Matthew Krebs

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.

Molecular analysis of circulating tumour cells—biology and biomarkers

Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test—and readily amenable to serial sampling—have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch® system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial–mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.

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.

Evaluation of Circulating Tumor Cells and Serological Cell Death Biomarkers in Small Cell Lung Cancer Patients Undergoing Chemotherapy

Serological cell death biomarkers and circulating tumor cells (CTCs) have potential uses as tools for pharmacodynamic blood-based assays and their subsequent application to early clinical trials. In this study, we evaluated both the expression and clinical significance of CTCs and serological cell death biomarkers in patients with small cell lung cancer. Blood samples from 88 patients were assayed using enzyme-linked immunosorbent assays for various cytokeratin 18 products (eg, M65, cell death, M30, and apoptosis) as well as nucleosomal DNA. CTCs (per 7.5 ml of blood) were quantified using Veridex CellSearch technology. Before therapeutic treatment, cell death biomarkers were elevated in patients compared with controls. CTCs were detected in 86% of patients; additionally, CD56 was detectable in CTCs, confirming their neoplastic origin. M30 levels correlated with the percentage of apoptotic CTCs. M30, M65, lactate dehydrogenase, and CTC number were prognostic for patient survival as determined by univariate analysis. Using multivariate analysis, both lactate dehydrogenase and M65 levels remained significant. CTC number fell following chemotherapy, whereas levels of serological cell death biomarkers peaked at 48 hours and fell by day 22, mirroring the tumor response. A 48-hour rise in nucleosomal DNA and M30 levels was associated with early response and severe toxicity, respectively. Our results provide a rationale to include the use of serological biomarkers and CTCs in early clinical trials of new agents for small cell lung cancer.

A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker

Background:Obtaining tissue for pancreatic carcinoma diagnosis and biomarker assessment to aid drug development is challenging. Circulating tumour cells (CTCs) may represent a potential biomarker to address these unmet needs. We compared prospectively the utility of two platforms for CTC enumeration and characterisation in pancreatic cancer patients in a pilot exploratory study.Patients and methods:Blood samples were obtained prospectively from 54 consenting patients and analysed by CellSearch and isolation by size of epithelial tumour cells (ISET). CellSearch exploits immunomagnetic capture of CTCs-expressing epithelial markers, whereas ISET is a marker independent, blood filtration device. Circulating tumour cell expression of epithelial and mesenchymal markers was assessed to explore any discrepancy in CTC number between the two platforms.Results:ISET detected CTCs in more patients than CellSearch (93% vs 40%) and in higher numbers (median CTCs/7.5 ml, 9 (range 0–240) vs 0 (range 0–144)). Heterogeneity observed for epithelial cell adhesion molecule, pan-cytokeratin (CK), E-Cadherin, Vimentin and CK 7 expression in CTCs may account for discrepancy in CTC number between platforms.Conclusion:ISET detects more CTCs than CellSearch and offers flexible CTC characterisation with potential to investigate CTC biology and develop biomarkers for pancreatic cancer patient management.

Tracking genomic cancer evolution for precision medicine: the lung TRACERx study.

TRACERx, a prospective study of patients with primary non-small cell lung cancer, aims to map the genomic landscape of lung cancer by tracking clonal heterogeneity and tumour evolution from diagnosis to relapse.

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.

Circulating Tumor Cells, Enumeration and Beyond

The detection and enumeration of circulating tumor cells (CTCs) has shown significant clinical utility with respect to prognosis in breast, colorectal and prostate cancers. Emerging studies show that CTCs can provide pharmacodynamic information to aid therapy decision making. CTCs as a ‘virtual and real-time biopsy’ have clear potential to facilitate exploration of tumor biology, and in particular, the process of metastasis. The challenge of profiling CTC molecular characteristics and generating CTC signatures using current technologies is that they enrich rather than purify CTCs from whole blood; we face the problem of looking for the proverbial ‘needle in the haystack’. This review summarizes the current methods for CTC detection and enumeration, focuses on molecular characterization of CTCs, unveils some aspects of CTC heterogeneity, describes attempts to purify CTCs and scans the horizon for approaches leading to comprehensive dissection of CTC biology.

Genetically Abnormal Circulating Cells in Lung Cancer Patients: An Antigen-Independent Fluorescence In situ Hybridization–Based Case-Control Study

Purpose: We performed a study to determine if a fluorescence in situ hybridization (FISH)–based assay using isolated peripheral blood mononuclear cells (PBMCs) with DNA probes targeting specific sites on chromosomes known to have abnormalities in non–small cell lung cancer (NSCLC) cases could detect circulating genetically abnormal cells (CACs). Experimental Design: We evaluated 59 NSCLC cases with stage I through IV disease and 24 controls. PBMCs and matched tumors were hybridized with 2 two-color [3p22.1/CEP3 and 10q22.3 (SP-A)/CEP10) and 2 four-color [CEP3, CEP7, CEP17, and 9p21.3 (URO); and EGFR, c-MYC, 6p11-q11, and 5p15.2 (LAV)] FISH probes. Percentages of cytogenetically abnormal cells (CACs) in peripheral blood and in matched tumor specimens were quantified by using an automated fluorescent scanner. Numbers of CACs were calculated based on the percentage of CACs (defined as PBMCs with genetic abnormalities) per milliliter of blood and expressed per microliter of blood. Results: Patients with NSCLC had significantly higher numbers of CACs than controls. Mean number of CACs ranged from 7.23 ± 1.32/μL for deletions of 10q22.3/CEP10 to 45.52 ± 7.49/μL for deletions of 3p22.1/CEP3. Numbers of CACs with deletions of 3p22.1, 10q22.3, and 9p21.3, and gains of URO, increased significantly from early to advanced stage of disease. Conclusions: We have developed a sensitive and quantitative antigen-independent FISH-based test for detecting CACs in peripheral blood of patients with NSCLC, which showed a significant correlation with the presence of cancer. If this pilot study can be validated in a larger study, CACs may have a role in the management of patients with NSCLC. Clin Cancer Res; 16(15); 3976–87. ©2010 AACR.