Colorectal cancer diagnostics: biomarkers, cellfree DNA, circulating tumor cells and defining heterogeneous populations by single-cell analysis

Expert Review of Molecular Diagnostics ISSN: 1473-7159 (Print) 1744-8352 (Online) Journal homepage: http://www.tandfonline.com/loi/iero20 Colorectal cancer diagnostics: biomarkers, cellfree DNA, circulating tumor cells and defining heterogeneous populations by single-cell analysis Cindy Kin, Evelyn Kidess, George A Poultsides, Brendan C Visser & Stefanie S Jeffrey To cite this article: Cindy Kin, Evelyn Kidess, George A Poultsides, Brendan C Visser & Stefanie S Jeffrey (2013) Colorectal cancer diagnostics: biomarkers, cell-free DNA, circulating tumor cells and defining heterogeneous populations by single-cell analysis, Expert Review of Molecular Diagnostics, 13:6, 581-599, DOI: 10.1586/14737159.2013.811896 To link to this article: https://doi.org/10.1586/14737159.2013.811896 Published online: 09 Jan 2014. Submit your article to this journal Article views: 2492 View related articles Citing articles: 23 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=iero20

THEMED ARTICLE Nanotechnology & Single-Cell Analysis Review Colorectal cancer diagnostics: biomarkers, cell-free DNA, circulating tumor cells and defining heterogeneous populations by single-cell analysis Expert Rev. Mol. Diagn. 13(6), 581 599 (2013) Cindy Kin*,, Evelyn Kidess, George A Poultsides, Brendan C Visser and Stefanie S Jeffrey* Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA *Author for correspondence: Tel.: +1 650 736 8406 Fax: +1 650 725 0791 cindykin@stanford.edu; ssj@stanford. edu Reliable biomarkers are needed to guide treatment of colorectal cancer, as well as for surveillance to detect recurrence and monitor therapeutic response. In this review, the authors discuss the use of various biomarkers in addition to serum carcinoembryonic antigen, the current surveillance method for metastatic recurrence after resection. The clinical relevance of mutations including microsatellite instability, KRAS, BRAF and SMAD4 is addressed. The role of circulating tumor cells and cell-free DNA with regards to their implementation into clinical use is discussed, as well as how single-cell analysis may fit into a monitoring program. The detection and characterization of circulating tumor cells and cell-free DNA in colorectal cancer patients will not only improve the understanding of the development of metastasis, but may also supplant the use of other biomarkers. Keywords: biomarkers cell-free DNA circulating tumor cells colorectal cancer colon cancer microsatellite instability plasma DNA single-cell profiling Authors contributed equally. Colorectal carcinoma is the third most common cancer diagnosed worldwide and the fourth most common cause of cancer death [1,2]. Oncologic outcomes have improved with advances in surgical technique and adjuvant therapy. Overall 5-year survival for colorectal cancer is excellent for disease localized to the colon (95% for stage I and 82% for stage II) (Table 1), but decreases to 61% for patients with regional spread to the lymph nodes (stage III) and only 8% for patients with distant metastases (stage IV) [3,4]. Fewer than 40% of the patients in the USA are diagnosed with stage I or II disease, for which surgery alone can be curative. Patients with stage III disease, comprising 36 54%, are candidates for adjuvant chemotherapy after surgical resection. Patients with stage IV disease comprise 20 31% and usually undergo first-line chemotherapy, potentially followed by surgical resection [5]. Metastasis occurs most commonly to the liver in a synchronous fashion in 14.5% and in a metachronous fashion in 60% [6,7]. Synchronous metastasis to the peritoneal surface occurs in 9 18% of the patients, to the lung in 10 14% and to the bone in 0.7 1.6% [8]. There are several regimens used for the treatment of stage III and IV colorectal cancer, including 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX); 5-fluorouracil, leucovorin and irinotecan (FOLFIRI); and capecitabine (the oral form of 5-fluorouracil, otherwise known as Xeloda ) plus oxaliplatin (XELOX). Advances in molecular diagnostics have allowed the development of more targeted therapeutic agents to reduce the risk of metastases and to treat metastases if they do occur. The most widely used tumor marker in colorectal cancer is serum carcinoembryonic antigen (CEA), which is helpful in the detection of liver metastases developing after tumor resection. In addition, tumor-specific markers such as KRAS www.expert-reviews.com 10.1586/14737159.2013.811896 2013 Informa UK Ltd ISSN 1473-7159 581

Review Kin, Kidess, Poultsides, Visser & Jeffrey Table 1. TNM staging of colon and rectal cancer, American Joint Committee on Cancer Manual, 7th edition. Primary tumor (T) Regional lymph nodes (N) Distant metastasis (M) TX: Primary tumor cannot be NX: Regional lymph nodes cannot be assessed M0: No distant metastasis assessed N0: No regional lymph node metastasis T0: No evidence of primary tumor N1: Metastasis in 1-3 regional lymph nodes M1: Distant metastasis Tis: Carcinoma in situ: intraepithelial or invasion of lamina propria N1a: Metastasis in one regional lymph node T1: Tumor invades submucosa N1b: Metastasis in 2-3 regional lymph nodes T2: Tumor invades muscularis propria N1c: Tumor deposit(s) in the subserosa, mesentery, or nonperitonealized pericolic or perirectal tissues without regional nodal metastasis T3: Tumor invades through the muscularis propria into pericolorectal tissues T4a: Tumor penetrates to the surface of the visceral peritoneum N2: Metastasis in 4 or more regional lymph nodes N2a: Metastasis in 4 6 regional lymph nodes T4b: Tumor directly invades or is N2b: Metastasis in 7 or more regional lymph nodes adherent to other organs or structures Stage T N M Dukes 0 Tis N0 M0 I T1 T2 N0 M0 A IIA T3 N0 M0 B IIB T4a N0 M0 B IIC T4b N0 M0 B IIIA T1 T2, T1 N1, N2a M0, M0 C, C IIIB T3 T4a, T2 T3, T1 T2b N1, N2a, N2b M0, M0, M0 C, C, C IIIC T4a, T3 T4a, T4b N2a, N2b, N1 N2 M0, M0, M0 C, C, C IVA Any T Any N M1a IVB Any T Any N M1b Adapted from [168]. and microsatellite instability (MSI) are useful for both prognosis and directing chemotherapy. Circulating tumor cells (CTCs) have been identified as a necessary component of the metastatic cascade. Numerous methods of CTC detection have been developed. Studies demonstrating that CTCs have prognostic and predictive value have spawned interest in potential clinical applications. Additionally, single-cell profiling of CTCs has opened a new frontier in personalized oncologic therapy. Cell-free DNA has also been studied for its prognostic and predictive value. Tumor markers in colorectal cancer CEA is the current serum biomarker used in clinical practice, with its main utility for indicating disease recurrence in the liver. Tumor biomarkers identified in colorectal cancer tissue have been used to guide chemotherapy regimens and include KRAS, BRAF, MSI and SMAD4 [9]. M1a: Metastasis confined to one organ or site M1b: Metastases in more than one organ/site or the peritoneum CEA Since its identification in colon adenocarcinoma in 1965, CEA has become the standard tumor marker in the clinical care of patients with colorectal carcinoma [10]. It is correlated to disease stage and preoperative CEA level is related to prognosis [11 16]. Following resection of the primary tumor, serial CEA levels are routinely obtained every 2 3 months for at least 2 years to monitor for disease recurrence or metastasis [17 19]. There is no consensus on what change in CEA level would trigger further evaluation for recurrence [20]. Increases of CEA by at least 50% has a sensitivity of 76% and specificity of 90% for predicting progression of disease in patients with metastatic disease undergoing palliative chemotherapy, and a drop of at least 30% can exclude disease progression [21]. MSI MSI occurs due to defects in DNA mismatch repair genes, and while it is typically associated with hereditary nonpolyposis 582 Expert Rev. Mol. Diagn. 13(6), (2013)

Colorectal cancer diagnostics Review colorectal cancer, most MSI-high tumors occur sporadically [22,23]. Despite their resistance to alkylating agents and cisplatin, MSI-high tumors have better recurrence-free and overall survival [23]. In patients with stage II disease, MSI-high status was found to confer the same advantage in long-term outcomes as that conferred by stage T3 over T4 [24]. KRAS Mutant KRAS is associated with resistance to anti-egfr monoclonal antibody immunotherapy with agents such as cetuximab or panitumumab. Randomized trials (CRYSTAL, OPUS and PRIME) demonstrated the efficacy of EGFR inhibitors when added to FOLFOX or 5-fluorouracil, leucovorin and irinotecan chemotherapy regimens for patients with KRAS wild-type tumors, especially with significantly improved progression-free survival, response rates and R0 resection rates compared with chemotherapy alone [25 28]. However, the MRC COIN and NORDIC-VII trials subsequently failed to show the same benefit, demonstrating increased response rate without improvement in progression-free or overall survival when cetuximab was added to first-line chemotherapy for metastatic disease [29,30]. Current clinical practice for patients with metastatic colorectal cancer screens tumor tissue for KRAS mutations so that only patients with wild-type KRAS tumors receive anti-egfr therapy. KRAS mutations occur more frequently in right-sided tumors, low-grade tumors, and MSI-low or microsatellite-stable (MSS) tumors. It does not appear to have prognostic value for recurrence-free and overall survival [31]. BRAF Efforts to find other tumor mutations that will allow more specific targeting of patients who will benefit from anti-egfr immunotherapy have shown that the presence of an activating mutation in BRAF in the KRAS wild-type population is associated with poor response [32,33]. However, the CRYSTAL and OPUS trials that supported the use of cetuximab in KRAS wild-type metastatic colorectal cancer also found that while BRAF mutation status could not be used in the same fashion as a predictive biomarker for chemotherapy, it did have an association with poor prognosis [34]. Similarly, the NORDIC-VII study that did not support the use of cetuximab in this same patient population also found that BRAF mutation was a strong negative prognostic factor [30]. Right-sided colon cancers with BRAF mutation and microsatellite stability have a particularly poor prognosis with regard to overall and disease-free survival. These tumors tend to have adverse histologic features such as lymphatic and perineural invasion, tumor budding and mucinous differentiation [35]. BRAF mutation in stage II and III disease has been shown to be prognostic for overall survival in the MSI-low or MSS population [24]. BRAF mutations occur more frequently in right-sided tumors, MSI-high tumors, high-grade tumors, patients over 60 years of age and female patients [31]. In preclinical studies, vemurafenib (PLX4032), a B-Raf kinase inhibitor approved by the US FDA for use in late-stage melanoma patients with BRAF mutations, showed poor efficacy in colorectal cancer (CRC) patients with a BRAF mutation [36]. This resistance to BRAF inhibitors can be overcome by using it in combination with phosphoinositide 3-kinase (PI3K) inhibitors, AKT inhibitors or standard regimens like capecitabine and bevacizumab or cetuximab and irinotecan; this has been demonstrated by in vitro as well as in vivo animal studies [37,38]. Loss of heterozygosity & SMAD4 The SMAD gene products are transcriptional mediators in the TGF-β signaling pathway. The genes encoding SMADs are located at chromosome 18q21. Initial studies demonstrated that loss of heterozygosity at chromosome 18q was a strong prognostic factor for worse survival rates in patients with stage II and III colorectal cancer [9,39,40]. Subsequent studies have demonstrated that loss of heterozygosity is less important in prognosis than previously thought, with no association with cancer-specific or overall survival, although it is independently associated with high tumor grade and KRAS mutation [24,41,42]. However, loss of SMAD4 expression, one of several genes encoded at 18q21, has been shown to be a significant independent prognostic factor for worse recurrence-free and overall survival, particularly in patients with stage III disease [24,43,44]. Mouse studies demonstrate that loss of SMAD4 expression changes the role of TGF-β from growth suppressor to growth promoter, thus increasing the tumorigenic and metastatic potential of colorectal cancer cells [45]. Patients with stage III disease and intact SMAD4 expression with MSI were found to have similar outcomes compared with patients with stage II disease, whereas patients with stage II disease and loss of SMAD4 expression without MSI status had outcomes similar to patients with stage III disease [24]. Retention of SMAD4 expression has also been found to be a predictive marker for a threefold increase in benefit from 5-fluorouracilbased chemotherapy [46]. Loss of SMAD activity occurs in 10% of the colorectal cancers and is associated with advanced-stage disease, the presence of lymph node metastases and shorter overall survival [47]. The role of CTCs in the metastatic cascade CTCs are defined as tumor cells that have separated from either the primary tumor or metastases, and are circulating in the peripheral blood. CTCs have been detected in almost all cancers, including colon, breast, prostate, lung, ovary, pancreas, liver, gastric, esophageal, renal, bladder, thyroid, nasopharyngeal and melanoma; they are extremely rare in patients without malignancy, and can be detected before the development of metastases [48,49]. An early step in the metastatic process is the sporadic shedding of malignant cells from the primary tumor, after which these cells must access the circulation [50]. It has been observed that up to 1 million cells enter the circulation daily per gram of tumor tissue [51]. However, tumor cell embolism does not necessarily lead to metastasis, due to a process of elimination of most of these cells, called metastatic inefficiency [52,53]. For example, trauma from mechanical shear forces causing lethal deformation can cause cancer cells in the peripheral www.expert-reviews.com 583

Review Kin, Kidess, Poultsides, Visser & Jeffrey blood to undergo apoptosis [54 56]. The majority of CTCs undergo anoikis, which is apoptosis induced by cell detachment; cells with anoikis resistance are more likely to survive in the circulation [57]. CTCs have been shown to possess physical properties that potentially aid their ability to intravasate into the peripheral blood, such as increased cell elasticity [58]. Even after hematogenous dissemination, tumor cells are highly inefficient in the processes of extravasation and subsequent growth. A small proportion of extravasated cells might form micrometastases, most of which disappear while only a few progress to form macroscopic tumors [53,56]. This process of extravasation and growth involves the attachment of tumor cells to the vascular endothelium followed by cancer cell aggregation at these sites of attachment [59,60]. Another contribution CTCs may make to the progression of malignancy is the development of highly metastatic phenotypes due to genetic exchange between tumor cells [61,62]. The epithelial mesenchymal transition (EMT) may also be a key step for metastatic spread to occur by way of CTCs [63 69]. Single-cell analysis has been used to identify and characterize CTCs, and in breast and prostate cancer, CTCs have been found to express EMT-related genes, with these cells more commonly associated with metastatic disease [58]. Interactions with platelets also play a role in allowing tumor cells to make the transition to an invasive mesenchymal phenotype leading to metastasis [70]. For formation of metastasis, cells undergo a reversed process referred to as mesenchymal epithelial transition (MET) [71]. However, it is not yet clearly established whether EMT is a necessary step leading to the development of metastases as there is evidence that cell invasion can occur in the absence of EMT, and whether MET plays an important role [72,73]. Nevertheless, recent work demonstrates that a small subset of CTCs is indeed capable of inducing metastasis. These cells express epithelial cell adhesion molecule (EpCAM), CD44, CD47 and MET in patients with luminal breast cancers [74]. Further investigation is needed to fully understand this process. As demonstrated by genomic analysis of single cells from primary and metastatic tumors, there is genetic diversity within primary tumors, and mutated tumor cells in the primary tumor can then undergo clonal expansion in the metastatic site [75,76]. There is substantial lack of concordance between the mutation phenotypes of primary and metastatic tumors in colorectal cancer, seen in up to 23% for KRAS mutations. KRAS and BRAF mutations can be identified in CTCs as well, although the mutation phenotype of CTCs has not been shown to necessarily correlate with that of metastatic tumors [77,78]. Mutational analysis of KRAS, BRAF and PIK3CA in single CTCs demonstrate heterogeneity among CTCs from the same patient, which may explain the inconsistent response to EGFR inhibitors and emphasizing the importance of single-cell analysis for identifying different subpopulations of CTCs that may respond to different therapies [79,80]. Not all CTCs are necessarily reflective of the population of cells that has successfully caused metastasis. Thus, the characteristics of CTCs may differ from those of the cells of metastatic tumors [81]. CTC enrichment & capture technologies The laboratory definition of a CTC is an epithelial cell found in a cancer patient s blood which has an intact nucleus and shows expression of cytokeratin, but not CD45 (a white blood cell marker) [82]. A wide variety of technologies have been developed to accomplish the feat of isolating a single CTC from approximately 5 10 6 nucleated leukocytes and 5 10 9 red blood cells per ml [48]. The potential sources of blood for CTC analysis in patients with colorectal cancer are mesenteric, portal or peripheral. In a large prospective study, the detection efficiency of CTCs in the central venous blood compartment and mesenteric venous blood compartment was compared, demonstrating that CTCs were found at a markedly higher rate in the mesenteric venous blood compartment than in the central venous blood compartment [83]. However, since a peripheral blood draw is much more convenient and can be obtained at different time points of treatment and disease progression, most studies analyze CTCs using this approach. The majority of CTC detection methods require an enrichment step, which may be in the form of red blood cell lysis and/or density-gradient centrifugation for elimination of red blood cells, followed by a separation from contaminating leukocytes prior to isolation and characterization [84,85]. CTC enrichment methods for colorectal cancer fall into two categories: detection according to expression of surface proteins which can be EpCAM-based or non-epcam-based, and detection according to other cell characteristics such as size and morphology, physical characteristics or functional properties (Tables 2 & 3). Enrichment according to expression of surface proteins EpCAM is a glycosylated membrane protein initially discovered as the dominant antigen on colon cancer cells and mediates epithelial-specific intercellular cell adhesion. High expression of EpCAM has been found in most adenocarcinomas, metastases and cancer stem cells and thus is considered an optimal tumor surface antigen for the detection of CTCs [86,87]. The CellSearch System (Veridex, NJ, USA) is the most widely used EpCAM-based system for CTC detection, and the only one that is FDA-approved for use in patients with metastatic colorectal, breast and prostate cancer [88 94]. For the purposes of prognostic and predictive value, the cut-off value separating favorable and unfavorable CTC in metastatic colorectal cancer is three CTCs per 7.5 ml of blood [94]. An EpCAM-based system developed by our group is the MagSweeper, which is an immunomagnetic cell separation device that isolates live cells with extremely high purity, allowing not only enumeration, but also downstream single-cell molecular characterization and growth of CTCs [95]. Multiple other systems based on EpCAM have been developed, including the Adna Test [96], microfluidic chips such as the Herringbone Chip or the newly developed ichip, which also can be used in a negative selection mode (non-epcam based) [97 101], the Isoflux system (Fluxion) [102], the GILUPI Nanodetector (insertable wire) [103], as well as magnetic-activated cell separation [104]. Furthermore, there are several non-epcam-based methods that allow enrichment of CTCs through negative selection by exclusion of CD45-positive leukocytes (Table 2) [105 107]. 584 Expert Rev. Mol. Diagn. 13(6), (2013)

Colorectal cancer diagnostics Review Table 2. Enrichment of circulating tumor cells by positive (using anti-epcam antibodies) and negative (non-epcam-based) selection. According to surface proteins Methods Mechanism Positive selection (EpCAM) CellSearch System Consists of CellTracks Autoprep system, Epithelial Cell Kit and CellSpotter Analyzer. CellTracks Autoprep: automated sample preparation system. Epithelial Cell Kit: for enrichment of cells expressing EpCAM immunomagnetically and for fluorescent labeling of cell nuclei with 4,6-diamidino-2-phenylindole (DAPI), of leukocytes with a monoclonal antibody against CD45, which is only expressed on these white blood cells (CD45-allophycocyan) and of epithelial cells (cytokeratins 8-, 18-, 19-phycoerythrin). CellSpotter analyzer: semiautomated fluorescence-based microscope for identification and enumeration of CTCs. Several groups showed prognostic effect of CTC numbers in cancer patients using this device: patients with metastatic breast, prostate and colorectal carcinoma have been shown to have a shorter progression-free survival if a high number of CTCs are detected; in colorectal cancer more than three cells per 7.5 ml of blood (breast and prostate cancer 5 cells/7.5 ml of blood [88 94]) MagSweeper Adna test CTC: Circulating tumor cell; MACS: Magnetic-activated cell separation. Consists of magnetic rods covered by plastic sheaths that are swept through the samples at a particular velocity and pattern, enabling the capture of EpCAM-positive cells. Preparation: red blood cell lysis; incubation with anti-epcam-coated beads; incubation with cell surface stainings (Alexa 488 antihuman CD45 and phycoerythrin antihuman EpCAM); MagSweeper for isolation of CTCs, three steps: Step 1: capture; Step 2: wash rods covered with plastic sheaths are moved to wash station filled with buffer solution to wash off contaminating and unlabeled cells; Step 3: release magnetic rods move out of plastic cover while at the same time a magnetic field is provided, so that all captured cells are released into a buffer solution containing membrane- impermeable DAPI; repetition of steps to remove further contaminating cells using Olympus inverted microscope equipped for epifluorescence: identification of CTCs as EpCAM positive, DAPI and CD45 negative cells [95] has been used to isolate live CTCs from breast cancer patients for characterization by gene expression analysis; single-cell analysis of CTCs has demonstrated significantly different gene expression patterns than usual breast cancer cell lines, as well as heterogeneity among CTCs captured from a single blood draw and among different patients [75] Uses antibodies against EpCAM and MUC-1 that are conjugated to magnetic beads. MUC-1 is a cell surface glycoprotein, often associated with epithelial cancers. Prelabelling of blood samples by incubation with a commercialized bead and conjugated antibody mixture; labeled cells are then extracted by a magnetic particle concentrator; in a second step, mrna isolated from lysed, enriched cells can be used for downstream analysis or isolated tumor cells can be stained immunohistochemically; this method for CTC isolation in combination with RT-PCR for detection of CTCs in peripheral blood of colorectal carcinoma patients yielded a sensitive approach for CTC detection; in comparison to the CEA levels from the same patients, the detection of CTCs was possible earlier than elevated CEA serum protein levels [96] www.expert-reviews.com 585

Review Kin, Kidess, Poultsides, Visser & Jeffrey Table 2. Enrichment of circulating tumor cells by positive (using anti-epcam antibodies) and negative (non-epcam-based) selection (cont.). According to surface proteins Methods Mechanism Positive selection (EpCAM) (cont.) Microchips (CTC-Chip, Herringbone-Chip, Ephesia-Chip) CTC-chip: contains an array of 78,000 microposts within an area of 970 mm 2 ; allows binding of EpCAM-positive cells to microposts without need for prelabeling of blood samples; due to minimal shear stress and lack of preprocessing of the samples, a high number of viable tumor cells (ranging from 42 to 375 per ml for colorectal cancer) are captured by the labeled microposts; visualization of CTCs by molecular analysis or by immunohistochemistry: cells positive for cytokeratin-staining and adherent to the EpCAM-positive microposts are CTCs, while CD45-positive cells are defined as contaminating hematologic cells [97]. Herringbone-chip: enhanced CTC-Chip, significantly increases number of captured cells by passive mixing of blood cells through generation of microvortices; these increase the interaction of cells with the antibody-coated surface of the chip; using this chip, clusters of CTCs were observed in patients with metastatic cancers; significance of clusters is not yet elucidated, but they may have developed from intravascular proliferation of CTCs or by embolizing from the main tumor mass into the circulation [98]. Ephesia chip: combines microfluidic and magnetic cell sorting; consists of an array of magnetic traps onto which columns of biofunctionalized beads are assembled in a microfluidic channel; allows isolation of CTCs in small sample volumes; in situ cultivation of captured cells possible when cell lines are used [99]. Chip combining microfluidics and electrophoresis: enables CTC selection in high-throughput microsampling unit followed by capture and enrichment by electrokinetic manipulation; in high-throughput microsampling unit is coated with anti-epcam antibodies, so CTCs are immunospecifically selected out of the blood and captured in this microfluidic channel; after enzymatical release of CTCs from EpCAMlabeled surface, they are transported through a pair of electrodes to allow conductivity-based enumeration; then cells are transported and subjected to electrokinetic enrichment as CTCs are negatively charged [100] GILUPI nanodetector Recently developed method for in vivo CTC detection. Consists of medical Seldinger wire functionalized with monoclonal antibody directed against EpCAM; wire is inserted through a medical venous cannula into the cubital vein (2 cm) and left for CTC capture for 30 min; total volume of blood passing the wire is approximately 1.5 3 l; after removal of the wire, CTCs can be identified by ICC method has been successfully tested on patients with advanced-stage lung and breast cancer without adverse effects [103] MACS Technology MACS separation column exposes immunomagnetically labeled blood samples to a strong magnet; labeled cells stay on the column while other cells flow through; depending on the magnetic nanoparticles used, both positive and negative selection can be performed. Prospective study comparing the MACS system to three other cytometric enrichment methods showed that the MACS system is highly reproducible and accurate method for CTC detection in patients with metastatic colorectal cancer and also demonstrated prognostic significance of CTC detection, as patients with metastatic colorectal cancer and more than one detectable CTC had a significantly shorter progression-free survival [104] IsoFlux System by Fluxion CTC: Circulating tumor cell; MACS: Magnetic-activated cell separation. Microfluidic technology for isolation of rare CTCs after prelabelling of blood sample with immunomagnetic capture beads (EpCAM labeled) sample is loaded into microfluidic cartridge on the device; sample passes through a microfluidic channel; cell separation: in the middle of this channel, passing cells are exposed to magnetic field in a cell isolation zone. Labeled target cells flow towards magnetic field, other cells flow through the device into waste container; target cells attach to removable disk at the roof of the isolation zone, so targeted cells can easily be aspirated and used for downstream analysis [102] 586 Expert Rev. Mol. Diagn. 13(6), (2013)

Colorectal cancer diagnostics Review Table 2. Enrichment of circulating tumor cells by positive (using anti-epcam antibodies) and negative (non-epcam-based) selection (cont.). According to surface proteins Methods Mechanism Negative selection (CD45) RosetteSep Applied Non-EpCAM-based method. CD45-positive cells are crosslinked to red Imaging Rare Event (RARE) blood cells by bispecific tetrameric complexes of antibodies; this leads to the formation of aggregates called rosettes with an increasing density gradient. After density gradient centrifugation, CD45-positive cells can be detected in the lower fraction of the tube and CD45-negative cells accumulate in a layer between a separation medium and the plasma [105] Quadrupole magnetic cell sorter CTC: Circulating tumor cell; MACS: Magnetic-activated cell separation. Flow-through separation device after red blood cell lysis, leukocytes are immunomagnetically labeled with an antibody against CD45; depletion of labeled cells as the sample flows through the device in a channel surrounded with four magnets [106] Enrichment according to other cell characteristics The following methods allow the detection of CTCs by using their different size, deformability, density and electric charges compared with normal blood cells. For isolation of CTCs by size, several different filtration methods have been developed (ISET [108,109], ScreenCell [110], the CREATV MicroTech filter [CellSieve ] [111] and a Parylene filter [112]) that enable rapid isolation of CTCs on removable filters, which can immediately be used for downstream analysis. Other technologies isolate CTCs according to their cellular functionality (Vitatex) [113], by microfluidics by the use of label-free biochips [114] or Dean Flow Fractionation [115], as well as by the use of physical cell properties using dielectrophoretic field flow fractionation [116,117]. The fiberoptic array scanning technology allows detailed cytomorphologic analysis of cells (Table 3) [118]. CTC detection methods Even after enrichment of CTCs, a considerable number of contaminating leukocytes remain in the CTC specimen, so a second step is needed for identification of CTCs. Detection and characterization of single CTCs can be carried out by performing immunocytochemistry (ICC), RT-PCR or the epithelial immunospot (EPISPOT) assay. ICC ICC analysis is used to identify CTCs, since there often is remaining leukocyte contamination after enrichment. Most methods use antibodies against cytokeratin, CD45 (leukocyte antigen used as a negative marker), and 4,6-diamidino-2-phenylindole (DAPI nuclear stain) for fluorescent staining of CTCs. RT-PCR Using RT-PCR, the gene expression analysis of at least one of the epithelial markers KRT-7, -8, -18 and/or -19 as well as reference genes (GAPDH, ACTB) confirms the existence of a CTC. Cells expressing CD45 have to be excluded [119,120]. RT-PCR has also been used to detect CEA and CK20 from peripheral blood before and after curative resection, and from mesenteric tumor drainage blood. Positive rates of marker genes in tumor drainage blood are 3.3-times higher than that of peripheral blood [121,122]. EPISPOT assay The EPISPOT assay is derived from the ELISA and can be used after any kind of enrichment method to detect only live CTCs by detecting proteins released from epithelial cancer cells. Enriched and captured cells are cultured for 48 h on an antibody-coated membrane. Those antibodies capture released proteins, which in a second step can be detected by adding secondary antibodies labeled with fluorochromes. Apoptotic tumor cells do not secrete measurable amounts of proteins and therefore are not detected [123]. CTCs are highly heterogeneous in their surface antigens and genetic characteristics. The only device that has received FDA approval is the CellSearch System. However, because this method is EpCAM-based, it has a large drawback in its inability to capture tumor cells undergoing EMT, as the expression of EpCAM and cytokeratins, used to identify CTCs, is downregulated, while markers of EMT (e.g., vimentin and N-cadherin) are expressed. Therefore, negative selection methods by eliminating leukocytes by CD45-depletion or label-free devices might seem more favorable for maximizing CTC enrichment. In addition, the development of novel markers of CTCs such as Plastin3 that is not repressed during EMT has successfully been shown to increase the yield of CTCs. CTCs identified by this method were also shown to be prognostically significant [124]. The large number of methods for enrichment and detection of CTCs and their use in various studies with different tumor entities renders fair comparison of results, specificity, sensitivity and reproducibility impossible. Assays successfully tested on cell-line tumor cells cannot be expected to show equal results to patient blood samples due to the heterogeneity of tumor cells even within a single patient. The use of anti-epcam antibodies for the isolation of CTCs in colorectal cancer is applicable as 98% of the colorectal cancers are adenocarcinomas [125]. Furthermore, in patients suffering from hepatocellular carcinoma, EpCAM-positive CTCs have a high tumorigenic potential compared with EpCAM-negative CTCs [126]. Nevertheless, a label-free device may be more effective in the isolation of CTCs undergoing phenotypic changes, and the analysis of these cells is likely to lead to a better understanding of tumor biology and the metastatic process. www.expert-reviews.com 587

Review Kin, Kidess, Poultsides, Visser & Jeffrey Table 3. Enrichment of circulating tumor cells by utilization of other cell characteristics. According to other cell Methods Mechanism characteristics Size ISET For enumeration, molecular and immunomorphological characterization of CTCs. Small sample volumes up to >1 ml can be used; filtration membrane is comprised of ten wells; allows processing of 10 ml blood at a time; filter membranes with pores 8 µm in diameter after red blood cell lysis, filtration of CTCs based on larger size than other blood cells [99]. Superior detection of CTCs in patients with metastatic prostate and lung cancer using the ISET device compared with CellSearch System in prospective study; may be due to additional capture of tumor cells in epithelial to mesenchymal transition, which leads to downregulation of EpCAM expression and upregulation of other mesenchymal markers; in addition, microemboli can frequently be detected using the ISET (but not with CellSearch method); for metastatic breast cancer, the CellSearch System had superior CTC detection than the ISET [108,109] Cell functionality ScreenCell CREATV MicroTech filter Parylene filter Vitatex (Vita-Cap, Vita- Assay ) Allows isolation of fixed or live cells and tumor cell clusters in only 3 min; consists of a polycarbonate filter with calibrated circular pores (7.5 ± 0.36 µm for fixed cells or 6.5 ± 0.33 µm for live cells [110]) Uses a CellSieve, which is a biocompatible polymer; achieves rapid and efficient isolation of CTCs; pore diameters are 8 µm [111] Contains 8-µm pores, has been compared with the CellSearch System and found to have higher recovery rates of CTCs from patients with metastatic breast, prostate, colorectal and bladder cancer [112] Enrichment of invasive CTCs by using a functional collagen adhesion matrix (CAM): patient samples are added to plates coated with CAM; only CTCs adhere. Captured CTCs are labeled by injection of fluorescently labeled CAM (e.g., FITC); can be used for downstream analysis; those cells are capable of propagation in culture. Using this method, CTCs were detected in 28 of 54 patients with stage I III breast cancer with an average mean CTC count of 61/ml; correlation of number of detected cells to the tumor stage, lymph node status and survival in patients with early stage breast cancer [113] Physical and morphological properties Label free biochips Isolation of CTCs by microfluidics, by exploiting the different physical properties of cancer cells compared with blood cells. Cancer cells are retained in physical structures placed in a microchannel which serve as traps, due to their larger size and lower deformability; successful isolation of CTCs has been demonstrated using cell line colon and breast cancer cells, and blood samples of metastatic lung cancer patients [114] Dean flow fractionation CTC: Circulating tumor cell; ISET: Isolation by size of epithelial tumor cells. Consists of spiral microchannel with inherent centrifugal forces; enables label-free, size-based isolation of CTCs; diluted whole blood and a sheath fluid are pumped through two different inlets at a certain velocity; cell separation occurs under influence of drag forces; as larger CTCs are exposed to strong inertial lift forces was shown to effectively isolate CTCs from metastatic lung cancer patients blood with high sensitivity [115] 588 Expert Rev. Mol. Diagn. 13(6), (2013)

Colorectal cancer diagnostics Review Table 3. Enrichment of circulating tumor cells by utilization of other cell characteristics (cont.). According to other cell characteristics Physical and morphological properties (cont.) Methods ApoStream technology dielectrophoresis field flow fractionation (DEP-FFF) Fiber-optic array scanning technology CTC: Circulating tumor cell; ISET: Isolation by size of epithelial tumor cells. Another limitation during CTC detection is the small volume of blood screened for rare CTCs in most assays. To overcome this problem, the newly developed GILUPI Nanodetector can be functionalized with antibodies, such as EpCAM, for a highly selective enrichment of CTCs in a blood volume as high as 1.5 l. Finally, CTC detection may also be affected by trauma of the tumor during resection that induces cell shedding; thus, surgical manipulation may be a confounding factor affecting the number of CTCs detected perioperatively [127 129]. This is further illustrated by the differences in CTC detection among different blood compartments within the same patient [83]. Further studies on larger patient cohorts are necessary to be able to determine which device can most effectively and reliably detect CTCs. Defining heterogeneous populations by single-cell analysis Single-cell profiling techniques have been developed to analyze the genome of CTCs, not only at the DNA level, but also at the RNA level, corresponding to gene expression. Comparative genome hybridization, for example, analyzes DNA copy number variation and can be performed in single cells using PCR-based whole genome amplification [130 132]. Next-generation sequencing, fluorescence-activating cell sorting and single-nucleus sequencing are other techniques that have been developed to not only enable the identification of tumor cells, but also the characterization of the genome of those individual cells [133,134]. The minute quantities of RNA within a single cell poses a great technical challenge for the sensitivity of detection. For transcriptional analysis, after converting RNA to cdna, amplification is necessary, after which gene expression analysis using a microarray or next-generation sequencing can be performed [82,135]. In a pioneering study, Dalerba et al. demonstrated the importance of single-cell expression analysis for improving the understanding of tumor heterogeneity and clonality [136]. Cells from normal and neoplastic human colon epithelia were isolated and Mechanism Utilizes difference in physical properties of tumor cells, namely the membrane capacitance, for separation. Chromatographic adaptation of microchip dielectrophoresis; DEP can discriminate between cells of similar size with different morphological features in contrast to size-based filtration devices; therefore, isolation of cancer cells from hematologic cells with similar size is possible [116,117] Automated microscope; allows continuous scanning to enumerate and characterize CTCs; allows detailed cytomorphologic analysis after using an enrichment-free immunofluorescent staining protocol. Comparison of morphologic characteristics of primary and metastatic tumor cells to CTCs from colorectal cancer patients has been performed: showed significant histological congruence, as CTCs represent a random sample of the pleomorphic cells in the primary tumor [118] fluorescence-activating cell sorting applied using established markers of differentiation to distinguish mature cells on the top of colon epithelial crypts from immature stem cells at the bottom of the crypt. In a second step, single-cell analysis by gene-expression profiling was performed. By principal component analysis, the gene expression data were then divided into different groups, defining the gene expression profiles of the differentiated enterocytes at the top of the crypts and of the immature, stem and goblet cells at the bottom of the crypts. Furthermore, a colon cancer mouse xenograft model was developed by injection of a single colorectal cancer cell into the flanks of the mice. Only cells highly expressing EpCAM and CD44, which are known to be cancer stem cell markers, resulted in tumors. By performing gene-expression profiling of these tumors and comparison with the primary tumor from which the implanted cells originated, a very similar heterogeneous cell population was found. In addition, the investigation and comparison of colorectal adenoma gene expression with healthy colon tissue demonstrated that the tumor was comprised of the exact cell populations, which shows that tumor heterogeneity is due to multilineage differentiation. In another recent study, CTCs from 15 patients with advancedstage colorectal cancer were isolated using the CellSearch device. The genomic profiles of the primary tumor were compared with the profiles of liver metastasis and CTCs in each patient by performing array comparative genome hybridization. This revealed that CTCs had some similarities in copy number changes to the primary tumor, as well as other similarities to the copy number changes in the liver metastasis, especially in known colorectal driver genes (e.g., KRAS, APC, PIK3CA), but that they also had additional different copy number changes. By deep sequencing of the primary and corresponding liver metastasis, it was discovered that the mutations that were found uniquely in CTCs were detectable in the primary and metastatic tissue at subclonal levels [137]. Analysis of the marker profiles of CTCs and primary tumors in nonmetastatic breast cancer patients using immunohistochemistry www.expert-reviews.com 589

Review Kin, Kidess, Poultsides, Visser & Jeffrey and FISH demonstrated no correlation between the two profiles [80]. These studies emphasize the importance of isolation and characterization of CTCs, as these cells can carry mutations that have developed in the clinical course of cancer patients and can serve as liquid biopsies to monitor therapeutic response and also help make decisions concerning therapeutic regimens for an individual patient. CTCs, however, do not necessarily carry the same mutations as the primary tumor, and this discordance may explain the current variability of treatment response to chemotherapy. The differences in profile between CTCs and primary tumors in breast cancer has been demonstrated and a similar difference in colorectal cancer is likely [80]. In the near future, it will be possible to use droplet-based microfluidics for isolation and high-throughput analysis of approximately 1 million single cells in parallel, as well as enable cell growth, which could significantly facilitate single-cell profiling. The droplets are only picoliters or nanoliters in size and are compartmentalized in the microfluidic system, leading to a faster accumulation and thus quicker detection of secreted molecules [138]. Clinical significance of CTCs CTCs have been shown in multiple studies to correlate not only with worse clinicopathologic features of disease, but also to have prognostic and predictive value. Prognosis refers to the likely course and outcome of the disease regardless of therapy, and is measured by cancer-specific survival, disease-free survival and overall survival. The predictive value of a biomarker refers to the effect of therapy on a patient s outcome [139]. As a marker of advanced disease The correlation of CTC levels with clinicopathologic features that indicate more advanced disease has been observed in both metastatic and nonmetastatic disease, at various sampling points before and after surgical or systemic treatment, and in both the peripheral and mesenteric compartment. CTC detection as determined by CEA/CK20 expression in both preoperative peripheral blood and preresection tumor drainage blood is significantly associated with depth of tumor invasion, venous invasion, lymph node metastasis, liver metastasis and stage [121]. CTC levels obtained from the peripheral blood after resection of the primary tumor have been shown to have a significant correlation with regional lymph node involvement and stage of disease [140]. Similarly, CTCs obtained from the venous drainage blood after curative CRC resection also correlate with lymph node positivity [141]. For patients with metastatic disease not undergoing resection, unfavorable baseline CTC levels (>3 CTCs per 7.5 ml blood using the CellSearch System) correlate with more advanced disease. Among patients with metastatic colorectal carcinoma, those with liver metastases and poorer performance status had higher baseline CTC levels [90,94]. Another marker of CTCs, Plastin3-, has recently been shown to have significant clinical relevance. Plastin3-positivity in the peripheral blood was found to be associated with clinicopathologic risk factors of greater depth of invasion (>=T3), lymph node metastasis, liver metastasis, peritoneal dissemination, recurrence rate and Dukes stage. Plastin3 expression was also detected in all patients with recurrent disease and at a higher level compared with prerecurrence levels and to patients without recurrence [124]. As a prognostic factor Detection of CTCs portends poor prognosis in patients with metastatic colorectal cancer. A meta-analysis of 16 studies to include 1491 patients with metastatic colorectal cancer demonstrated that patients in whom circulating tumor cells are detected have a 2.5-fold increased mortality risk and a twofold increased risk of disease progression or recurrence [142,143]. A higher percentage of patients with metastatic colorectal cancer who had disease progression or death had unfavorable CTCs at 3 5 weeks after treatment than patients whose disease did not progress. Baseline CTC is an independent prognostic factor in metastatic colorectal cancer. Patients with unfavorable levels of CTCs at baseline had significantly shorter median disease-free and overall survival than patients with fewer CTCs [90,144,145]. The effect of favorable baseline CTCs was seen in significantly longer progression-free survival time for patients receiving irinotecan, as well as in a near doubling of overall survival time for all patients regardless of whether they received oxaliplatin, irinotecan or bevacizumab. This overall effect was more pronounced in patients over 65 years of age with poor ECOG performance status [144]. For patients with nonmetastatic disease (Dukes stage B or C), overall and disease-free survival of patients in whom CTCs were detected were significantly worse than those of patients in whom CTCs were not detected. This difference was not seen in patients with Dukes stage A disease [146]. In combination with lymph node staging, CTC detection 24 h after curative resection has even greater sensitivity for predicting recurrence [122]. Patients with metastatic disease and high baseline CTC levels who demonstrate a decrease to favorable CTC levels after 3 5 weeks of chemotherapy experience the same disease-free survival as those who started with favorable baseline CTC levels, although this effect was not seen in overall survival. Patients with persistently unfavorable CTC levels prior to and after treatment have significantly worse disease-free survival and overall survival than those in whom the CTCs converted to favorable levels after treatment [90,147]. Plastin3-positivity was also associated with worse prognosis. Stage for stage, patients in whom Plastin3-positive CTCs were detected had significantly shorter overall survival than Plastin3-negative patients; patients without synchronous distant metastasis had significant shorter disease-free survival. For Dukes stage B and C disease, Plastin3-expression was independently associated with decreased overall survival and disease-free survival, whereas clinicopathological factors such as tumor size, lymphatic invasion, venous invasion, histologic type and depth of invasion were not [124]. In addition to CTC detection in the peripheral blood, CTC detection in tumor drainage venous blood before and after resection is also associated with prognosis. CTC detection in tumor drainage blood obtained just prior to resection has been demonstrated to correlate with overall and disease-free survival [121]. CTC levels obtained from tumor drainage venous blood after resection in Dukes stage B and C colorectal cancer patients was 590 Expert Rev. Mol. Diagn. 13(6), (2013)