >90% target engagement in vivo. Taken together, tumor infiltrating CD8+ T cells express low levels of CEACAM1 and CC1 Ab mediates no or minimal anti-tumor effects in vivo, as a monotherapy or in combination with anti-PD-1 treatment.

PD-L1 in tumor microenvironment mediates resistance to oncolytic immunotherapy.

Intralesional therapy with oncolytic viruses (OVs) leads to the activation of local and systemic immune pathways, which may present targets for further combinatorial therapies. Here, we used human tumor histocultures as well as syngeneic tumor models treated with Newcastle disease virus (NDV) to identify a range of immune targets upregulated with OV treatment. Despite tumor infiltration of effector T lymphocytes in response to NDV, there was ongoing inhibition through programmed death ligand 1 (PD-L1), acting as a mechanism of early and late adaptive immune resistance to the type I IFN response and T cell infiltration, respectively. Systemic therapeutic targeting of programmed cell death receptor 1 (PD-1) or PD-L1 in combination with intratumoral NDV resulted in the rejection of both treated and distant tumors. These findings have implications for the timing of PD-1/PD-L1 blockade in conjunction with OV therapy and highlight the importance of understanding the adaptive mechanisms of immune resistance to specific OVs for the rational design of combinatorial approaches using these agents.

Multiplexed quantification of proteins and transcripts in single cells.

We present a tool to measure gene and protein expression levels in single cells with DNA-labeled antibodies and droplet microfluidics. Using the RNA expression and protein sequencing assay (REAP-seq), we quantified proteins with 82 barcoded antibodies and >20,000 genes in a single workflow. We used REAP-seq to assess the costimulatory effects of a CD27 agonist on human CD8+ lymphocytes and to identify and characterize an unknown cell type.

Characterization of MK-4166, a Clinical Agonistic Antibody That Targets Human GITR and Inhibits the Generation and Suppressive Effects of T Regulatory Cells.

GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregsin vitro In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378-88. ©2017 AACR.

A colitogenic memory CD4+ T cell population mediates gastrointestinal graft-versus-host disease.

Damage to the gastrointestinal tract is a major cause of morbidity and mortality in graft-versus-host disease (GVHD) and is attributable to T cell-mediated inflammation. In this work, we identified a unique CD4+ T cell population that constitutively expresses the ß2 integrin CD11c and displays a biased central memory phenotype and memory T cell transcriptional profile, innate-like properties, and increased expression of the gut-homing molecules α4ß7 and CCR9. Using several complementary murine GVHD models, we determined that adoptive transfer and early accumulation of ß2 integrin-expressing CD4+ T cells in the gastrointestinal tract initiated Th1-mediated proinflammatory cytokine production, augmented pathological damage in the colon, and increased mortality. The pathogenic effect of this CD4+ T cell population critically depended on coexpression of the IL-23 receptor, which was required for maximal inflammatory effects. Non-Foxp3-expressing CD4+ T cells produced IL-10, which regulated colonic inflammation and attenuated lethality in the absence of functional CD4+Foxp3+ T cells. Thus, the coordinate expression of CD11c and the IL-23 receptor defines an IL-10-regulated, colitogenic memory CD4+ T cell subset that is poised to initiate inflammation when there is loss of tolerance and breakdown of mucosal barriers.

Interleukin-23-Induced Transcription Factor Blimp-1 Promotes Pathogenicity of T Helper 17 Cells.

Interleukin-23 (IL-23) is a pro-inflammatory cytokine required for the pathogenicity of T helper 17 (Th17) cells but the molecular mechanisms governing this process remain unclear. We identified the transcription factor Blimp-1 (Prdm1) as a key IL-23-induced factor that drove the inflammatory function of Th17 cells. In contrast to thymic deletion of Blimp-1, which causes T cell development defects and spontaneous autoimmunity, peripheral deletion of this transcription factor resulted in reduced Th17 activation and reduced severity of autoimmune encephalomyelitis. Furthermore, genome-wide occupancy and overexpression studies in Th17 cells revealed that Blimp-1 co-localized with transcription factors RORγt, STAT-3, and p300 at the Il23r, Il17a/f, and Csf2 cytokine loci to enhance their expression. Blimp-1 also directly bound to and repressed cytokine loci Il2 and Bcl6. Taken together, our results demonstrate that Blimp-1 is an essential transcription factor downstream of IL-23 that acts in concert with RORγt to activate the Th17 inflammatory program.

TSLP is differentially regulated by vitamin D3 and cytokines in human skin.

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic diseases and is highly expressed in keratinocytes in human lesional atopic dermatitis (AD) skin. In nonlesional AD skin TSLP expression can be induced by applying house dust mite allergen onto the skin in the atopy patch test. Several studies have demonstrated that the induction of TSLP expression in mouse skin does not only lead to AD-like inflammation of the skin, but also predisposes to severe inflammation of the airways. In mice, TSLP expression can be induced by application of the 1,25-dihydroxyvitamin D3 (VD3) analogue calcipotriol and results in the development of eczema-like lesions. The objective is to investigate the effect of VD3 (calcitriol) or calcipotriol on TSLP expression in normal human skin and skin from AD patients. Using multiple ex vivo experimental setups, the effects of calci(po)triol on TSLP expression by normal human skin, and skin from AD patients were investigated and compared to effects of calcipotriol on mouse and non-human primates (NHP) skin. No induction of TSLP expression (mRNA or protein) was observed in human keratinocytes, normal human skin, nonlesional AD skin, or NHP skin samples after stimulation with calcipotriol or topical application of calcitriol. The biological activity of calci(po)triol in human skin samples was demonstrated by the increased expression of the VD3-responsive Cyp24a1 gene. TSLP expression was induced by cytokines (IL-4, IL-13, and TNF-α) in skin samples from all three species. In contrast to the findings in human and NHP, a consistent increase in TSLP expression was confirmed in mouse skin biopsies after stimulation with calcipotriol. VD3 failed to induce expression of TSLP in human or monkey skin in contrast to mouse, implicating careful extrapolation of this often-used mouse model to AD patients.

Histone H3 Phosphorylation in Human Skin Histoculture as a Tool to Evaluate Patient's Response to Antiproliferative Drugs.

Evaluation of patient's response to chemotherapeutic drugs is often difficult and time consuming. Skin punch biopsies are easily accessible material that can be used for the evaluation of surrogate biomarkers of a patient's response to a drug. In this study, we hypothesized that assessment of phosphorylated histone H3 in human skin punch biopsies could be used as a pharmacodynamics biomarker of patient's response to the kinesin spindle protein inhibitor SCH2047069. To test this hypothesis, we used a human skin histoculture technique that allows culturing intact human skin in the presence of the drug. Human melanoma and skin histocultures were treated with SCH2047069, and the effect of the drug was assessed by increasing histone H3 phosphorylation using immunohistochemistry. Our results demonstrate that SCH2047069 has a significant effect on cell proliferation in human melanoma and skin histoculture and justify using human skin punch biopsies for evaluation of the pharmacodynamic changes induced by SCH2047069. ACRONYMS: Histone subunit H3 (H3), Kinesin spindle protein (KSP), 5-ethynyl-2'-deoxyuridine (EDU), Dimethyl sulfoxide (DMSO), Formalin-fixed paraffin embedded (FFPE).

Autoimmune memory T helper 17 cell function and expansion are dependent on interleukin-23.

Interleukin-23 (IL-23) is essential for the differentiation of pathogenic effector T helper 17 (Th17) cells, but its role in memory Th17 cell responses is unclear. Using the experimental autoimmune encephalomyelitis (EAE) model, we report that memory Th17 cells rapidly expanded in response to rechallenge and migrated to the CNS in high numbers, resulting in earlier onset and increased severity of clinical disease. Memory Th17 cells were generated from IL-17+ and RORγt+ precursors, and the stability of the Th17 cell phenotype depended on the amount of time allowed for the primary response. IL-23 was required for this enhanced recall response. IL-23 receptor blockade did not directly impact IL-17 production, but did impair the subsequent proliferation and generation of effectors coexpressing the Th1 cell-specific transcription factor T-bet. In addition, many genes required for cell-cycle progression were downregulated in Th17 cells that lacked IL-23 signaling, showing that a major mechanism for IL-23 in primary and memory Th17 cell responses operates via regulation of proliferation-associated pathways.

Gene-expression profiling of microdissected breast cancer microvasculature identifies distinct tumor vascular subtypes.

INTRODUCTION: Angiogenesis represents a potential therapeutic target in breast cancer. However, responses to targeted antiangiogenic therapies have been reported to vary among patients. This suggests that the tumor vasculature may be heterogeneous and that an appropriate choice of treatment would require an understanding of these differences. METHODS: To investigate whether and how the breast tumor vasculature varies between individuals, we isolated tumor-associated and matched normal vasculature from 17 breast carcinomas by laser-capture microdissection, and generated gene-expression profiles. Because microvessel density has previously been associated with disease course, tumors with low (n = 9) or high (n = 8) microvessel density were selected for analysis to maximize heterogeneity for this feature. RESULTS: We identified differences between tumor and normal vasculature, and we describe two subtypes present within tumor vasculature. These subtypes exhibit distinct gene-expression signatures that reflect features including hallmarks of vessel maturity. Potential therapeutic targets (MET, ITGAV, and PDGFRß) are differentially expressed between subtypes. Taking these subtypes into account has allowed us to derive a vascular signature associated with disease outcome. CONCLUSIONS: Our results further support a role for tumor microvasculature in determining disease progression. Overall, this study provides a deeper molecular understanding of the heterogeneity existing within the breast tumor vasculature and opens new avenues toward the improved design and targeting of antiangiogenic therapies.

Human Th17 cells comprise heterogeneous subsets including IFN-gamma-producing cells with distinct properties from the Th1 lineage.

Th17 cells have been named after their signature cytokine IL-17 and accumulating evidence indicates their involvement in the induction and progression of inflammatory diseases. In addition to IL-17 single-producing T cells, IL-17/IFN-gamma double-positive T cells are found in significantly elevated numbers in inflamed tissues or blood from patients with chronic inflammatory disorders. Because IFN-gamma is the classical Th1-associated cytokine, the origin and roles of these subsets remain elusive. In this paper, we show that not only IL-17(+)/IFN-gamma(+) but also IFN-gamma(+) (IL-17(-)) cells arise under Th17-inducing condition and have distinct properties from the Th1 lineage. In fact, these populations displayed characteristics reminiscent to IL-17 single-producing cells, including production of IL-22, CCL20, and induction of antimicrobial gene expression from epithelial cells. Live sorted IL-17(+) and Th17-IFN-gamma(+) cells retained expression of IL-17 or IFN-gamma after culture, respectively, whereas the IL-17(+)/IFN-gamma(+) population was less stable and could also become IL-17 or IFN-gamma single-producing cells. Interestingly, these Th17 subsets became "Th1-like" cells in the presence of IL-12. These results provide novel insights into the relationship and functionality of the Th17 and Th1 subsets and have direct implications for the analysis and relevance of IL-17 and/or IFN-gamma-producing T cells present in patients' peripheral blood and inflamed tissues.

Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation.

The C-type lectin-like receptor CD161, which has recently been described to promote T cell expansion, is expressed on a discrete subset of human CD4 T cells. The function of such cells, however, has remained elusive. We now demonstrate that CD161(+) CD4 T cells comprise a circulating and gut-resident T helper 17 (Th17) cell population. During Crohn's disease (CD), these CD161(+) cells display an activated Th17 phenotype, as indicated by increased expression of interleukin (IL)-17, IL-22, and IL-23 receptor. CD161(+) CD4 T cells from CD patients readily produce IL-17 and interferon gamma upon stimulation with IL-23, whereas, in healthy subjects, priming by additional inflammatory stimuli such as IL-1beta was required to enable IL-23-induced cytokine release. Circulating CD161(+) Th17 cells are imprinted for gut homing, as indicated by high levels of CC chemokine receptor 6 and integrin beta7 expression. Supporting their colitogenic phenotype, CD161(+) Th17 cells were found in increased numbers in the inflammatory infiltrate of CD lesions and induced expression of inflammatory mediators by intestinal cells. Our data identify CD161(+) CD4 T cells as a resting Th17 pool that can be activated by IL-23 and mediate destructive tissue inflammation.

Stromal gene expression predicts clinical outcome in breast cancer.

Although it is increasingly evident that cancer is influenced by signals emanating from tumor stroma, little is known regarding how changes in stromal gene expression affect epithelial tumor progression. We used laser capture microdissection to compare gene expression profiles of tumor stroma from 53 primary breast tumors and derived signatures strongly associated with clinical outcome. We present a new stroma-derived prognostic predictor (SDPP) that stratifies disease outcome independently of standard clinical prognostic factors and published expression-based predictors. The SDPP predicts outcome in several published whole tumor-derived expression data sets, identifies poor-outcome individuals from multiple clinical subtypes, including lymph node-negative tumors, and shows increased accuracy with respect to previously published predictors, especially for HER2-positive tumors. Prognostic power increases substantially when the predictor is combined with existing outcome predictors. Genes represented in the SDPP reveal the strong prognostic capacity of differential immune responses as well as angiogenic and hypoxic responses, highlighting the importance of stromal biology in tumor progression.

Gene expression signatures of morphologically normal breast tissue identify basal-like tumors.

INTRODUCTION: The role of the cellular microenvironment in breast tumorigenesis has become an important research area. However, little is known about gene expression in histologically normal tissue adjacent to breast tumor, if this is influenced by the tumor, and how this compares with non-tumor-bearing breast tissue. METHODS: To address this, we have generated gene expression profiles of morphologically normal epithelial and stromal tissue, isolated using laser capture microdissection, from patients with breast cancer or undergoing breast reduction mammoplasty (n = 44). RESULTS: Based on this data, we determined that morphologically normal epithelium and stroma exhibited distinct expression profiles, but molecular signatures that distinguished breast reduction tissue from tumor-adjacent normal tissue were absent. Stroma isolated from morphologically normal ducts adjacent to tumor tissue contained two distinct expression profiles that correlated with stromal cellularity, and shared similarities with soft tissue tumors with favorable outcome. Adjacent normal epithelium and stroma from breast cancer patients showed no significant association between expression profiles and standard clinical characteristics, but did cluster ER/PR/HER2-negative breast cancers with basal-like subtype expression profiles with poor prognosis. CONCLUSION: Our data reveal that morphologically normal tissue adjacent to breast carcinomas has not undergone significant gene expression changes when compared to breast reduction tissue, and provide an important gene expression dataset for comparative studies of tumor expression profiles.