Jm Reuben

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.

Characterization of Metastatic Breast Cancer Patients with Non-Detectable Circulating Tumor Cells.

Background: Circulating tumor cells (CTC) are independent predictor of progression free and overall survival in metastatic breast cancer patients, with superior prognosis for patients with CTC Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3006.

Abstract OT2-3-01: Phase Ib pilot study to evaluate reparixin in combination with chemotherapy with weekly paclitaxel in patients with HER-2 negative metastatic breast cancer (MBC)

Background: Breast cancer stem cells (BCSCs) have the ability to self renew and generate the full range of cells that make up a bulk tumor. Chemokine receptor 1 (CXCR1) is almost exclusively expressed in the aldehyde dehydrogenase positive (ALDH+) BCSC population compared with its expression in bulk tumor cells. CXCR1 is a receptor for CXCL8 (previously IL8), a proinflammatory chemokine implicated in the metastasis and progression of multiple malignancies, including breast cancers. CXCL8 has also been shown to stimulate self-renewal of BCSCs in vitro. Tissue damage induced by chemotherapeutic agents may induce CXCL8 as part of the injury response. This suggests that strategies aimed at interfering with the CXCL8/CXCR1 axis, activated by conventional chemotherapy (CT), may be able to target BCSCs and increase the efficacy of current therapies. Reparixin, a low molecular weight blocker of CXCL8 biological activity, reduced ALDH-1+ cells in a human breast cancer xenograft when administered alone or in combination with taxane chemotherapy, and reduced metastases. Based on these findings, a Phase Ib study to determine the safety of paclitaxel plus reparixin therapy, and to explore the effects of reparixin on BCSCs and the tumor microenvironment, was initiated. Design and Treatment: Patients will receive a three-day run-in with reparixin oral tablets 3 times/day (tid) followed by paclitaxel 80 mg/m2/week (Days 1, 8, and 15 for 28-day cycle) + reparixin oral tablets tid for 21 days. Three dose levels of 3–6 subjects will be explored in total: Dose level 1 = 400 mg oral reparixin tid; Dose level 2 = 800 mg reparixin tid; and Dose level 3 = 1200 mg reparixin tid. An additional 6 subjects will be enrolled at the highest tolerated dose. Safety will be assessed following one cycle. Treatment continues as long as clinical benefit is observed. Main Eligibility Criteria: Patients must be female aged > 18 years with HER-2 negative MBC, eligible for treatment with paclitaxel (not taxane-refractory), have had up to 3 prior CT lines for advanced breast cancer (not including neo/adjuvant chemotherapy), must have measurable disease according to RECIST criteria version 1.1, have ECOG PS of 0–1, have adequate organ function, and have no brain metastases. Objectives: Primary: To evaluate the safety and pharmacokinetic (PK) profile of the combination treatment. Secondary: 1) To evaluate tumor biopsies for the effects of reparixin on BCSC markers and tumoral microenvironment; 2) To evaluate blood samples for a) enumeration and molecular characterization of circulating tumor cells (CTCs), biomarker profiling for BCSC and epithelial-mesenchymal transition (EMT), and b) inflammatory cytokines; 3) To assess disease response for indication of efficacy. Statistical Methods: All tumor data collected will be reviewed and listed. No formal statistical analysis of this data is planned. Safety and tolerability analysis will be applied on the safety population with descriptive statistics for the safety variables. Summary statistics will be performed for each PK parameter and correlative study parameter. Accrual status: To date, four subjects have been enrolled at the first dose level. Target accrual is 15–24 subjects. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT2-3-01.

Abstract OT2-6-03: A single arm, preoperative, pilot study to evaluate the safety and biological effects of orally administered reparixin in early breast cancer patients who are candidates for surgery

Background. Experimental models and retrospective clinical observations point to Cancer Stem Cells (CSC) as responsible for tumor initiation, treatment resistance, disease recurrence and metastasis. CXCR1, one of the receptors for CXCL8, was identified on breast cancer (BC) CSC. Reparixin, an investigational allosteric inhibitor of CXCR1, administered as monotherapy reduced the metastatic spread of human BC cells and the CSC content of human BC xenograft in mice (Ginestier C et al., JCI 2010).Methods. In this single arm, monotherapy trial (NCT01861054) patients were female aged ? 18 years with HER-2 negative operable BC with a clinical diameter of >1 cm not eligible for neoadjuvant treatment. It was planned to enrol 20 patients with ER+ and/or PgR+ and 20 patients with ER-/PgR- BC (i.e., Triple-Negative BC, TNBC). Patients received reparixin oral tablets 1000 mg 3 times daily (t.i.d.) for 21 days prior to surgery. Patients underwent core biopsies at baseline and at the completion of therapy. Primary objectives were evaluation of the effects of reparixin on CSC and tumor microenvironment and the safety of reparixin. The secondary objective was to define the pharmacokinetic (PK) profile of orally administered reparixin. Signal of Activity (SoA) was defined as a reduction by at least 20% in ALDH-1+ or CD24-/CD44+ CSC by flow cytometry (FC), with consistent reduction by immunohistochemistry (IHC).Results. 18 patients with ER+ and/or PgR+ BC and 2 with TNBC were enrolled. The trial was terminated due to slow enrolment in TNBC cohort. Safety: adverse reaction of grade ?2 (none serious) were experienced by 10/20 patients. All patients completed study treatment. PK: reparixin was rapidly absorbed (median Tmax 1h) and metabolized with median t1/2 of about 1.5 hr on both day 1 and 21. Efficacy: 12/18 and 2/2 patients in the ER+ and/or PgR+ BC group and TNBC group, respectively, had a sizeable ALDH+ and/or CD24-/CD44+ cell population at baseline and a second biopsy evaluated by FC. 11 evaluable patients achieved SoA (9/12 ER+ and/or PgR+ and 2/2 TNBC). The possibility to confirm FC results in tissue sections was hindered by very low numbers of CSC. A reduction in total CXCR1+ cells was found by FC in 7/13 evaluable patients and a reduction in p62 by IHC in 8/13 evaluable patients, suggesting target engagement and induction of autophagy.Conclusions. Enrolment of patients with TNBC in preoperative trials with single non cytotoxic agents is limited by the widespread use of neoadjuvant treatment. Reparixin 1000 mg t.i.d. for 21 consecutive days appeared to be well tolerated. SoA was achieved in 11/14 evaluable patients. The clinical relevance of a ?20% reduction of CSC following a single 21-day course of reparixin in this patient population is unknown. A randomized phase 2 study of reparixin plus paclitaxel versus paclitaxel in frontline treatment of metastatic TNBC is ongoing (NCT02370238). Citation Format: Lori J. Goldstein, Raymond P. Perez, Denise A. Yardley, Linda K. Han, James M. Reuben, Susan McCanna, Beth Butler, Pier Adelchi Ruffini, Jenny C. Chang. A single-arm, preoperative, pilot study to evaluate the safety and biological effects of orally administered reparixin in early breast cancer patients who are candidates for surgery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT057.

Predictive Value of Circulating Tumor Cells (CTCs) in Metastatic Breast Cancer Patients Treated by Bevacizumab-Based Therapy.

Background:Circulating tumor cells (CTC) are involved in cancer dissemination and are an independent prognostic factor in metastatic breast cancer (MBC). Antiangiogenic, bevacizumab-based chemotherapy improves response rate and progression free survival in patients with metastatic breast cancer (MBC), without impact on overall survival. Preclinical data suggest the possibility of increased metastatic potential of tumor cells pretreated by anti-angiogenic therapy (Ebos et al. Cancer Cell 2009,15: 232–9). The aim of this study was to determine the prognostic value of CTC in MBC patients treated by bevacizumab-based therapy.Patients and Methods: This retrospective study included 48 MBC treated with bevacizumab combined chemotherapy regimens and 46 patients treated with chemotherapy alone between January 2004 and December 2008 at M.D.Anderson Cancer Center. CTCs were detected and enumerated before patients started therapy using the CellSearch™ system (Veridex, LLC, NJ, USA). Progression free survival (PFS) and overall survival (OS) were calculated from the date of CTC measurement, estimated by the Kaplan-Meier product limit method, and compared between groups with the log-rank test.Results: At a median follow up of 10.1 months (range: 1-26 months), 22 patients (45.8%) had died. The estimated medians of PFS in bevacizumab-treated patients were 8.1 vs. 5.2 months (p = 0.42) in patients with baseline Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3013.

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.

Abstract PD03-08: Statin use and improved outcome in primary inflammatory breast cancer: retrospective cohort study

Background Inflammatory breast cancer (IBC) is the most aggressive type of breast cancer. HMG-CoA reductase inhibitors (statins) are cholesterol reducing agents with pleiotropic effects, including antitumorigenic and anti-inflammatory properties. We hypothesized that statins reduce the metastatic potential in primary IBC. Methods We retrospectively reviewed 724 patients diagnosed with and treated for primary IBC at The University of Texas MD Anderson Cancer Center between Jan. 12, 1995 and Jan. 27, 2011. Patients with records indicating statin use at the time of IBC diagnosis on the electronic medical record were compared with those without. We further compared outcomes stratified by statin type (hydrophilic [H] versus lipophilic [L]). We used the Kaplan-Meier method to estimate the median disease-free survival (DFS) after surgery, overall survival (OS), and disease specific survival (DSS), followed by Cox proportional hazards regression model to test statistical significance of several potential prognostic factors. Results For primary IBC patients who had information on their statin use status at IBC diagnosis, the median DFS time were 4.88 years, 2.47 years and 1.76 years (P= 0.04); the median OS time 5.05 years, 3.79 years and 4.32 years (P= 0.35); and the median DSS time 5.10 years, 3.79 years and 4.52 years (P= 0.37), for patients who took “ H”, “L” and no statin, respectively. In multivariable Cox model stratified by radiation therapy, ER/PR status and HER2 status, statin “H” use was associated with significantly improved DFS compared to no statin use (HR=0.49; 95% CI: 0.28–0.84; p Conclusions Hydrophilic statin use was associated with improved DFS. There was a trend for reduced HR in OS and DSS among primary IBC patient who used hydrophilic statins. A prospective randomized study to evaluate the potential survival benefits of statins in primary IBC population is warranted. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-08.

Novel inflammatory breast cancer cell line, MDA-IBC-1 expresses and secretes WISP3, a putative tumor suppressor in inflammatory breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #1051 Wnt-1-induced secreted protein 3 (WISP3) is a member of the CCN family of proteins, which also includes connective tissue growth factor (CTGF), Cy61, Nov, WISP1, and WISP2. WISP3, as well as the other CCN family members, plays important roles in cellular processes such as proliferation, migration, and survival. WISP3 was originally isolated as Lost in Inflammatory Breast Cancer (LIBC) based on its expression in only 20% of human IBC tumors. Lack of WISP3 expression has also been reported in the IBC-derived cell line SUM-149. In SUM149 cells WISP3 has been reported to function as a tumor suppressor capable of decreasing proliferation and maintaining an epithelial phenotype. Here we examine the expression of WISP3 in a novel aggressive IBC cell line with epithelial-mesenchymal transition (EMT) morphology and abundant WISP3 expression. MDA-IBC-1 cells were isolated from the pleural fluid of a patient with ER+, PR-, HER2/neu- IBC. In 2-dimensional (2D) adherent culture, MDA-IBC-1 cells exhibit fibroblast-like EMT morphology and express EMT-associated proteins such as N-cadherin, vimentin, and fibronectin. Correspondingly, E-cadherin protein expression is absent in 2D cultures. MDA-IBC-1 cells derived from 2D cultures form mammospheres when cultured in 3-dimensional (3D) progenitor promoting conditions. Interestingly, the expression of EMT molecular markers is absent in 3D progenitor promoting mammosphere culture. MDA-IBC-1 cells exhibit robust WISP3 protein expression in both 2D and 3D culture (1.8-fold higher in 3D). In addition, MDA-IBC-1 cells secrete WISP3 as indicated by the presence of WISP3 protein in MDA-IBC-1 derived conditioned media. WISP3 binding is reported to decrease insulin-like growth factor-1 (IGF-1) activation of the IGF1R signaling cascade in SUM-149 cells. IGFR1 protein is present in 2D and 3D cultured MDA-IBC-1 cells. IGF-1 protein overlay assays using conditioned media demonstrate a WISP3:IGF-1 interaction, suggesting functional folding. Serum-free MDA-IBC-1 conditioned media did not increase basal SUM149 proliferation and it inhibited IGF-1 stimulated proliferation to serum-free (basal) levels (p≤0.05). In addition, culturing SUM-149 cells in conditioned media obtained from IBC-1 cells significantly alters SUM-149 to a completely fibroblastic morphology. These data demonstrate that MDA-IBC-1 cells represent an IBC cell line with novel expression and regulation of WISP3. MDA-IBC-1 cells may better represent the cohort of IBC patients whose tumors express WISP3. This data suggest WISP3 may not function as a tumor suppressor in all IBC patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1051.

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.

Circulating tumor cells and FDG-PET/CT: biological and functional methods for therapeutic monitoring in metastatic breast cancer.

Abstract #6052 Introduction: The combination of Computed Tomography (CT) and [18F] Fluorodeoxyglucose - Positron Emission Tomography (FDG-PET) scanning technologies provides a more complete picture of disease activity than CT alone. Circulating tumor cell (CTC) levels were shown to be more predictive than standard imaging (CT) when used to monitor disease progression in women with metastatic breast cancer (MBC). We performed a retrospective study to compare the ability of combined FDG-PET/CT to CTC to predict clinical outcomes in patients treated for MBC.
 Methods: One hundred and two MBC patients with either measurable or evaluable disease starting a new line of therapy had CTC counts and FDG-PET/CT scans done at baseline (BL) and at mid-therapy. CTC: 7.5mL of blood collected in CellSave tubes at both time points was assayed for CTC using the FDA approved CellSearch® System. Patients were categorized as having a favorable ( 50%. Changes in CTC and SUV at mid-therapy were compared to progression free survival (PFS) and overall survival (OS).
 Results: CTC: 50% (51/102) patients had ≥5 CTC at baseline (BL). At mid-therapy (median 2.5 months from BL), 21/102 progressed (≥5CTC) with a median PFS of 2.8 months vs. 7.8 months for those with no progression ( 2nd line chemotherapy and triple-negative disease.
 Conclusion: ≥5 CTC and/or no response at FDG-PET/CT at mid-therapy accurately predicted significantly shorter OS. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6052.