Detection of the Circulating Tumor Cells in Cancer Patients

Transcription

1 Detection of the Circulating Tumor Cells in Cancer Patients Athanasios Armakolas; Zacharoula Panteleakou; Adrianos Nezos; Aikaterini Tsouma; Maria Skondra; Peter Lembessis; Nikolaos Pissimissis; Michael Koutsilieris Authors and Disclosures Posted: 02/21/2011; Future Oncology. 2010;6(12): Future Medicine Ltd. Abstract and Introduction Abstract As the presence of tumor cells circulating in the blood is associated with systemic disease and shortened survival, the establishment of a method to detect circulating tumor cells (CTCs) is of critical importance for a more concise staging and follow-up of cancer patients. Recently, the most robust strategies for the determination of CTCs are the PCR-based methods and the CellSearch system that exploits the immunofluorescent characterization and isolation of cancer cells. Herein, we analyzed the experimental strategies used for determining CTCs with respect to accuracy, sensitivity and reproducibility in cancers of the breast, colon, prostate and melanoma. Introduction The major cause of cancer-related death in patients with solid tumors is the presence of metastatic disease. [1] The existence of malignant cells of epithelial origin in the blood circulating tumor cells (CTCs) has been known for over a century and has been associated with metastasis. It has been suggested that the hallmark of the 'invasive behavior' of a proportion of cancer cells is associated with escaping from the primary tumor mass and colonizing new terrain in the body where, at least initially, nutrients and space are not limited, and there form metastases. Although there is a large number of different methods developed for determining DNA or protein concentrations in the blood or plasma of cancer patients and genetic or epigenetic alterations specific to tumors, all aiming at different biomarkers such as circulating nucleic acids and proteins with various techniques, [2 4] recent evidence suggesting that the major cause of metastasis is the presence of CTCs in cancer patients holds tremendous promise for the identification of the potential for metastatic disease at very early stages, managing risk stratification in the adjuvant setting, monitoring responses to treatment, assessing disease recurrence and the prospective development of more effective and better tailored anticancer therapies for individual patients. [5 7] The rationale for measuring CTCs for disease that is not clinically or radiographically identifiable could be a major challenge for oncologists, as the cancer patient treatment arena is becoming congested with numerous types of therapies relative to specific clinicopathological parameters. Over the past few years, different approaches have been developed, prior to the detection of CTCs in different tumors. Each of these approaches has distinct advantages and disadvantages, with the most notable being sensitivity and specificity. Our aim is to critically analyze the major methodologies involved in the detection of CTCs, namely the CellSearch system (Veridex, NJ, USA) and the PCR-based methods, and to discuss the advantages and drawbacks concerning the requirements of high sensitivity, high specificity and affordability. Detection of Circulating Tumor Cells by PCR Real-rime PCR (RT-PCR) is the method most widely used and most extensively studied thus far for the identification of CTCs. The advent of PCR technology has provided a specific and sensitive way to distinguish cells based on differential gene expression and genetic profiling. [8,9] In summary, this technique is based on the isolation of patient RNA from blood (the amount of blood used usually varies from 3 to 10

2 ml depending on the laboratory) followed by reverse transcription on this cdna product; it is then applied to the PCR reaction for the target gene, usually a gene that is expressed uniquely in the CTCs we are aiming for. The RT-PCR is set in such a way so as to allow for the detection of a certain number of CTCs in the total cell population in the circulation by targeting different genes that are only expressed in cancer cells and by varying the conditions of the technique. This is a rather qualitative method for detecting CTCs in cancer patients, although by introducing a housekeeping gene or 18S mrna in this reaction and by estimating the ratio between the expression of the gene of interest and the expression of the housekeeping gene, this method can be considered to be semiquantitative. Alternatively, a purely quantitative technique has also been applied for the detection of CTCs: the quantitative RT-PCR. A commonly used variation of this method is the multiplex RT-PCR. In this case, more than one locus is simultaneously amplified in the same reaction by including more than one pair of primers. This technique is a conceptually practical, simple, safe, cost effective and full automation-amenable alternative to traditional methods of diagnosing disease recurrence and/or systemic spreading. The limitations of using a single PCR to detect CTCs include cost, since many markers have to be examined, and sometimes the availability of adequate quantities of test samples. [10] Multiplex PCR has the potential to produce considerable savings of time and effort; in line with the requirements of the speed and low cost of these methods, an increasing number of targets can be analyzed throughout each run on a thermocycler without compromising test utility. In addition, multiplex PCR allows for the coamplification of internal control, thus eliminating variability and providing more reliable results. In addition, it has reduced reagent costs, since the targets are coamplified instead of undergoing separate amplification, and ensures conservation of precious clinical samples. The major drawback of this technique is the extensive planning required to optimize the reaction conditions and determine primer compatibility, as well as defining the positivity of the analysis vis à vis disease volume (i.e., number of CTCs in the sample mainly obtained by spiking experiments with a known number of respective cell lines in blood samples). The CellSearch System A recently developed technology to identify CTCs is the CellSearch system. The CellSearch system identifies and, more importantly, counts CTCs in a blood sample, giving a prediction of progression-free survival and overall survival in patients with metastatic cancer. The results of serial testing for CTCs with the CellSearch system, in conjunction with other clinical methods for cancer monitoring, have been suggested to help inform physicians' treatment decisions. [11 13] In summary, this CTC detection system is based on a combination of immunomagnetic labeling and automated digital microscopy, which is currently approved by the US FDA for predicting the prognosis and monitoring the clinical outcome of patients with metastatic breast, prostate and colorectal cancers. [11 13] Peripheral blood is mixed with iron particles coated with anti-epithelial cell adhesion molecule (EpCAM) to confer magnetic properties to all the epithelial cells, including epithelial carcinoma cells. Anti-cytokeratin (CK) antibodies are then used for the identification of these epithelial cells, while anti-cd45 antibodies are utilized to rule out lymphocyte presence, and the nuclear dye DAPI is applied to fluorescently label cell nuclei for microscopic visualization of the enriched cell population. Following incubation, washing, magnetic separation and fixation, the immunomagnetically separated cell population can be viewed and enumerated by automated digital fluorescent microscopy. [11] Currently, the CellSearch system can analyze a 7.5-cc tube of blood and is considered to be the first diagnostic test to automate the detection and enumeration of CTCs. This method accurately provides information regarding the presence and number of CTCs in the well by exploiting the specificity of the three different antibodies used (EpCAM, CK and CD45). Therefore, the success of the assay depends on the level of expression of the EpCAM and CK target antigens that are used as surface CTC-specific markers. [6] Applications in Breast Cancer Patients

3 Assessment of CTCs by CellSearch suggested that it is a superior surrogate end point than the current radiology imaging methods for predicting overall survival in patients with metastatic breast cancer. [14] Moreover, in the same cohort of patients with measurable metastatic breast cancer, CTC detection at different follow-up times during therapy was reported to predict progression-free survival and overall survival. [15] The data on CTC monitoring with CellSearch apply mostly to patients with measurable metastatic breast cancer beginning chemotherapy but not hormonal therapy. In order to provide further evidence that CTC detection can improve outcomes in metastatic breast cancer patients, the Southwest Oncology Group (SWOG) has launched a randomized Phase III trial to test the strategy of changing chemotherapy compared with continuing the same chemotherapy for metastatic breast cancer patients who have elevated CTC levels at their first follow-up assessment. [101] Apart from CellSearch, the presence of CTCs can also be indirectly assessed by RT-PCR. RT-PCR has been used to amplify epithelial-specific or breast cancer-specific mrna transcripts. Although it is a very sensitive detection method, issues of specificity have been a concern. Commonly used RT-PCR markers to detect CTCs, such as CK19, EGF receptor (EGFR) and mammaglobin were found to be expressed in normal mitogen-stimulated peripheral blood mononuclear cells. [16] Thus, it was suggested that RT-PCR assays should be validated in activated lymphoid cells and not in normal peripheral blood mononuclear cells, especially if they are used in the metastatic setting, which is often accompanied by inflammatory reactions. [16] Applications in Colorectal Cancer The CellSearch system has been recently applied in colorectal cancer (CRC) patient, with increasing data arising in relation to its potential utility; it has been applied to patients with metastatic colon cancer and gastrointestinal cancer and has led to the association of the presence of CTCs with prognosis and survival. [12,17 19] The benefits of multiple transcript examination for the detection of CTCs in CRC patients have been previously reported. [20] Among the various studies using multiple molecular markers for the detection of occult CRC, Shen et al. examined the detection of CEA, CK20 and survivin mrnas by RT- PCR and found that the combination of these markers enhances detection sensitivity and correlates with stage and lymph node metastasis. [21] Commonly used markers examined by RT-PCR in CDC patients included CEA, CK20, CK19 and GCC. Analysis of each marker separately produces a sensitivity rate of 63.3%, while combined analysis of all four markers results in a sensitivity boost of up to 87.7%. [22] Comparable results have also been observed by two other groups, which suggested that increased sensitivity was associated with the detection of CEA and CK20 mrnas in combination, [23] while the second group reached the conclusion that combined RT-PCR evaluation of six mrna markers (CK20, CEA, GCC, EGFR, MMP-7 and HLM) improves the sensitivity of the assay. [24] In conclusion, it can be suggested that the multiplex RT-PCR for two or more markers is more sensitive compared with the use of a single marker for CTC detection. Applications in Prostate Cancer In many studies, the detection of CTCs in the peripheral blood of patients with metastatic prostate cancer has been correlated with poorer outcome with regards to both progression-free and overall survival. Tumor markers, such as the prostate-specific antigen (PSA), are upregulated by androgens and might be more difficult to detect after hormonal therapy, and prostate-specific membrane antigen (PSMA) is highly expressed in hormone-refractory patients. Therefore, it is more accurate and clinically relevant to evaluate both PSA and PSMA simultaneously. The clinical utility of positive molecular staging as defined by detection of both PSA and PSMA cdna transcripts in the peripheral blood of patients with clinically localized prostate cancer has been validated as a staging tool and predictor of response to therapy. [10] The aim of multiplex assays for prostate cancer is the detection of micrometastases before surgery, which are not identifiable with images. When considering surgery as a curative option, it is important to identify patients with clinically confined disease over those who have locally advanced disease and consequently are at higher risk of relapse and less likely to benefit from surgery. [10] We have documented that an increased rate of biochemical failure-free survival after curative therapy is associated with positive RT-PCR

4 patients. [25] A 40-fold higher chance for the occurrence of prostate cancer was observed in 73 prostatic tissue samples from patients with prostate cancer and benign hyperplasia when at least three out of five markers (AR, SRD5A2, KLK2, PSMA and PCA3) analyzed by multiplex RT-PCR were positive and serum PSA levels were greater than or equal to 4 ng/ml. [26] A multiplex PCR assay (GOLPH2, SPINK1, PCA3 and TMPRSS2 ERG fusion) on sedimented urine from patients presenting for prostate biopsy or prostatectomy outperforms serum PSA. [27] According to Helo et al., RT-PCR and CellSearch CTC results based on KLK3, KLK2 and PSCA mrna for patients with metastatic castration-refractory prostate cancer were strongly concordant (80 85%) and were closely associated with clinical evidence of bone metastases and with poor survival. [28] However, in the subset of patients with localized prostate cancer, such a relationship was not found. [28] At present, not enough evidence is available regarding how CTC detection might prove useful for the clinical management of the early-stage cancer patients. [29,30] Applications in Melanoma The detection of CTCs in melanoma is expected to differentiate primary melanomas of low metastatic potential from primary melanomas of high metastatic potential. Several melanoma markers have been identified to date, some of which specifically detect tumor cells derived from the melanocyte lineage, whereas others simply indicate the presence of malignant cells that may be derived from other cancers as well. These markers can be grouped into four categories. The first category includes melanocytic or melanoma-specific markers, such as tyrosinase, MITF, PAX3, TRP-1, TRP-2/Dct and gp100, which are involved in melanin production or reveal the presence of melanin metabolites. [31] The second category includes melanomaassociated antigens such as MART-1/Melan-A, p97, GalNAc-T, MIA and MUC18/MCAM. The third category includes less specific tumor markers that may also be associated with other cancers such as prostate, breast and colorectal cancers. This category includes the markers MAGE- A1, -A3 and -A6, PRAME, NY-ESO GAGE, S100b, YKL-40, CRP, CRT-MAA and E/P-cadherin. The fourth category of tumor markers emphasizes their association with tumor growth, cell proliferation and migration. Such markers include VEGF, EGFR, NF-kB, ATF-2, FOS, JUN, MK167, TOP2A, BIRC5, STK6 and 11, HTERT, SPP1, ERBB3 and MMP-2. [32,33] The detection of these markers in peripheral blood (or in tissue) may again be due to the presence of cancers other than melanoma, such as prostate, liver, lung or breast cancer. The CellSearch system has recently been used to assess whether it could be applicable for the detection of circulating melanoma cells (CMCs) in malignant melanoma patients. [34] CMCs were defined as being positive for EpCAM and CK8, 18 and 19. [34] These cell populations should also be CD45-negative to rule out lymphocyte presence. However, the CellSearch system has so far only been shown to detect CMCs in stage IV melanoma patients. [34] Peripheral blood mrna expression of tumor-specific genes is the current strategy applied by the RT-PCR-based techniques for metastatic melanoma. Discussion Despite important advances in early diagnosis and treatment, metastatic disease occurs in approximately half of cancer patients. [35] Staging of carcinoma patients in clinical practice is still based on tumor characteristics obtained by the patients' clinicopatholgical data at the time of primary surgery, steroid receptor status and human EGFR2 amplification. [36] The assessment of CTCs in peripheral blood samples is not considered to be a routine procedure in the clinical management of cancer as it is very difficult to draw firm conclusions from this assessment method owing to interlaboratory procedure differences, mainly in the volume of blood analyzed, the quality of sensitivity and specificity of tests, the number of patients versus controls and data interpretation. Although in general approaches based on RT-PCR have a high sensitivity for the detection of CTCs, an important limitation of these methods is that these cannot quantify the number of CTCs and no morphological evaluation of cells can be obtained. Furthermore, it remains unclear whether the minute tumor dissemination detected by PCR is capable of causing clinically relevant distant metastasis. [10,37] To date, few data have been published concerning the role of CTC detection by the CellSearch system in cancer. In

5 contrast to RT-PCR assays, the CellSearch CTC test allows for the counting of target cells. The advantages of the CellSearch system are its capability of standardizing preanalytical preparations of CTCs, the use of CTC preservative tubes that allow stabilization of CTCs for up to 96 h and the inclusion of a positive control for assuring proper performance on a daily or run-by-run basis. All these features are beneficial in a multicenter setting in clinical trials. However, one of the limitations of the CellSearch system is the anti-epcam antibody-based enrichment strategy. Several authors reported the heterogeneous expression of EpCAM in mammary carcinomas, [38] and downregulation of EpCAM has been reported for disseminated tumor cells in bone marrow and CTCs in peripheral blood. [39] Cytokeratins are commonly used to distinguish epithelial cells from a heterogeneous cell population but they do not confer tissue-type specificity, nor do they distinguish the origin of the epithelial cells. Therefore, in the case of sample contamination by epithelial cells (during the venipuncture) the use of epithelial markers carries a risk of false-positive results. Interestingly, although the majority of leukocytes do not express epithelial markers, they have occasionally been observed to become positive for such antigens when activated by cytokines. [40,41] Several studies have shown CKs to be expressed in samples from normal volunteers and from patients with hematologic malignancies, yielding false-positive results. [42 47] The presence of epithelial cells in the peripheral blood [46] has been associated with inflammation, [16] often accompanying cancer development, [16,48] tissue trauma and surgical manipulation [49,50] or biopsy intervention. [51] Epithelial cell adhesion molecule is regulated negatively by TNF-α, resulting in diminished EpCAM protein at the cell surface of tumor cells. [52] EpCAM is not expressed in all histological tumor types (soft-tissue tumors and all lymphomas were EpCAM-negative [53] ) and its utility is hampered by the fact that subpopulations of hematopoietic cells and erythroid progenitors express EpCAM and coexist with tumor cells in the sample. Tumor cell detection by the CellSearch antibody-based method is clearly dependent on the ability of antibodies to distinguish between cells of different tissue origin. [54] If epithelial nontumor cells can be spread in the peripheral blood, then the actual number of tumor cells cannot be determined based on antibodies specific to epithelial cells such as EpCAM and CKs. However, the intrinsic advantage of this system is that it is reproducible across different laboratories and can identify CTCs in different cancer types. [14,55] The combination of markers in the multimarker assay can compensate for individual marker expression, [56] thus increasing the specificity of detection, reducing false-negative results and reducing the likelihood of falsepositive tests. The fact is that tumors are inherently heterogeneous, and there are currently no markers known that are consistently and specifically expressed by all tumor cells. [57] Furthermore, tumors continuously evolve genetically over time in response to host pressures and treatment interventions. [58] One must acknowledge that the process of metastasis is highly selective, and the metastatic lesion represents the end point of many destructive events that only a few cells can survive. [59] Tumor cell shedding may be intermittent and unpredictable as well. [60] Recent studies have shown that the same patient can oscillate between being PCR-positive and PCR-negative for CTCs depending on the timing of blood draws within the same day. [34,61] Another limitation is illegitimate transcription, transcripts of various tissue-specific genes expressed at low levels in human nonspecific cells. [62] For example, the possibility that hematopoietic cells illegitimately synthesize CK19 transcripts [63,64] or that the increased secretion of cytokines induces its expression [37] have been reported. The heterogeneity and genetic instability of tumors [65] make it unlikely that such an ideal single marker even exists. Because of these limitations, many researchers are developing new methods for the detection of CTCs. Prior to proceeding in individualized therapy, knowledge regarding the presence and the number of CTCs is not the only prerequisite. Multiplex RT-PCR and standard RT-PCR, although less automatized and more laborious in comparison with the CellSearch, may be more informative regarding CTCs and the prediction of patient outcomes and responses to chemotherapy by correlation with certain specific markers rather than CTC number. Strategies that combine both techniques (the PCR and the immunocytochemical characterization

6 and isolation) such as multiplex RT-PCR and multiplex RT-PCR in combination with magnetic beads have also be performed with promising results; [15,48] however, the cost is significantly increased. In conclusion, detection and characterization of CTCs may provide new insights in the field of cancer biology and metastasis, aiming towards individualized and more specific therapies depending on the number and the type of CTCs; of course, consideration regarding the cost of such methods is another important issue in the clinical settings. Conclusion & Future Perspective Although large-scale clinical data is still lacking with regard to how molecular characterization of CTCs could be used as a clinical decision-making tool, this type of analysis holds tremendous promise with regard to gaining a better biological understanding of the metastatic process, improving stratification of patients, identifying novel therapeutic targets and monitoring systemic anticancer therapies aiming towards the prospective development of individualized, targeted therapies. Nevertheless, it is becoming increasingly apparent that disease progression depends on numerous complex pathways, and that the analysis of one single CTC marker is unlikely to precisely predict progression of disease with sufficient accuracy and reproducibility. In addition, available studies are hampered by small sample sizes, heterogeneous patient populations and, most importantly, the lack of standardized methodologies for the detection and characterization of CTCs. Quality control at all stages, as well as improved standardization with regard to the time of sampling in a given cohort of patients, the number of samples required to reliably detect CTC and the type of test performed for CTC detection, are needed to overcome some of these technical limitations.