RESEARCH ARTICLE
Improved Detection of Circulating Tumor Cells in Metastatic Colorectal Cancer by the Combination of the CellSearch1 System and the AdnaTest1 Tobias M. Gorges1☯, Alexander Stein2☯, Julia Quidde2, Siegfried Hauch3, Katharina Röck1, Sabine Riethdorf1, Simon A. Joosse1, Klaus Pantel1*
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1 Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2 Department of Internal Medicine II and Clinic (Oncology Center), University Medical Center HamburgEppendorf, Hamburg, Germany, 3 AdnaGen GmbH, Langenhagen, Germany ☯ These authors contributed equally to this work. *
[email protected]
OPEN ACCESS Citation: Gorges TM, Stein A, Quidde J, Hauch S, Röck K, Riethdorf S, et al. (2016) Improved Detection of Circulating Tumor Cells in Metastatic Colorectal Cancer by the Combination of the CellSearch1 System and the AdnaTest1. PLoS ONE 11(5): e0155126. doi:10.1371/journal.pone.0155126 Editor: Min-Hsien Wu, Chang Gung University, TAIWAN Received: November 3, 2015 Accepted: April 25, 2016 Published: May 16, 2016 Copyright: © 2016 Gorges et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: TMG, SR, and KP were supported by the City of Hamburg, Landesexzellenzinitiative Hamburg (LEXI 2012; Tumor targeting via cell surface molecules essential in cancer progression and dissemination). These authors were also supported by the ERC Advanced Investigator Grant DISSECT, TRANSCAN ERA-Network: Grant CTC-SCAN. TMG and KP were also supported from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115749, resources of which are
Abstract Colorectal cancer (CRC) is one of the major causes of cancer-related death and reliable blood-based prognostic biomarkers are urgently needed. The enumeration and molecular characterization of circulating tumor cells (CTCs) has gained increasing interest in clinical practice. CTC detection by CellSearch1 has already been correlated to an unfavorable outcome in metastatic CRC. However, the CTC detection rate in mCRC disease is low compared to other tumor entities. Thus, the use of alternative (or supplementary) assays might help to itemize the prognostic use of CTCs as blood-based biomarkers. In this study, blood samples from 47 mCRC patients were screened for CTCs using the FDA-cleared CellSearch1 technology and / or the AdnaTest1. 38 samples could be processed in parallel. We demonstrate that a combined analysis of CellSearch1 and the AdnaTest1 leads to an improved detection of CTCs in our mCRC patient cohort (positivity rate CellSearch1 33%, AdnaTest1 30%, combined 50%). While CTCs detected with the CellSearch1 system were significantly associated with progression-free survival (p = 0.046), a significant correlation regarding overall survival could be only seen when both assays were combined (p = 0.013). These findings could help to establish improved tools to detect CTCs as on-treatment biomarkers for clinical routine in future studies.
Introduction Cancer-related death is usually caused by the outgrowth of aggressive cancer cells at new locations in the body (metastasis formation) that have been disseminated from the primary tumors. Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies and one of the leading causes of cancer related deaths [1]. Approximately one quarter of patients with CRC
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composed of financial contribution from the European Union's Seventh ramework Programme (FP7/20072013) and EFPIA companies’ in kind contribution. The funders provided support in the form of salaries for the author TMG, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. SH is a full time employee of the AdnaGen GmbH, Langenhagen, Germany. The AdnaGen GmbH provided support in the form of salary for author SH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section. Competing Interests: SH is the Site Manager Research & Development of the AdnaGen GmbH (Qiagen). All other authors have declared that no competing interests exist. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.
exhibit metastases (mCRC) at the time of diagnosis (synchronous disease) and further patients will develop metastases during the course of their disease, resulting in the relatively high mortality rate associated with CRC [2]. Different prognostic markers can currently be used in mCRC, e.g., analyses of white blood cells, lactate dehydrogenase amount, performance status, localization of primary tumor and metastases, molecular markers (e.g., mutations in the BRAF gene), or advanced integrative clustering) [3–7]. Besides, tumor growth dynamics have been shown to offer on-treatment information about future prognosis [8, 9]. Enumeration and molecular characterization of tumor cells captured from a minimally invasive blood test (circulating tumor cells, CTCs) is increasingly used in clinical practice for disease monitoring and discovering prognostic relevance [10]. CTCs are easy to obtain by lessinvasive peripheral blood sampling allowing a continuous “real-time monitoring” of tumor progression. So far, the CellSearch1 system is the only approach which has been cleared by the U.S. American FDA (Food and Drug Administration) for CTC detection in metastatic breast, colon, and prostate cancer [11–13]. Detection of CTCs using CellSearch1 has recently been correlated to an unfavorable outcome in mCRC [14]. However, studies performed in mCRC patients using CellSearch1 demonstrated a much lower yield of CTCs in this tumor type compared with breast or prostate cancer [11–13]. It is expected that “only” 30–40% of patients with mCRC harbor 3 or more CTCs per 7.5 ml of blood [15]. Therefore, the use of other (or supplementary) assays might improve our understanding of the mechanisms of cancer biology and itemize the use of CTCs as cancer biomarkers in mCRC. For example, significant discordance between CellSearch1 and the AdnaTest1 in the detection of CTCs from mCRC patients has already been observed [16]. The CellSearch1 system uses immunostaining and defines CTCs as cells expressing both EpCAM and pankeratins and not expressing CD45, as well as having a nucleus stained with DAPI (40 ,6-diamidino-2-phenylindole). In contrast, the AdnaTest1 uses an RT-PCR platform targeting three different transcripts (EpCAM, EGFR, CEA) to identify the tumor cells within the EpCAM-enriched cell fraction. A combined analysis of both assays could therefore improve the sensitivity of CTC detection by applying two methodological approaches (immunocytochemistry and RT-PCR) and increasing the range of CTC markers. This is an important advantage in view of the wellknown phenotypic heterogeneity of CTCs [10]. The purpose of this study was to analyze the clinical relevance of CTCs by comparing and combining two different assays for CTC detection in a small cohort of mCRC patients (n = 47). For this aim, the AdnaTest1 and the CellSearch1 system were employed in parallel. Our data shows that a combined analysis of both assays leads to increased detection rates of CTCs with additional prognostic information. These findings could help to establish new diagnostic tools to use of CTCs as on-treatment biomarkers for clinical routine in future studies.
Material and Methods Patient series Consecutive patients scheduled for palliative chemotherapy for CRC from the out-patient clinic from the Department of Oncology and Hematology at the University Medical Center Hamburg-Eppendorf were recruited. Patient characteristics are summarized in Table 1. The majority of patients were already extensively pretreated, receiving the 3rd (median) line treatment (range: 1–8). Overall, more than 80% of patients received fluoropyrimidines, oxaliplatin, irinotecan, and bevacizumab and about 40% of patients EGFR antibodies. The demographics and patterns of metastasis were as expected, although the overall population was slightly younger than the median metastatic CRC population, likely related to the university hospital
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Table 1. Patient characteristics. Patient characteristics at first diagnosis (n = 47) Age (years)
Median 56 (range 37–79)
Gender
male (n = 29) / female (n = 15) / n.d. (n = 3)
T stage
I (n = 0) / II (n = 2) / III (n = 20) / IV (n = 9) / n.d. (n = 16)
N stage
0 (n = 8) / I (n = 13) / II (n = 11) / n.d. (n = 16)
M stage
0 (n = 11) / 1 (n = 28) / n.d. (n = 8)
KRAS staus
Wild type (n = 25) / Mutated (n = 21) Patient characteristics at blood withdrawal
Liver metastases
Positive (n = 39) / Negative (n = 7)
Lung metastases
Positive (n = 26) / Negative (n = 20)
Lymph node metastases
Positive (n = 10) / Negative (n = 36)
Bone metastases
Positive (n = 4) / Negative (n = 42)
Therapy line
1st (n = 4) / 2nd (n = 9) / 3rd (n = 9) / 4th (n = 6) / 5th (n = 9) / 6th (n = 4) / 7th (n = 2) / 8th (n = 1)
Patient characteristics at time point of diagnosis and blood withdrawal. doi:10.1371/journal.pone.0155126.t001
background. The study was carried out in accordance with the World Medical Association Declaration of Helsinki and the guidelines for experimentation with humans by the Chambers of Physicians of the State of Hamburg (“Hamburger Ärztekammer”). The experimental protocol was approved (Approval No. PVN-3779) by the Ethics Committee of the Chambers of Physicians of the State of Hamburg (“Hamburger Ärztekammer”). All participants gave written informed consent before the study began. In total, blood samples (5 ml and 7.5 ml) from 47 patients were collected into AdnaCollect1 blood collection tubes (AdnaGen1) or CellSave1 preservation tubes (Janssen Diagnostics), and processed within 24 h (AdnaTest1) or 96 h (CellSearch1) according to the guidelines of the vendors.
AdnaTest1 For the enrichment and analysis of circulating tumor cells (CTC) the AdnaTest ColonCancerSelect and the AdnaTest ColonCancerDetect, (AdnaGen GmbH, Langenhagen) were used to prepare mRNA, followed by a RT-PCR for a later multiplex PCR according to the manufacturer’s instructions [16]. All required information regarding sample processing can be found on the webpage http://www.adnagen.com. Briefly, 5 ml of blood was taken for an enrichment of CTC by using antibody-coated magnetic particles consisting of a mixture of antibodies against different EpCAM epitopes The enriched cells were subsequently lysed and mRNA was purified by means of oligo-dT beads contained in the kit followed by reverse transcription (Sensiscript, Qiagen, Hilden). The resulting cDNA was processed in a multiplex PCR for tumor-associated transcripts (epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA) and EpCAM) as well as Actin as housekeeping control. PCR was performed using the HotStarTaq Master Mix (QIAGEN GmbH, Hilden, Germany). Visualization of the PCR fragments was carried out with a 2100 Bioanalyzer using the DNA1000 assay (Agilent Technologies, Waldbronn, Germany). CTCs were positively identified if at least one of the multiplex PCR markers was detected.
CellSearch1 For isolation of CTCs using CellSearch1, CTC detection was performed as described elsewhere [17]. The criteria for an event to be defined as CTC were: a round to oval morphology, a visible
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nucleus (DAPI-positive), and a positive staining pattern for an epithelial specific cell (Keratinpositive and CD45-negative). For EGFR determination on CTCs, the CellSearch1 Tumor Phenotyping Reagent EGFR was applied in the fourth channel of the system [18].
Statistical analysis In order to compare the results of the CellSearch1 system and the AdnaTest1 experiments, CTC counts from CellSearch1 data were transformed to positive (3 CTCs) or negative (
