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BACKGROUND: Excess body fatness and hyperinsulinemia are both associated with an increased risk of postmenopausal breast cancer. However, whether women with high body fatness but normal insulin levels or those with normal body fatness and high levels of insulin are at elevated risk of breast cancer is not known. We investigated the associations of metabolically defined body size and shape phenotypes with the risk of postmenopausal breast cancer in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition.METHODS: Concentrations of C-peptide-a marker for insulin secretion-were measured at inclusion prior to cancer diagnosis in serum from 610 incident postmenopausal breast cancer cases and 1130 matched controls. C-peptide concentrations among the control participants were used to define metabolically healthy (MH; in first tertile) and metabolically unhealthy (MU; >1st tertile) status. We created four metabolic health/body size phenotype categories by combining the metabolic health definitions with normal weight (NW; BMI RESULTS: Women classified as MUOW/OB were at higher risk of postmenopausal breast cancer compared to MHNW women considering BMI (OR = 1.58, 95% CI = 1.14-2.19) and WC (OR = 1.51, 95% CI = 1.09-2.08) cut points and there was also a suggestive increased risk for the WHR (OR = 1.29, 95% CI = 0.94-1.77) definition. Conversely, women with the MHOW/OB and MUNW were not at statistically significant elevated risk of postmenopausal breast cancer risk compared to MHNW women.CONCLUSION: These findings suggest that being overweight or obese and metabolically unhealthy raises risk of postmenopausal breast cancer while overweight or obese women with normal insulin levels are not at higher risk. Additional research should consider the combined utility of anthropometric measures with metabolic parameters in predicting breast cancer risk.

Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) are more aggressive than other breast cancer subtypes, and up until recently lacked therapy options that maintain acceptable quality of life. While chemotherapy is a treatment option for all subtypes of breast cancer, it is used long-term as maintenance therapy for metastatic TNBC (mTNBC) and often produces cumulative toxicity resulting in discontinuation of treatment. Moreover, responses are rarely durable after discontinuation of chemotherapy. Immune checkpoint blockade has the potential to maintain therapy. To determine factors associated with response to anti-PD-1 therapy (pembrolizumab), we performed minimally invasive blood-based analyses of T-cell repertoire and phenotype to evaluate their association with progression-free survival (PFS) in 15 patients with metastatic IBC (mIBC) or mTNBC. Materials and Methods: Fifteen patients with mIBC (n=6) or mTNBC (n=9) were enrolled on an ongoing phase II study to receive pembrolizumab as maintenance therapy after achieving a clinical response or stable disease to systemic chemotherapy for metastatic disease. We performed analyses of T-cell repertoire and phenotype on peripheral blood mononuclear cells (PBMC) from samples obtained post induction therapy but before initiation of pembrolizumab (baseline) therapy. Expression of T-cell exhaustion markers 2B4, CTLA4, BTLA, Lag3, PD-1, and Tim3 was evaluated on PBMC by flow cytometry. T-cell receptor (TCR) beta chain CDR3 DNA sequencing by ImmunoSEQ and richness (diversity) and clonality (reactivity) were evaluated. Patients were followed through 5 months of treatment with pembrolizumab to evaluate the association between T-cell repertoire and phenotype with PFS. Results: Seven patients had stable disease (SD) and 8 had progressive disease (PD) by 5 months. The median follow-up was 14.2 months (range: 4.5 to 27.7 months). Among the patients who progressed within 5 months, the earliest and the latest time to progression was at 1.38 months and 4.82 months, respectively. CTLA4 expression in CD4+ T cells at baseline was significantly higher in patients with PD than in patients with SD (p = 0.040). Patients with a low percentage of CD4+ T cells expressing exhaustion markers (CTLA4, Tim3, and 2B4) at baseline were more likely to have SD (chi-square p = 0.041) and significantly longer median PFS than patients with PD (median time to progression was not reached in SD vs. 4.1 months in PD, p = 0.018). Additionally, patients with high clonality and low CD4+ T exhaustion markers were more likely to have SD (chi-square p = 0.041) and a longer median time to progression (median time to progression was not reached in SD vs. 4.1 months in PD, p = 0.015). Conclusions: Baseline T-cell clonality and T-cell exhaustion markers have significant translational relevance and can help to explain variable responses to immune checkpoint blockade. Our data suggest that T-cell reactivity at baseline, and a lower percentage of CD4+ T cells expressing CTLA4/Tim3/2B4 were favorable prognostic factors for pembrolizumab maintenance therapy. This proof-of-concept provides compelling data that T-cell clonality and phenotyping of T-cell exhaustion markers can be useful and should be applied to future trials with immune checkpoint inhibitors. Citation Format: Hui Gao, Kumiko Kida, Evan N Cohen, Angela Alexander, Bora Lim, Charla Parker, Sanda Tin, Vicente Valero, Debu Tripathy, Alexandre Reuben, Naoto T Ueno, James M Reuben. Peripheral T cell clonality and exhaustion as novel biomarkers for anti-PD-1 (pembrolizumab) maintenance therapy in patients with metastatic inflammatory breast cancer (mIBC) and non-IBC triple negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-12.

PURPOSE: The carbohydrate Sialyl Lewis(X) (sLe(X)) mediates cell adhesion, is critical in the normal function of immune cells, and is frequently over-expressed on cancer cells. We assessed the association, differential levels, and prognostic value of sLe(X) and inflammatory cytokines/chemokines in breast cancer sera. METHODS: We retrospectively measured sLe(X) and a panel of cytokines/chemokines in the sera of 26 non-invasive ductal carcinoma in situ (DCIS), 154 invasive non-metastatic breast cancer (non-MBC), 63 metastatic breast cancer (MBC) patients, and 43 healthy controls. Differences in sLe(X) and inflammatory cytokines among and between patient groups and healthy controls were assessed with nonparametric tests and we performed survival analysis for the prognostic potential of sLe(X) using a cut-off of 8 U/ml as previously defined. RESULTS: Median serum sLe(X) was significantly higher than controls for invasive breast cancer patients (MBC and non-MBC) but not DCIS. In univariate analysis, we confirmed patients with serum sLe(X) >8 U/ml have a significantly shorter progression free survival (PFS) (P=0.0074) and overall survival (OS (P=0.0003). Similarly, patients with high serum MCP-1 and IP-10 had shorter OS (P=0.001 and P

Purpose To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. Methods and Materials The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an “antitumor” (M1) or a “protumor” (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. Results Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. Conclusions These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.

Background:Circulating tumor cells (CTCs) are an independent predictor of survival in patients with breast cancer. In addition, mesenchymal (EMT-CTC) and stem-like (Stem-CTC) CTCs contribute to disease progression. The objective of the overall study is to determine whether a comprehensive lifestyle intervention program started prior to radiotherapy can modulate changes in CTC subsets that are correlated with disease recurrence and progression. For these analyses we examined the association between medical and treatment-related factors and CTCs. Patients and Methods: Seventy-eight patients with stage II/III breast cancer were recruited and randomized to either the intervention group or a standard care group. The intervention group (n=42) had in-person lifestyle counseling across the 4-6 weeks of radiotherapy (XRT) followed by video counseling for the subsequent 12 months. The standard care group (n=36) was provided patient-education materials for cancer prevention including information on diet, exercise, and stress management, without counseling. Blood samples were collected prior to initiation of XRT, end of XRT, and at 3-month intervals thereafter for up to 5 years. CTC subsets were detected by AdnaTest EMT2 kit (Qiagen, Venlo, Netherlands). Samples were considered positive for CTCs if any one of breast (EPCAM, MUC1, and HER2), EMT (TWIST1), or stem cell-related (ALDH1, AKT2, and PI3Kalpha) genes were detected by PCR above the manufacturer's suggested threshold. Results: The median age of patients was 49 years (range 26-82 years). Thirty-four patients were overweight (BMI 24.4-30) and 44 patients were obese (BMI >30). Forty-five patients were HR+Her2-, 12 patients were HR+Her2+, 5 patients were HR-Her2+, and 16 patients were TNBC. Sixteen patients were stage IIA or IIB, 34 patients were stage IIIA or IIIB, 27 patients were stage IIIC, and 1 was stage IV. Sixty-seven of 78 patients received neoadjuvant chemotherapy (NACT); 13 patients achieved a complete pathological response (pCR). The median follow-up was 21.6 months. CTC data of both intervention and standard groups were similar at baseline. Presence of CTCs at baseline or follow-up time points was not correlated to HR/Her2 status, stage, obesity, or pCR, but was significantly correlated with receiving NACT. Patients without NACT had significantly higher CTCs than patients who underwent NACT (Fisher Exact Test p=0.010). Furthermore, CTCs by the detection of any gene 3 months after completing XRT was associated with shorter PFS (log-rank p=0.016) and OS (p=0.03). Conclusions:This is an interim analysis of the prognostic potential of CTCs detected by AdnaTest EMT2 kit in non-metastatic breast cancer. We observed a lower proportion of patients with CTCs following neoadjuvant chemotherapy. However, the relative small sample size and short follow-up time preclude drawing conclusions to the efficacy of using CTCs as surrogate measures for lifestyle interventions, although the presence of CTCs in peripheral blood of patients 3 months after radiation therapy can be a promising indicator of disease relapse and overall survival. Citation Format: Gao H, Cohen EN, Yang P, Austin TA, Haddad R, Wu Q, Basen-Engquist KM, Ochoa JM, Arun BK, Perkins GH, Tin S, Vallone VS, Mallaiah SG, West CB, Thompson AS, Chaoul A, Cohen L, Reuben JM. Circulating tumor cell subset analysis to assess lifestyle interventions for breast cancer patients after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-15.

BACKGROUND AND RATIONALE Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer and patients frequently present with metastases at the time of their diagnosis. Although a robust IBC-specific molecular signature remains elusive, the disease is frequently characterized by persistent expression of the adhesion molecule, E-cadherin. This is highly counterintuitive as epithelial to mesenchymal transition (EMT), frequently associated with metastasis, results in decreased E-cadherin expression and highly aggressive cancers frequently express low levels of E-cadherin. We hypothesized that persistent inflammation, mediated by immune activation, increases the plasticity of IBC cells, inducing EMT and allowing the re-acquisition of epithelia characteristics once removed from the inflammatory foci. In support of this hypothesis, previous in vitro work showed that soluble factors from activated immune cells induce EMT-related transcripts in both IBC and non-IBC cell lines. However, uniquely in 3 of 4 IBC cell lines but none of the non-IBC cell lines, this program included an increase of E-cadherin expression. RESULTS We used real-time cell analysis (RTCA) from Acea Biosciences (San Diego, CA) to probe the effect of immune conditioned media, produced by stimulating healthy donor peripheral blood mononuclear cells through the T-cell receptor or through toll-like receptor-4, on SUM149 inflammatory breast cancer cells. Consistent with the increased expression of E-cadherin, we observed rapid and strong increases in cellular adhesion as measured by the RCTA cell-index following culture with immune inflammatory factors. However, using the CIM chip, the same cells also showed strong increases in invasion and migration. To determine the inflammatory factors involved in this process, we screened the immune conditioned media using a Luminex array (Millipore, Billerica, MA). TGF-b, TNF-α, and IL-6, previously shown to induce EMT, were all found at elevated levels. In 5 culture supernatants of healthy donor PBMC activated for 48h with anti-CD3 antibody, TGF-β had a modest 1.6-fold increase; TNF-α had an average 101-fold increase; while IL-6 had an average 347-fold increase. When added to cultures of SUM149 cells, these factors recapitulated the EMT gene expression signature in SUM149 including the increase in E-cadherin expression. Furthermore, the addition of neutralizing antibodies against TNF-α, TGF-β, and IL-6 to immune conditioned media prior to exposure to SUM149 cells resulted in less EMT. CONCLUSIONS Inflammatory factors may induce both the migratory ability and the characteristic persistent E-cadherin expression of IBC cells. This is mediated in part by TNF-α, TGF-β, and IL-6. However, the molecular basis for this unique IBC response requires further study hindering the development of optimal therapies. Ongoing studies at MD Anderson are exploring both the tumor and stromal components of inflammatory breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-07.

Background: Cytokines and chemokines are known to be involved in tumor growth and progression of disease. Sialyl LewisX (sLeX), a ligand for adhesion molecule E-selectin, is known to affect inflammatory processes and an elevated level is associated with tumor metastasis. Therefore, we assessed serum levels of sLeX and cytokines/chemokines in patients with non-invasive ductal carcinoma in situ (DCIS), early invasive breast cancer (EBC), or metastatic breast cancer (MBC). Patients and Methods: Sera from 250 patients (26 DCIS, 157 EBC, 67 MBC) and 43 healthy donors (HD) were assayed for sLeX using an immunoassay kit (CSLEX; Nittobo Medical Co. Ltd., Japan) and a panel of cytokines and chemokines using a multiplex assay kit. Differences in serum markers between patients and HD, and among patient groups were determined using the Kruskal-Wallis and Mann-Whitney tests. Spearman's correlation determined the non-parametric correlation between the serum levels of sLeX and the inflammatory mediators. The receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analyses were used to determine the sensitivity and specificity of a given cut-off value for a particular serum marker. Results: The median sLeX level tended to increase with the stage of disease: MBC > EBC > DCIS albeit without significant differences among the disease stages. Among MBC patients, patients with sLeX below 1.75 U/mL had significantly improved overall survival (OS, mean survival 11.1 vs. 33.7 months, P = 0.002) and progression-free survival (PFS, mean survival 9.7 vs. 20.9 months, p = 0.042). The Hazard Ratio of high sLeX for OS was 5.5 (95% CI 1.6 to 18.9, p = 0.007) and 2.3 for PFS (95% CI 1.0 to 5.2, P = 0.048). EBC and MBC patients have significantly higher serum levels of IL-1, IL-1RA, IL-6, IL-8, MCP-1, MCP-3, and MIP-1βthan those of HD. In addition, there were positive correlations between the serum levels of sLeX and cytokines IL-1β, IL-1RA, IL-2, IL-8, MIP-1β, and MCP-3. The AUC for sLeX was 0.598 (P = 0.016), and a cut-off of 3.13 pg/mL distinguished hormone receptor (HR)-positive from HR-negative patients (χ2 = 4.0, P = 0.045). Likewise, the AUC for TNF-α was 0.620 (P = 0.003), and a cut-off 7.18 pg/mL distinguished HR-positive from HR-negative patients (χ2 = 12.6, P < 0.001). Using a cut-off value established by ROC curves, few MBC patients (9 of 66, 13.6%) had a serum IL-2 level > 7.1 pg/mL compared to 57 of 185 (30.8%) non-MBC patients (χ2 = 7.4, P = 0.007), suggesting that metastatic disease may be associated with immune suppression related to low serum IL-2. Conversely, 31of 66 (47%) MBC patients had a serum MCP-1 level > 750 pg/mL vs. 37 of 185 non-MBC patients (20%) (χ2 = 23.8, P < 0.0001), suggesting that a high level of MCP-1 may play an important role in metastasis. Conclusion: Serum levels of sLeX were able to distinguish HR-positive from HR-negative patients and predict overall survival in metastatic patients. Serum sLeX and some inflammatory mediators tended to increase with the severity of disease, and together may facilitate local invasion of tumor cells. Furthermore, serum levels of MCP-1 and IL-2 may have prognostic value in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-32.

CTCs are involved in tumor dissemination and are an independent prognostic factor in primary and metastatic breast cancer patients. Dendritic cells (DCs) are the most efficient antigen presenting cells and are comprised of plasmacytoid-(pDC) and myeloid-(mDC) derived DC subsets. This study aimed to correlate CTC counts with the peripheral blood DC immunophenotypes and functions of inflammatory breast cancer (IBC) patients. This study included 65 IBC patients. Peripheral blood (PB) was obtained from patients prior to starting a new line of chemotherapy for CTCs enumeration by CellSearch® and DC phenotype and function by flow cytometry; the characteristics of DCs were then correlated with CTC counts and clinical outcome. Twenty-one (32.3%) patients with CTCs ≥5 had a significantly inferior overall survival (OS) compared to patients with

Background: Impaired immunosurveillance and immune dysregulation contribute to the pathogenesis and progression of breast cancer (BC). Upon activation, T cells synthesize inflammatory cytokines such as TNF-α that can promote or inhibit tumor growth. We therefore investigated T-cell cytokine syntheses as a predictor of disease progression. Methods: We recruited 115 BC patients [25 with locally advanced breast cancer (LABC), 21 with metastatic breast cancer (MBC), 25 with non-metastatic inflammatory breast cancer (IBC), and 44 with metastatic IBC (mIBC)] and 31 healthy donors (HD) for this ongoing study. The tumor phenotype consisted of 69 hormone receptor (HR) positive (including 26 patients with HER2 positive disease), 16 HR negative but HER2+, 30 triple negative BC (TNBC). To evaluate T cell function, peripheral blood mononuclear cells from patients and HD were stimulated overnight with immobilized anti-CD3 and soluble anti-CD28 antibodies and assessed for the percentage of T cells that synthesized cytokines by multi-parameter flow cytometry. The associations of T cell cytokine production profile with patient progression free survival (PFS) were analyzed by Kaplan Meier Test. Results: The median follow-up (FU) of 113 evaluable patients was 14.1 months with a median time to relapse of 10.5 months; 54 patients had stable disease (SD) and 59 patients had progression of disease (PD). In the entire cohort, on univariate analysis, metastasis, IBC, stage, and previous treatment predicted for worse PFS (p< 0.05). In non-metastatic patients (LABC+IBC), absolute count of anti-CD3 activated CD8+ T cells producing IL-17 was significantly higher in the SD patients compared with patient with PD (p=0.038), but it did not predict PFS (p=0.073). Similarly in metastatic patients, anti-CD3 activated CD4+ T cells producing TNF-α were significantly higher in patients with SD (p=0.025) and was predictive of longer PFS (p=0.033). Considering all patients with IBC (IBC + mIBC), although patients with PD had significantly fewer (percent and absolute number) anti-CD3 activated T cells capable of producing cytokines, this immune impairment was mostly related to metastasis and previous treatment. However, the percentage of anti-CD3 activated CD8+ T cells producing TNF-α was an independent positive prognostic indicator of PFS (p=0.002). Conclusion: Higher than average cytokine syntheses by anti-CD3 activated T cells are significantly associated with longer PFS. These data are consistent with the hypothesis that an adaptive immune response can control disease progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-20-03.

Background: Deficiencies in innate and adaptive immune responses by plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) have been linked to poor clinical outcome in breast cancer (BC) (Treilleux, Clin Cancer Res, 2004, PMID 15569976). pDC produce IFN-a and pro-inflammatory cytokines that regulate innate and adaptive immunity in breast cancer. mDC present in blood and secondary lymphoid organs secrete IL-12 and induce inflammatory cytokine production by T cells. Therefore, we studied DC activity in the peripheral blood and assessed their function with clinical outcome in breast cancer patients. Methods: We recruited 115 BC patients [25 with locally advanced non-IBC (LABC), 25 with IBC, 21 with metastatic breast cancer (MBC), and 44 with metastatic IBC (mIBC)] and 31 healthy donors (HD) for this study. Peripheral blood pDC and mDC were activated through toll-like receptor (TLR)-7 to assess IFN-α and IL-10 production whereas mDC were activated through TLR-8 to assess production of IL-12 and TNF-α by multi-parameter flow cytometry. Associations between cytokine production by TLR-activated pDC and mDC with progression free survival (PFS) and overall survival (OS) of patients were analyzed by Kaplan Meier Test. Results: The median follow-up (FU) of 113 evaluable patients was 14.1 months with a median time to progression of 10.5 months; 54 patients had stable disease (SD) and 59 had progression of disease (PD). Metastasis, previous treatments, and IBC contributed to shorter PFS and OS. Compared to HD, BC patients had significantly fewer total DC (p=0.008), mDC (p=0.008), and pDC (p=0.003) per μL. In general, the number of TLR-7-activated pDC per μL that produced IFN-a(p=0.023) or IL-10 (p=0.027) and the number of TLR-8-activated mDC per μL that produced IL-12 (p Conclusion: BC patients had significantly fewer pDC and mDC in peripheral blood than HD. IBC patients with above average numbers of TLR-activated DC capable of producing proinflammatory cytokines had a significantly shorter PFS or OS. Disease progression in IBC is related to an increased number of activated dendritic cells producing inflammatory cytokines. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-20-04.

Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G(1) cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element-Sp1 composite element within the hTERT promoter. I3C inhibited 17β-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1-DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.

Background: Given the benefits in using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), clinical practice guidelines recommend EGFR mutation testing of nonsquamous non–small cell lung cancer (NSCLC). However, not all patients get tested, which may have an impact on the prevalence of EGFR mutation previously estimated. Aim: To determine the trends of EGFR mutation testing in patients with nonsquamous NSCLC in New Zealand, and to explore the possible associations between the proportions tested, selectivity and the prevalence of EGFR mutation. Methods: This population-based study involves all patients who were diagnosed with nonsquamous NSCLC in the four health regions of New Zealand between January 2010 and July 2016. We identified eligible patients from the New Zealand Cancer Registry and obtained information on EGFR testing from TestSafe, a clinical information sharing service. We then calculated the proportions of patients tested for EGFR mutation and computed selectivity indices for eleven periods. We used a log-linear model to assess the associations between the proportions tested, selectivity and the prevalence of EGFR mutation. Results: Of the 2986 patients involved in this analysis, 1280 (42.9%) were tested for EGFR mutation. The proportion tested increased from 3.7% in 2010 to 74.0% in 2016. Testing was more prevalent in younger age group, female, Asian and patients with adenocarcinoma, and when specimens for testing was available. Such selectivity, however, decreased from 2010 to 2016. The prevalence of EGFR mutation varied widely across the periods, ranging from 16.8% in January-June 2014 to 43.8% in 2010. It was negatively associated with the proportion tested ( P = 0.02), and positively associated with the selectivity of testing ( P = 0.03). The log linear models estimated that the prevalence of EGFR mutation would be at most 16.1% (95% CI: 9.5%–27.1%) if 100% of patients were tested. Conclusion: In New Zealand, the uptake of EGFR mutation testing has improved over time but there is still room for improvement. Incomplete and selective testing may result in an overestimation of the prevalence of EGFR mutation in patients with nonsquamous NSCLC.

Background: Women diagnosed with breast cancer, their doctors, and their families, would find a valid estimate of her prognosis helpful in planning treatment and support. Assessing prognosis is complex as many factors influence it. Several predictive models have been produced, but none has been developed or tested on patients in New Zealand (NZ). Aim: We aimed to develop and validate a NZ predictive model (NZPM) for breast cancer, and compare its performance to a widely used UK-developed model, the Nottingham Prognostic Index (NPI). Methods: We developed a model to predict 10-year breast cancer-specific survival, using data collected prospectively in the largest population-based breast cancer registry in NZ (Auckland, 9182 patients), and assessed its performance in this data set (internal validation) and in an independent NZ population-based series of 2625 patients in Waikato (external validation). The data included all women with primary invasive breast cancer diagnosed from 1 June 2000 to 30 June 2014, with follow-up to death or to 31 December 2014. We used multivariate Cox proportional hazards regression to assess predictors and to estimate the probability of breast cancer mortality within 10 years, and therefore 10-year survival, for each patient. We assessed observed survival by the Kaplan-Meier method. We assessed discrimination by the C-statistic, and calibration by comparing predicted and observed survival rates for patients in 10 groups ordered by predicted 10-year survival. We compared this NZPM with the NPI in the validation data set. Results: The final NZPM used continuous variables of age, tumor size, and number of positive lymph nodes, and categorical variables of ethnicity, tumor stage, tumor grade, ER and PR receptors, HER2 status, and histologic type of tumor. Discrimination was good: C-statistics were 0.84 for internal validity and 0.83 for independent external validity. For calibration, for both internal and external validity, the predicted 10-year survival probabilities in 10 groups of patients, ordered by predicted survival, were all within the 95% confidence intervals (CI) of the observed Kaplan-Meier survival probabilities. The NZPM showed good discrimination even within the prognostic groups defined by the NPI. Conclusion: These results for the NZPM show good internal and external validity, transportability, potential clinical value, and its clear superiority over the NPI. Further research will assess other potential predictors, other outcomes, performance in specific subgroups of patients, and compare the NZPM to other models, which have been developed in other countries and have not yet been tested in NZ.

Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-06.

Background: Expression of cancer related genes and proteins in clinical specimens are the mainstay of personalized targeted therapy; however, a diagnostic signature for inflammatory breast cancer (IBC) remains elusive. In this study, we employed a blood-based, non-invasive, sensitive technology to map biomarkers in patients with IBC at the protein level. Proximity Extension Assay consists of a harmonious blending of immunoassay and PCR to amplify protein expression signal, thereby enabling multiplexing with small sample input (1 μl). Other multi-platform assays require a large amount of clinical material, multi-step sample processing and complicated data analysis. Materials and methods: Serum samples (n= 159) from patients with primary IBC (IBC, n= 30), metastatic IBC (MIBC, n= 54), locally advanced breast cancer (LABC, n= 24) and metastatic breast cancer (MBC, n= 27) were prospectively collected from subjects prior to starting a new therapy (treatment naive) or a new line of therapy between 2009 and 2012. Sera from 24 healthy normal donors (HD) were included in the analysis for comparison. The samples were analyzed using two panels: Proseek Multiplex Oncology II and Proseek Multiplex Inflammation I (Olink Proteomics, Uppsala, Sweden) for simultaneous detection of 92 human protein biomarkers in each panel. In the assay, each protein biomarker is detected by a matched pair of antibodies coupled to unique DNA-tags. Upon binding to the proteins, the correctly hybridized DNA-tags form an amplicon that can be measured by PCR.For initial analysis, sample populations were compared using the Mann-Whitney-U test. Results: In comparison with HD sera, sera of breast cancer patients had 41 proteins from the oncology panel and 28 from the inflammation panel that were significantly higher, whereas 5 from the inflammation panel were significantly lower. From the inflammation panel, 11 proteins (PD-L1, IL-2, IL-7, IL-18, uPA, CCL4, CCL23, CXCL9, CXCL10, CXCL11 and TNF-alpha) showed significant differential expression between IBC and non-IBC derived samples (irrespective of metastatic status); for each marker, levels were higher in IBC than in non-IBC. In contrast, 9 proteins from the oncology panel (CRNN, CTSV, ERBB4, FR-gamma, ITGAV, MIA, PODXL, SCF and SEZ6L) were differentially expressed; however, each of these proteins was higher in non-IBC than in IBC. Among the aforementioned proteins, CCL4, IL-2, IL-7, PD-L1, TNF-alpha, uPA, CRNN, CTSV, FR-gamma, ITGAV, MIA, SCF and SEZ6L did not differentiate cancer and HD, but were uniquely characteristic of the IBC vs non-IBC comparison. Conclusion: These preliminary data suggest that it is possible to distinguish between cancer patients and healthy normal donors, and also to delineate between IBC and non-IBC patients based on expression of serum proteins. Validation of this serum protein signature is planned in a larger patient cohort. Citation Format: Cohen EN, Jayachandran G, Gao H, Tin S, Alvarez RH, Valero V, Lim B, Woodward WA, Ueno NT, Reuben JM. Circulating protein biomarker profile for inflammatory breast cancer using a multiplexed proximity extension assay [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-04.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

Background Nucleostemin is a GTPase residing in the nucleolus that is considered to be an important cancer stem/progenitor cell marker protein due to its high expression levels in breast cancer stem cells and its role in tumor initiation of human mammary tumor cells. It has been proposed that nucleostemin may represent a valuable therapeutic target for breast cancer; however, to date evidence supporting the cellular mechanism has not been elucidated. Results Expression of exogenous HER2, a member of the EGF receptor gene family, in the human MCF-10AT preneoplastic mammary epithelial cell line, formed a new breast cancer cell line, 10AT-Her2, which is highly enriched in cells with stem/progenitor cell-like character. 10AT-Her2 cells display a CD44+/CD24-/low phenotype with high levels of the cancer stem/progenitor cell marker proteins nucleostemin, and active aldehyde dehydrogenase-1 (ALDH-1). The overall expression pattern of HER2 protein and the stem/progenitor cell marker proteins in the 10AT-Her2 cell population is similar to that of the luminal HER2+ SKBR3 human breast cancer cell line, whereas both MCF-7 and MDA-MB-231 cells display reduced levels of nucleostemin and no detectable expression of ALDH-1. Importantly, in contrast to the other well-established human breast cancer cell lines, 10AT-Her2 cells efficiently form tumorspheres in suspension cultures and initiate tumor xenograft formation in athymic mice at low cell numbers. Furthermore, 10AT-Her2 cells are highly sensitive to the anti-proliferative apoptotic effects of indole-3-carbinol (I3C), a natural anti-cancer indole carbinol from cruciferous vegetables of the Brassica genus such as broccoli and cabbage. I3C promotes the interaction of nucleostemin with MDM2 (murine double mutant 2), an inhibitor of the p53 tumor suppressor, and disrupts the MDM2 interaction with p53. I3C also induced nucleostemin to sequester MDM2 in a nucleolus compartment, thereby freeing p53 to mediate its apoptotic activity. Small interfering RNA knockdown of nucleostemin functionally documented that nucleostemin is required for I3C to trigger its cellular anti-proliferative responses, inhibit tumorsphere formation, and disrupt MDM2–p53 protein–protein interactions. Furthermore, expression of an I3C-resistant form of elastase, the only known target protein for I3C, prevented I3C anti-proliferative responses in cells and in tumor xenografts in vivo, as well as disrupting the I3C-stimulated nucleostemin–MDM2 interactions. Conclusions Our results provide the first evidence that a natural anti-cancer compound mediates its cellular and in vivo tumor anti-proliferative responses by selectively stimulating cellular interactions of the stem/progenitor cell marker nucleostemin with MDM2, which frees p53 to trigger its apoptotic response. Furthermore, our study provides a new mechanistic template that can potentially be exploited for the development of therapeutic strategies targeted at cancer stem/progenitor cells.

Currently, there is extensive information about circulating tumor cells (CTC) and their prognostic value; however, little is known about other characteristics of these cells. In this prospective study, we assessed the gene transcripts of epithelial-to-mesenchymal transition—inducing transcription factors (EMT-TF) and cancer stem cell (CSC) features in patients with HER2+ metastatic breast cancer (MBC). Epithelial cells were enriched from peripheral blood mononuclear cells (PBMC) using antibody-coated anti-CD326 antibody (CD326+) magnetic beads, and the residual CD326− PBMCs were further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326−CD45−). RNA was extracted from all cell fractions, reverse transcribed to cDNA, and subjected to quantitative reverse transcription PCR to detect EMT-TFs (TWIST1, SNAIL1, ZEB1, and TG2) as a measure of CTCs undergoing EMT (EMT-CTCs). In addition, PBMCs were analyzed using multiparameter flow cytometry for ALDH activity and CSCs that express CD24, CD44, and CD133. Twenty-eight patients were included in this study. At least one EMT-TF mRNA was elevated in the CTCs of 88.2% of patients and in the CD326−CD45− cell fraction of 60.7% of patients. The CD326−CD45− fraction of patients with elevated SNAIL1 and ZEB1 transcripts also had a higher percentage of ALDH+/CD133+ cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P = 0.038). Our data indicate that patients with HER2+ MBCs have EMT-CTCs. Moreover, an enrichment of CSCs was found in CD326−CD45− cells. Additional studies are needed to determine whether EMT-CTCs and CSCs have prognostic value in patients with HER2+ MBCs treated with trastuzumab-based therapy. Mol Cancer Ther; 11(11); 2526–34. ©2012 AACR.

Artemisinin, a sesquiterpene phytolactone derived from Artemisia annua, is a potent antimalarial compound with promising anticancer properties, although the mechanism of its anticancer signaling is not well understood. Artemisinin inhibited proliferation and induced a strong G1 cell cycle arrest of cultured MCF7 cells, an estrogen-responsive human breast cancer cell line that represents an early-stage cancer phenotype, and effectively inhibited the in-vivo growth of MCF7 cell-derived tumors from xenografts in athymic nude mice. Artemisinin also induced a growth arrest of tumorigenic human breast cancer cell lines with preneoplastic and late stage cancer phenotypes, but failed to arrest the growth of a nontumorigenic human mammary cell line. Concurrent with the cell cycle arrest of MCF7 cells, artemisinin selectively downregulated the transcript and protein levels of the CDK2 and CDK4 cyclin-dependent kinases, cyclin E, cyclin D1, and the E2F1 transcription factor. Analysis of CDK2 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK2 gene expression was accounted for by the loss of CDK2 promoter activity. Chromatin immunoprecipitation revealed that artemisinin inhibited E2F1 interactions with the endogenous MCF7 cell CDK2 and cyclin E promoters. Moreover, constitutive expression of exogenous E2F1 prevented the artemisinin-induced cell cycle arrest and downregulation of CDK2 and cyclin E gene expression. Taken together, our results demonstrate that the artemisinin disruption of E2F1 transcription factor expression mediates the cell cycle arrest of human breast cancer cells and represents a critical transcriptional pathway by which artemisinin controls human reproductive cancer cell growth.

Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter.

Background: CTCs play a crucial role in tumor dissemination and are prognostic factor in primary and metastatic breast cancer patients. Immune cells in peripheral blood (PB) contribute to an unfavorable microenvironment for the CTCs survival. As such, effective host innate and adaptive immune surveillance systems could adversely influence tumor dissemination whereas dysfunctional immune systems could provide a favorable microenvironment for the dissemination of CTCs and cancer progression. This study aimed to correlate CTCs with the functions of innate [natural killer (NK) cells] and adaptive (T-cells) immune effector cells in PB of IBC patients. Methods: This prospective study included 65 IBC (21 non-metastatic, 14 de novo metastatic and 30 recurrent metastatic) patients treated between October 2008 and April 2012 at the MD Anderson Cancer Center. CTCs were enumerated before patients started a new line of chemotherapy using the CellSearch® system, 33 (50.8%) of patients were treatment naïve at the time of blood collection. The phenotype of T cells, their ability to secrete cytokines following activation through the T-cell receptor (TCR) and the NK cell subsets were analyzed by multiparameter flow cytometry and the results were correlated with CTCs and clinical outcome. For survival analysis immune cell counts were dichotomized to low or high category using the median count. Results: At least 1 CTC (≥ 1) or ≥ 5 CTCs per 7.5 mL of PB was detected in 40 (61.5%) or 21 (32.3%) of patients, respectively. Patients with at least 1 CTC or ≥ 5 CTCs had a significantly inferior overall survival (OS) [HR=2.48, p=0.003 and HR=1.85, p=0.045] than patients with no CTCs or with Conclusions: IBC patients with CTCs in PB had abnormalities in both innate and adaptive immunity as evidenced by low percentages of NK cell subsets, and low percentage of TCR-activated CD8+ T cells producing TNF-α, respectively. These data illustrate an inverse relationship between CTCs and both innate and adaptive immune cells in the PB microenvironment that could potentially impact tumor cell dissemination and initiation of the metastatic cascade. Moreover, immune cell profiling could add further prognostic value to CTCs in IBC patients. Citation Format: Michal Mego, Hui Gao, Evan N Cohen, Simone Anfossi, Antonio Giordano, Sanda Tin, Tamer M Fouad, Wendy A Woodward, Ricardo H Alvarez, Vicente Valero, Naoto T Ueno, Gabriel N Hortobagyi, Massimo Cristofanilli, James M Reuben. Circulating tumor cells (CTC) are associated with defects in innate and adaptive immunity in inflammatory breast cancer (IBC) patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-04.

Background: Cancer stem cells (CSCs) in bone marrow (BM) are epithelial tumor cells that express CD44+CD24−/lo. CSCs can be further divided into those that have ALDH activity (Aldefluor+) and those that do not. We hypothesized that if CSCs are responsible for tumor dissemination, their presence in BM would be prognostic in early stages of breast cancer (EBC) patients. Therefore, using multiparameter flow cytometry (FACS), we assessed epithelial cells for expression of CD44+CD24−/low and Aldefluor in BM of EBC patients and correlated these findings with circulating tumor cells (CTC), disseminated tumor cells (DTC) and clinical outcome. Methods: This is a prospective laboratory study (Protocol 04–0657) conducted in the Departments of Surgical Oncology and Hematopathology, at The University of Texas MD Anderson Cancer Center in Houston, TX, USA, and was approved by the institutional review board. BM aspirates were collected at the time of surgery from 108 patients with EBC. BM was analyzed for CSCs and ALDH activity by FACS analysis. Ten cytospin slides of the bone marrow mononuclear cells were prepared from each BM aspirate and reacted with a cocktail of antibodies, including AE1/AE3, CAM5.2, MNF116, CK8 and CK18, to identify epithelial cells (DTC) by immunohistochemistry (IHC). In addition, the CellSearch system (Veridex, Raritan, NJ) was used to enumerate CTCs in each of 3 tubes of 7.5 mL of peripheral blood per patient. Overall survival (OS) and disease-free survival (DFS) were calculated from the date of diagnosis and analyzed with Kaplan-Meier survival plots. Cox multivariate proportional hazards model was also performed. Results: Of the 103 evaluable patients, 45 patients received neoadjuvant chemotherapy, 35 adjuvant chemotherapy and 23 no chemotherapy. The median follow-up was 40 months (range 7–66 months). Patients with CSCs in BM had a hazard ratio (HR) of 8.8 for DFS (p = 0.002); patients with Aldefluor+ CSCs had a HR of 5.9 (p = 0.052) for DFS. Seven patients have expired and all of them had CSCs in BM. Moreover, we evaluated the presence of DTCs by FACS (CD326+CD45−) in 104 patients. Sixty-one (75%) of patients had positive BM samples for DTC (CD326+CD45- BM cells ≥ 0.53%). No association was shown between DTC in BM as determined by FACS and CTCs in PB. In a multivariate model, after adjusting for age (< 45 years old), clinical T stage, N stage, ER, PR, HER2 status, and nuclear grading, the presence of CSCs was found to be an independent predictor of DFS (HR = 15.8, P = 0.017) as was the presence of CTCs (HR = 13.9, P = 0.007). In this subset of patients, the presence of Aldefluor+ CSCs and DTCs was not found to be predictive of DFS in the multivariate model including the same factors as listed above. Conclusions: Our data indicate that the presence of BM metastasis is correlated to CSCs, and CSCs irrespective of ALDH activity are independent adverse prognostic factors in EBC patients. Moreover, the presence of CTCs was a strong independent predictor of DFS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-03-01.

Rationale: Inflammatory breast cancer (IBC) is the most insidious form of locally advanced disease. Emerging evidence suggests that host factors in the microenviromement may interact with underlying IBC genetics to promote the aggressive nature of the tumor. An integral part of the metastatic process involves epithelial to mesenchymal transition (EMT) where primary breast cancer cells gain motility and stem cell features that allow distant seeding. Interestingly, the IBC consortium microarray data found no clear evidence for EMT in IBC tumor tissues. However, it is unknown if soluble factors secreted by activated immune cells mediate EMT in the IBC microenvironment that may account for the absence of EMT in studies of the tumor cells themselves. Therefore, we tested whether the conditioned media of activated immune cells were capable of inducing EMT in IBC cells. Methods: Conditioned media (CM) were generated using healthy donor peripheral blood mononuclear cells that were activated with anti-CD3 antibody immobilized to plastic and soluble anti-CD28 antibody to activate T cells through the T-cell receptor (TCR) or left unstimulated for 48 hours. Thereafter, CM from each of the cultures was harvested and filtered. Next, 48-hour pre-seeded SUM149 IBC cells were grown in culture medium consisting of 25% CM and 75% IBC culture medium for an additional 2 days. Unconditioned media and TGF-β were used as negative and positive controls, respectively for EMT. Following treatment with CM, RNA was extracted from the target cells and analyzed for the presence of EMT-related transcription factors (EMT-TF) and markers of epithelial and mesenchymal states by TaqMan® qRT-PCR. Subsequently, a panel of 24 genes was tested on 4 IBC cell lines (SUM149, SUM190, KPL4 and IBC-3) and 5 non-IBC cell lines (MCF-10a, MCF-7, MDA-231, and MDA-453) treated with immune-activated CM using the Fluidigm® Dynamic Array integrated fluidic circuit (“chip”) gene expression platform which allows for the simultaneous quantification of 2,304 data points using TaqMan® assays. Formalin-fixed, paraffin embedded blocks were prepared from trypsinized cells for immunohistochemical (IHC) staining to detect E-cadherin and vimentin expression. Results: SUM149 cells cultured in the presence of TCR-activated CM for two days showed upregulation in EMT-TFs (SNAIL1, ZEB1, and TG2), vimentin and fibronectin by qRT-PCR. IHC staining showed increases in both vimentin and E-cadherin expression after 48-hour exposure to anti-TCR CM. Fluidigm® gene expression analysis of multiple cell lines exposed to anti-TCR CM showed that E-cadherin expression was unchanged or slightly decreased in non-IBC cell lines, whereas 3 of 4 IBC cell lines showed an increase in E-cadherin. Discussion: These data suggest that soluble factors secreted by activated immune cells are capable of inducing EMT in IBC cells and may mediate the persistent E-cadherin expression observed in IBC. Such processes may contribute to the highly aggressive nature of the disease; however, an immune competent in vivo model is warranted to fully understand the implications of these findings. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-06.

e11061 Background: Sialyl Lewis X (sLeX), a carbohydrate antigen on tumor cells and ligand for E-selectin, mediates tethering and rolling along vascular endothelium. sLeX on tumor cells is purporte...

Background: Circulating tumor cells (CTCs) are an independent predictor of survival in metastatic breast cancer patients and patients with ≥5 CTC per 7.5 mL of whole blood (WB) have a poor prognosis and overall survival than patients with fewer than 5 CTC. Dendritic cells (DCs), the most efficient antigen presenting cells (APC), are essential for a robust host immune response. Therefore, we investigated the relationship between CTCs and the immunophenotype and function of DCs in breast cancer (BC) patients. Methods: This ongoing prospective study consists of 47 patients, 26 with metastatic breast cancer, 19 with inflammatory breast cancer; and 2 with locally advanced breast cancer, with a median age of 52 years (range: 34-71 years). Primary tumor characteristics of patients included 28 hormone receptor positive, 18 HER2 amplified, and 12 triple receptor negative. Fluorescence-activated cell sorting analysis was used to enumerate circulating DC subsets, myeloid (mDC) and plasmacytoid (pDC), that express costimulatory molecules (CD40, CD80, and CD86). Additionally, we assessed the ability of DCs to synthesize interleukin (IL)-10, IL-12, interferon-alpha (IFN-a) and tumor necrosis factor (TNF)-a constitutively and following activation with toll-like receptor (TLR) agonists. CTCs were enumerated in 7.5 mL of WB using CellSearch (Raritan, NJ). Results: CTCs were detected in 26 patients (range from 1 to 1009) including 12 patients with ≥5 CTCs. Compared with patients with < 5 CTCs, those with ≥5 CTCs have a significantly higher median number of mDC (P = 0.007) and higher median percentages (%) of TLR-activated mDC that expressed CD80 (P = 0.047) and CD86 (P = 0.063). Additionally, compared with patients who had < 5 CTCs, patients with ≥5 CTCs had a lower median % of TLR-activated mDC that synthesized IL-12 (P=0.028), TNF-α (P=0.006), or IL-10 (P = 0.031); and there was a higher median % of TLR-activated pDC that synthesized IL-10 (P = 0.000). Conclusions: Even though BC patients with ≥5 CTCs have more mDC, activated mDCs are less capable of synthesizing inflammatory factors (TNF-a and IL-12) responsible for adaptive immunity. Moreover, there was increased % of activated pDC that synthesized IL-10, a cytokine responsible for downregulating adaptive immune responses. These preliminary data suggest that activated DCs from BC patients with ≥5 CTCs are likely to have a suboptimal ability to sustain an efficient adaptive immune response compared with activated DCs of BC patients with < 5 CTCs. This is the first report linking CTCs, a proven poor prognostic indicator, with potential dysfunctional APC in BC patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-04.

Background Transforming growth factor (TGF)-b1 has pleiotropic effects in cancer. In the early stages of breast cancer, TGF-b may be responsible for immune tolerance through the activation of T-Regulatory cells (TR). On the other hand, in the late stages of disease, it may induce angiogenic factors [vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) (in Bonnomet J Mammary Gland Biol Neoplasia 2010); and IL-17 (Pickens J Immunol 2010)], and epithelial to mesenchymal transition (EMT) which may lead to an increase in the number of circulating tumor cells (CTC) (Kim, Cell 2009). Therefore, we investigated the possible correlation between TGF-b1, CTC count, angiogenic factors and T-cell function of patients with locally advanced or metastatic breast cancer. Methods As an interim analysis of an on-going prospective study, sera and peripheral blood mononuclear cells (PBMC) were collected from breast cancer patients starting a new line of therapy. At analysis, study enrollment included 78 patients with breast cancer [19 with locally advance disease (LABC), 23 with non-metastatic inflammatory breast cancer (IBC), and 32 with metastatic disease (MBC) including 19 with IBC features], and 28 healthy donors (HD). Serum TGF-b1, VEGF, and IL-8 levels were measured by Milliplex Luminex kits (Millipore, Billerica, MA). CD4+CD25+CD127dim TR cells were enumerated in whole blood by FACS (BD Biosciences, San Jose, CA). PBMC were used to study the ability of T cells to synthesize cytokines following activation of the T-cell receptor by immobilized anti-CD3 antibodies. CTC were enumerated by CellSearch (Veridex, Raritan, NJ). The Spearman Rho was calculated for nonparametric correlations and the Mann-Whitney U test was used to determine significant differences between median values. Results LABC patients and HD had a median serum TGF-b1 that was significantly higher than that of MBC patients (P = 0.042). There were statistically significantly positive correlations between serum TGF-b1 and the number of CD4+ T cells (rho = .226, P = .029) and IL-10 produced by %CD4+ (rho = .388, P = .002) and %CD8+ (rho = .459, P < .001) T cells. Furthermore, there was a positive correlation between serum TGF-b1 and the % of CD8+, but not CD4+, T cells that produced IL-17 (rho = .250, P = .022). Serum TGF-b1 levels did not correlate with either % or number of TR cells. Although serum TGF-b1 level of MBC patients was independent of CTCs (24.4 ng/mL vs. 24.0 ng/mL, P = .317), MBC patients with CTC had significantly higher serum levels of angiogenic factors such as VEGF (530 ng/mL vs. 240 ng/mL, P=0.037) and IL-8 (45.6 pg/ml vs. 20.0 pg/ml P= .006) than those of patients with no CTC. Even so, MBC patients with or without CTC have similar % of CD4 and CD8 T-cell subsets that could be activated to produce IFN-g, IL-2, TNF-a, IL-17, or IL-10. Conclusion: The concomitant presence of elevated serum TGF-ß1 levels and IL-10 producing T cells suggest immune suppression could facilitate disease progression of breast cancer. T-cell function is independent of CTC in MBC patients; however, serum VEGF and IL-8 levels were significantly elevated in MBC patients with CTC suggesting that vascular changes can facilitate tumor dissemination. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-07.

Background: Impaired immunosurveillance and immune dysregulation contribute to the pathogenesis and progression of breast cancer. We recently reported that breast cancer (BC) patients have compromised immunity with significantly fewer T cells, B cells, and dendritic cells compared to normal healthy donors. To further investigate immune function, T cells were assessed for their ability to synthesize cytokines prior to initiation of therapy and 3 months later. Methods: We recruited 46 BC patients [3 with locally advanced breast cancer (LABC), 13 with metastatic breast cancer (MBC), 15 have newly diagnosed inflammatory breast cancer (IBC), and 15 with metastatic IBC (MIBC)] and 13 healthy donors (HD) for this ongoing study. The tumor phenotype consisted of 28 hormone receptor (HR) positive (including 10 patients with HER2 positive disease), 7 HR negative but HER2+, 11 triple negative (TN). Thirty-nine patients are alive and have both baseline and follow up data at 3 months; 7 deceased patients had baseline data only. Peripheral blood mononuclear cells (PBMC) from patients and HD were stimulated overnight with immobilized anti-CD3 and soluble anti-CD28 antibodies and cytokine (IL-2, IL-4, IL-10, and IL-17, TNF-α, and IFN-γ) syntheses by activated (CD69+) CD4 and CD8 T cells were measured by multi-color flow cytometry. Statistical differences between the immunoresponses of patients and HD were analyzed by Mann-Whitney U test. Results: Compared with HD, IBC and MIBC patients had a statistically significantly lower percentage (%) of TNF-α+ CD8+ T cells (p=0.041 and p=0.037 respectively) at baseline. MIBC patients also had significantly lower % IFN-γ+ CD8+ T cells (p=0.046) than HD at baseline visit. But there were no significant differences among LABC, IBC, MBC, and MIBC patients. TN patients had statistically significantly lower % of IL-2+ CD4+ T cells (p=0.047) and TNF-α+ CD8+ T cells (p=0.041) at baseline than HD. The 7 deceased patients (3 IBC, 3 MIBC, and 1 MBC) had significantly lower %'s of CD4+ and CD8+ T cells that synthesized IL-2, TNF-α, IFN-γ, IL-4, and IL-10 than the 39 living patients (all p < 0.05) at baseline. At 3 months, all patient groups except IBC had T-cell responses that were similar to those of HD. Specifically, IBC patients had persistently significantly lower % of TNF-α+ CD8+ T cells than HD (p=0.048). Conclusion: In summary, T cell function is essential for maintaining immunosurveillance against tumor progression. Our data suggest that BC patient, especially IBC patients, have impaired T-cell responses and that therapeutic intervention may restore immune function only in a subset of those patients. Immunomodulatory therapy can play a role in IBC and contribute to improve patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5589.

Background: Inflammatory breast cancer (IBC) is a rare but highly aggressive form of the disease but the immune profile associate with pathology and disease progression is poorly characterized. The cells of the innate immune system including natural killer (NK) and dendritic cells (DC) can establish the conditions for an adaptive response to the tumor and can actively kill tumor cells. Alternatively, the inflammatory conditions created by these cells can promote processes like metastasis and invasion. Here we report that immune activation in IBC is associated with a poor prognosis. Methods: A total of 76 breast cancer (BC) patients starting a new line of therapy (22 IBC, 23 metastatic IBC, 8 locally advanced breast cancer, and 23 metastatic) were evaluated for immune phenotypes and cytokine synthesis by DC activated through toll-like receptors (TLR) 7 and 8. Results: Among evaluable patients, 39 (58%) had hormone receptor positive (HR+) primary tumors, 20 (31%) were Her2 positive, and 18 (26%) were triple negative (TN). Among IBC patients, 23 (49%) were HR+, 15 (33%) were Her2+, and 14 (31%) were TN. Among non-IBC patients, 16 (73%) were HR+, 5 (22%) were Her2+, and 4 (17%) were TN. With a mean follow-up time of 7.8 months, 11 (16%) of the patients (10 IBC) died of disease. Most strikingly, BC survivors had a higher median CD4 count than deceased patients (p = .023) and was a significant predictor of outcome (p = 0.01). CD3 and CD19 lymphocytes of BC survivors had higher expression of CXCR4 than deceased patients (p = .038 and p = .029). In contrast, survivors also had lower lymphocyte percentages of all NK sub-types including ADCC NK (p = .008), non-ADCC NK (p = .001), exhausted (p = .014) and non-exhausted NK (p = .005) than deceased patients. Although DC counts were not significantly different, the percentage of TLR-induced pDC producing IFN-α was lower in BC survivors than deceased patients (p = 0.025). Conversely, the percentage of pDC constitutively producing the anti-inflammatory cytokine IL-10 was higher in survivors compared to non-survivors (p = .007). Compared with IBC non-survivors, IBC survivors had a higher % of pDC producing IL-10 (p = 0.005) and fewer mDC producing TNF-α (p = .030) following TLR activation. With respect to antigen presentation, mDC of BC survivors had lower expressions of the co-stimulatory molecules CD40 (p= .036), CD80 (p = .013) and CD86 (p = .006) than mDC of deceased patients. Conclusion: We observed low CD4 counts in IBC that were associated with a poor prognosis. Additionally, immune activation as evidenced by DC pro-inflammatory cytokine production, expression of costimulatory molecules, and increased NK counts was associated with poor prognosis. Together, these data suggest that the innate immune system is highly active in the late stages of disease but the adaptive response is severely compromised by factors still unknown that can contribute to the peculiar clinical presentation and aggressive features of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5583.

Background: Inflammatory breast cancer (IBC) is an insidious form of breast cancer. Cytokines and chemokines direct the metastatic potential of disease and serve as biomarkers for disease progression. Circulating tumor cells (CTC) are an independent prognostic factor in metastatic disease. Further, TGF-β is involved in the induction of the epithelial-mesenchymal transition (EMT) which regulates the metastatic ability of IBC. As prognostic factors of aggressive disease, we measured serum cytokines and leukocyte phenotypes, and correlated the values with known CTC results of IBC and non-IBC patients.Methods: Peripheral blood (PB) from 35 IBC patients (18 non-metastatic and 17 metastatic) and 19 non-IBC patients (7 LABC, 12 MBC) was collected prior to starting a new therapy to measure a panel of 33 cytokines, chemokines, and growth factors in serum by Luminex; CTC by CellSearch™; and PB leukocyte immunophenotype by FACS. The Kruskal-Wallis and Mann-Whitney U tests determined the differences in cytokine levels between IBC, non-IBC and healthy donors (HD) and presence of CTCs.Results: There were no differences in serum cytokine, chemokine or growth factor levels between IBC and non-IBC patients. EGF, IP-10, MIG, Eotaxin, MCP-1 and TNF-RI were significantly elevated in breast cancer patients compared to HD. TNF-RI, EGF, HGF, IP-10, MIG, Eotaxin, MCP-1 and interleukin (IL)-10 were higher in IBC patients than in HD. Compared to IBC patients, non-IBC patients had fewer dysregulated cytokines relative to HD including higher EGF, Eotaxin, MCP-1 and IL-8.Perhaps counter-intuitively, non-metastatic IBC patients had higher plasma levels of IL-2, -2R, -4, -5, -10, -12p70, -15, -17, FGF-b, IFN-γ, GM-CSF, and MIP1-α than metastatic IBC patients. Moreover, these differences were not observed between LABC and MBC patients. Furthermore, compared to HD, metastatic IBC had lower IL-4, -7, -17, -12p70, IFN-γ, RANTES, but higher levels of IP-10, Eotaxin, MCP-1, and TNF-RI. These data suggest that non-metastatic IBC patients are more immune competent than metastatic IBC patients. Finally, there were differences in the immunophenotype as well as cytokine levels between IBC patients with and without CTCs. IBC patients with CTC had a lower %T-cells (p=0.003) and higher %B-cells (p=0.008) and TNF-RI (p=0.01) than IBC patients without CTCs which may lead to a decrease in cellular immunity. Cell-mediated immunity may be further compromised by the elevated levels of serum TGF-β (p= 0.064) that can also promote EMT and metastatic progression.Conclusion: We report a comprehensive analysis of the serum cytokine and chemokine profiles in IBC patients. More importantly, this is the first report of potential interactions between soluble factors, CTC, and immune parameters in IBC patients. Non-metastatic IBC patients are more immune competent than metastatic IBC patients as evidenced by the high levels of pro- and anti-inflammatory factors; however, the presence of CTC in IBC tends to shift the immune response to a TH2 polarization with a decrease in T-cells, and a concomitant increase in B-cells and serum TGF-β and TNF-RI levels. Additional studies are needed to determine the role of soluble factors in the pathogenesis and progression of IBC and the impact on clinical outcome. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2135.

Background: Circulating tumor cells (CTCs) are an independent predictor of survival in metastatic breast cancer (BC) patients. CTC are readily detected by CellSearch System based on their expression of EpCAM. Epithelial-mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased chemotherapy resistance. Human mammary epithelial cells (HMEC) transformed by the EMT transcription factor TWIST1 and spiked into normal peripheral blood (PB) are not detected by EpCAM enrichment based conventional detection methods compared to non-transformed HMECs. We hypothesize that CTCs undergoing EMT and resultant loss of epithelial markers may escape detection by conventional detection methods. The aim of this study was to detect CTCs based on expression of EMT genes in breast cancer patient's peripheral blood.Methods: This prospective ongoing study of breast cancer patients consisted of 16 (57.1%) patients with metastatic disease, 19 (67.9%) patients with inflammatory breast cancer (IBC) and 12 (42.9%) patients with primary, non-IBC breast cancer, respectively. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using anti-CD45 coated magnetic beads. RNA extracted from CD45-depleted (CD45-) PBMC were interrogated for expression of TWIST1, SNAIL1, SLUG, ZEB1, FOXC2 and EpCAM gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of healthy donors (HD). Expression of one or more gene transcripts was considered a positive result. Concurrently, a 7.5 mL PB sample was collected for determination of CTC by CellSearch.Results: Median age was 54 year (range: 34-72 years). Overall, the median CTC count by CellSearch was 2 (range; 0-750) per 7.5 mL of PB. TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2 were overexpressed in CD45- PBMC in 7%, 4 %, 4%, 0% and 14 % of patients, respectively. At least one of the EMT genes was overexpressed in 6 (21%) of patients. TWIST1 and SLUG were overexpressed only in IBC patients (10.5% and 5.3% of patients, respectively). Patients with triple negative breast cancer more commonly overexpressed EMT genes compared to non-triple negative patients (30.8% vs. 13.3%). There was no correlation between expression of EMT genes, EpCAM expression or CTC count measured by CellSearch, respectively.Conclusions: These data suggest that EMT genes may be involved in the dissemination of CTCs. Loss of epithelial antigen on CTC due to EMT, triggered by high expression of these genes, may be responsible for their undetection by conventional methods in a fraction of patients with early or advanced breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3011.

Background: Inflammatory breast cancer (IBC) is characterized by an acute inflammation of the skin of the affected breast due to blockage of the dermal lymphatics by tumor emboli. Cytokines and chemokines affect the migration of tumor cells and immune cell function that regulates the pathogenesis of IBC. Preliminary data suggest that sera of IBC patients contain several inflammatory cytokines and chemokines capable of regulating innate and adaptive cellular immune responses. Since there is a paucity of data on the characteristics and function of immune cells of IBC patients, we determined the immunophenotype and cytokine production by T cells and dendritic cells.Methods: From October 2008 through May 2009, peripheral blood (PB) from 34 IBC patients (18 non-metastatic and 16 metastatic), 18 non-IBC patients and 24 healthy donors (HD) were analyzed to determine the immunophenotype of T-cell subsets, activated and regulatory T-cells, B-cells, natural killer cell subsets (NKC), and dendritic cell (DC) subsets. Additionally, we assessed the ability of T-cells and DCs to synthesize cytokines following activation through the T-cell receptor (TCR) and toll-like receptors (TLR), respectively. The Kruskal-Wallis and Mann-Whitney U tests determined the differences between IBC patients, non-IBC patients, and HD.Results: IBC and non-IBC patients were well-matched in terms of ER, PR, Her2, high-grade tumor, tumor size, and menopause status. Both IBC and non-IBC patients had significantly fewer lymphocytes, total T-cells (CD3+), T-helper (CD4+), T-cytotoxic/suppressor (CD8+), and B (CD19+) than HD (all p < 0.01). Non-IBC patients had a significantly lower CD4/CD8 ratio than HD (p = 0.016) while IBC patients had significantly fewer T-regulatory (CD4+CD25hiCD127-) cells than HD (p = 0.02) and non-IBC patients (p < 0.05). With respect to innate immunity markers, IBC patients also had significantly lower percentages of ADCC/NK (CD16+CD56+, p = 0.034) and non-exhaustive NK (CD56+CD57+, p = 0.02) than HD and a significantly higher percentage of non-ADCC/NK cells than non-IBC patients (p = 0.03). Non-IBC patients and HD had similar innate immunity markers. Even though all groups had similar percentages of myeloid (mDC) and plasmacytoid (pDC) dendritic cells, mDC of IBC patients displayed a higher level of constitutive activation than non-IBC with increased expression of CD40 and CD80, and decreased CCR5 expression. IBC patients had more mDC that constitutively produced TNF-a (p 0.041) and IL-10 (p = 0.029) than HD, and more mDC than non-IBC patients that produced IL-10 (p = 0.028).Conclusion: The immune profile of patients with breast cancer suggests multiple abnormalities involving T-, B-, NK- and dendritic cells. Furthermore, IBC patients exhibited constitutive immune activation as measured by increased expression of co-stimulatory receptors (CD80, CD40) and constitutive production of TNF-a and IL-10 by mDC as well as fewer T-regulatory cells. Taken together, these preliminary data suggest that IBC patients are more likely than non-IBC patients to have a dysregulated immune function. Additional studies on T-cell activation and function are warranted to better understand factors associated with host defense mechanisms of IBC patients and possible therapeutic strategies to restore immune function. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4129.