Editorial
Emerging techniques in molecular detection of circulating tumor cells Expert Rev. Mol. Diagn. 14(2), 131–134 (2014)
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
William CS Cho Hospital Authority, Kowloon, Hong Kong [email protected]
Circulating tumor cells (CTCs) are increasingly recognized for their potential utility in cancer diagnosis and prognosis. Emerging technologies in the past decade have allowed possible isolation and characterization of CTCs in the peripheral blood of cancer patients, including immunomagnetic technique coupled with immunofluorescence methodology, microfluidic platform, x-ray imaging technique and flow cytometry, filter-adapted FISH and miRNA microarray. Although there are still a number of challenges associated with the identification and molecular characterization of CTCs, the analysis of CTCs carries important prognostic and therapeutic implications for personalized cancer management.
Metastatic cancer patients have approximately one circulating tumor cell (CTC) per billion background cells. It has been reported that metastatic breast cancer patients with more than five CTCs per 7.5 ml of blood is predictive of shorter survival than those with fewer CTCs [1]. Indeed, CTCs are increasingly recognized for their potential utility in disease diagnosis and therapeutic response monitoring [2]. For example, two clinical trials of PARP inhibitor have used the gH2AX levels in CTCs as monitoring marker for treatment response [3,4]. However, as CTCs are rare cells found in the blood of cancer patients, their presence is always dwarfed by blood cells. Nevertheless, emerging technologies in the past decade have allowed possible isolation and characterization of CTCs in the peripheral blood of cancer patients.
“…as CTCs are rare cells found in the blood of cancer patients, their presence is always dwarfed by blood cells.” Immunomagnetic technique
From 2004 to 2008, the US FDA has subsequently cleared the CellSearch CTC tests for the enumeration of CTCs of epithelial origin (CD45-, epithelial
cell-adhesion molecule [EpCAM]+ and cytokeratins 8, 18+ and/or 19+) in the blood of metastatic breast, colorectal and prostate cancer patients. With the use of immunomagnetic selection and immunofluorescence methodology, the automated technology captures, identifies and classifies rare CTCs in the bloodstream that detach from the solid tumor. A recent study has shown that the CellSearch System is also predictive of the pathological effects of induction chemoradiotherapy for non-small-cell lung cancer (NSCLC) patients [5].
“With the use of immunomagnetic selection and immunofluorescence methodology, the automated technology captures, identifies and classifies rare CTCs in the bloodstream.” A strategy for rapid quantification of CTCs in colon adenocarcinoma cells using antibody-modified gold nanoparticles (AuNPs) has also been reported. This strategy combines the capturing capability of anti-EpCAM functionalized magnetic beads and the specific labeling through AuNPs, with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction detection technique [6]. Another study has reported a novel
KEYWORDS: cancer • circulating tumor cells • fluorescent in situ hybridization • immunomagnetic • microfluidic • miRNA
informahealthcare.com
10.1586/14737159.2014.868308
2014 Informa UK Ltd
ISSN 1473-7159
131
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
Editorial
Cho
method for the isolation of rare CTCs from blood cells using gas-filled buoyant immuno-microbubbles. The immunomicrobubbles are prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-EpCAM antibody. This technique could efficiently (88%) isolate rare CTCs of plasma-depleted blood [7]. On the other hand, a CTC-enrichment technique has been recently developed based on red blood cell lysis to remove erythrocytes, followed by depletion of CD45+ leukocytes using a magnetic bead separation method and subsequent isolation of CTCs by virtue of their larger size compared with leukocytes. This technique allows isolation and characterization of CTCs that do not express classical epithelial antigens [8]. Microfluidic platform
To capture and analyze the rare CTC population, the antibody-based surface marker positive selection technique is not the only method. A recent study has demonstrated a negative selection method that leverages a microfluidic cell concentrator to allow collection and analysis of rare CTC population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the microfluidic cell concentrator is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population [9].
“…microfluidic platform has the potential to be developed into a valuable clinical tool for isolation, enumeration and analysis of rare CTCs.” Another work has reported a novel spiral microfluidic device with a trapezoidal cross-section for ultra-fast, label-free enrichment of CTCs from clinically relevant blood volumes. Smaller hematologic components are trapped in the Dean vortices skewed toward the outer channel walls and eventually removed at the outer outlet, while the larger CTCs equilibrate near the inner channel wall and are collected from the inner outlet. This single spiral microchannel has successfully isolated and recovered >80% of the tested cancer cells spiked in 7.5 ml of blood within 8 min [10]. This encouraging result suggests that microfluidic platform has the potential to be developed into a valuable clinical tool for isolation, enumeration and analysis of rare CTCs. X-ray imaging technique & flow cytometry
Previous research has shown the existence of hybrid CTCs with an epithelial–mesenchymal transition (EMT) phenotype in cancer patients [11]. Indeed, recent studies have demonstrated the clinical significance of EMT-associated CTCs in several malignancies, including lung, prostate, breast and colorectal cancers [12]. Detailed information on the location and the size of these CTCs may contribute to improve the treatment of metastatic cancer. 132
Synchrotron x-ray micro-imaging technique enables highresolution images of individual flowing tumor cells and the selectively incorporation of AuNPs into CTCs can enhance the image contrast. This new in vivo imaging technology is able to capture the images and temporal movements of individual CTCs and thus may be useful to cancer diagnosis and prognosis [13]. Apart from the detection of CTCs, x-ray technique can play a role in cancer therapy as well. A study modifies nanoparticles to conjugate with CTCs through overexpressed folate receptors and uses a permanent micromagnet to collect tumor cells in whole blood. The characteristic x-ray emissions from collected bismuth nanoparticles, upon excitation with collimated x-rays, are used to detect CTCs with a detection limit of 100 CTCs/ml. They further enhance the dose of primary x-rays to kill the localized CTCs by radiation-induced DNA damage [14].
“Synchrotron x-ray micro-imaging techniques enable high-resolution images of individual flowing tumor cells” In vivo flow cytometry also has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and non-invasively. To enhance the sensitivity, a study combines in vivo flow cytometry technique and a green fluorescent protein-transfected hepatocellular carcinoma orthotopic metastatic tumor model to monitor CTC dynamics. With 1.8-fold higher sensitivity than conventional flow cytometry, this novel approach shows that CTC dynamics is correlated with tumor growth in the orthotopic tumor model while the number and size of distant metastases correspond to CTC dynamics [15]. Filter-adapted FISH
Some studies have focused on the analysis of genetic mutations or rearrangements of CTCs. Subsequent to the identification of somatic mutations (exon 19 deletion or L858R) in the EGFR gene in 15% of NSCLC, the fusion of echinoderm microtubule-associated protein-like 4 with anaplastic lymphoma kinase (ALK) has also been discovered to be a potent oncogenic driver in 3–7% of NSCLC patients [16]. These have led to the development of several reversible EGFR (gefitinib and erlotinib) and ALK (crizotinib) tyrosine kinase inhibitors.
“…the FA-FISH method has reported the same sensitivity and specificity compared with ALK FISH on paraffin-embedded tumor samples.” The Vysis ALK Break Apart FISH Probe is the current FDA-approved test to identify ALK rearrangements, which requires tissue acquisition in the form of a biopsy, aspirate or cell block preparation. A study group has recently developed a new technique to isolate rare CTCs and analyze their in situ protein expression by the use of detachable beads and high-pore-density filters [17]. Simultaneously, the Expert Rev. Mol. Diagn. 14(2), (2014)
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
Emerging techniques for CTCs
FA-FISH assay has also been developed which consists a filter to capture CTCs enriched by blood filtration, followed by preparation of the filter spot to fixate cells and finally the analysis of cells using the FISH assay. Applying a cutoff value of more than four ALK-rearranged CTCs/ml of blood, the FA-FISH method has reported the same sensitivity and specificity compared with ALK FISH on paraffinembedded tumor samples. This blood-based assay can also be used to monitor quantitative and qualitative changes of CTCs from ALK-rearranged NSCLC while patients receiving crizotinib [18]. miRNA microarray
Circulating cancer-associated miRNAs are readily measured in peripheral blood and they can robustly distinguish between good-prognosis and poor-prognosis patients [19]. miRNA microarray is the most commonly used platform for miRNA expression profiling. This technology is a powerful high-throughput tool capable of monitoring the expression of thousands of miRNAs at once within tens of samples processed in parallel in a single experiment. Using miRNA microarray, a recent study has identified a four-serum miRNAs (miR-22, miR-572, miR-638 and miR-1234) signature that can predict survival in patients with nasopharyngeal carcinoma [20]. It has been hypothesized that circulating miRNAs may predict the CTC status of cancer patients. Profiled by TaqMan human miRNA microarrays, plasma of CTC-positive metastatic breast cancer patients were found to have significantly higher level of miR-200b than CTC-negative metastatic breast cancer [21]. Another study also identified 10 miRNAs that were more abundantly expressed in samples from breast cancer patients with at least five CTCs in 7.5 ml of blood compared with samples from patients without detectable CTCs [22]. These studies show that miRNA profiling of low numbers of CTCs in a high background of leukocytes is feasible. Prospective & challenges
CTCs are surrogate markers that allow real-time biopsy of a tumor’s biological activity. They can be used to assess the existence of EMT, cancer stem cell markers as well as genetic mutations and epigenetic changes in the tumor. The detection of CTCs has a number of potential applications in personalized cancer management, including diagnosis, prognosis and the prediction of treatment response. It is envisioned that the analysis of CTCs will allow real-time monitoring of disease status and tailoring personalized therapy for cancer patients. Indeed, molecular detection and characterization of CTCs proves to be an emerging tool for diagnosing, stratifying and monitoring patients with metastatic cancer. Evaluating the presence of CTCs in the bloodstream has become a minimally invasive and useful approach for cancer prognosis. Most assays detect CTCs relying on EpCAM expression in tumor cells, but some normal-like CTCs are EpCAM-negative informahealthcare.com
Editorial
and thus cannot be captured with EpCAM-based technology. Combined use of EpCAM and other biomarkers (e.g., CD146 and CD49f) can improve CTC detection in cancer patients [23].
“circulating miRNAs may predict the CTC status of cancer patients” On the other hand, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC enumeration and specific molecular characterization extremely challenging. Many of the current techniques, including the sole US FDA-approved test for CTC enumeration, destroy CTCs during the isolation process. As a treatment tool, CTC isolation should avoid cellular damage and contamination by other cell types to enable further development for personalized therapies. A combined micromagnetic-microfluidic device has been developed to isolate, detect and culture viable CTCs from whole blood. CTCs can be easily removed from the device and expand in culture for additional analytical studies or potential drug sensitivity testing [24]. Identification of gene-rearranged CTCs also provides important information for treatment response assessment. The development of FA-FISH assay shines new light on the molecular characterization of CTCs. Further large clinical trials are needed to establish whether this is indeed a feasible and reliable approach for cancer management. The successful detection of ALK-rearranged CTCs by FA-FISH is just the beginning; similar assays for other oncogenic drivers should be developed.
“…the identification and molecular characterization of CTCs carries important prognostic and therapeutic implications for personalized cancer management.” On the other hand, miRNA profiling of CTCs shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Determination of CTC-associated miRNAs in peripheral blood is expected to become important in oncology, especially when interpreted together with CTC status. However, the correlation between CTCs and miRNAs is as-yet unknown. Continued researches and clinical trials should be carried out to investigate the utility of circulating miRNAs as prognostic markers for predicting CTC status. Emerging technologies in the past decade have developed rapidly to enable CTC detection in various cancers. However, a recent study using microfluidic-based single cell transcriptional profiling of cancer-associated genes has shown that there is heterogeneity among individual CTCs even from one single patient [25]. Further studies are required to evaluate the heterogeneity among single CTCs. Nevertheless, there is no doubt that the identification and molecular characterization of CTCs carries important prognostic and therapeutic implications for personalized cancer management. 133
Editorial
Cho
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This
enumeration of circulating tumor cells. Methods 64(2), 137–134 (2013).
References
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
1
2
Cristofanilli M, Budd GT, Ellis MJ et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N. Engl. J. Med. 351(8), 781–791 (2004). Kang Y, Pantel K. Tumor cell dissemination: emerging biological insights from animal models and cancer patients. Cancer Cell 23(5), 573–581 (2013).
3
Kummar S, Chen A, Ji J et al. Phase I study of PARP inhibitor ABT-888 in combination with topotecan in adults with refractory solid tumors and lymphomas. Cancer Res. 71(17), 5626–5634 (2011).
4
Kummar S, Ji J, Morgan R et al. A phase I study of veliparib in combination with metronomic cyclophosphamide in adults with refractory solid tumors and lymphomas. Clin. Cancer Res. 18(6), 1726–1734 (2012).
5
6
7
8
9
Tarumi S, Gotoh M, Kasai Y et al. Innovative method using circulating tumor cells for prediction of the effects of induction therapy on locally advanced non-small cell lung cancer. J. Cardiothorac. Surg. 8, 175 (2013). Maltez-da Costa M, de la Escosura-Mun˜iz A, Nogue´s C, Barrios L, Iba´n˜ez E, Merkoc¸i A. Simple monitoring of cancer cells using nanoparticles. Nano Lett. 12(8), 4164–4171 (2012). Shi G, Cui W, Benchimol M et al. Isolation of rare tumor cells from blood cells with buoyant immuno-microbubbles. PLoS ONE 8(3), e58017 (2013). Wu S, Liu Z, Liu S, Lin L, Yang W, Xu J. Enrichment and enumeration of circulating tumor cells by efficient depletion of leukocyte fractions. Clin. Chem. Lab. Med. 1–9 (2013). Casavant BP, Mosher R, Warrick JW et al. A negative selection methodology using a microfluidic platform for the isolation and
134
includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
10
11
Warkiani ME, Guan G, Luan KB et al. Slanted spiral microfluidics for the ultra-fast, label-free isolation of circulating tumor cells. Lab Chip doi:10.1039/C3LC50617G (2013) (Epub ahead of print). Lecharpentier A, Vielh P, Perez-Moreno P, Planchard D, Soria JC, Farace F. Detection of circulating tumour cells with a hybrid (epithelial/mesenchymal) phenotype in patients with metastatic non-small cell lung cancer. Br. J. Cancer 105(9), 1338–1341 (2011).
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Yu M, Bardia A, Wittner BS et al. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 339(6119), 580–584 (2013).
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Jung SY, Ahn S, Seo E, Lee SJ. Detection of circulating tumor cells via an X-ray imaging technique. J. Synchrotron. Radiat. 20(Pt 2), 324–331 (2013).
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Hossain M, Luo Y, Sun Z et al. X-ray enabled detection and eradication of circulating tumor cells with nanoparticles. Biosens. Bioelectron. 38(1), 348–354 (2012). Fan ZC, Yan J, Liu GD et al. Real-time monitoring of rare circulating hepatocellular carcinoma cells in an orthotopic model by in vivo flow cytometry assesses resection on metastasis. Cancer Res. 72(10), 2683–2691 (2012).
16
Marchesi V. Lung cancer: ALK status of NSCLC reflected in CTCs. Nat. Rev. Clin. Oncol. 10(7), 366 (2013).
17
Lee HJ, Oh JH, Oh JM et al. Efficient isolation and accurate in situ analysis of circulating tumor cells using detachable beads and a high-pore-density filter. Angew. Chem. Int. Ed. Engl. 52(32), 8337–8340 (2013).
18
Pailler E, Adam J, Barthe´le´my A et al. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer. J. Clin. Oncol. 31(18), 2273–2281 (2013).
19
Cho WC. Circulating microRNAs as minimally invasive biomarkers for cancer theragnosis and prognosis. Front. Genet. 2, 7 (2011).
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Liu N, Cui RX, Sun Y et al. A four-serum microRNA signature predicts survival in patients with nasopharyngeal carcinoma. Int. J. Cancer doi:10.1002/ijc.28468 (2013) (Epub ahead of print).
21
Madhavan D, Zucknick M, Wallwiener M et al. Circulating miRNAs as surrogate markers for circulating tumor cells and prognostic markers in metastatic breast cancer. Clin. Cancer Res. 18(21), 5972–5982 (2012).
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Sieuwerts AM, Mostert B, Bolt-de Vries J et al. mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients. Clin. Cancer Res. 17(11), 3600–3618 (2011).
23
Mostert B, Kraan J, Sieuwerts AM et al. CD49f-based selection of circulating tumor cells (CTCs) improves detection across breast cancer subtypes. Cancer Lett. 319(1), 49–55 (2012).
24
Kang JH, Krause S, Tobin H, Mammoto A, Kanapathipillai M, Ingber DE. A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells. Lab. Chip 12(12), 2175–2181 (2012).
25
Powell AA, Talasaz AH, Zhang H et al. Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS ONE 7(5), e337788 (2012).
Expert Rev. Mol. Diagn. 14(2), (2014)
Emerging techniques in molecular detection of circulating tumor cells Expert Rev. Mol. Diagn. 14(2), 131–134 (2014)
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
William CS Cho Hospital Authority, Kowloon, Hong Kong [email protected]
Circulating tumor cells (CTCs) are increasingly recognized for their potential utility in cancer diagnosis and prognosis. Emerging technologies in the past decade have allowed possible isolation and characterization of CTCs in the peripheral blood of cancer patients, including immunomagnetic technique coupled with immunofluorescence methodology, microfluidic platform, x-ray imaging technique and flow cytometry, filter-adapted FISH and miRNA microarray. Although there are still a number of challenges associated with the identification and molecular characterization of CTCs, the analysis of CTCs carries important prognostic and therapeutic implications for personalized cancer management.
Metastatic cancer patients have approximately one circulating tumor cell (CTC) per billion background cells. It has been reported that metastatic breast cancer patients with more than five CTCs per 7.5 ml of blood is predictive of shorter survival than those with fewer CTCs [1]. Indeed, CTCs are increasingly recognized for their potential utility in disease diagnosis and therapeutic response monitoring [2]. For example, two clinical trials of PARP inhibitor have used the gH2AX levels in CTCs as monitoring marker for treatment response [3,4]. However, as CTCs are rare cells found in the blood of cancer patients, their presence is always dwarfed by blood cells. Nevertheless, emerging technologies in the past decade have allowed possible isolation and characterization of CTCs in the peripheral blood of cancer patients.
“…as CTCs are rare cells found in the blood of cancer patients, their presence is always dwarfed by blood cells.” Immunomagnetic technique
From 2004 to 2008, the US FDA has subsequently cleared the CellSearch CTC tests for the enumeration of CTCs of epithelial origin (CD45-, epithelial
cell-adhesion molecule [EpCAM]+ and cytokeratins 8, 18+ and/or 19+) in the blood of metastatic breast, colorectal and prostate cancer patients. With the use of immunomagnetic selection and immunofluorescence methodology, the automated technology captures, identifies and classifies rare CTCs in the bloodstream that detach from the solid tumor. A recent study has shown that the CellSearch System is also predictive of the pathological effects of induction chemoradiotherapy for non-small-cell lung cancer (NSCLC) patients [5].
“With the use of immunomagnetic selection and immunofluorescence methodology, the automated technology captures, identifies and classifies rare CTCs in the bloodstream.” A strategy for rapid quantification of CTCs in colon adenocarcinoma cells using antibody-modified gold nanoparticles (AuNPs) has also been reported. This strategy combines the capturing capability of anti-EpCAM functionalized magnetic beads and the specific labeling through AuNPs, with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction detection technique [6]. Another study has reported a novel
KEYWORDS: cancer • circulating tumor cells • fluorescent in situ hybridization • immunomagnetic • microfluidic • miRNA
informahealthcare.com
10.1586/14737159.2014.868308
2014 Informa UK Ltd
ISSN 1473-7159
131
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
Editorial
Cho
method for the isolation of rare CTCs from blood cells using gas-filled buoyant immuno-microbubbles. The immunomicrobubbles are prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-EpCAM antibody. This technique could efficiently (88%) isolate rare CTCs of plasma-depleted blood [7]. On the other hand, a CTC-enrichment technique has been recently developed based on red blood cell lysis to remove erythrocytes, followed by depletion of CD45+ leukocytes using a magnetic bead separation method and subsequent isolation of CTCs by virtue of their larger size compared with leukocytes. This technique allows isolation and characterization of CTCs that do not express classical epithelial antigens [8]. Microfluidic platform
To capture and analyze the rare CTC population, the antibody-based surface marker positive selection technique is not the only method. A recent study has demonstrated a negative selection method that leverages a microfluidic cell concentrator to allow collection and analysis of rare CTC population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the microfluidic cell concentrator is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population [9].
“…microfluidic platform has the potential to be developed into a valuable clinical tool for isolation, enumeration and analysis of rare CTCs.” Another work has reported a novel spiral microfluidic device with a trapezoidal cross-section for ultra-fast, label-free enrichment of CTCs from clinically relevant blood volumes. Smaller hematologic components are trapped in the Dean vortices skewed toward the outer channel walls and eventually removed at the outer outlet, while the larger CTCs equilibrate near the inner channel wall and are collected from the inner outlet. This single spiral microchannel has successfully isolated and recovered >80% of the tested cancer cells spiked in 7.5 ml of blood within 8 min [10]. This encouraging result suggests that microfluidic platform has the potential to be developed into a valuable clinical tool for isolation, enumeration and analysis of rare CTCs. X-ray imaging technique & flow cytometry
Previous research has shown the existence of hybrid CTCs with an epithelial–mesenchymal transition (EMT) phenotype in cancer patients [11]. Indeed, recent studies have demonstrated the clinical significance of EMT-associated CTCs in several malignancies, including lung, prostate, breast and colorectal cancers [12]. Detailed information on the location and the size of these CTCs may contribute to improve the treatment of metastatic cancer. 132
Synchrotron x-ray micro-imaging technique enables highresolution images of individual flowing tumor cells and the selectively incorporation of AuNPs into CTCs can enhance the image contrast. This new in vivo imaging technology is able to capture the images and temporal movements of individual CTCs and thus may be useful to cancer diagnosis and prognosis [13]. Apart from the detection of CTCs, x-ray technique can play a role in cancer therapy as well. A study modifies nanoparticles to conjugate with CTCs through overexpressed folate receptors and uses a permanent micromagnet to collect tumor cells in whole blood. The characteristic x-ray emissions from collected bismuth nanoparticles, upon excitation with collimated x-rays, are used to detect CTCs with a detection limit of 100 CTCs/ml. They further enhance the dose of primary x-rays to kill the localized CTCs by radiation-induced DNA damage [14].
“Synchrotron x-ray micro-imaging techniques enable high-resolution images of individual flowing tumor cells” In vivo flow cytometry also has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and non-invasively. To enhance the sensitivity, a study combines in vivo flow cytometry technique and a green fluorescent protein-transfected hepatocellular carcinoma orthotopic metastatic tumor model to monitor CTC dynamics. With 1.8-fold higher sensitivity than conventional flow cytometry, this novel approach shows that CTC dynamics is correlated with tumor growth in the orthotopic tumor model while the number and size of distant metastases correspond to CTC dynamics [15]. Filter-adapted FISH
Some studies have focused on the analysis of genetic mutations or rearrangements of CTCs. Subsequent to the identification of somatic mutations (exon 19 deletion or L858R) in the EGFR gene in 15% of NSCLC, the fusion of echinoderm microtubule-associated protein-like 4 with anaplastic lymphoma kinase (ALK) has also been discovered to be a potent oncogenic driver in 3–7% of NSCLC patients [16]. These have led to the development of several reversible EGFR (gefitinib and erlotinib) and ALK (crizotinib) tyrosine kinase inhibitors.
“…the FA-FISH method has reported the same sensitivity and specificity compared with ALK FISH on paraffin-embedded tumor samples.” The Vysis ALK Break Apart FISH Probe is the current FDA-approved test to identify ALK rearrangements, which requires tissue acquisition in the form of a biopsy, aspirate or cell block preparation. A study group has recently developed a new technique to isolate rare CTCs and analyze their in situ protein expression by the use of detachable beads and high-pore-density filters [17]. Simultaneously, the Expert Rev. Mol. Diagn. 14(2), (2014)
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
Emerging techniques for CTCs
FA-FISH assay has also been developed which consists a filter to capture CTCs enriched by blood filtration, followed by preparation of the filter spot to fixate cells and finally the analysis of cells using the FISH assay. Applying a cutoff value of more than four ALK-rearranged CTCs/ml of blood, the FA-FISH method has reported the same sensitivity and specificity compared with ALK FISH on paraffinembedded tumor samples. This blood-based assay can also be used to monitor quantitative and qualitative changes of CTCs from ALK-rearranged NSCLC while patients receiving crizotinib [18]. miRNA microarray
Circulating cancer-associated miRNAs are readily measured in peripheral blood and they can robustly distinguish between good-prognosis and poor-prognosis patients [19]. miRNA microarray is the most commonly used platform for miRNA expression profiling. This technology is a powerful high-throughput tool capable of monitoring the expression of thousands of miRNAs at once within tens of samples processed in parallel in a single experiment. Using miRNA microarray, a recent study has identified a four-serum miRNAs (miR-22, miR-572, miR-638 and miR-1234) signature that can predict survival in patients with nasopharyngeal carcinoma [20]. It has been hypothesized that circulating miRNAs may predict the CTC status of cancer patients. Profiled by TaqMan human miRNA microarrays, plasma of CTC-positive metastatic breast cancer patients were found to have significantly higher level of miR-200b than CTC-negative metastatic breast cancer [21]. Another study also identified 10 miRNAs that were more abundantly expressed in samples from breast cancer patients with at least five CTCs in 7.5 ml of blood compared with samples from patients without detectable CTCs [22]. These studies show that miRNA profiling of low numbers of CTCs in a high background of leukocytes is feasible. Prospective & challenges
CTCs are surrogate markers that allow real-time biopsy of a tumor’s biological activity. They can be used to assess the existence of EMT, cancer stem cell markers as well as genetic mutations and epigenetic changes in the tumor. The detection of CTCs has a number of potential applications in personalized cancer management, including diagnosis, prognosis and the prediction of treatment response. It is envisioned that the analysis of CTCs will allow real-time monitoring of disease status and tailoring personalized therapy for cancer patients. Indeed, molecular detection and characterization of CTCs proves to be an emerging tool for diagnosing, stratifying and monitoring patients with metastatic cancer. Evaluating the presence of CTCs in the bloodstream has become a minimally invasive and useful approach for cancer prognosis. Most assays detect CTCs relying on EpCAM expression in tumor cells, but some normal-like CTCs are EpCAM-negative informahealthcare.com
Editorial
and thus cannot be captured with EpCAM-based technology. Combined use of EpCAM and other biomarkers (e.g., CD146 and CD49f) can improve CTC detection in cancer patients [23].
“circulating miRNAs may predict the CTC status of cancer patients” On the other hand, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC enumeration and specific molecular characterization extremely challenging. Many of the current techniques, including the sole US FDA-approved test for CTC enumeration, destroy CTCs during the isolation process. As a treatment tool, CTC isolation should avoid cellular damage and contamination by other cell types to enable further development for personalized therapies. A combined micromagnetic-microfluidic device has been developed to isolate, detect and culture viable CTCs from whole blood. CTCs can be easily removed from the device and expand in culture for additional analytical studies or potential drug sensitivity testing [24]. Identification of gene-rearranged CTCs also provides important information for treatment response assessment. The development of FA-FISH assay shines new light on the molecular characterization of CTCs. Further large clinical trials are needed to establish whether this is indeed a feasible and reliable approach for cancer management. The successful detection of ALK-rearranged CTCs by FA-FISH is just the beginning; similar assays for other oncogenic drivers should be developed.
“…the identification and molecular characterization of CTCs carries important prognostic and therapeutic implications for personalized cancer management.” On the other hand, miRNA profiling of CTCs shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Determination of CTC-associated miRNAs in peripheral blood is expected to become important in oncology, especially when interpreted together with CTC status. However, the correlation between CTCs and miRNAs is as-yet unknown. Continued researches and clinical trials should be carried out to investigate the utility of circulating miRNAs as prognostic markers for predicting CTC status. Emerging technologies in the past decade have developed rapidly to enable CTC detection in various cancers. However, a recent study using microfluidic-based single cell transcriptional profiling of cancer-associated genes has shown that there is heterogeneity among individual CTCs even from one single patient [25]. Further studies are required to evaluate the heterogeneity among single CTCs. Nevertheless, there is no doubt that the identification and molecular characterization of CTCs carries important prognostic and therapeutic implications for personalized cancer management. 133
Editorial
Cho
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This
enumeration of circulating tumor cells. Methods 64(2), 137–134 (2013).
References
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Osaka University on 10/16/14 For personal use only.
1
2
Cristofanilli M, Budd GT, Ellis MJ et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N. Engl. J. Med. 351(8), 781–791 (2004). Kang Y, Pantel K. Tumor cell dissemination: emerging biological insights from animal models and cancer patients. Cancer Cell 23(5), 573–581 (2013).
3
Kummar S, Chen A, Ji J et al. Phase I study of PARP inhibitor ABT-888 in combination with topotecan in adults with refractory solid tumors and lymphomas. Cancer Res. 71(17), 5626–5634 (2011).
4
Kummar S, Ji J, Morgan R et al. A phase I study of veliparib in combination with metronomic cyclophosphamide in adults with refractory solid tumors and lymphomas. Clin. Cancer Res. 18(6), 1726–1734 (2012).
5
6
7
8
9
Tarumi S, Gotoh M, Kasai Y et al. Innovative method using circulating tumor cells for prediction of the effects of induction therapy on locally advanced non-small cell lung cancer. J. Cardiothorac. Surg. 8, 175 (2013). Maltez-da Costa M, de la Escosura-Mun˜iz A, Nogue´s C, Barrios L, Iba´n˜ez E, Merkoc¸i A. Simple monitoring of cancer cells using nanoparticles. Nano Lett. 12(8), 4164–4171 (2012). Shi G, Cui W, Benchimol M et al. Isolation of rare tumor cells from blood cells with buoyant immuno-microbubbles. PLoS ONE 8(3), e58017 (2013). Wu S, Liu Z, Liu S, Lin L, Yang W, Xu J. Enrichment and enumeration of circulating tumor cells by efficient depletion of leukocyte fractions. Clin. Chem. Lab. Med. 1–9 (2013). Casavant BP, Mosher R, Warrick JW et al. A negative selection methodology using a microfluidic platform for the isolation and
134
includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
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