EpCAM-independent isolation of circulating tumor cells with epithelial-to-mesenchymal transition and cancer stem cell phenotypes using ApoStream® in patients with breast cancer treated with primary systemic therapy.

Background: Tumor cells with a mesenchymal phenotype and/or cancer stem-like cells (CSCs) are known to contribute to metastasis and drug resistance. Circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) and CTCs reflecting a dedifferentiated CSC phenotype may not be detected using only an anti-EpCAM antibody to capture them. We used an antibody-independent CTC enrichment platform, ApoStream®, which does not rely on any antibody, including anti-EpCAM, to capture EMT- and CSC-CTCs in breast cancer patients who received neoadjuvant chemotherapy and correlated them to pathological complete response (pCR).

A phase Ib study of entinostat plus lapatinib with or without trastuzumab in patients with HER2-positive metastatic breast cancer that progressed during trastuzumab treatment.

Background: Human epidermal growth factor 2 (HER2) is an effective therapeutic target in breast cancer; however, resistance to anti-HER2 agents such as trastuzumab and lapatinib develops. In a preclinical model, an HDAC inhibitor epigenetically reversed the resistance of cancer cells to trastuzumab and showed synergistic efficacy with lapatinib in inhibiting growth of trastuzumab-resistant HER2-positive (HER2+) breast cancer.

Methods: A phase 1b, dose escalation study was performed to assess maximum tolerated dose, safety/toxicity, clinical efficacy and explored pharmacodynamic biomarkers of response to entinostat combined with lapatinib with or without trastuzumab.

Change in Topoisomerase 1-Positive Circulating Tumor Cells Affects Overall Survival in Patients with Advanced Breast Cancer after Treatment with Etirinotecan Pegol.

Preplanned exploratory analyses were performed to identify biomarkers in circulating tumor cells (CTC) predictive of response to the topoisomerase 1 inhibitor etirinotecan pegol (EP). The BEACON trial treated patients with metastatic breast cancer (MBC) with EP or treatment of physician's choice (TPC). Blood from 656 of 852 patients (77%) was processed with ApoStream to enrich for CTCs.

Isolation of Circulating Tumor Cells from Multiple Epithelial Cancers with ApoStream(®) for Detecting (or Monitoring) the Expression of Folate Receptor Alpha.

This study describes our efforts to further the field of noninvasive diagnostics, specifically in the area of liquid biopsies in oncology. We employed laser scanning cytometry using highly selective antibodies to interrogate circulating tumor cells (CTCs) that were isolated using ApoStream(®) technology to identify folate receptor alpha (FRα)-positive cells. We demonstrate that FRα(+) CTCs can be isolated from patients with metastatic cancers, including NSCLC adenocarcinoma, breast cancer, and ovarian cancer, whereas squamous cell lung cancer and normal healthy controls were devoid of FRα(+) CTCs.

Dual inhibition of the vascular endothelial growth factor pathway: a phase 1 trial evaluating bevacizumab and AZD2171 (cediranib) in patients with advanced solid tumors.

Background: The current study was conducted to evaluate the safety and biological activity of dual inhibition of the vascular endothelial growth factor (VEGF) pathway with combined bevacizumab and cediranib (a VEGF receptor tyrosine kinase inhibitor).

Methods: This was a 3 + 3 dose escalation study in patients with advanced solid tumors. Cediranib was given orally daily for 21 days and bevacizumab intravenously every 2 weeks.

ApoStream(™), a new dielectrophoretic device for antibody independent isolation and recovery of viable cancer cells from blood.

Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream(™) that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber.

Epithelial cell adhesion molecule-positive circulating tumor cells as predictive biomarker in patients with prostate cancer.

Objective: To assess the use of circulating tumor cells (CTCs) as a longitudinal endpoint factor for clinical monitoring of patients with prostate cancer and to evaluate the association among the baseline CTC number, various clinical characteristics, and survival.

Materials And Methods: The CTCs were enumerated using the CellSearch Food and Drug Administration-cleared CTC kit in 202 patients with prostate cancer. Variables, including metastatic site, prostate-specific antigen level, Gleason score, testosterone level, and use of androgen treatment, were tested for association with the CTC number.

A phase II study of lapatinib in recurrent/metastatic squamous cell carcinoma of the head and neck.

Purpose: This study sought to determine the efficacy and safety profile of lapatinib in patients with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN).

Experimental Design: This phase II multiinstitutional study enrolled patients with recurrent/metastatic SCCHN into two cohorts: those without (arm A) and those with (arm B) before exposure to an epidermal growth factor receptor (EGFR) inhibitor. All subjects were treated with lapatinib 1,500 mg daily.

Erlotinib and bevacizumab in patients with recurrent or metastatic squamous-cell carcinoma of the head and neck: a phase I/II study.

Background: Epidermal growth factor receptor (EGFR) is a validated target in squamous-cell carcinoma of the head and neck, but in patients with recurrent or metastatic disease, EGFR targeting agents have displayed modest efficacy. Vascular endothelial growth factor (VEGF)-mediated angiogenesis has been implicated as a mechanism of resistance to anti-EGFR therapy. In this multi-institutional phase I/II study we combined an EGFR inhibitor, erlotinib, with an anti-VEGF antibody, bevacizumab.

Quantitative analysis of melanocytic tissue array reveals inverse correlation between activator protein-2alpha and protease-activated receptor-1 expression during melanoma progression.

The identification of molecular markers of melanoma progression is needed to more accurately stage and identify treatments for patients with malignant melanoma. Previously, we demonstrated that loss of the activator protein-2alpha (AP-2alpha) expression results in overexpression of the protease-activated receptor-1 (PAR-1) in human melanoma cell lines. Here, we used a tissue microarray platform that consisted of 64 melanocytic lesions, including dysplastic nevi (N=21), primary melanoma (N=20), and metastatic melanoma (N=23).

Automated quantitative analysis of activator protein-2alpha subcellular expression in melanoma tissue microarrays correlates with survival prediction.

The activator protein-2alpha (AP-2) transcription factor plays a key role in regulating expression of genes involved in tumor growth and metastasis of human melanoma. We sought to assess the prognostic significance of AP-2 expression and its role in the transition of nevi to metastatic melanoma. Two cohorts were analyzed.

The vascular-targeting fusion toxin VEGF121/rGel inhibits the growth of orthotopic human bladder carcinoma tumors.

Vascular endothelial growth factor (VEGF) and its receptors (FLT-1 and KDR) are overexpressed by human bladder cancer cells and tumor endothelial cells, respectively. Strategies that target VEGF receptors hold promise as antiangiogenic therapeutic approaches to bladder cancer. A fusion protein of VEGF121 and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity.

Pharmacodynamic analysis of target inhibition and endothelial cell death in tumors treated with the vascular endothelial growth factor receptor antagonists SU5416 or SU6668.

Purpose: To determine the effects of small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR)-2 (SU5416 and SU6668) on receptor phosphorylation in tumor xenografts and in paired tumor biopsies obtained in three clinical trials in patients with advanced solid malignancies.

Experimental Design: The dose-dependent effects of SU6668 on angiogenesis and tumor growth were investigated in orthotopic L3.6pl pancreatic tumors.

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts.

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.

Phase II study of the antiangiogenic agent SU5416 in patients with advanced soft tissue sarcomas.

Purpose: SU5416 (semaxanib) is a small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor-2 and KIT receptor tyrosine kinases. This Phase II study was conducted to investigate the safety and efficacy of SU5416 for patients with soft tissue sarcomas.

Experimental Design: Thirteen patients with locally advanced or metastatic soft tissue sarcomas were treated with SU5416 via intravenous infusion at a dose of 145 mg/m(2) twice weekly.

Regional effects of an antivascular endothelial growth factor receptor monoclonal antibody on receptor phosphorylation and apoptosis in human 253J B-V bladder cancer xenografts.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor in a variety of solid tumors, making it one of the most attractive therapeutic targets. VEGF promotes the proliferation, survival, and differentiation of vascular endothelial cells by stimulating autophosphorylation and activation of VEGF receptor-2 (VEGFR-2, fetal liver kinase-1, and kinase insert domain-containing receptor). We developed fluorescence-based, quantitative methods to measure total VEGFR-2, VEGFR-2 phosphorylation, apoptosis, and microvessel density and size within whole tumor cross-sections using a laser scanning cytometer.

Direct effects of recombinant human endostatin on tumor cell IL-8 production are associated with increased endothelial cell apoptosis in an orthotopic model of human pancreatic cancer.

Purpose: Recombinant human endostatin (rhES) is an antiangiogenic agent derived from collagen XVIII which inhibits tumor growth in subcutaneous models of various human malignancies. However, its effectiveness in an orthotopic xenograft model of an abdominal neoplasm has not been demonstrated.

Design: An orthotopic model of pancreatic cancer was established in 6-week-old male athymic mice from either of 2 human cell lines (L3.

The proteasome inhibitor bortezomib synergizes with gemcitabine to block the growth of human 253JB-V bladder tumors in vivo.

Bortezomib (PS-341, Velcade) is a dipeptidyl boronic acid inhibitor of the 20S proteasome that was developed as a therapeutic agent for cancer. Here, we investigated the effects of bortezomib on the growth of human 253JB-V bladder cancer cells. Although the drug did not stimulate significant increases in levels of apoptosis, it inhibited cell growth in a concentration-dependent fashion and augmented the growth inhibitory effects of gemcitabine in vitro.

Quantitative analysis of biomarkers defines an optimal biological dose for recombinant human endostatin in primary human tumors.

Purpose: In a recent study, we presented preliminary evidence for biological activity in a Phase I dose-finding study (15-600 mg/m(2)) of recombinant human endostatin in patients with refractory solid tumors. Here, we conducted additional biomarker analyses to correlate changes in tumor biology with dose.

Experimental Design: Excisional tumor biopsies were obtained at baseline and after 56 days of endostatin therapy.

Automated quantification of apoptosis after neoadjuvant chemotherapy for breast cancer: early assessment predicts clinical response.

Purpose: A large body of evidence implicates apoptosis in the effects of cancer chemotherapeutic agents on tumor cells in vitro and tumor xenografts in vivo, but the predictive value of apoptosis as an early marker for clinical response in cancer patients remains unclear.

Experimental Design: We developed an automated, laser scanning cytometer-based method to quantify the percentage of tumor cells containing DNA fragmentation characteristic of apoptosis in tumor sections. We measured levels of apoptosis in a panel of 15 matched, 18-gauge core breast cancer biopsies obtained before and 48 h after neoadjuvant therapy with docetaxel plus doxorubicin or paclitaxel as part of two prospective clinical trials.

Chemotherapy-induced apoptosis and Bcl-2 levels correlate with breast cancer response to chemotherapy.

Purpose: The relevance of apoptosis to breast cancer response to chemotherapy is unclear. We investigated whether changes in tumor cell apoptosis and Bcl-2 expression immediately after chemotherapy correlated with response to breast cancer treatment.

Patients And Methods: Serial core biopsies of 25 breast cancer primary tumors were performed at either two or three time points: before treatment (N = 24) and approximately 24 hours (N = 22) and/or 48 hours (N = 19) after the initiation of the first cycle of chemotherapy.

Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin.

Purpose: Angiogenesis is a target for the treatment of cancer and other diseases, and its complex biology suggests that establishing the appropriate dose and schedule for antiangiogenic treatment will require extensive study. We present the initial results of a dose-finding clinical trial of recombinant human endostatin (rh-Endo) that examined potential surrogates for response to antiangiogenic therapy.

Patients And Methods: Twenty-five patients were treated with escalating doses of rh-Endo.

Phase I study of recombinant human endostatin in patients with advanced solid tumors.

Purpose: Endostatin, a 20-kd fragment of collagen XVIII, is a potent inhibitor of angiogenesis. We evaluated recombinant human endostatin (rh-Endo) in a phase I trial designed to assess safety, pharmacokinetics, and serum markers of angiogenesis in patients with solid tumors.

Patients And Methods: Twenty-six patients were enrolled onto a dose-finding trial of rh-Endo administered as an intravenous bolus over a 20-minute period once daily.