Circulating Cancer Cells and Their Clinical Applications

Minimal residual cancer is defined as “the presence of tumor cells that are not detectable by the current routine diagnostic procedures used for tumor staging in cancer patients after surgical removal of the primary tumor.” Data from European and North American groups have demonstrated the prognostic impact of disseminated tumor cells (DTCs)9 in the bone marrow of breast cancer patients. Circulating tumor cell (CTC) detection and enumeration in peripheral blood have been examined in prospective multicenter studies of metastatic breast, colorectal, and prostate cancers and have been associated with decreased progression-free and overall survival. An increasing number of clinical research studies are validating these observations and extending them to other cancers and to earlier disease stages. CTCs are highly heterogeneous, and their molecular characterization is important, not only to confirm their malignant origin but also to follow immune-phenotypic changes with tumor progression and identify diagnostically and therapeutically relevant targets that will help stratify cancer patients for individualized therapies. The rarity of CTCs—and thus the very limited amount of available sample—presents a formidable analytical and technical challenge. Recent technical advances in CTC detection and characterization include reverse-transcription quantitative PCR (RT-qPCR) methods, image-based approaches, and microfilter and microchip devices. CTCs represent a promising new diagnostic field for advanced-stage patients in that the sensitive CTC-detection platforms allow monitoring of disease and treatment efficacy. The development of single-cell technologies might allow profiling of these cells for the purpose of adapting treatment regimens. CTC-detection and- characterization techniques hold promise for playing a role as a “liquid biopsy” that will allow physicians to follow cancer changes over time and to tailor treatment. Current research on CTCs is focusing on the identification of novel diagnostic and therapeutic biomarkers produced by these cells. CTCs are promising as novel tumor biomarkers because they are well-defined targets for understanding tumor biology and tumor cell dissemination that can open new avenues for the early detection of metastasis and its successful treatment. We discuss CTCs and their diagnostic potential with 4 leading scientists and clinicians in this field. A suggested reading list on this topic is provided in the Data Supplement that accompanies the online version of this Q&A at http://www.clinchem.org/content/vol57/issue11. What are the current analytical methods for detecting CTCs? How reliable are these methods? Does it matter which method is used clinically? Klaus Pantel: Current analytical methods for detecting CTCs always include an enrichment step and a detection step. Enrichment of CTCs can be based on size (filtration devices), density (e.g., Ficoll centrifugation), ability to invade a collagen matrix, and positive immunoselection [e.g., epithelial cell adhesion molecule (EpCAM) antibody–based enrichment of CTCs] or negative immunoselection (i.e., depletion of leukocytes by CD45 antibodies). The subsequent approaches used to detect CTCs are (i) immunocytochemistry with anticytokeratin (anti-CK) antibodies; (ii) RT-PCR targeting various epithelial mRNAs, including CK-19 mRNA; and (iii) epithelial immunospot (EPISPOT) assays detecting tumor-specific proteins released by CTCs [e.g., prostate-specific antigen (PSA)]. All enrichment methods are biased because tumor cells are heterogeneous and some fraction of the CTCs might be lost (e.g., immunoselection with EpCAM antibodies cannot catch EpCAM-negative CTCs). Detection with anti-CK antibodies is currently the most validated and standardized approach, which also allows morphological interpretation of positive events. Different detection methods lead to different results, as shown by the comparative analysis of the same patient samples with different technologies. Thus, the clinical results largely depend on the technology used to detect CTCs. The technology that has produced the largest amount of clinical data on the prognostic relevance of CTCs in breast, prostate, and colon cancers is the US Food and Drug Administration (FDA)-approved CellSearch system (Veridex). Howard I. Scher: At this point, there is no standardized definition of a CTC, and the various techniques used to enrich and characterize these cells do not measure or report the same “CTC” biomarker. The broad range of assays and devices in use and in development include those based on: physical differences, e.g., density gradient centrifugation, filtration, or the plasticity of CTCs relative to nonmalignant cells; cell surface antibodies conjugated to magnetic beads, microposts, or ferrofluids to “positively select/capture” tumor cells; and depleting the nonmalignant cell population first, leaving CTCs behind—a process called “negative selection.” The various enrichment steps are then followed by different methods to detect and characterize the cells via various molecular or cytometric techniques. Assay reliability must consider 2 components. The first is the analytical performance of the assay. In most reports, details of the analytical validation steps that have been performed are lacking. Many of the assays have been studied only in a single-laboratory setting, and most of these assays are not CLIA certified. Knowing both the capability of the assay and what it is actually measuring and reporting is critical before proceeding to clinical testing. The second component is the clinical evaluation, i.e., the level of evidence that has been generated to date with respect to the context in which the test might be used in the clinic. Does it matter which method is being used clinically? Yes, and the first consideration is the “context of use” for which the test is being developed. Stated differently, what is the medical decision that the test result is needed to inform? Contexts of use include diagnosis, prognostication, prediction, response indicators, or efficacy/response surrogates. As examples, knowledge of a cell count does not inform the choice of one specific therapy over another, whereas detection of a kinase mutation in CTCs could. No single test will provide information on all these contexts. Leon Terstappen: At present, the CellSearch system is the only validated system for CTC enumeration and as such is the only system that can be used in the clinic. Although a variety of analytical methods are being explored for the detection of CTCs, only a few are available for routine laboratory use. The definitions used to define CTCs vary greatly between the different methods and result in a large range in the reported numbers of detected CTCs, as well as in the proportion of patients in which CTCs are detected. As a consequence, not only does a CTC-detection method need to be accurate and reproducible, but prospective clinical studies also will need to be conducted to determine the implications of the detected CTCs for each analytical method. Evi Lianidou: Currently there is a plethora of analytical methods for detecting CTCs. However, as indicated earlier, the main analytical approach toward the detection of CTCs always includes 2 steps: (a) isolation/enrichment and (b) detection. CTCs are rare events that follow a Poisson distribution, and this fact has to be taken into account for their detection. The sample volume of peripheral blood used for their isolation is critical, especially in the case of early disease. The most widely used enrichment approaches are based on (a) the different density of CTCs, a feature exploited with such methods as centrifugation in the presence of Ficoll; (b) filtration; and (c) immunomagnetic isolation (positive or negative) through antibodies specific for epithelial markers such as EpCAM or leukocytes (CD45), respectively. A combination of enrichment methods is also used, e.g., filtration devices in combination with EpCAM-positive isolation, Ficoll enrichment, and then positive immune-magnetic isolation. Detection approaches are based on (a) imaging (immunocytochemistry and immunofluorescence) through the use of specific markers for CTCs such as CKs (mainly CK-8, −18, and −19), leukocytes such as CD45, and cell viability via 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining—mainly by the FDA-approved CellSearch system; (b) molecular methods based on gene expression of specific markers such as CK-19; and (c) methods based on the detection of proteins secreted by immobilized CTCs, such as the EPISPOT assay. Despite the fact that most of these methods are highly specific and sensitive, thus far there are no extensive studies designed to compare their efficacy when using the same clinical samples. This is an important issue for their clinical use since, especially in early disease, differences in analytical sensitivity between these methods play a very critical role. Is there a need for a quality-control system for CTC enumeration? Klaus Pantel: There is a clear need for a quality-control system for CTC enumeration. Automated CTC detection systems (e.g., CellSearch) have included built-in positive and negative controls that have allowed the distribution of images among participating laboratory centers. Since microscopical detection systems are observer dependent, the development of international standards for CTC enumeration and characterization is of utmost importance. Howard I. Scher: There is more than a need; it is essential. Without quality control, the reported results and the clinical data that might be derived from them are virtually uninterpretable and are of limited to no value. At this time, as indicated earlier, the only CTC assay that has been FDA-cleared for use, CellSearch, defines a CTC as a cell that is morphologically intact, has a nucleus surrounded by cytoplasm after DAPI staining, expresses CK-8, −18, or −19, and is CD45 negative. Noteworthy is that as a part of the validation process, which showed the reproducibility and consistency of the assay in the reference and local laboratories, over 450 breast cancer patient samples, in addition to control samples, were evaluated. Leon Terstappen: Each CTC system should be validated and accompanied with a quality-control system. Evi Lianidou: Numerous single-institutional studies suggest that CTCs can play an important role in risk stratification and monitoring of therapeutic efficacy. These findings need to be evaluated in trials to verify this concept in the clinical setting. Agreement on the standardized detection of CTCs is absolutely necessary. Critical issues include: (a) the standardization of the preanalytical phase, such as sampling itself (e.g., sample volume, avoidance of epidermal epithelial cell cosampling in case epithelial markers such as CK-19 will be used later for CTC detection), sample shipping (stability of CTCs under different conditions), and storage conditions (use of preservatives or anticoagulants); (b) standardization of CTC isolation through the use of spiking controls in peripheral blood; (c) standardization of detection systems; and (d) interlaboratory- and intralaboratory-comparison studies for the same samples. The development of international standards for CTC enumeration and characterization is also very important, especially in imaging detection systems that are observer dependent. A recent study has shown the feasibility of external quality assurance of CTC enumeration using the CellSearch system. RT-qPCR–based molecular methods can be used in routine clinical laboratories and can be standardized, since the required quality issues, such as quantification cycle (Cq) values, limit of detection, precision, accuracy, and recovery experiments, have been clearly described. Studies that have compared molecular RT-PCR–based methods and immunocytochemistry have shown a significant correlation, whereas in a recent comparison study of the CellSearch assay and a molecular test (AdnaTest BreastCancer), concordant results regarding HER2 (human epidermal growth factor receptor 2) positivity were obtained in only 50% of the patients. In conclusion, a universal internal and external quality-control system for CTC detection and enumeration is urgently needed before their application in the clinic. which cancer has the most been with CTCs and Klaus Pantel: cancer has been the of the international on CTCs because it is that early dissemination of tumor cells an important role in breast as by the on the clinical relevance of in bone data on monitoring CTCs in prostate cancer has provided important of this approach as a “liquid in the context of new therapies. Howard I. Scher: A for tumor cells and tumor produced the numbers of breast, prostate, colorectal, and and The number of is only one of the in a disease important is the which to the analytical of the used in the and the level of evidence that has been generated to a specific context of use. issue is in the detection step. using CellSearch, one cell in of of patients with metastatic for this is In detection are new assays are needed to the proportion of patients in CTC biomarkers can be evaluated. The for CTC enumeration with CellSearch presence of CTC in the peripheral as detected by the Circulating is associated with decreased progression and decreased overall in patients for metastatic breast, or prostate The test is to be used as an in the monitoring of patients. for CTC should be used in with other clinical methods for does not that the results can be used as an efficacy response for in can be only by the in an is in and CTC enumeration in the of a of in the of and in the of and A this has been with the for Drug and of the and of the of CTC enumeration with is The prognostic of CTC number after treatment is also being studied in breast cancer as a response to treatment. in and cancers is the ability to detect kinase that treatment both at the of therapy and on these the test for an blood sample for profiling tumor at the time a treatment is the need for an and that is to CellSearch as currently cannot be used to detect CTCs in patients with and cell because these do not detect CTCs in these some groups are CTC detection using for the of promising 2 trials are the level of research will be by the need that the test will be used to the technical performance of the assays and the results from the of trials required to evidence to routine use in a setting. Leon Terstappen: multicenter studies have been conducted for metastatic breast, colorectal, and prostate The to studies in the most Evi Lianidou: CTCs are studied in breast The is that the clinical relevance of in the bone marrow of breast cancer patients has been clearly Since early dissemination of tumor cells an important role in breast a of has been in this of cancer with both bone marrow and peripheral blood samples. using has shown that in early breast cancer the detection of peripheral blood CK-19 cells is an prognostic factor for a and overall for breast cancer patients. the detection of peripheral blood and cells before whereas the detection of CK-19 CTCs in the blood after is an risk factor the presence of residual disease. The of CTC enumeration in breast cancer has been shown by using the CellSearch system. data on monitoring CTCs in other of such as prostate have provided important as a “liquid in the context of new therapies. What are the main clinical issues and to be with CTCs? Klaus Pantel: (i) of the risk for metastatic or metastatic progression (ii) stratification and monitoring of (iii) identification of therapeutic targets and Howard I. Scher: on prostate cancer in issues in development and patient are the of treatment This is because which are to represent the most of metastatic and one of the of and do there are a patient be when the is or not be when it is that can be in 2 trials are both for the as well as to inform the decision to to testing. An is that it is to metastatic tumor for molecular profiling from the most of and when it is few of the assays used have been a therapy a that the is when treatment is are and to all prostate are to but to a to as prostate or which is are used as an of disease as well as a response for both and However, changes are not a for overall survival. These findings suggest the need to identify and define of efficacy that more the clinical Leon Terstappen: A to determine which therapy or combination of to be the most for the A to determine the of the treatment Evi Lianidou: clinical (a) clinical studies to that CTC detection can lead to a in the of cancer patients that results in an clinical is especially designed to test the of therapy therapy for metastatic breast cancer patients have CTC at the first and is to be and (b) the use of CTCs for stratification of patients and monitoring of therapies. to be (a) of findings between (b) molecular characterization of CTCs will the identification of novel that will and their to cancer cells. Is detection of among CTCs Klaus Pantel: CTCs and a in of and determine this is important, e.g., to the of the residual tumor and to information on the of CTC therapy (e.g., HER2 of CTCs therapy with Howard I. Scher: and are an issue with specific to of the tumor in cell and some tumor cell is to of the enrichment are to be heterogeneous, and CTCs a molecular of this The of markers in detecting CTCs is In a mutation that is for response to a specific be detected in a population of CTCs, but the mutation is in only a proportion of CTCs, the clinical be The same consideration to Leon Terstappen: Yes, with respect to the presence of treatment targets in important to the of therapies. Evi Lianidou: CTCs are highly heterogeneous, as has been shown through and molecular This is highly important, when therapeutic targets are in CTCs but not in the primary Since CTCs have been detected in a number of patients with primary of HER2 by of HER2 expression on CTCs is a with potential clinical However, the of CTC thus far has not been exploited is it to CTCs? or after primary Klaus Pantel: The of CTCs is Thus, it can be that the of CTCs after primary therapy (e.g., after of information on whereas the detection at primary surgical removal of the might be largely by the of the primary Although data the clinical relevance of CTCs at these 2 time are the enumeration of CTCs that therapy might be more relevant for the than the detection of CTCs at primary Howard I. Scher: the to the a assay disease in only or of patients for primary it be more to for new assays that detection because the to CTCs does not a there are data from tumor that the detection of cells at is associated with an but there are no data to on the treatment of a primary tumor on the of the presence or of cells. the context of using CTCs for molecular profiling to treatment it is to have a whereas for the context of response both are Leon Terstappen: of the presence and number of CTCs should be used to and determine the presence or of treatment the first cycle of CTCs should be used to determine or not the therapy is Evi Lianidou: The presence of in bone marrow has been clearly shown to be of prognostic in patients with breast cancer before primary treatment. has shown that in early breast the detection of CK-19 CTCs is an prognostic factor both before and after What is the role of CTCs in Klaus Pantel: monitoring and molecular characterization of CTCs, for therapeutic targets (e.g., or to [e.g., to a of cancer patients from with and these also allow an early to in patients. Howard I. Scher: tumor the that to progression over In such molecular of the primary tumor obtained to a or that is as therapy not be In these it is to tumor at the time of first and all subsequent it is that molecular can also on a tumor for profiling is essential. most performance of a after and treatment is not a part of routine as is also and to CTCs obtained from a sample obtained in the context of routine patient can this studies the presence or of CTCs will be shown to provide of treatment that are a patient to be and those that are not to be Leon Terstappen: In patients in which CTCs can be detected and CTCs can a Evi Lianidou: characterization of CTCs can provide information on the expression of specific such as such as and specific such as in the epidermal growth factor that sensitivity to therapies. CTC molecular analysis the of monitoring changes the of treatment and can as a to in the Is the currently available evidence to use CTCs in the for which Klaus Pantel: In patients with cancer breast and prostate there is evidence that enumeration of CTCs prognostic information and to be more sensitive than the current imaging technologies or markers (e.g., used to measure In the prognostic role of CTCs is under CTC are part of clinical trials new in breast and prostate and the of these trials will determine CTC detection will a as a (i.e., for therapy response or Howard I. Scher: Yes, the evidence to date does the use of CTC enumeration for the clinical use for which it is The data are to that the test can be used in the of or as a to this is being in Leon Terstappen: Yes, for the of and the monitoring of therapy of patients for metastatic breast and prostate Evi Lianidou: There is evidence that enumeration of CTCs prognostic information in patients with of cancer CTC detection to be more sensitive than current imaging technologies or the tumor biomarkers used to detect early In patients with breast has shown that detection of CK-19 positive CTCs is an prognostic both before and after However, this is a that has not been in other laboratories, the prognostic role of CTCs in early disease is under CTC can play a role for the of the efficacy of novel in breast and prostate This is being in clinical and their will determine CTCs can be used as markers for therapy What is for the clinical of CTCs from Klaus Pantel: monitoring of CTCs to therapeutic efficacy will current of tumor progression by imaging technologies or of blood In the molecular analysis of CTCs for therapeutic targets in to molecular to tailor to the of a cancer Howard I. Scher: In addition to the in the groups are the ability to cells and cell The are used to test the of specific to inform the choice of a therapy most to that as well as to of and the biology of the disease. are also under and the in this will the analysis of numbers of cells in a setting. In addition to the it is that new be and to the This is to and to the use of studies that studies not only are and but also have the of development and patients to treatment. Leon Terstappen: CTCs will be used in routine to cancer treatment and will be used to determine what might be in the Evi Lianidou: A combination of imaging systems and molecular characterization of CTCs will be very to define treatment and or the risk of The use of technologies such as will the of molecular in CTCs and lead to the of novel molecular that CTCs CTCs might the method to the efficacy of cancer therapies. disseminated tumor cell tumor cell reverse-transcription quantitative PCR epithelial cell adhesion molecule epithelial immunospot prostate-specific antigen US Food and Drug Administration 4′,6-diamidino-2-phenylindole dihydrochloride quantification cycle epidermal growth factor receptor 2 for Drug and

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