Sanda Tin

Epithelial–Mesenchymal Transition and Stem Cell Markers in Patients with HER2-Positive Metastatic Breast Cancer

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.

Prognostic Value of EMT-Circulating Tumor Cells in Metastatic Breast Cancer Patients Undergoing High-Dose Chemotherapy with Autologous Hematopoietic Stem Cell Transplantation.

Background: Circulating tumor cells (CTCs) are an independent prognostic factor in metastatic breast cancer (MBC) patients treated by conventional dose chemotherapy. The aim of this study was to determine the role of CTCs and CTCs undergoing epithelial-mesenchymal transition (EMT) in metastatic breast cancer. We used the platform of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation (AHSCT) to study the CTCs and CTCs with EMT. Patients and methods: CTCs were enumerated in 21 MBC patients before apheresis and 1 month after AHSCT. CD34-depleted apheresis products were analyzed for CD326+ epithelial and Aldefluor+ cancer stem cells (CSC) by flow cytometry and were depleted of CD45+ cells and assessed for EMT-inducing transcription factors (EMT-TF) by quantitative RT-PCR. Results: Patients with ≥ 5 CTCs/7.5 mL of peripheral blood 1 month after AHSCT had shorter progression-free survival (PFS) (P=0.02) and overall survival (OS) (P=0.02). Patients with apheresis products containing high percentages of CD326+ epithelial cells or overexpressing EMT-TF had shorter PFS. In multivariate analysis, low percentage of CD326+ epithelial cells and response to HDCT with AHSCT were associated with longer PFS, whereas lower CTCs after AHSCT was associated with longer OS. High CTCs, 1 month after AHSCT correlated with shorter PFS and OS in MBC patients undergoing HDCT and AHSCT, while CTCs with EMT and CSCs phenotype in apheresis products are associated with relapse. Conclusion: Our data suggest that CTC and CTCs with EMT are prognostic in MBC patients undergoing HDCT followed by AHSCT.

High serum miR-19a levels are associated with inflammatory breast cancer and are predictive of favorable clinical outcome in patients with metastatic HER2+ inflammatory breast cancer

Introduction Altered serum microRNA (miRNA) levels may be correlated with a dysregulated expression pattern in parental tumor tissue and reflect the clinical evolution of disease. The overexpression of miR-21, miR-10b, and miR-19a is associated with the acquisition of malignant characteristics (increased tumor cell proliferation, migration, invasion, dissemination, and metastasis); thus, we determined their utility as serum biomarkers for aggressive breast cancer (HER2-overexpressed or -amplified [HER2+] and inflammatory breast cancer [IBC]). Experimental Design In this prospective study, we measured miR-21, miR-10b, and miR-19a levels using quantitative reverse transcriptase-polymerase chain reaction in the serum of 113 breast cancer patients and determined their association with clinicopathologic factors and clinical outcome. Thirty healthy donors with no history of cancer were enrolled as controls. Results Patients with non-metastatic HER2+ breast cancer had higher serum miR-21 median levels than patients with non-metastatic HER2− disease (p = 0.044); whereas patients with metastatic HER2+ breast cancer had higher serum miR-10b median levels than patients with metastatic HER2− disease (p = 0.0004). There were no significant differences in serum miR-19a median levels between HER2+ and HER2− groups, regardless of the presence of metastases. High serum miR-19a levels were associated with IBC (p = 0.039). Patients with metastatic IBC had significantly higher serum miR-19a median levels than patients with metastatic non-IBC (p = 0.019). Finally, high serum miR-19a levels were associated with longer progression-free survival time (10.3 vs. 3.2 months; p = 0.022) and longer overall survival time (median not reached vs. 11.2 months; p = 0.003) in patients with metastatic HER2+ IBC. Conclusion High levels of miR-21 and miR-10b were present in the serum of patients with non-metastatic and metastatic HER2+ breast cancer, respectively. High levels of serum miR-19a may represent a biomarker for IBC that is predictive for favorable clinical outcome in patients with metastatic HER2+ IBC.

Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells

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.

Clinical relevance of cancer stem cells in bone marrow of early breast cancer patients

BACKGROUND Cancer stem cells (CSCs) are epithelial tumor cells that express CD44(+)CD24(-/lo). CSCs can be further divided into those that have aldehyde dehydrogenase (ALDH) activity (Aldefluor(+)) and those that do not. We hypothesized that if CSCs are responsible for tumor dissemination, their presence in bone marrow (BM) would be prognostic in early stages of breast cancer (EBC) patients. PATIENTS AND METHODS BM aspirates were collected at the time of surgery from 108 patients with EBC. BM was analyzed for CSCs and ALDH activity by flow cytometry. Overall survival 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 carried out. RESULTS 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. All deceased patients (n = 7) had CSCs in BM. In multivariate analysis, the presence of CSCs in BM was a prognostic factor of DFS (HR = 15.8, P = 0.017). CONCLUSIONS The presence of BM metastasis is correlated with CSCs and these CSCs irrespective of ALDH activity are an independent adverse prognostic factor in EBC patients.

The Role of Thalidomide and Placebo for the Treatment of Cancer-Related Anorexia-Cachexia Symptoms: Results of a Double-Blind Placebo-Controlled Randomized Study

OBJECTIVES To determine the effects of thalidomide and placebo on anorexia-cachexia and its related symptoms, body composition, resting metabolic rate, and serum cytokines and their receptors in patients with advanced cancer. METHODS Included in the study were patients with advanced cancer with weight loss greater than 5% in 6 months and who reported anorexia, fatigue, and one of the following: anxiety, depression, or sleep disturbances. Patients on chemotherapy within 2 weeks prior or during the study were excluded from the study. Patients were randomly assigned to either 100 mg thalidomide or placebo once a day for 14 days. The Edmonton Symptom Assessment Scale (ESAS), Functional Assessment of Anorexia/Cachexia Therapy (FAACT), Functional Assessment of Cancer Illness Therapy (FACIT-F), Hospital Anxiety Depression Scale (HADS) Pittsburgh Sleep Quality Index (PSQI) were utilized, and in addition body composition, Resting Energy Expenditure (REE), and serum cytokine levels were assessed. RESULTS Of the 31 patients entered in the study, 15 were assigned to the thalidomide group and 16 to the placebo group. However only 21/31 patients were able to complete the study. Compared with their baseline values, both the thalidomide and the placebo groups showed significant reduction in cytokines. Tumor necrosis factor (TNF)-α (p=0.04) and its receptors TNFR1 (p=0.04), TNFR2 (p=0.04), and interleukin (IL)-8 (p=0.04) were statistically significant in the thalidomide group. In the placebo group, TNF-α (p=0.008), TNFR1 (p=0.005), TNFR2 (p=0.005), IL-RA (p=0.005), IL-6 (p=0.005), and IL-8 (p=0.005) were statistically significant. However, improvement in these symptoms and cytokine levels were not significantly different in the thalidomide group compared with the placebo group. None of the patients withdrew from the study because of toxicity of either thalidomide or placebo. CONCLUSIONS Based on the poor accrual rate and attrition observed in this study, it is important that future research on thalidomide as a treatment for cancer-related anorexia-cachexia symptoms (ACS) in patients with advanced cancer use less stringent entry criteria and less exhaustive outcome measures.

Circulating Tumor Cells (CTCs) and Epithelial Mesenchymal Transition (EMT) in Breast Cancer: Describing the Heterogeneity of Microscopic Disease.

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 patient9s 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.

Circulating tumour cells are linked to plasma D-dimer levels in patients with metastatic breast cancer

Cancer is a risk factor for venous thromboembolism (VTE). Elevated plasma D-dimer and fibrinogen levels are also risk factors for VTE. Furthermore, in patients with metastatic breast cancer (MBC), the presence of circulating tumour cells (CTCs) is a risk factor for VTE. The relationship between CTCs and D-dimer is unknown. The aim of this study was to determine whether CTCs correlate with plasma D-dimer level, fibrinogen level, and risk of VTE in MBC. This prospective study included 47 MBC patients treated from July 2009 through December 2010 at the MD Anderson Cancer Center. CTCs in peripheral blood were detected and enumerated using the CellSearch system. D-dimer and fibrinogen were measured in plasma at the time of CTC detection. Thirty-three patients (70 %) had ≥ 1 CTC, and 22 patients (47 %) had ≥ 5 CTCs. Patients with ≥ 1 CTC or ≥ 5 CTCs had significantly higher mean plasma D-dimer levels (µg/mL) than patients with no CTCs and < 5 CTCs (2.48 and 3.31 vs 0.80 and 0.84, respectively; p=0.006 for cut-off ≥ 1 CTC and p=0.003 for cut-off ≥ 5 CTCs). In multivariate analysis, presence of CTCs and number of metastases were positively associated with plasma D-dimer level. CTCs were not associated with plasma fibrinogen level. At median follow-up of 13.5 months, three of 33 patients (9 %) with ≥ 1 CTC had VTE, vs no patients with undetectable CTCs. In conclusion, the presence of CTCs was associated with higher levels of plasma D-dimer in MBC patients. This study further confirms an association between CTCs and risk of VTE.

Circulating tumor cells (CTCs) are associated with abnormalities in peripheral blood dendritic cells in patients with inflammatory breast cancer

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 <5 CTCs (p=0.045). In addition, patients with ≥5 CTCs had a lower percentage of mDCs capable of producing TNF-α before or after activation through the toll-like receptor (TLR), as well as a lower percentage of mDCs producing IL-12 after TLR-activation. There was a positive correlation between CTCs counts and expression of the activation (CCR7) and costimulatory (CD86) receptors on TLR-activated mDCs and pDCs, respectively. Moreover, presence of high percentage of mDC capable to produce increased levels of TNF-α was independently associated with inferior OS (p = 0.0006). An increase in the percentage of mDC producing TNF-α might induce a pro-inflammatory environment that could play a role in determining the poor clinical outcome in IBC patients and could add further prognostic value to CTCs.

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.