B-N Lee

Soluble Factors and Circulating Tumor Cells in Inflammatory Breast Cancer.

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.

Immune Profile of Inflammatory Breast Cancer Patients.

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 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4129.

Abstract P1-06-07: Immune-induced epithelial to mesenchymal transition in inflammatory breast cancer induces unique increases in E-cadherin, adhesion and migration through TNF-a, IL-6 and TGF-b

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.

Abstract P5-04-06: Soluble factors from activated immune cells induce epithelial mesenchymal transition in inflammatory breast cancer cells

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.

Abstract P2-10-32: Sialyl LewisXand inflammatory mediators in breast cancer patients: biological correlations and prognostic value

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. Spearman9s 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 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 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.

P1-02-07: Epithelial-Mesenchymal Transition Correlated with Serum Cytokine Profiling in Breast Cancer Patients.

Background: Inflammation contributes to the increased invasiveness and poor prognosis in breast cancer (BC) patients. Specifically, the expression of the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα) and interleukin-1 (IL-1) have all been linked to increased invasiveness and poor prognosis. Interestingly, the increased invasiveness was associated with an increase in the acquisition of markers of epithelial-mesenchymal transition (EMT). Therefore, we determined whether the levels of circulating proinflammatory cytokines (IL-1, IL-6, TNFα) and antiinflammatory cytokines (IL10) were correlated with the induction of EMT transcription factors (TFs), Snail1, Zeb1, Twist1, in breast cancer patients. Materials and Methods: From two laboratory-based ongoing studies at the MD Anderson Cancer Center, 41 BC patients were assessed for EMT-TFs in circulating CD45-ve cells (EMT-CTCs) and for serum proinflammatory cytokines before starting any treatment. 32 of 41 patients assessed for EMT had metastatic BC. EMT-CTCs were detected by qRT-PCR for the EMT-TFs Snail1, Zeb1 and Twist1 (Mego 2011; PMID 21387303) and serum cytokines were measured by Luminex bead array assay (MILLIPLEX™ MAP Human Cytokine/Chemokine Panel). Cytokine serum concentrations were compared with the median cytokine levels of healthy donors (HD). We examined the association of serum cytokines above the median HD levels and the presence of EMT-CTCs by non-parametric Mann-Whitney test with a statistical significance for p Results: Of the 41 patients assessed for both serum cytokines and EMT-CTCs, 14 (34%) were positive for at least one EMT-TF, including 3 of 9 (33%) patients with no-metastatic BC and 11 of 32 (34%) patients with metastatic BC. We found that serum levels of IL1a, IL2, TGFα, and TNFβ in patients that were above the median levels of HD sera were higher in patients with EMT-CTCs in the blood (higher IL1a concentration in patients with over expression of Snail1, Zeb1, and Twist1; IL2 with Zeb1; TGFα with Snail1; TNFβ with Zeb1, and Twist1). Further, the higher ratio of proinflammatory/anti-inflammatory cytokines, was associated with the presence of at least one EMT-TF, e.g., IL8/IL10 (p=.005) and TNFα/IL10 (p=.037). Discussion: Patients with proinflammatory cytokine (IL1a, IL2, TGFα, and TNFβ) levels above the median levels of HD or who had a predominantly proinflammatory cytokine profile were more likely to have at least one EMT-TF in their blood. These data are consistent with the hypothesis that proinflammatory cytokines promote EMT, which may be involved in tumor aggressiveness and disease progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-07.

P4-20-04: Cytokine Synthesis by Activated Dendritic Cells in Relation to Disease Progression in Inflammatory Breast Cancer (IBC).

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.

P3-03-02: Higher Expression Levels of Circulating miR-21, miR-19a and miR-10b Are Associated with High Risk Features in Breast Cancer.

Background : MicroRNAs (miRs) have oncogenic and tumor-suppressor functions. MiR-21, miR-19a and miR-10b are overexpressed in breast cancer and regulate tumor cell migration, invasion and angiogenesis. We assessed the levels of miR-21, -19a, and -10b in sera of breast cancer patients and their association with the stage, histological type, hormonal receptor (HR) status and HER2 amplification in the primary tumor. Since circulating tumor cells (CTC) detected by CellSearch® are an independent and strong predictor of overall survival in metastatic breast cancer (MBC), we assessed the relationship between circulating miRs and CTCs. Methods : The study consisted of 30 healthy donors (HD) and 95 breast cancer (BC) patients. Patients’ sera were collected before starting a new line of treatment. Total RNA was isolated, reverse transcribed to cDNA and then subjected to qRT-PCR for the detection of miR-21, -19a, -10b and -192 using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). Mir-192 was used to normalize the expression levels of the other miRs. Fold-changes in expression of miRs were calculated using the 2 −DCt method, where DCt= mean CT target-miRNA -mean CT miR-192 . CTCs were enumerated using CellSearch™ (Veridex LLC, Warren, NJ). Mann-Whitney U test was used to determine differences in serum miR expression levels between patients and HD. Results : Of the 95 BC patients, 39 were non-MBC and 56 MBC. Patients grouped according to the receptor expression by immunohistochemical staining consisted of 27 HR+HER2 − , 30 HR+HER2+, 20 HRHER2+, and 18 HRHER2 − triple negative BC (TNBC). MiR-21 and miR-19a were higher in non-MBC patients than in HD (p=.001, p − HER2+, TNBC) had significantly higher median levels of both miR-21 (p=.018; p=.009; p=.045) and miR-10b (p=.011; p=.014; p=.03) compared with HR+HER2 − BC. HER2+ patients had higher median levels of both miR-21 and miR-10b than those of HER2 − BC (p=.033; p=.01) and HD (p Discussion : High expression levels of miR-21, miR-19a and miR-10b in sera are observed in breast cancer patients, especially with advanced disease. HER2+ BC patients had higher serum levels of miR-21 and miR-10b than HER2 − . IBC patients had a higher serum level of miR-19a than non-IBC patients. Moreover, patients with Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-02.

Abstract P3-02-08: Detection of Circulating Tumor Cells Prior to Autologous Stem Cell Transplantation Is Predictive of Shorter Progression Free Survival in Metastatic Breast Cancer

Background: High dose chemotherapy (HDCT) in patients with metastatic breast cancer (MBC) followed by autologous hematopoietic stem cell transplantation (ASCT) offers high complete response rates compared to standard therapy. However, the bone marrow (BM) is a reservoir for disseminated tumor cells (DTC) and the inadvertent recruitment from BM of DTC, cancer stem cells (CSC), and non-leukocytes undergoing epithelial mesenchymal transition (EMT) during the CD34+ hematopoietic progenitor cell (HPC) mobilization could adversely impact clinical outcome. As epithelial cells in EMT possess stem-like properties, we evaluated the apheresis product for CSC and expression of EMT to ascertain if they had an impact on clinical outcome in the transplant setting. Methods: Twenty-one MBC patients treated with HDCT underwent apheresis to harvest HPC and subsequent ASCT. A peripheral blood (PB) sample was collected before apheresis (baseline) and again after ASCT (post-treatment) for enumeration of CTC by CellSearch (Veridex, LLC, Raritan, NJ). Furthermore, mononuclear cells (MNC) were isolated from 19 HPC-depleted apheresis products. An aliquot of MNC was further depleted of CD45 + leukocytes and then interrogated for expression of the EMT-inducing transcription factor TWIST1 by quantitative RT-PCR. Another aliquot of each MNC sample was analyzed by 6-color flow cytometry for the presence of CSC (CD326 + CD44 hi CD24 lo ) and with ALDH-1 activity (Aldefluor+ CSC), measured by Aldefluor (StemCell Technologies, Vancouver, BC). The logrank test was used to determine if progression-free survival (PFS) was associated with the number of CTCs at baseline and post-ASCT; baseline percentages of CSC and Aldefluor + CSC; and expression of TWIST1 by non-leukocytes. Results: The median follow-up was 17 months with a median time to disease progression of 9.4 months. The median overall survival was 12.9 months. At follow-up, 7 patients had non-progressive disease (NPD) and 12 had progressive disease (PD). CTCs were detected in 6 pts (range, 1-115) at baseline and in 9 patients post-ASCT (range, 1-131). PD patients had statistically significantly higher % of CD326+ epithelial cells but not CSC within the HPC-depleted apheresis products when compared with identical apheresis products of NPD patients (0.20% vs. 0.11%; P=0.033). Aldefluor test was performed in 12 of 19 samples. Four samples with TWIST1 transcripts contained a significantly higher % of CD326+ Aldefluor+ CSC than the samples without TWIST (9.58% vs. 1.58%; P =0.007). The presence of CTC at baseline was associated with shorter progression-free survival (PFS) (P =0.027). However, PFS was not associated with the TWIST1 expression or with % of Aldefluor+ CSC detected in apheresis products. Conclusion: Baseline CTC number was predictive of PFS. HDCT followed by ASCT did not reduce the number of CTC in patients with MBC. Finally, leukocyte-depleted apheresis products that expressed EMT-inducing TWIST1 transcripts contained higher % CSCs suggesting a strong linkage between circulating epithelial cells undergoing EMT and the production of cancer stem cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-08.

Abstract P3-02-04: Dendritic Cells from Breast Cancer Patients with ≥5 CTCs Are Functionally Impaired

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 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-04.