Purpose: Chemokine receptor 1 (CXCR1) is recognized as an actionable receptor selectively expressed by breast cancer stem cells (BCSCs). Reparixin is an investigational allosteric inhibitor of chemokine receptors 1 and 2 (CXCR1/2), and demonstrates activity against BCSCs in human breast cancer xenografts. This phase Ib clinical trial examined dose, safety, and pharmacokinetics of paclitaxel plus reparixin therapy, and explored effects of reparixin on BCSCs in patients with metastatic breast cancer (MBC) (trial registration ID: NCT02001974). Experimental Design: Eligible patients had MBC and were candidates for paclitaxel therapy. Study treatment included a 3-day run-in with reparixin oral tablets three times a day, followed by paclitaxel 80 mg/m²/week (days 1, 8, and 15 for 28-day cycle) + reparixin tablets three times a day for 21/28 days; three dose cohorts were examined in a 3+3 dose escalation schema. Additional patients were recruited into an expansion cohort at the recommended phase II dose to further explore pharmacokinetics, safety, and biological effects of the combination therapy. Results: There were neither G4–5 adverse events nor serious adverse events related to study therapy and no interactions between reparixin and paclitaxel to influence their respective pharmacokinetic profiles. A 30% response rate was recorded, with durable responses >12 months in two patients. Exploratory biomarker analysis was inconclusive for therapy effect on BCSCs. Conclusions: Weekly paclitaxel plus reparixin in MBC appeared to be safe and tolerable, with demonstrated responses in the enrolled population. Dose level 3, 1200 mg orally three times a day, was selected for further study in a randomized phase II trial (NCT02370238). Clin Cancer Res; 23(18); 5358–65. ©2017 AACR.

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.

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.

Epithelial cancer cells are likely to undergo epithelial–mesenchymal transition (EMT) prior to entering the peripheral circulation. By undergoing EMT, circulating tumor cells (CTCs) lose epithelial markers and may escape detection by conventional methods. Therefore, we conducted a pilot study to investigate mRNA transcripts of EMT‐inducing transcription factors (TFs) in tumor cells from the peripheral blood (PB) of patients with primary breast cancer (PBC). PB mononuclear cells were isolated from 52 patients with stages I–III PBC and 30 healthy donors (HDs) and were sequentially depleted of EpCAM⁺ cells and CD45⁺ leukocytes, henceforth referred to as CD45⁻. The expression levels of EMT‐inducing TFs (TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2) in the CD45⁻ cells were determined using quantitative real‐time polymerase chain reaction. The highest level of expression by the CD45⁻ cell fraction of HD was used as “cutoff” to determine if samples from patients with PBC overexpressed any EMT‐inducing TFs. In total, 15.4% of patients with PBC overexpressed at least one of the EMT‐inducing TF transcripts. Overexpression of any EMT‐inducing TF transcripts was more likely to be detected in patients with PBC who received neoadjuvant therapies (NAT) than patients who received no NAT (p = 0.003). Concurrently, CTCs were detected in 7 of 38 (18.4%) patients by CellSearch® and in 15 of 42 (35.7%) patients by AdnaTest™. There was no association between the presence of CTCs measured by CellSearch® or AdnaTest™. In summary, our results demonstrate that CTCs with EMT phenotype may occur in the peripheral circulation of patients with PBC and that NAT is unable to eliminate CTCs undergoing EMT.

BACKGROUND: T‐regulatory (T_R) cells expressing cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) maintain peripheral immune tolerance and negatively affect host immune responses against cancer. The immunobiologic effects of ticilimumab, a human monoclonal antibody against CTLA‐4, was administered to patients with metastatic melanoma who participated in a Phase I/II clinical trial.METHODS: Thirty patients who received ticilimumab at a dose of 10 mg/kg monthly (n = 20) or 15 mg/kg every 3 months (n = 10) were studied at study entry and at 14‐day intervals thereafter to assess lymphocyte immunophenotypes, interleukin (IL)‐2 and IL‐10 production, and the expression of T_R‐related genes in peripheral blood mononuclear cells (PBMC) from a subset of patients was studied by real‐time polymerase chain reaction.RESULTS: Four of 12 patients with immune‐related adverse events (IRAE) attained objective antitumor responses (ATR), whereas only 1 of 18 patients without IRAE attained ATR (χ² = 4.0; P = .0455). Patients with ATR had significant reductions in T_R cells and constitutive IL‐10 production accompanied by a significant increase in IL‐2 production by activated T cells. Although IRAE⁺/ATR⁺ patients demonstrated a positive correlation between CTLA‐4 and glucocorticoid‐induced tumor necrosis factor receptor (GITR) transcripts (Spearman rho = .522; P = .015), IRAE⁻/ATR⁻ patients had a positive correlation between the transcripts of CTLA‐4 and program death‐1 (PD‐1) receptor (Spearman rho = .891; P = .000).CONCLUSIONS: Antitumor responses in patients with metastatic melanoma who were treated with ticilimumab were found to be correlated with reductions in T_R cells and constitutive secretion of IL‐10, an increase in IL‐2 production, and a positive correlation between transcripts of CTLA‐4 and GITR. Conversely, a lack of ATR was found to be correlated with steady levels of T_R cells and constitutive IL‐10 secretion, and a positive correlation between the transcripts of CTLA‐4 and PD‐1. Cancer 2006. © 2006 American Cancer Society.