Introduction
CTCs: A background
Major questions to be answered concerning CTCs
The biomarker development process and development of CTCs as biomarkers
Qualitative Rankings of Data Sources and Study Designs | |
---|---|
Most Reliable | Collaborative studies that use large panels of well characterized samples; summary data from well-designed external proficiency testing schemes; inter-laboratory comparison programs |
Reliable | Other data from proficiency testing schemes, well-designed peer-reviewed studies, and expert panel-reviewed FDA summaries |
Less Reliable | Less well designed peer-reviewed studies |
Least Reliable | Unpublished and/or non-peer reviewed research; clinical laboratory, or manufacturer data; studies on performance of the same basic methodology, but used to test for a different target |
Criteria for Assessing the Internal Validity of the Studies Used to Obtain Data
| |
• Adequate descriptions of the assay platform and test procedures, including the reproducibility of test results, quality control measures, and comparison to a “gold standard” reference assay. | |
• Samples representative of the study population, blinded testing and interpretation. | |
• Data analysis including point estimates of sensitivity and specificity with 95% confidence intervals, and sample size/power calculations. | |
• Studies graded as convincing, adequate, or inadequate based on their ability to provide confident estimates of analytic sensitivity and specificity using intended sample types from representative populations. |
-
Materials at study site, e.g., sample collection tubes, shipping containers
-
Materials at analytic laboratory, e.g., assay-specific kit (w/instructions for use, reagents, buffers, controls, etc.), additional equipment (centrifuge, test tubes, micropipettes, vortex mixer, etc.)
-
Reagent storage conditions (temperature, time, etc.)
-
Specimen collection, e.g., timing of specimen collection relative to study treatments and procedures, phlebotomy procedure (patient positioning, needle size, blood draw site, sample size and number of samples, collection tube, tube inversion for clot prevention)
-
Specimen handling e.g., storage time and temperature, shipping time, etc.
-
Type of analysis (enrichment/enumeration/molecular characterization of target cells)
-
What is the range of specimens tested?
-
Sensitivity of assay (enrichment/enumeration/molecular characterization of all cells)
-
Specificity of assay (enrichment/enumeration/molecular characterization of only target cells)
-
How often does the assay give a useable result?
-
How similar are the results with repeated measurements?
-
What are the analytical range, reproducibility, and clinical applicability of the test?
-
How similar are the results obtained within the same or in multiple laboratories using the same or different technology?
-
Assay-specific controls should be run each day of analysis or when a new assay lot is used to check overall performance, including instrument, reagents, and operator technique
-
Assay-specific controls should be run regularly (monthly or quarterly) to show performance at the lower limit of analysis
-
Assay-specific samples should be run semi-annually to document the lower limit of quantitation
-
Calibration/calibration verification (at least semi-annually)
-
All participating laboratories in the same study should use common controls to document continuously across laboratory performance.
-
Splitting samples across laboratories prior to the study is recommended to document assay performance across laboratories.
-
Clinical sensitivity and specificity of assay
-
Are there methods to resolve clinical false positive assay results in a timely manner?
-
Prevalence (how often are the target cells detectable in the study population?)
-
What is the relationship between detectable target cells and the study disease?
-
Positive predictive value (what is the probability that a positive assay result means that the subject has the target disease?)
-
What are the genetic, environmental, or other modifiers for detection of the target cells?
Potential contexts of use for CTCs
Assay/Technology Name | Manufacturer/Developer | Assay Outcome | Target Cancer(s) | Technology/Process |
---|---|---|---|---|
AdnaTest
| AdnaGen, Langenhagen, Germany | Enrichment/Characterization | Breast, Prostate, Colon | Immunomagnetic-based EpCAM enrichment using labelled beads incubated with the whole blood sample. Unlabeled cells are removed; labelled cells are then lyzed. RNA is isolated, followed by multiplex RT-PCR (GA733-2, HER2, MUC1) to detect specific tumor biomarkers. |
Anti-EpCAM/Anti-CK Antibody CTC Enrichment
| Glenn Deng, Stanford University, Stanford, CA | Enrichment/Enumeration | Metastatic Breast Cancer | CTC enrichment assay using the combination of anti-CK and anti-EpCAM antibodies. Image analysis performed using the Ariol® system. CTC identification with brightfield and fluorescence labelled anti-CK, anti-CD45, and 4′,6-diamidino-2-phenylindole (DAPI) images. |
ApoStream
TM
| ApoCell | Enrichment | Prostate, Lung | Isolation of CTCs in a whole blood sample using dielectrophoretic field flow fractionation (DFFF), which separates cells based on differing dielectric properties. See also DFFF technology entry below. |
autoMACS/MACS (Magnetic Activated Cell Sorting System)
| Mitenyi Biotec, Bergisch Gladbach, Germany | Enrichment | ---------- | Utilizes an immunomagnetic column to capture cells with various antigens (EpCAM, pan-CK, HER2/neu, or CD45). Manual or semi-automated system. These viable cells are available for subsequent analysis following enrichment. |
Bioflux
| Fluxion Biosciences, South San Francisco, CA | Enumeration | ---------- | Well Plate Microfluidic™ technology to obtain physiologically-relevant data from cell-based assays. Data acquisition obtained in brightfield, phase, fluorescence, and confocal high-resolution microscopy. |
CEER (Collaborative Enzyme Enhanced Reactive) Immunoassay
| Prometheus Laboratories Inc. | Characterization | ---------- | Multiplexed protein microarray platform that measures the expression and activation of specific cancer pathways with high levels of sensitivity and specificity. |
Bayer Schering Pharma AG, Germany | ||||
CELLection™ Epithelial Enrich
| Traci Libby, Invitrogen, Carlsbad, California | Enrichment | ---------- | Positive isolation. Obtain up to 5 log enrichment of viable epithelial tumor cells that are suitable for immunocytochemical staining or any other downstream application. |
CellSearch™
| Veridex | Enrichment/Enumeration | Metastatic Breast, Colon, Prostate, Lung, Melanoma, Urothelial Cancer | Automated immunomagnetic enrichment and staining system for quantification of CTCs in whole blood samples. CTCs are enriched using ferrofluids coupled to anti-EpCAM antibodies and identified by cytokeratin staining using fluorescent anti-CK antibodies, as well as counterstaining with anti-CD45 antibodies. Currently, the only diagnostic test cleared by the FDA. |
ClearCell System (CTChip and Clearcell Unit) | Clearbridge Biomedic, Singapore | Enrichment | ---------- | Detects and isolates intact, viable CTCs from small quantities of whole, unprocessed blood. Isolated CTCs can then be stained directly on the CTChip® for identification, or retrieved for further molecular analysis. |
CTC Chip
| Dan Haber and Mehmet Toner, Dana Farber and MGH, Boston, MA | Enrichment/Enumeration | Breast, Colon, Lung, Prostate, Pancreas | Enrichment using microfluidic technology—whole blood is pumped across a silicon-etched chip that contains 78,000 microposts fitted with anti-EpCAM antibodies. EpCAM-positive cells attach to the microposts and are then detected by a camera. Includes a chamber to enclose the fluid and chip and a pneumatic pump. |
Developers: | ||||
On-Q-ity, Waltham, MA | ||||
ICx Biosystems, San Diego, CA | ||||
Johnson & Johnson | ||||
CTC Membrane Microfilter
| Richard Cote, Ram Datar, University of Miami, FL | Enumeration | Prostate | Stepwise photolithography process that produces controlled-size pores designed to exploit cell size differences between tumor and normal blood cells. Combined with quantum dot-based immunofluorescence detection for CTC characterization. |
Cytoscale CTC Assay
| Hsian-Rong Tseng, University of California, Los Angeles | Enumeration | Prostate, Breast, Colon and Kidney | Antibody cell-surface marker capture enhanced by nanostructures; immunohistochemistry staining for cell identification. |
DEPArray
| Nicolo Manares, Silicon Biosystems, SpA, Bologna, Italy | Enumeration | ---------- | Cell microarray for individual cell manipulation and detection. The base is a microelectronic active silicon substrate embedding control circuitry for addressing each individual dielectrophoretic (DEP) cage (cage size can be set to accommodate a single cell). The system allows detection and sorting of rare cells and sorting by morphological parameters such as shape, nucleus-to-cytoplasm ratio, fluorophores co-localization (by image-based selection). |
Dielectrophoretic Field Flow Fractionation
(DFFF)
| Peter Gascoyne, MD Anderson Cancer Center, Houston, TX | Enumeration | ---------- | Cell-separation technique that exploits the differences in density and dielectric properties of cells to aid isolation of CTCs from clinical blood specimens. See also ApoStream™ assay above. |
Dylight Technology
| Medical University Graz, Austria | Enumeration | Breast | Immunofluorescence method for identifying CTCs that utilizes staining for multiple markers, including CD44, ALDH1, and CK using DyLight dyes; and subsequent analysis by novel DyLight technology. |
Dynabeads® CD45
| Traci Libby, Invitrogen, Carlsbad, California | Enrichment | ---------- | Dynabeads® are coated with anti-CD45 monoclonal antibody for efficient depletion of human leucocytes in whole blood samples to enrich epithelial tumor cells. |
Dynabeads® Epithelial Enriched
| Traci Libby, Invitrogen, Carlsbad, California | Enrichment | ---------- | Dynabeads® are coated with the monoclonal antibody BerEP4 against the human epithelial antigen, EpCAM. Enriched tumor cells are lysed for mRNA isolation and RT-PCR amplification. |
Epic HD-CTC Assay
| Epic Sciences, Inc. | Enumeration/Characterization | Prostate, Breast, Pancreas | CTC detected in peripheral blood through red blood cells lysis and fluorescentlylabeled antibodies. See also FAST Cytometer entry. |
EPISPOT (EPithelial ImmunoSPOT)
| Catherine Alix-Panabieres and Klaus Pantel, Laboratoire de Virologie, Hôpital Lapeyronie, CHU Montpellier, France & UKE, Hamburg, Germany | Characterization | Breast, Prostate, Colon | After depletion of CD45 positive cells, remaining cells in a whole blood sample are cultured for 24 hours on a membrane coated with antibodies that detect proteins shed from viable CTCs by secondary antibodies labelled with fluorochromes. |
FAST Cytometer
(Fiberoptic Array Scanning Technology)
| Peter Kuhn, Scripps Institute, La Jolla, CA | Enumeration/Characterization | Metastatic Breast Cancer | Fluorescence cytometry combined with an automated digital microscopy imaging system. Immunofluorescently labelled CTCs are detected on a glass slide using laser-printing optics, which can scan 300,000 cells per second. See also Epic HD-CTC Assay entry. |
Robert Bruce, Scripps Palo Alto Research Center, Palo Alto, CA | ||||
Flow Cytometry
| Jeannie Gaylor, Becton-Dickinson, San Jose CA | Enrichment/Enumeration | ---------- | Multiple reagents and systems adaptable to analysis or sorting of CTCs. |
HB-CTC (Herringbone-Chip)
| Mehmet Toner and Daniel Haber, Massachusetts General Hospital (MGH) and Harvard Medical School | Enrichment/Enumeration | ---------- | A high-throughput microfluidic mixing device which provides an enhanced platform (over the CTC-chip) for CTC isolation where microvortices are utilized to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. |
ISET (Isolation by Size of Epithelial Tumor cells)
| Metagenex, Paris, France | Enrichment | ---------- | CTCs are separated from other cells in whole blood by size via vacuum filtration. This technique is gentle and produces viable cells that can be further analyzed following enrichment. |
IsoFlux™ Rare Cell Access System
| Fluxion Biosciences, South San Francisco, CA | Enrichment | ---------- | Proprietary microfluidic technology to isolate rare cells with high efficiency. The system incorporates CellSpot™ Technology to produce a highly concentrated sample that is optimized for downstream molecular analyses. |
Laser Scanning Cytometry
| Maintrac, Bayreuth Germany | Enumeration/Characterization | Breast, Colon, Prostate, Sarcoma | Custom laboratory analysis service performed on slides using a variety of fluorochrome-labelled antibodies or other techniques (e.g., estrogen receptor, HER2, prostate-specific antigen, FISH, terminal dUTP nick end labelling). Traceable single cell detection within 1 million cells. |
Laser Scanning Cytometry (LSC)
| ApoCell | Enumeration | ---------- | Proprietary microscope-based immunofluorescent image analysis. |
Lymphoprep™ (Ficoll-Isopaque)
| Axis-Shield PoC, Oslo, Norway | Enrichment | ---------- | Separates mononuclear cells from other cells in whole blood based on cell density. |
MagSweeper
| Stephanie Jeffrey and Ronald W. Davis, Stanford University, Stanford, CA | Enrichment/Enumeration | Metastatic Breast | Automated immunomagnetic enrichment―gently enriches target cells and eliminates cells that are not bound to magnetic particles. Isolated cells can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. Processes 9 mL of blood per hour and captures >50% of circulating epithelial cells as measured in spiking experiments. |
Multiphoton Intravital Flow Cytometry
| Philip Low, Purdue University, West Lafayette, IN | Enumeration | Prostate | Noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves intravenous injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. |
Nanodetector
| Gilupi, Potsdam, Germany | Enrichment/Enumeration | Breast, lung, prostate | The nanodetector (functionalized structured medical wire, FSMW) is inserted into the patient’s arm vein via a standard 20-gauge needle. The nanodetector consists of a medical stainless steel wire, coated with a gold layer and a hydrogel functionalized with an anti-EpCAM antibody. During the 30 min application in the vein, up to 1,500 mL of blood including the respective CTC pass the nanodetector and enable a high number of CTC to be bound by the anti-EpCAM antibody. |
Negative Enrichment OMS
| Jeffrey Chalmers, Cleveland Clinic, Cleveland, OH | Enrichment/Enumeration | Head & Neck, Breast | Red cell lysis, immunomagnetic labelling and subsequent depletion of CD45+ cells (leukocytes). Remaining cells may be further characterized (epithelial cells, cells undergoing EMT). |
PerCelleon, LLC | ||||
Stem Cell Technologies | ||||
Nucleopore Assay
| Whatman International Ltd., UK | Enrichment | ---------- | CTCs are separated from other cells in whole blood by size via vacuum filtration. |
OncoCEE-BR™
| Biocept, Inc. and Clarient, Inc. | Enumeration/Characterization | Breast | OncoCEE™ captures CTCs via a microfluidic system that uses multiple antibodies for capture followed by detection using CEE-Enhanced staining and then detects their HER2/neu status via FISH. |
OncoQuick
| Greiner Bio-One, Germany, North Carolina | Enrichment | Breast, Colon, Others | Centrifugal separation using optimized liquid media based on tumor cell buoyant density only. Achieves enrichment of up to 6 logs from approximately 104 total mononuclear cells. Validated with spiking studies. |
Optofluidic Intracavity Spectroscopy (OFIS)
| David Kisker eOptra, Longmont, CO | Enumeration | ---------- | OFIS has been used to investigate the properties of several tumor cell lines and compared the results to cells from peripheral blood. The results suggest that a unique optical signature may be a characteristic of many tumor cells. This signature may offer a complementary tool to molecular methods for detection and enumeration of CTCs. In addition, by using dielectrophoresis to trap and steer cells, it is possible that induced changes in the OFIS spectrum may detect other characteristics of tumor cells, as well as transport and sort them according to those characteristic properties. |
Photoacoustic Detection
| John Viator, University of Missouri, Columbia, MO | Enumeration | Melanoma, Breast Cancer | Flowmetry system in which blood samples are irradiated with laser light, and photoacoustic waves from cancer cells are detected and counted (uses melanin in melanoma, gold-tagging of other cancer cells). |
RARE (RosetteSep-Applied Imaging Rare Event)
| StemCell Technologies, Vancouver, BC | Enrichment | ---------- | Negative selection technique where tetrameric antibody complexes crosslink CD45-expressing leukocytes to red blood cells in whole blood. These complexes pellet to the bottom of the tube when centrifuged due to increased density, enriching CD45-negative cells (CTCs). |
RoboSep/EasySep™
| Stem Cell Technologies, Vancouver | Enrichment | Myeloma, Epithelial Tumors, CD45 depletion | Immunomagnetic nanoparticle-cell complexes are captured in tubes and unlabeled cells are poured off. Adaptable to custom CTC antibody surface antigens. Manual or semi-automated systems. |
ScreenCell® Cyto, ScreenCell® CC, ScreenCell® MB
| ScreenCell Company Biopark 12 rue J-A de Baïf 75013, Paris | Enrichment | ---------- | The ScreenCell® Cyto device isolates rare tumor cells, with a high recovery rate. The ScreenCell® CC device allows isolation of either fixed or live cells. Fixed cells are well preserved morphologically. Immunocytochemistry and FISH assays can be performed directly on the filter. Isolated live cells are able to grow in culture. High-quality genetic materials (DNA, RNA) can be obtained directly from tumor cells isolated on the ScreenCell® MB device filter. The ScreenCell® devices may be able to simplify and improve noninvasive access to tumor cells due to their reduced size, versatility, and capacity to isolate CTCs within minutes. |
Single Cell Gene Expression with BioMark™ Real-Time PCR System
| Fluidigm Corporation, South San Francisco, CA | Characterization | ---------- | Allows high-throughput cell-line studies to determine individual cell behavior and is suited to determine single-gene cell expression levels in CTCs. Results are presented as a heat map, with individual assays on the X-axis and individual cell samples on Y-axis. The intersection of each assay and sample is an individual real-time qPCR reaction. |
Supervised* Automated Microscopy
| Iqbal Habib, Ariol, Genetix, Boston MA, San Jose CA | Enumeration/Characterization | Breast, Others? | Commercial component to automatically track, review, and enumerate immunocytochemically stained candidate CTCs. Nuclear, shape factor morphology image analysis system with computer display. |
TelomeScan (OBP-401)
| Oncolys BioPharma, Tokyo | Enumeration/Characterization | Gastric, Breast | Uses a virus vector for CTC detection. The virus is incubated with whole blood sample for 24 hrs and replicates with cancer cells, incorporating the GFP marker into them. CTCs are then detectable by fluorescence system analysis of cell preparation on slide. Potential for in vivo transfection and detection of GFP + CTCs in capillary bed of patient. |
Vita-Assay
[Functional Collagen Adhesion Matrix (CAM)]
| Wen-Tien Chen, Vitatex, Stony Brook, NY | Enumeration | Prostate | CAM ingestion. Enables molecular characterization of captured cells. |
Selection of CTC technologies for clinical development
Practical application
Assay Validation | |
---|---|
Pre-Analytic
| How is specimen collected (venous route, body position, draw order, tourniquet time, needle bore, tube type)? |
When is specimen collected (time of day, relative to treatment, relative to infusates)? | |
How is specimen stored (time and temperature)? | |
How is specimen handled (shipping, transfers)? | |
Analytic
| Sensitivity (lower limit of quantitation)? |
Reportable range? | |
Specificity? | |
Reproducibility? | |
Robustness? | |
Post-Analytic
| How is data reported? |
How is data analyzed? | |
What are the reference intervals? | |
Clinical Feasibility
| |
• Are there analytically valid results when tested in appropriate preclinical models? | |
○ with use of clinically relevant/feasible specimen acquisition? | |
○ with use of clinically relevant specimen handling procedures (both at the point of acquisition and in the receiving laboratory)? These processes should be tracked and recorded. | |
○ with use of clinically relevant collection scheduling? | |
Therapeutic Relevance
| |
• For predictive biomarkers, is there a relationship between dose/exposure, quantifiable target modulation, and disease outcome? | |
• For prognostic biomarkers, is there a relationship between baseline levels and survival? |
Standards
-
Sample collection kit with instructions for use
-
Shipping container
-
Temperature monitoring strips
-
Appropriate analytical instrumentation
-
Analytical reagents
-
Standards
-
Multiple levels of controls
-
Reagent storage temperature
-
Reagent storage time
-
Patient preparation—for example, fasting status
-
Timing of sample collection in study protocol
-
Sample collection method
-
Number of specimen collected, including duplicates
-
Subject positioning during sample collection
-
Specimen transport time
-
Specimen storage temperature
-
Specimen storage time
-
Assay-specific SOPs
-
Lot-specific control testing
-
Levels of quality control testing (daily, monthly)
-
Lab certification
-
Assay platform training