• Association between circulating tumor cells and peripheral blood monocytes in metastatic breast cancer

      De Giorgi, U; Mego, M; Scarpi, E; Giordano, A; Giuliano, M; Valero, V; Alvarez, RH; Ueno, NT; Cristofanilli, M; Reuben, JM; Giordano, Antonio|0000-0002-5959-016X (2019-08-01)
      © The Author(s), 2019. Background: We retrospectively evaluated the correlation between a baseline measurement of circulating tumor cells (CTCs) and inflammation-based scores in patients with metastatic breast cancer (MBC). Methods: The optimal value of inflammation-based scores as the neutrophil–lymphocyte ratio (NLR), platelet–lymphocyte ratio (PLR), monocyte–lymphocyte ratio (MLR) and systemic immune-inflammation index (SII) to predict survival was determined and compared with CTC <5 or ⩾5 per 7.5 ml of blood. Results: In the overall population of 516 women with MBC, CTCs correlated with peripheral blood monocytes (p = 0.008) and neutrophils (p = 0.038). In triple-negative tumors, CTCs correlated with monocyte count (p = 0.009); in HER2+ tumors, CTCs correlated with neutrophil count (p = 0.009), with a trend versus monocyte count (p = 0.061), whereas no correlation was found in HER2– estrogen receptor-positive (ER+) tumors. In multivariate analysis only monocytes were associated with ⩾5 CTCs (OR = 2.72, 95% CI 1.09–6.80, p = 0.033). In multivariable analysis for predictors of overall survival, CTC (⩾5 versus <5), number of metastatic sites (>1 versus 1), tumor subtypes (triple-negative versus HER2– ER+ tumors) and MLR only remained significant. Conclusions: CTC and MLR are predictors of overall survival in MBC. CTC correlates with monocytes, in particular in triple-negative tumors.
    • Circulating tumor cells (CTC) are associated with defects in adaptive immunity in patients with inflammatory breast cancer

      Mego, M; Gao, H; Cohen, EN; Anfossi, S; Giordano, A; Sanda, T; Fouad, TM; De Giorgi, U; Giuliano, M; Woodward, WA; Alvarez, RH; Valero, V; Ueno, NT; Hortobagyi, GN; Cristofanilli, M; Reuben, JM; Giordano, Antonio|0000-0002-5959-016X (2016-06-03)
      © Ivyspring International Publisher. Background: Circulating tumor cells (CTCs) play a crucial role in tumor dissemination and are prognostic in primary and metastatic breast cancer. Peripheral blood (PB) immune cells contribute to an unfavorable microenvironment for CTC survival. This study aimed to correlate CTCs with the PB T-cell immunophenotypes and functions of patients with inflammatory breast cancer (IBC). Methods: This study included 65 IBC patients treated at the MD Anderson Cancer Center. PB was obtained from patients prior to starting a new line of chemotherapy for CTCs enumeration by CellSearch®, and T cell phenotype and function by flow cytometry; the results were correlated with CTCs and clinical outcome. Results: At least 1 CTC (≥1) or ≥5 CTCs was detected in 61.5% or 32.3% of patients, respectively. CTC count did not correlate with total lymphocytes; however, patients with ≥1 CTC or ≥5 CTCs had lower percentages (%) of CD3+ and CD4+ T cells compared with patients with no CTCs or <5 CTCs, respectively. Patients with ≥1 CTC had a lower percentage of T-cell receptor (TCR)-activated CD8+ T cells synthesizing TNF-α and IFN-γ and a higher percentage of T-regulatory lymphocytes compared to patients without CTCs. In multivariate analysis, tumor grade and % CD3+ T-cells were associated with ≥1 CTC, whereas ≥5 CTC was associated with tumor grade, stage, % CD3+ and % CD4+ T cells, and % TCR-activated CD8 T-cells synthesizing IL-17. Conclusions: IBC patients with CTCs in PB had abnormalities in adaptive immunity that could potentially impact tumor cell dissemination and initiation of the metastatic cascade.
    • Circulating tumor cells (CTCs) are associated with abnormalities in peripheral blood dendritic cells in patients with inflammatory breast cancer

      Mego, M; Gao, H; Cohen, EN; Anfossi, S; Giordano, A; Tin, S; Fouad, TM; Giorgi, UD; Giuliano, M; Woodward, WA; Alvarez, RH; Valero, V; Ueno, NT; Hortobagyi, GN; Cristofanilli, M; Reuben, JM; Giordano, Antonio|0000-0002-5959-016X (2017-01-01)
      © Mego et al. 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-a 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-a was independently associated with inferior OS (p = 0.0006). An increase in the percentage of mDC producing TNF-a might induce a proinflammatory environment that could play a role in determining the poor clinical outcome in IBC patients and could add further prognostic value to CTCs.
    • Circulating tumor cells as early predictors of metastatic spread in breast cancer patients with limited metastatic dissemination

      Giuliano, M; Giordano, A; Jackson, S; De Giorgi, U; Mego, M; Cohen, EN; Gao, H; Anfossi, S; Handy, BC; Ueno, NT; Alvarez, RH; De Placido, S; Valero, V; Hortobagyi, GN; Reuben, JM; Cristofanilli, M; Giordano, Antonio|0000-0002-5959-016X (2014-09-16)
      © 2014 Giuliano et al.; licensee BioMed Central Ltd. Introduction: Traditional factors currently used for prognostic stratification do not always adequately predict treatment response and disease evolution in advanced breast cancer patients. Therefore, the use of blood-based markers, such as circulating tumor cells (CTCs), represents a promising complementary strategy for disease monitoring. In this retrospective study, we explored the role of CTC counts as predictors of disease evolution in breast cancer patients with limited metastatic dissemination. Methods: A total of 492 advanced breast cancer patients who had a CTC count assessed by CellSearch prior to starting a new line of systemic therapy were eligible for this analysis. Using the threshold of 5 CTCs/7.5 ml of blood, pretreatment CTC counts were correlated in the overall population with metastatic site distribution, evaluated at baseline and at the time of treatment failure, using Fisher's exact test. Time to visceral progression and time to the development of new metastatic lesions and sites were estimated in patients with nonvisceral metastases and with single-site metastatic disease, respectively, by the Kaplan-Meier method. Survival times were compared between groups according to pretreatment CTC count by logrank test. Results: In the overall population, a pretreatment level ≥5 CTCs/7.5 ml was associated with an increased baseline number of metastatic sites compared with <5 CTCs/7.5 ml (P = 0.0077). At the time of treatment failure, patients with ≥5 CTCs/7.5 ml more frequently developed new metastatic lesions and sites compared with those with <5 CTCs/7.5 ml (development of new lesions: P = 0.0002; development of new sites: P = 0.0031). Among patients with disease originally confined to nonvisceral sites, ≥5 CTCs/7.5 ml was associated with remarkably shorter time to visceral metastases (P = 0.0021) and overall survival (P = 0.0006) compared with <5 CTCs/7.5 ml. In patients with single-site metastatic disease, ≥5 CTCs/7.5 ml was associated with a significant reduction of the time to development of new metastatic sites (P = 0.0051) and new lesions (P = 0.0002) and with worse overall survival (P = 0.0101). Conclusion: Our results suggest that baseline CTC counts can be used as an early predictor of metastatic potential in breast cancer patients with limited metastatic dissemination.
    • Circulating tumor cells as prognostic and predictive markers in metastatic breast cancer patients receiving first-line systemic treatment

      Giuliano, M; Giordano, A; Jackson, S; Hess, KR; De Giorgi, U; Mego, M; Handy, BC; Ueno, NT; Alvarez, RH; De Laurentiis, M; De Placido, S; Valero, V; Hortobagyi, GN; Reuben, JM; Cristofanilli, M; Giordano, Antonio|0000-0002-5959-016X (2011-06-15)
      Introduction: Circulating tumor cells (CTCs) represent an independent predictor of outcome in patients with metastatic breast cancer (MBC). We assessed the prognostic impact of CTCs according to different first-line systemic treatments, and explored their potential predictive value in MBC patients.Methods: We retrospectively evaluated 235 newly diagnosed MBC patients, treated at the University of Texas MD Anderson Cancer Center. All patients had a baseline CTC assessment performed with CellSearch ®. Progression-free survival and overall survival were compared with the log-rank test between groups, according to CTC count (< 5 vs. ≥ 5) and type of systemic therapy. We further explored the predictive value of baseline CTCs in patients receiving different treatments.Results: At a median follow-up of 18 months, the CTC count was confirmed to be a robust prognostic marker in the overall population (median progression-free survival 12.0 and 7.0 months for patients with CTC < 5 and ≥ 5, respectively; P < 0.001). Conversely, in patients with human epidermal growth factor receptor-2-overexpressed/amplified tumors receiving trastuzumab or lapatinib, the baseline CTC count was not prognostic (median progression-free survival 14.5 months for patients with CTC < 5 and 16.1 months for those with CTC ≥ 5; P = 0.947). Furthermore, in patients with human epidermal growth factor receptor-2 normal tumors, a baseline CTC count ≥ 5 identified subjects who derived benefit from more aggressive treatments, including combination chemotherapy and chemotherapy plus bevacizumab.Conclusions: This analysis suggests that the prognostic information provided by CTC count may be useful in patient stratifications and therapeutic selection, particularly in the group with positive CTCs, in which various therapeutic choices may procure differential palliative benefit. © 2011 Giuliano et al.; licensee BioMed Central Ltd.
    • Circulating tumor cells in newly diagnosed inflammatory breast cancer

      Mego, M; Giordano, A; De Giorgi, U; Masuda, H; Hsu, L; Giuliano, M; Fouad, TM; Dawood, S; Ueno, NT; Valero, V; Andreopoulou, E; Alvarez, RH; Woodward, WA; Hortobagyi, GN; Cristofanilli, M; Reuben, JM; Giordano, Antonio|0000-0002-5959-016X (2015-01-15)
      © Mego et al. Introduction: Circulating tumor cells (CTCs) are an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. The prognostic value of a CTC count in newly diagnosed IBC has not been established. The aim of this study was to assess the prognostic value of a baseline CTC count in patients with newly diagnosed IBC. Methods: This retrospective study included 147 patients with newly diagnosed IBC (77 with locally advanced and 70 with metastatic IBC) treated with neoadjuvant therapy or first-line chemotherapy during the period from January 2004 through December 2012 at The University of Texas MD Anderson Cancer Center. CTCs were detected and enumerated by using the CellSearch system before patients were started with chemotherapy. Results: The proportion of patients with ≥1 CTC was lower among patients with stage III than among patients with metastatic IBC (54.5% versus 84.3%; P=0.0002); the proportion of patients with ≥5 CTCs was also lower for stage III than for metastatic IBC (19.5% versus 47.1%; P=0.0004). Patients with fewer than five CTCs had significantly better progression-free survival (PFS) (hazard ratio (HR)=0.60; P=0.02) and overall survival (HR=0.59; P=0.03) than patients with five or more CTCs. Among patients with stage III IBC, there was a nonsignificant difference in PFS (HR=0.66; 95% confidence interval (CI), 0.31 to 1.39; P=0.29) and OS (HR=0.54; 95% CI, 0.24 to 1.26; P=0.48) in patients with no CTCs compared with patients with one or more CTCs. In multivariate analysis, CTC was prognostic for PFS and OS independent of clinical stage. Conclusions: CTCs can be detected in a large proportion of patients with newly diagnosed IBC and are a strong predictor of worse prognosis in patients with newly diagnosed IBC.
    • High serum miR-19a levels are associated with inflammatory breast cancer and are predictive of favorable clinical outcome in patients with metastatic HER2<sup>+</sup> inflammatory breast cancer

      Anfossi, S; Giordano, A; Gao, H; Cohen, EN; Tin, S; Wu, Q; Garza, RJ; Debeb, BG; Alvarez, RH; Valero, V; Hortobagyi, GN; Calin, GA; Ueno, NT; Woodward, WA; Reuben, JM; Giordano, Antonio|0000-0002-5959-016X (2014-01-08)
      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 nonmetastatic 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 HER22 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. © 2014 Anfossi et al.
    • Inflammation mediated metastasis: Immune induced epithelial-to-mesenchymal transition in inflammatory breast cancer cells

      Cohen, EN; Gao, H; Anfossi, S; Mego, M; Reddy, NG; Debeb, B; Giordano, A; Tin, S; Wu, Q; Garza, RJ; Cristofanilli, M; Mani, SA; Croix, DA; Ueno, NT; Woodward, WA; Luthra, R; Krishnamurthy, S; Reuben, JM; Giordano, Antonio|0000-0002-5959-016X (2015-07-24)
      © 2015 Cohen et al. 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 adhesionmolecule, 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.
    • Prognostic value of EMT-circulating tumor cells in metastatic breast can-cer patients undergoing high-dose chemotherapy with autologous hematopoietic stem cell transplantation

      Mego, M; Gao, H; Lee, BN; Cohen, EN; Tin, S; Giordano, A; Wu, Q; Liu, P; Nieto, Y; Champlin, RE; Hortobagyi, GN; Cristofanilli, M; Ueno, NT; Reuben, JM; Giordano, Antonio|0000-0002-5959-016X (2012-12-10)
      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 epitheli-al-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+ epi-thelial 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 overex-pressing EMT-TF had shorter PFS. In multivariate analysis, low percentage of CD326+ epi-thelial 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. © Ivyspring International Publisher.