Int J Clin Oncol (2015) 20:878–890 DOI 10.1007/s10147-015-0798-3
ORIGINAL ARTICLE
Detection of circulating tumor cell‑specific markers in breast cancer patients using the quantitative RT‑PCR assay Hye‑young Wang · Sungwoo Ahn · Sunghyun Kim · Sunyoung Park · Dongju Jung · Sangjung Park · Hyunju Han · JooHyuk Sohn · SeungIl Kim · Hyeyoung Lee
Received: 9 December 2014 / Accepted: 29 January 2015 / Published online: 24 February 2015 © Japan Society of Clinical Oncology 2015
Abstract Background Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within breast cancer tumors is used to determine the prognosis and select therapies, additional markers need to be identified. Circulating tumor cells (CTCs) are constituent cells that have detached from a primary tumor to circulate in the bloodstream. CTCs are considered the main source of breast cancer metastases; therefore, detection of CTCs could be a promising diagnostic method for metastatic breast cancer. Methods In this study, the CircleGen CTC RT-qDx assay was used to analyze the mRNA expression levels of six CTC-specific markers including EpCAM, CK19, HER2, Ki67, hTERT, and vimentin with a total of 692 peripheral whole blood samples from 221 breast cancer patients and 376 healthy individuals. Results This assay showed high specificity with multiple markers; none of the healthy controls were detected
positive, whereas 21.7 and 14 % of breast cancer patients were positive for EpCAM and CK19, respectively. Of the 221 breast cancer patients, 84 (38 %), 46 (20.8 %), 83 (37.6 %), and 39 (17.6 %) were positively for HER2, Ki67, hTERT, and vimentin mRNA, respectively. Of the 84 patients who were HER2 positive, nine (4 %) were also positive for EpCAM, CK19, Ki67, hTERT, and vimentin. Of the 139 breast cancer patients who were HER2 negative, 65 (29.1 %) were negative for EpCAM, CK19, Ki67, hTERT, and vimentin. Furthermore, the EpCAM-positive population decreased from 21.5 to 8.3 % after completion of anti-tumor treatment (TP4). Similarly, the CK19, HER2, hTERT, and vimentin positives also decreased from 13.9 to 9.5 %, from 37.7 to 21.4 %, from 37.2 to 33.3 %, and from 17.5 to 14.3 %, respectively, after completion of anti-tumor treatment. In contrast, the Ki67 positives increased from 20.6 to 41.7 % after completion of anti-tumor treatment. Conclusions mRNA overexpression of six CTC-specific markers was detected by the CircleGen CTC RT-qDx assay
H. Wang M&D, Inc., Wonju Eco Environmental Technology Center, Wonju, Gangwon, Republic of Korea
S. Park Department of Clinical Laboratory Science, College of Medical Science, Daegu Haany University, Daegu, Republic of Korea
S. Ahn · S. Kim · S. Park · H. Lee (*) Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, 1 Yonseidae‑gil, Wonju, Gangwon 220‑710, Republic of Korea e-mail:
[email protected]
H. Han Avison Biomedical Research Center, Yonsei University Medical Center, Seoul, Republic of Korea
S. Kim Institute for Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea D. Jung Department of Biomedical Laboratory Science, College of Natural Sciences, Hoseo University, Chungnam, Republic of Korea
13
J. Sohn Division of Medical Oncology, Severance Breast Cancer Clinic, Yonsei Cancer Center, Seoul, Republic of Korea S. Kim (*) Department of Surgery, Yonsei University College of Medicine, Yonsei University, 50 Yonsei‑ro, Seodaemun‑gu, Seoul 120‑752, Republic of Korea e-mail:
[email protected]
Int J Clin Oncol (2015) 20:878–890
with high specificity, and the obtained mRNA expression levels of CTC-specific markers might provide useful criteria to select appropriate anti-tumor treatment for breast cancer patients. Keywords Circulating tumor cells (CTCs) · Breast cancer · RT-qPCR · Anti-tumor treatment · Molecular diagnosis
Introduction Breast cancer is a major cause of death in women throughout the world; it is the most frequent cancer in women and the second leading cause of death [1]. Despite the continuous improvement in survival rate over the past 40 years, the median age of women who die from breast cancer is 68 years, which is still young. Early cancer detection and application of improved treatment has extended the survival rate in three-quarters of breast cancer patients by at least 10 years [2]. The main cause of cancer-associated deaths is not the primary tumor, but metastasis to other organs. Breast cancer is considered a systemic disease because of micrometastasis, which indicates the presence of small tumors even at the early stage of cancer development. The problem is that the small size tumors are extremely difficult to detect using common imaging-based laboratory examinations [3]. Therefore, despite the considerable advances in developing diagnostic methods and treating therapies, metastases remain the principal cause of death in breast cancer patients. Cancer cells disseminate into distant organs through blood vessels and capillaries formed within the tumor during the early stage of tumor formation [4]. By changing to mesenchymal cells from epithelial cells (epithelial-to-mesenchymal transition), metastatic cells start to express adhesion molecules and proteases that allow the cells to enter the circulatory system [5]. The presence of disseminated tumor cells (DTCs) in the bone marrow or circulating tumor cells (CTCs) in the peripheral blood of patients are prognostic factors for disease recurrence and disease-related death. In particular, the presence of CTCs in the bloodstream may also be an important indicator of potential metastatic disease and poor prognosis for cancers at both an early stage and an advanced stage [6]. EpCAM and CK19 are recognized tumor cell markers for epithelial cancers [7]. Antigen Ki67 is a nuclear protein that is associated with cellular proliferation, and hTERT is the catalytic subunit of the enzyme telomerase, which is associated with immortalization of cancer cells. Ki67 and hTERT are tumor cell markers indicating cancer cell proliferation in the blood of cancer patients [8]. Vimentin is expressed in mesenchymal cells, lymphomas,
879
stromal connective tissue, blood vessel walls, and nerves. Vimentin expression indicates poor prognosis in nodenegative infiltrating ductal breast cancer and hormone receptor negativity [9]. Diverse analytical systems to isolate and detect CTCs in whole blood samples from cancer patients have been developed based on immunocytochemistry (ICC), immunofluorescence (IF), fluorescence-activated cell sorting (FACS), and reverse transcriptase-polymerase chain reaction (RT-PCR)-based molecular assays. Although these assays use different analytical methods, they have a common separation process in which tumor markers are purified by size or biological characteristics [10–13]. To date, the Cell Search® System (Veridex LLC, Warren, NJ, USA) has been approved by the US Food and Drug Administration (US FDA) for detection of CTCs in patients with metastatic cancers. In the system, epithelial cells are immunomagnetically separated and labeled with fluorescence, and then the number of nucleated (DAPI+) cells having the EpCAM+, CK 8/18/19+ and CD45 phenotype are identified as CTCs [14]. Another commercial CTC detection assay is the Adna Test Breast Cancer Select/Detect (AdnaGen AG, Langenhagen, Germany) assay. In this system, immunomagnetic separation is followed by multiplex RT-PCR of the genes of three tumor markers—human epidermal receptor (HER) 2, mucin (Muc)-1 and GA773-2—to identify cancer cells [15]. The Cell Search® System indicates a poor prognosis for metastatic breast cancer patients with a basal CTC count ≥5 in 7.5 mL of blood. RT-PCR method-based assays including the Adna Test Breast Cancer Select/Detect need reliable marker genes because they have low positive rates (low sensitivity) for patients with metastatic breast cancer [16]. In the present study, the CircleGen CTC RT-qDx assay (Syantra, Calgary, Canada), a commercial diagnostic kit based on the RT-qPCR assay, was employed to detect multiple CTC-specific markers, such as EpCAM, CK19, HER2, Ki67, hTERT, and vimentin in blood samples from patients with breast cancer. The assay was evaluated using 692 blood samples from 221 patients with breast cancer and 376 healthy individuals.
Materials and methods Clinical samples Blood samples from 221 breast cancer patients (stage I– IV) were used in the experiments. The patients had been under care at the Yonsei University Severance Hospital (Seoul, Republic of Korea) from 2013−2014. Blood samples from 376 healthy donors who had never been
13
880
Int J Clin Oncol (2015) 20:878–890
Table 1 Population characteristics Characteristics
No. of patients (%)
Age (median = 49.8, SD ± 9.9 years) ≥20 30 40 50 ≥60 Cancer stage I II III IV Unknown Therapy option Adjuvant Neoadjuvant Metastasis
141 (63.8 %) 60 (27.1 %) 20 (9.0 %)
Lymph node invasion status (n = 141) 0 1 2
89 (63.1 %) 17 (12.1 %) 6 (4.3 %)
Unknown HER2 immunohistochemistry Positive Negative Unknown ER immunohistochemistry Positive (≥10 %) Negative (20 % >5 mm increase in target lesion diameter sum No complete response, partial response, or stable disease documented before increased disease New lesion and confirmed on contemporaneous or follow-up computed tomography Unequivocal progression of nontarget lesions Does not meet other criteria; neither partial response nor progressive disease criteria met
0 (0 %) 2 (10 %) 8 (40 %)
Stable disease Unknown Total
8 (40 %) 2 (10 %) 20 (100 %)
RECIST Response evaluation criteria in solid tumors
Discussion The detection of circulating tumor cells is a promising and powerful tool for cancer diagnosis and disease monitoring. The clinical significance of the detection of CTCs in low stages of non-metastatic breast cancer in patients treated with short-term preoperative therapy is so far unknown. CTCs are shed from primary or secondary tumors into the blood, and the presence of CTCs in the bloodstream is critical for establishing metastases. Despite significant advances in treating and screening breast cancer, 5 % of patients have clinically detectable metastases, and 30–40 % has occult metastases that can lead to a poor prognosis and disease relapse [17, 18]. Early identification of patients at risk of developing metastatic disease after therapy may therefore be of great importance for improving clinical outcome. Growing evidence suggests that cancer cells are shed from a primary tumor into blood circulation before clinical symptoms appear. These CTCs may colonize distant sites and form metastatic lesions [18, 19]. Applying an easy and sensitive method targeting circulating tumor cell markers might be a solution to reduce diagnostic cost and for early detection of cancers before it grows to systemic diseases. Most of the recent techniques developed to isolate and detect CTCs use antibodies against epithelial markers such as EpCAM and CK, a protein that sticks on the surface of CTCs but never found on the surface of normal blood cells. One of the commercial products targeting EpCAM and CK is the CellSearch® System developed by Veridex LLC. The US FDA-approved Cell Search® System uses antibodies for EpCAM and CK attached to magnetic beads to pull out targeting cells from solution in a magnetic field [20]. In addition to using the CTCs for detection markers, understanding CTC characteristics would be valuable for increasing efficiency of the current breast cancer-treating therapies. The presence of the CTC-specific marker, a new commercial RT-qPCR-based molecular assay, which can detect
13
overexpression of CTC-specific markers, such as EpCAM, CK19, HER2, Ki67, hTERT, HER2, and vimentin, was clinically evaluated with blood samples from breast cancer patients and healthy donors. The RT-qPCR assay has been considered a useful method for screening expressions of tumor-associated genes in CTCs because PCR-based methods can handle multiple samples at once. The RT-qPCR methods also have the potential to be standardized in terms of performance, speed, accuracy, sensitivity, specificity, and capacity for high throughput [21]. In addition, a PCR monitoring system incorporating fluorescent labels offers advantages over conventional PCR methods for quantification. The fluorescence-based monitoring RT-qPCR methods have better accuracy because PCR amplification and quantification are performed in the same reaction tube by eliminating the need to manipulate the PCR product after amplification [21]. Data from this study showed that 59 breast cancer patients who were detected as HER2 positive by conventional IHC methods, 26 (44.1 %) were determined to be HER2 positive by the RT-qPCR method. Of the 100 breast cancer patients who were detected as HER2 negative by IHC methods, 38 (38 %) were determined HER2 positive by the RT-qPCR method. Analysis of HER2 expression in CTC-specific markers may have clinical significance for HER2-targeted therapy because HER2-positive CTCs and DTCs can be present in patients with HER2-negative primary tumors. These patients currently do not have access to HER2-targeted therapy [22]. Furthermore, blood samples from the breast cancer patients having high levels of HER2 mRNA expressed CTC-specific markers. HER2 mRNA expression levels in blood samples from breast cancer patients correlated with Ki67 (Pearson r = 0.2370, P
