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+CD44hiCD24lo) 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.
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<.05. 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.
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<0.001) or TNF-α (p=0,008) were significantly lower in BC patients than in HD. However, patients with DC that produced these cytokines above the median levels of HD had shorter PFS or OS. In IBC patients, higher numbers of TLR-8-activated mDC that produced TNF-α (p=0.025) or IL-12 (p=0.003) predicted shorter OS. In mIBC patients, a higher number of TLR-7-activated pDC producing IFN-α (p=0.024) or IL-10 (p=0.034) predicted shorter PFS. 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.
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.
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 (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.
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.
Background MicroRNAs (miRNAs) play an important role in the regulation of tumorigenesis and metastatic process. MiR-21 is an oncomiR that is overexpressed by breast cancer tumor tissue and is associated with high tumor grade and negative hormone receptor status. MiR-21 can control angiogenesis by regulating vascular endothelial growth factor (VEGF) as well as the expression and secretion of basic fibroblast growth factor (bFGF). MiR-19a is a member of the miR-17-92 family that was shown to suppress breast cancer cell proliferation. As angiogenesis factors act as attractants of circulating tumor cells (CTCs), a strong predictor of overall survival in metastatic breast cancer (MBC), we investigated the relationship between CTC count, levels of VEGF and bFGF, and the expression of miR-21 and miR-19a in sera of MBC patients. Methods RNA was extracted from sera collected from 33 MBC patients and 10 healthy donors (HD) using Total RNA purification Kit (Norgene Biotek Corporation, ON, Canada). The expression levels of miR-21, miR-19a and miR-192 were evaluated in triplicate by qRT-PCR using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). Mir-192 was used to normalize the expression levels of miR-21 and miR-19a. The fold-change values were calculated using 2-DCt, where DCt= mean CTtarget-miRNA - mean CTmiR-192. CTCs were detected by CellSearch System (Veridex LLC, Warren, NJ). Serum VEGF and bFGF were determined using a multiplex bead assay (Millipore, Billerica, MA). Mann-Whitney U test was used to compare the miRNAs expression level between MBC patients and HD. Spearman's correlation was used to determine the association between the expression of miRNAs and growth factors. Results There was a significantly higher median level of miR-21 in MBC patients compared with HD (22.8 versus 7.2, P=0.003), while the expression level of miR-19a was similar for both groups. On average, the relative abundance of miR-21 in sera (ratio of MBC versus HD) is 3.1-fold overexpression. Furthermore, levels of miR-21 was positively correlated with the serum levels of VEGF (Spearman Rho= 0.364; P= 0.038) and bFGF (Spearman Rho= 0.367; P = 0.036). CTCs were detected in 23 patients, ranged from 1 to 134 per 7.5 mL of peripheral blood. Patients with no CTCs had a significantly higher median level of miR-19a than patients with no CTC (6.5 vs 2.8, P =0.047). Conclusion Our results support the notion that miR-21 overexpression may play a role in angiogenesis and metastasis by upregulating VEGF and bFGF in MBC. Undetectable CTC count may be related to overexpression of miR-19a, an epigenetic suppressor of breast tumor proliferation. Whereas overexpression of miR-19a may limit the tumor cells from entering the peripheral circulation, increasing miR-21 in the serum could accelerate the disease progression by promoting angiogenesis in MBC patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-01.
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