In most industrialized countries, primary lung cancer is. Circulating Tumor Cells in Pulmonary Venous Blood of Primary Lung Cancer Patients

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1 Circulating Tumor Cells in Pulmonary Venous Blood of Primary Lung Cancer Patients Yoshitomo Okumura, MD, Fumihiro Tanaka, MD, PhD, Kazue Yoneda, Masaki Hashimoto, MD, Teruhisa Takuwa, MD, Nobuyuki Kondo, MD, and Seiki Hasegawa, MD, PhD Department of Thoracic Surgery, Hyogo College of Medicine, Nishinomiya, Japan Background. Circulating tumor cells in peripheral blood (CTC) is a potential surrogate of distant metastasis, which is the critical factor influencing decision making regarding therapy and prognosis of primary lung cancer patients. After our preliminary study showing that CTCs were detected in peripheral blood in 29.4% of resectable lung cancer patients, we conducted a prospective study on CTC in pulmonary vein (PV) blood because tumor cells apart from the primary tumor may circulate after passing through the drainage PV. Methods. A total of 30 consecutive lung cancer patients who underwent thoracotomy were included. The CTCs in peripheral blood and in PV blood from the primary tumor site were quantitatively examined with the CellSearch system, and the numbers of CTCs per 7.5 ml peripheral and PV blood in each patient were represented as perictc count and pvctc count, respectively. Results. Circulating tumor cell was detected in peripheral blood in 5 patients (16.7%; the perictc count was 1 in 2 patients; and 2, 3, and 16 in 1 patient each), and the incidence of positive perictc was higher in squamous carcinoma patients than in adenocarcinoma patients (p 0.028). Circulating tumor cell was detected in PV blood in most patients (29 of 30, 96.7%), and the mean and median pvctc counts were 1,195 and 81, respectively (range, 0 to 10,034). There was no significant correlation between pvctc count and any other patient characteristic, including perictc count. Conclusions. In resectable lung cancer, CTC was positive in peripheral blood of some patients and in PV blood of most patients. A long-term follow-up study to clarify the clinical significance of pvctc status is warranted. (Ann Thorac Surg 2009;87: ) 2009 by The Society of Thoracic Surgeons In most industrialized countries, primary lung cancer is the leading cause of cancer death. Approximately 40% of lung cancer patients have distant metastases symptomatic or detectable with current diagnostic modalities such as computed tomography and positron emission tomography scanning [1]. In addition, even in patients without distant metastasis at the time of initial diagnosis, distant metastasis frequently occurs during treatment or after complete resection, which may lead to cancer death. Thus, in most lung cancer patients, tumor cells originating from the primary tumor may circulate in peripheral blood with or without apparent distant metastatic disease, and detection of circulating tumor cells (CTCs) may be important in the diagnosis and treatment of lung cancer [2]. Although the clinical significance of CTCs in lung cancer patients remains unclear, our preliminary study in which the CellSearch system (Veridex LLC, Raritan, NJ) was employed to capture CTCs revealed promising results, showing that CTC was detected in peripheral blood in 29.4% of resectable lung cancer Accepted for publication March 25, Presented at the Poster Session of the Forty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Francisco, CA, Jan 26 28, Address correspondence to Dr Tanaka, Department of Thoracic Surgery, Hyogo College of Medicine, Mukogawa 1-1, Nishinomiya, , Japan; ftanaka@hyo-med.ac.jp. patients and in 64.7% of unresectable lung cancer patients [3]. Accordingly, in parallel with prospective studies on CTC in peripheral blood (perictc) of lung cancer patients, we initiated a prospective study on CTC in pulmonary venous (PV) blood (pvctc) because tumor cells apart from the primary tumor may circulate after passing through the drainage PV. Here, we report the preliminary results of the pvctc study first showing that CTCs were detected in PV blood of most resectable lung cancer patients. Material and Methods Study Design Among patients in whom perictc was evaluated in a study to assess the prevalence of perictc [3] (and in a submitted manuscript), a total of 30 consecutive patients who underwent lobectomy, bilobectomy, or pneumonectomy during thoracotomy from November 1, 2007, through April 30, 2008, at the Department of Thoracic Surgery, Hyogo College of Medicine Hospital were prospectively evaluated in the study (Table 1). For lung resection, the pulmonary artery (PA) was first ligated and dissected, followed by ligation and dissection of the PV, and finally the bronchus was dissected and closed. Most 2009 by The Society of Thoracic Surgeons /09/$36.00 Published by Elsevier Inc doi: /j.athoracsur

2 1670 OKUMURA ET AL Ann Thorac Surg PULMONARY VENOUS CTC IN LUNG CANCER PATIENTS 2009;87: Table 1. Patient Characteristics No. of Patients (%) Sex Male 21 (70.0) Female 9 (30.0) Age Mean (years) 65.2 Range Histology Squamous cell carcinoma 7 (23.3) Adenocarcinoma 18 (60.0) Large cell carcinoma 2 (6.7) Adenosquamous cell carcinoma 1 (3.3) Pleomorphic carcinoma 1 (3.3) Small cell carcinoma 1 (3.3) Pathologic stage I 20 (66.7) II 3 (10.0) III 5 (16.7) IV 2 (6.7) Site of primary tumor Hilar 2 (6.7) Peripheral 28 (93.3) Extent of lung resection Pneumonectomy 2 (6.7) Lobectomy 28 (93.3) Vessel invasion of primary tumor Negative 8 (26.7) Positive 22 (73.3) Lymphatic invasion of primary tumor Negative 9 (30.0) Positive 21 (70.0) Total 30 (100) patients (27 of 30, 90.0%) had clinical (c) stage I and II diseases before thoracotomy; 3 patients migrated from c-stage I to pathologic (p) stage IIIA owing to mediastinal nodal involvement, 1 patient went from c-stage I to p-stage IIIB owing to malignant effusion, and 1 patient went from c-stage I to stage IV because of intrapulmonary metastases after thoracotomy. Complete clinical data including history, physical examination, and radiographic studies were collected. For the evaluation of tumor progression, whole-body computed tomography, brain computed tomography or magnetic resonance imaging, and positron emission tomography scanning were routinely conducted before thoracotomy. The p-stage was determined according to the current tumor-node-metastasis (TNM) classification as revised in 1997 [4]. Vessel invasion and lymphatic invasion of primary tumor in the resected lung were evaluated under light microscopy. Follow-up of postoperative clinical course was conducted by outpatient medical records and by inquiries by telephone. The median follow-up time from thoracotomy was 13 months (with a minimal follow-up time of 12 months for patients alive). This study was approved by the Institutional Review Board of Hyogo College of Medicine Hospital, and written informed consent for the study was obtained from each patient. Evaluation of CTC A pulmonary blood sample was taken from the resected lung just after resection as follows: 2.5 ml blood was gently drawn from the lobar PV of the primary tumor site, and was collected in CellSaver tube (Veridex LLC) containing cell preservatives. The dilution buffer, 5.0 ml, was added to the PV blood sample, and a total of 7.5 ml diluted sample was served for CTC preparation. A peripheral blood sample, 7.5 ml, was also taken just before thoracotomy. The CTCs were captured and quantitatively evaluated using a semiautomated system, the CellSearch system (Veridex LLC), following the manufacturer s protocol [5]. In brief, epithelial cells that were captured using ferroparticles coupled to a monoclonal antibody against epithelial cell adhesion molecule (EpCAM) were separated in a magnetic field, and the enriched samples were then stained with 4=,6-diamidino-2-phenylindole (DAPI) and an anticytokeratin antibody conjugated with phycoerythrin (CK-PE). Contaminated white blood cells were excluded by negative selection for CD45. Stained cells were then analyzed on a florescent microscope using the Cell Track Analyzer II (Veridex LLC). The criteria for each cell to be defined as a CTC are as follows: round to oval morphology, a visible DAPI-positive nucleus, positive cytokeratin staining in the cytoplasm, and negative staining for CD45 (Fig 1). All evaluations were performed without knowledge of the clinical characteristics of the patients. The number of CTCs in 7.5 ml of blood was represented as the CTC count; in the case of the PV blood sample, the number of CTCs detected in each sample was triplicated. Statistics Counts were compared by the 2 test. Continuous data were compared using Student s t test if the distribution of samples was normal, or using Mann-Whitney U test if the sample distribution was asymmetrical. Spearman correlation coefficients (two-tailed) were used to evaluate whether pvctc count was correlated with perictc count or other patient characteristics. Differences were considered significant when a p value was less than All statistical manipulations were performed using the SPSS for Windows software system (SPSS, Chicago, IL). Results Distribution of Peripheral and Pulmonary Venous CTCs Circulating tumor cells were detected in peripheral blood in 5 patients (16.7%; Table 2), and the distribution of perictc count was as follows: 1 in 2 patients (1 p-stage IB patient with small-cell lung carcinoma and 1 p-stage IIIA patient with squamous cell carcinoma); 2 in 1 p-stage IA

3 Ann Thorac Surg OKUMURA ET AL 2009;87: PULMONARY VENOUS CTC IN LUNG CANCER PATIENTS 1671 Fig 1. Circulating tumor cells (CTCs) displayed by the CellSearch system. Cells captured with an anti epithelial cell adhesion molecule (Ep- CAM) antibody were stained with (4=,6-diamidino-2-phenylindole (DAPI), an anti-cytokeratin antibody conjugated with phycoerythrin (CK- PE), and an anti-cd45 antibody conjugated with allophycocyanin (CD45-APC). Cells with round to oval morphology, a visible DAPI-positive nucleus, positive CK-PE staining in the cytoplasm, and negative staining for CD45-APC were judged as CTCs (cells 1, 2, and 3). Contaminated leukocytes were identified as DAPI /CK /CD45 cells (cell 5). The CD45-positive cells were not judged as CTCs even when cells were positively stained for DAPI and CK-PE (cell 4). patient with adenocarcinoma; 3 in 1 p-stage IB patient with squamous cell cancer, and 16 in 1 p-stage IIA patient with squamous cell cancer. The incidence of positive perictc (perictc count, 1 or more) was significantly higher in squamous cell carcinoma than in adenocarcinoma (p 0.028), but there was no significant correlation between the incidence of positive perictc and any other patient characteristic, such as the site of primary tumor (Table 2). In PV blood, CTC was detected in most patients (29 among all patients, 96.7%), and the mean and median pvctc counts were 1,195 and 81, respectively (range, 0 to 10,034). Distribution of pvctc count in correlation with perictc count is shown in Figure 2, and no correlation between pvctc count and perictc count was documented. When patients were classified into lower pvctc count or into higher pvctc count based on the median pvctc count (81) as a cut-off, 80% (4 of 5) of positive perictc patients had a higher pvctc, whereas only 44% (11 of 25) of negative perictc patients had a higher pvctc (p 0.330; Table 2). Patient Characteristics and Pulmonary Venous CTCs Distribution of pvctc in correlation with histologic type, p-stage, primary tumor site, and microscopic vessel and lymphatic invasion of the primary tumor are shown in Figure 3. There was no significant correlation between pvctc count and any patient characteristic, and there was no significant difference in the incidence of higher pvctc count according to any patient characteristic (Table 2). Short-Term Clinical Outcome Correlation With Pulmonary Venous CTC Status Short-term clinical outcomes with a median follow-up of 13 months are shown in Table 3. There was no significant difference in the incidence of recurrence or death according to the status of pvctc.

4 1672 OKUMURA ET AL Ann Thorac Surg PULMONARY VENOUS CTC IN LUNG CANCER PATIENTS 2009;87: Table 2. Circulating Tumor Cells (CTC) in Peripheral Blood (PeriCTC) and in Pulmonary Venous Blood (PvCTC) According to Patient Characteristics PeriCTC Count Positive (1 or More) PvCTC Count Higher (81 or More) Patient Characteristics n (%) p Value n (%) p Value Sex Male (n 21) 2 (9.5) 9 (42.9) Female (n 9) 3 (33.3) 6 (66.7) Histology Squamous cell carcinoma (n 7) 3 (42.9) 6 (85.7) Adenocarcinoma (n 18) 1 (5.6) 6 (33.3) Pathologic stage I(n 20) 3 (15.0) 11 (55.0) II (n 3) 1 (33.3) 1 (33.3) III (n 5) 1 (20.0) 2 (40.0) IV (n 2) 0 (0.0) 1 (50.5) Site of primary tumor Hilar (n 2) 1 (50.0) 2 (100.0) Peripheral (n 28) 4 (14.3) 13 (46.4) Extent of lung resection Pneumonectomy (n 2) 0 (0.0) 1 (50.0) Lobectomy (n 28) 4 (17.9) 14 (50.0) Vessel invasion of primary tumor Negative (n 8) 1 (12.5) 4 (50.0) Positive (n 22) 4 (18.2) 11 (50.9) Lymphatic invasion of primary tumor Negative (n 9) 0 (0.0) 5 (55.6) Positive (n 21) 5 (23.8) 10 (46.7) PeriCTC count (0, negative; 1 or more, positive) Not indicated Negative (n 25) 11 (44.0) Positive (n 5) 4 (80.0) Total (n 30) 5 (16.7) 15 (50.0) CTC circulating tumor cell. Comment In spite of many efforts to develop new techniques to capture CTCs in peripheral blood, no CTC test had been established as a clinical marker, mainly because of lack of its reproducibility and specificity for detection of CTCs in malignant and nonmalignant diseases [2]. The CellSearch system is a semiautomated system for the capture and quantitative evaluation of CTCs [3]. Owing to its reproducibility across different laboratories as well as its specificity documented in breast cancer and colorectal cancer [5 10], the CTC test using the CellSearch system has been approved in the United States by the Food and Drug Administration for monitoring blood from metastatic breast cancer and colorectal cancer patients. In addition, several studies suggest that the CTC test is clinically useful in the diagnosis and therapy of other malignant tumors such as prostate cancer [11 13], but little has been reported with regard to the CTC test in lung cancer [3, 5, 14]. In the present study, we documented that tumor cells circulated in peripheral blood in some lung cancer patients, and first revealed that higher Fig 2. Distribution of circulating tumor cells (CTCs) in pulmonary venous blood (pvctc count) in correlation with CTCs in peripheral blood (perictc count). The number of CTCs per 7.5 ml of each blood sample was represented as the CTC count, and pvctc count was shown in log 10 scale.

5 Ann Thorac Surg OKUMURA ET AL 2009;87: PULMONARY VENOUS CTC IN LUNG CANCER PATIENTS 1673 Fig 3. Distribution of circulating tumor cells (CTCs) in pulmonary venous blood (pvctc) according to (A) histologic type, (B) pathologic (p) stage, (C) primary tumor site, (D) microscopic vessel invasion of primary tumor, and (E) microscopic lymphatic invasion of primary tumor. The number of CTCs per 7.5 ml of each pulmonary blood sample was represented as the pvctc count, and pvctc count was shown in log 10 scale. number of tumor cells passed through the PV in most lung cancer patients. The most important findings are probably that CTC can be detected in peripheral blood as well as in PV blood, even in resectable patients, most of whom are c-stage I and II patients. In the present study, epithelial cells in peripheral and PV blood were captured with an anti-epcam antibody by using the CellSearch system, and it is likely that these epithelial cells identified by this assay represent tumor cells, because epithelial cells other than tumor cells may not be present in blood. In addition, many clinical studies have shown that the status of CTCs detected in peripheral blood with the CellSearch system is a significant prognostic and predictive factor in metastatic breast cancer and colorectal cancer [7 11], possibly suggesting that epithelial cells captured with the CellSearch system

6 1674 OKUMURA ET AL Ann Thorac Surg PULMONARY VENOUS CTC IN LUNG CANCER PATIENTS 2009;87: Table 3. Short-Term Clinical Outcomes According to Status of Circulating Tumor Cells (CTC) in Peripheral Blood (PeriCTC) and in Pulmonary Venous Blood (PvCTC) PeriCTC Count (No. of Patients and Percentage) PvCTC Count (No. of Patients and Percentage) Clinical Outcomes Negative Positive p Value Lower Higher p Value Recurrence Recurrence ( ) Recurrence ( ) 6 (24.0%) 1 (20.0%) 4 (26.7%) 3 (20.0%) Local only Distant only Local and distant Survival Alive Dead 3 (12.0%) 0 2 (13.3%) 1 (6.7%) Lung cancer Other diseases Total (n 30) CTC circulating tumor cell. are true tumor cells. However, there remain some limitations in the detection of circulating tumor cells with the CellSearch system, as follows: there is no definite evidence showing that epithelial cells captured with an anti-epcam antibody are true tumor cells; and it remains unclear whether circulating tumor cells detected with the CellSearch system are viable and can grow in distant organs to form metastases. To address answers to these questions, further studies on the molecular and functional characterization of captured epithelial cells should be conducted, and we are now performing both molecular analyses of genes extracted from epithelial cells and in vitro cell culture of captured epithelial cells. It is generally recognized that tumor cells isolating from the primary tumor first pass through the PV and thereafter circulate in peripheral blood. Thus, pvctc should be positively correlated with perictc in theory, which we failed to demonstrate in the present study (Spearman r and p 0.415). Possible reasons for the failure are the very small number of patients (n 30) included in the present study and the low incidence of positive perictc (16.7%, 5 of 30). As shown in Figure 1, 4 of 5 patients with positive perictc also had a higher pvctc count (101, 435, 1,470, and 1,593), whereas only 11 of 25 patients with negative perictc had a higher pvctc. Anyway, the present study showed no apparent correlation between pvctc and perictc. In addition to continuing patient accrual, we will continue longer follow-up of clinical outcomes (postoperative recurrence and survival) of patients in correlation with the status of pvctc. At present, with a shorter follow-up with a median of 13 months, there is no difference in the incidence of recurrence or cancer death according to the status of pvctc. However, if a longer follow-up study reveals that higher pvctc is correlated with early recurrence or poor survival, patients with higher pvctc count may be candidates for postoperative adjuvant chemotherapy even if they have p-stage I disease. Moreover, a difference in pvctc count according to an operation procedure, that is, PA ligation first followed by PV ligation, as in the present study, or PV ligation first followed by PA ligation, should be assessed. In addition, changes in CTCs before and after thoracotomy may be more important. In fact, Sawabata and coworkers [14] conducted a pilot study of changes in perictc count evaluated with the CellSearch system, and showed that 2 of 7 nonsmall-cell lung cancer patients with negative perictc before thoracotomy had positive perictc after thoracotomy. These results suggest that circulation of tumor cells is promoted by lung manipulation during thoracotomy. Thus, perictc and pvctc before and after lung manipulation during thoracotomy would be informative, and that will be examined in planned future studies. One major limitation of the CellSearch system may be a low sensitivity for capturing CTCs, as shown in the present study. Rolle and coworkers [15] have documented higher numbers (more than 1,000/10 ml blood in most patients) of circulating epithelial cells in peripheral blood of lung cancer patients by using another system (MAINTRAC) of capturing cells with an anti-epcam antibody [15]. However, the clinical value of circulating epithelial cells identified the MAINTRAC system remains unclear, because there has been no accumulating report. A more sensitive detection system of CTCs using a microfluidic device (CTC chip) has been recently developed [16, 17]. This CTC chip consists of an array of 78,000 microposts that are coated with anti-epcam antibodies, and the selection of target CTC is mediated by the interaction of these cells with the EpCAM-coated microposts under precisely controlled laminar flow conditions. With the CTC chip, CTC was detected in most patients (115 of 116) with lung, prostate, pancreatic, breast, and colon cancer. When the CTC chip is be available in future, a comparative study between the

7 Ann Thorac Surg OKUMURA ET AL 2009;87: PULMONARY VENOUS CTC IN LUNG CANCER PATIENTS CellSearch system and the CTC chip system may be conducted. In conclusion, tumor cells circulate in peripheral blood of some (16.7%) resectable lung cancer patients, and higher numbers of tumor cells are detected in PV blood of most (96.7%) resectable lung cancer patients. To clarify the clinical significance of pvctc status, a long-term follow-up study should be conducted. We thank Dr Seiji Matsumoto for helpful discussion, Dr Shakibur Rahman for helpful assistance with sample collection, and also Mrs Masami Shiiba for helpful assistance with the preparation of the manuscript. This work was supported by Grant-in-Aid (to Dr Okumura) for Scientific Research (C) and Grants-in-Aid (to Dr Tanaka) for Scientific Research (B) from the Japan Society for the Promotion of Science. References 1. Socinski MA, Crowell R, Hensing TE, et al. Treatment of non-small cell lung cancer, stage IV. Diagnosis and management of lung cancer: evidence-based clinical practice guidelines (2nd edition). Chest 2007;132:277s 89s. 2. Alix-Panabières C, Riethdorf S, Pantel K. Circulating tumor cells and bone marrow micrometastasis. Clin Cancer Res 2008;14: Tanaka F, Hashimoto M, Takuwa T, et al. Circulating tumor cells (CTCs) and endothelial cells (CECs) in malignant pleural mesothelioma (MPM) and primary lung cancer (LC). J Clin Oncol 2008;26:582s. 4. Mountain CF. Revisions in the international system for lung cancer. Chest 1997;111: Alland WJ, Matera J, Miller MC, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy or patients with nonmalignant diseases. Clin Cancer Res 2004;10: Riethdort S, Fritsche H, Muller V, et al. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res 2007;13: Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004;351: Cristofanilli M, Hayes DF, Budd GT, et al. Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 2005;23: Cohen SJ, Punt CJ, Iannotti N, et al. Relationship of circulating tumor cells to tumor response, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol 2008;26: Sastre J, Maestro ML, Puente J, et al. Circulating tumor cells in colorectal cancer: correlation with clinical and pathological variables. Ann Oncol 2008;19: de Bono JS, Scher HI, Montgomery RB, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res 2008;14: Naoe M, Ogawa Y, Morita J, et al. Detection of circulating urothelial cancer cells in the blood using the CellSearch system. Cancer 2007;109: Hiraiwa K, Takeuchi H, Hasegawa H, et al. Clinical significance of circulating tumor cells in blood from gastrointestinal cancers. Ann Surg Oncol 2008;15: Sawabata N, Okumura M, Utsumi T, et al. Circulating tumor cells in peripheral blood caused by surgical manipulation of non-small cell lung cancer: pilot study using an immunocytology method. Gen Thorac Surg 2007;55: Rolle A, Gunzel R, Pachmann U, et al. Increase in number of circulating disseminated epithelial cells after surgery for non-small cell lung cancer monitored by MAINTRAC is a predictor for relapse: a preliminary report. World J Surg Oncol 2005;3: Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 2007;450: Maheswaran S, Sequist LV, Nagrath S, et al. Detection of mutations in EGFR in circulating tumor cells. N Engl J Med 2008;359: