Timothy H Ward

Evaluation and Prognostic Significance of Circulating Tumor Cells in Patients With Non–Small-Cell Lung Cancer

PURPOSE Lung cancer is the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) lacks validated biomarkers to predict treatment response. This study investigated whether circulating tumor cells (CTCs) are detectable in patients with NSCLC and what their ability might be to provide prognostic information and/or early indication of patient response to conventional therapy. PATIENTS AND METHODS In this single-center prospective study, blood samples for CTC analysis were obtained from 101 patients with previously untreated, stage III or IV NSCLC both before and after administration of one cycle of standard chemotherapy. CTCs were measured using a semiautomated, epithelial cell adhesion molecule-based immunomagnetic technique. RESULTS The number of CTCs in 7.5 mL of blood was higher in patients with stage IV NSCLC (n = 60; range, 0 to 146) compared with patients with stage IIIB (n = 27; range, 0 to 3) or IIIA disease (n = 14; no CTCs detected). In univariate analysis, progression-free survival was 6.8 v 2.4 months with P < .001, and overall survival (OS) was 8.1 v 4.3 months with P < .001 for patients with fewer than five CTCs compared with five or more CTCs before chemotherapy, respectively. In multivariate analysis, CTC number was the strongest predictor of OS (hazard ratio [HR], 7.92; 95% CI, 2.85 to 22.01; P < .001), and the point estimate of the HR was increased with incorporation of a second CTC sample that was taken after one cycle of chemotherapy (HR, 15.65; 95% CI, 3.63 to 67.53; P < .001). CONCLUSION CTCs are detectable in patients with stage IV NSCLC and are a novel prognostic factor for this disease. Further validation is warranted before routine clinical application.

Circulating tumor cells as a window on metastasis biology in lung cancer.

Circulating tumor cell (CTC) number in metastatic cancer patients yields prognostic information consistent with enhanced cell migration and invasion via loss of adhesion, a feature of epithelial-to-mesenchymal transition (EMT). Tumor cells also invade via collective migration with maintained cell-cell contacts and consistent with this is the circulating tumor microemboli (CTM; contiguous groups of tumor cells) that are observed in metastatic cancer patients. Using a blood filtration approach, we examined markers of EMT (cytokeratins, E-cadherin, vimentin, neural cadherin) and prevalence of apoptosis in CTCs and CTM to explore likely mechanism(s) of invasion in lung cancer patients and address the hypothesis that cells within CTM have a survival advantage. Intra-patient and inter-patient heterogeneity was observed for EMT markers in CTCs and CTM. Vimentin was only expressed in some CTCs, but in the majority of cells within CTM; E-cadherin expression was lost, cytoplasmic or nuclear, and rarely expressed at the surface of the cells within CTM. A subpopulation of CTCs was apoptotic, but apoptosis was absent within CTM. This pilot study suggests that EMT is not prosecuted homogeneously in tumor cells within the circulation of lung cancer patients and that collective migration and enhanced survival of cells within CTM might contribute to lung cancer metastasis. Multiplex analysis and further detailed exploration of metastatic potential and EMT in CTCs/CTM is now warranted in a larger patient cohort.

Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches.

Introduction: Epithelial circulating tumor cells (CTCs) are detectable in patients with non-small cell lung cancer (NSCLC). However, epithelial to mesenchymal transition, a widely reported prerequisite for metastasis, may lead to an underestimation of CTC number. We compared directly an epithelial marker-dependent (CellSearch) and a marker-independent (isolation by size of epithelial tumor cells [ISET]) technology platform for the ability to identify CTCs. Molecular characteristics of CTCs were also explored. Methods: Paired peripheral blood samples were collected from 40 chemonäive, stages IIIA to IV NSCLC patients. CTCs were enumerated by Epithelial Cell Adhesion Molecule-based immunomagnetic capture (CellSearch, Veridex) and by filtration (ISET, RareCell Diagnostics). CTCs isolated by filtration were assessed by immunohistochemistry for epithelial marker expression (cytokeratins, Epithelial Cell Adhesion Molecule, epidermal growth factor receptor) and for proliferation status (Ki67). Results: CTCs were detected using ISET in 32 of 40 (80%) patients compared with 9 of 40 (23%) patients using CellSearch. A subpopulation of CTCs isolated by ISET did not express epithelial markers. Circulating tumor microemboli (CTM, clusters of ≥3 CTCs) were observed in 43% patients using ISET but were undetectable by CellSearch. Up to 62% of single CTCs were positive for the proliferation marker Ki67, whereas cells within CTM were nonproliferative. Conclusions: Both technology platforms detected NSCLC CTCs. ISET detected higher numbers of CTCs including epithelial marker negative tumor cells. ISET also isolated CTM and permitted molecular characterization. Combined with our previous CellSearch data confirming CTC number as an independent prognostic biomarker for NSCLC, we propose that this complementary dual technology approach to CTC analysis allows more complete exploration of CTCs in patients with NSCLC.

Biomarker method validation in anticancer drug development

Over recent years the role of biomarkers in anticancer drug development has expanded across a spectrum of applications ranging from research tool during early discovery to surrogate endpoint in the clinic. However, in Europe when biomarker measurements are performed on samples collected from subjects entered into clinical trials of new investigational agents, laboratories conducting these analyses become subject to the Clinical Trials Regulations. While these regulations are not specific in their requirements of research laboratories, quality assurance and in particular assay validation are essential. This review, therefore, focuses on a discussion of current thinking in biomarker assay validation. Five categories define the majority of biomarker assays from ‘absolute quantitation’ to ‘categorical’. Validation must therefore take account of both the position of the biomarker in the spectrum towards clinical end point and the level of quantitation inherent in the methodology. Biomarker assay validation should be performed ideally in stages on ‘a fit for purpose’ basis avoiding unnecessarily dogmatic adherence to rigid guidelines but with careful monitoring of progress at the end of each stage. These principles are illustrated with two specific examples: (a) absolute quantitation of protein biomarkers by mass spectrometry and (b) the M30 and M65 ELISA assays as surrogate end points of cell death.

Circulating tumour cells: their utility in cancer management and predicting outcomes

Recent advances in technology now permit robust and reproducible detection of circulating tumour cells (CTCs) from a simple blood test. Standardization in methodology has been instrumental in facilitating multicentre trials with the purpose of evaluating the clinical utility of CTCs. We review the current body of evidence supporting the prognostic value of CTC enumeration in breast, prostate and colorectal cancer, using standardized approaches, and studies evaluating the correlation of CTC number with radiological outcome. The exploitation of CTCs in cancer management, however, is now extending beyond prognostication. As technologies emerge to characterize CTCs at the molecular level, biological information can be obtained in real time, with the promise of serving as a ‘surrogate tumour biopsy’. Current studies illuminate the potential of CTCs as pharmacodynamic and predictive biomarkers and potentially their use in revealing drug resistance in real time. Approaches for CTC characterization are summarized and the potential of CTCs in cancer patient management exemplified via the detection of epidermal growth factor receptor mutations from CTCs in patients with non-small cell lung cancer. The opportunity to learn more about the biology of metastasis through CTC analysis is now being realized with the horizon of CTC-guided development of novel anticancer therapies.

Phase I Trial of AEG35156 Administered as a 7-Day and 3-Day Continuous Intravenous Infusion in Patients With Advanced Refractory Cancer

PURPOSE To establish the maximum-tolerated dose and evaluate tolerability, pharmacokinetics, pharmacodynamic effects, and antitumor activity of AEG35156, a second-generation antisense to X-linked inhibitor of apoptosis (XIAP) protein, in patients with advanced refractory malignant tumors. PATIENTS AND METHODS This was a first-in-man, open-label, phase I dose-escalation study. AEG35156 was administered by continuous intravenous infusion over 7 days (7DI) or 3 days (3DI) of a 21-day treatment cycle. Dose escalation started at 48 mg/m(2)/d and continued until consistent dose-limiting toxicity (DLT) was observed. RESULTS Thirty-eight patients were entered in seven cohorts. Grade 3 to 4 adverse events were uncommon and were predominantly abnormal laboratory values: elevated ALT, thrombocytopenia, and lymphopenia. DLTs comprised elevated hepatic enzymes, hypophosphatemia, and thrombocytopenia. The maximum-tolerated doses were defined as 125 mg/m(2)/d for the 7DI regimen and < or = 213 mg/m(2)/d for the 3DI schedule. AEG35156 area under the plasma concentration curve and peak plasma concentration increased proportionally with dose. Suppression of XIAP mRNA levels was maximal at 72 hours (mean suppression, 21%), and this coincided with a dramatic decrease in circulating tumor cells in a patient with non-Hodgkins lymphoma. Two further patients had unconfirmed partial responses. Circulating biomarkers of cell death and apoptosis altered in association with drug infusion and toxicity. CONCLUSION In this first-in-man study, AEG35156 was well tolerated, with predictable toxicities, pharmacokinetic properties, and clinical evidence of antitumor activity in patients with refractory lymphoma, melanoma, and breast cancer. Phase I/II trials of AEG35156 chemotherapy combinations are ongoing in patients with pancreatic, breast, non-small-cell lung cancer, acute myeloid leukemia, lymphoma, and solid tumors for which docetaxel is indicated.

Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST–XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at −80°C for at least 60 days. M30-Apoptosense™ plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at −80°C, while at 37°C it had a half-life of 80–100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.

Optimisation of circulating biomarkers of cell death for routine clinical use

BACKGROUND M30 and M65 enzyme-linked immunosorbent assays detect circulating cytokeratin 18 fragments released during caspase-dependent or total cell death, respectively, and have potential as biomarkers in epithelial cancers. While these assays have been validated, their robustness for routine clinical use is unknown. PATIENTS AND METHODS M30 and M65 were measured in matched serum and plasma samples from 31 lung cancer patients and 18 controls. RESULTS Time allowable between sample acquisition and processing is critical for assays in clinical use. A 4-h delay in processing at room temperature increased M30 (P < 0.0001), an effect minimised by incubation on ice. M30 and M65 in serum were resistant to processing variations including delays. Serum and plasma measurements correlated well although M30 but not M65 was lower in serum (P < 0.0005). Less variation between duplicate assays was observed in serum. Prolonged storage (-80 degrees C) led to increased M30 (12%, 6 months; 34%, 1 year). Sample dilution in the supplied assay diluent proved non-linear, whereas dilution in donor serum or porcine plasma restored linearity up to a ratio of 1 : 6. CONCLUSION We present recommendations that improve the reliability of these assays for clinical use and recommend serum as the preferred matrix with data more resistant to variations in collection.

Biomarkers of apoptosis.

Within the era of molecularly targeted anticancer agents, it has become increasingly important to provide proof of mechanism as early on as possible in the drug development cycle, especially in the clinic. Selective activation of apoptosis is often cited as one of the major goals of cancer chemotherapy. Thus, the present minireview focuses on a discussion of the pros and cons of a variety of methodological approaches to detect different components of the apoptotic cascade as potential biomarkers of programmed cell death. The bulk of the discussion centres on serological assays utilising the technique of ELISA, since here there is an obvious advantage of sampling multiple time points. Potential biomarkers of apoptosis including circulating tumour cells, cytokeratins and DNA nucleosomes are discussed at length. However, accepting that a single biomarker may not have the power to predict proof of concept and patient outcome, it is clear that in the future more emphasis will be placed on technologies that can analyse panels of biomarkers in small volumes of samples. To this end the increased throughput afforded by multiplex ELISA technologies is discussed.

Quantitative multiplexed quantum dot immunohistochemistry

Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.