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 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.
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: Increased expression of miR-19a plays an important role in tumor progression by regulating angiogenesis and apoptosis. As miRs can be a valuable diagnostic and prognostic serum biomarker, we assessed if high serum levels of miR-19a were associated with the shorter overall survival (OS) of patients with metastatic inflammatory breast cancer (MIBC) and metastatic non-inflammatory breast cancer (MNIBC). Methods: We analyzed 27 MNIBC (10 HER2 normal [HER2−] and 17 HER2 amplified [HER2+]), 37 MIBC (18 HER2− and 19 HER2+) patients, and 30 healthy donors (HDs). Patients' sera were collected before starting a new line of treatment. MiR-19a levels were measured in patients' sera and cell lines (MCF-7, SKBR3, MA-231, KPL-4, SUM-149) by qRT-PCR to assess differences in expression levels between IBC and non-IBC. MiR-192 and U6 snRNA were used to normalize miR-19a expression levels in serum and cell lines, respectively. Fold-changes in expression of miR-19a were calculated using the 2−DCt method. Mann-Whitney U test, ROC curve analysis, Kaplan-Meier, and Student's t-test were used for statistical analysis. Results: Median levels of miR-19a were higher in sera of both MNIBC and MIBC patients than in HDs (p = .001, p < 001, respectively). Median serum level of miR-19a was higher in MIBC than in MNIBC (p = .013). ROC curve analysis revealed that miR-19a could differentiate MNIBC from MIBC (AUC = 0.683; p = 0.013). With a cutoff value of 1.62 set by ROC, the sensitivity, specificity and positive predictive value (PPV) were 56.7%, 85.2%, and 84.0%, respectively. The triple receptor negative (TN) IBC cell line SUM-149 had the highest level of miR-19a and it was higher than the non-IBC: MDA-231 (TN; p = .031); SKBR3 (HER2+; p = .021); and MCF-7 (ER/PR+; p = .003). KPL-4 (IBC, HER2+) had the second highest miR-19a levels after SUM-149. MIBC patients had a shorter median OS than MNIBC patients (18.8 vs 27.8 months; p = .041; range, 0.7–40.3 months; median follow-up 19.5 months). MIBC patients with HER2− tumor (n = 11) and high serum levels of miR-19a (>1.62) had a shorter median OS than MNIBC patients with HER2− tumors (n = 9) and low serum level of miR-19a (<1.62) (16.15 vs 33.45 months; p = .025). There was no difference in the OS between HER2+ MIBC patients with high miR-19a (> 1.62) and HER2+ MNIBC patients with low miR-19a (<1.62). Discussion: In this pilot study, miR-19a may be a potential biomarker for inflammatory breast cancer, as significantly higher miR-19a levels were present in the sera of MIBC compared with MNIBC as well as in IBC compared with non-IBC cell lines. Furthermore, miR-19a may be predictive of OS, as HER2− MIBC patients with high serum levels of miR-19a had shorter OS than HER2− MNIBC patients with low serum levels of miR-19a. Our data suggest that high expression of miR-19a: is a feature of IBC tumor cells; can be detected in the serum; may favor tumor progression and predict poor outcome of HER2− MNIBC patient. A larger cohort of patients is required to confirm these observations and examine predictive potential. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-02.
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