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Early ligation of the pulmonary vein can reduce the dissemination of shed tumor cells during thoracoscopic lobectomy

  • Xinchun Duan
    Affiliations
    Department of Thoracic Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing, China
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  • Zhenrong Yang
    Affiliations
    State Key Laboratory of Molecular Oncology, Department of Etiology and Carcinogenesis, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Xuefeng Hao
    Affiliations
    Department of Thoracic Surgery, Beijing Chest Hospital, Capital Medical University, Beijing, China
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  • Shijie Zhou
    Affiliations
    Department of Thoracic Surgery, Beijing Chest Hospital, Capital Medical University, Beijing, China
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  • Zhidong Liu
    Correspondence
    Zhidong Liu, MD, Department of Thoracic Surgery, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China.
    Affiliations
    Department of Thoracic Surgery, Beijing Chest Hospital, Capital Medical University, Beijing, China
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  • Kaitai Zhang
    Correspondence
    Kaitai Zhang, PhD, State Key Laboratory of Molecular Oncology, Department of Etiology and Carcinogenesis, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
    Affiliations
    State Key Laboratory of Molecular Oncology, Department of Etiology and Carcinogenesis, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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  • Yong Cui
    Correspondence
    Addresses for reprints: Yong Cui, MD, Department of Thoracic Surgery, Beijing Friendship Hospital, Capital Medical University, No 95, Yongan Rd, Xicheng District, Beijing 100050, China.
    Affiliations
    Department of Thoracic Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing, China
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Open AccessPublished:April 20, 2022DOI:https://doi.org/10.1016/j.jtcvs.2022.03.038

      Abstract

      Objective

      The sequence of vessel ligation in lobectomy can significantly affect the hematogenous spread of circulating tumor cells (CTCs). Vein-first ligation substantially reduces CTC dissemination and achieves favorable survival compared with artery-first ligation. In this study, we further explored whether the timing of pulmonary vein (PV) ligation determined according to the early and late PV ligation technique is associated with CTC dissemination.

      Methods

      A total of 44 patients who underwent uniform 2-port video-assisted thoracoscopic surgery lobectomy were enrolled; the subjects were divided into the early ligation group (n = 18) and late ligation group (n = 26) according to whether PV ligation was prioritized during surgery. PV blood was obtained before PV ligation and after lobe resection. CTCs were detected using telomerase reverse transcriptase-based CTC detection and validated using FlowSight and fluorescence in situ hybridization.

      Results

      The median postoperative PV CTC (Post-PVCTC) count was 9 (interquartile range [IQR], 6-18), which was higher than the median preoperative PV CTC (Pre-PVCTC) count of 1 (IQR, 0-3; P < .001). Clinicopathologic correlation analysis showed that the Pre-PVCTC count correlated positively with TNM stage (P = .002) and lymph node metastasis (P = .002) and that the Post-PVCTC count correlated positively with tumor density (P = .043) and vessel/lymphatic invasion (P < .030). Interestingly, although no statistical difference in the median Pre-PVCTC count was observed, the median Post-PVCTC count in the early ligation group was 16 (IQR, 9.5-36.75), whereas that in the late ligation group it was 8 (IQR, 4.75-12.25), showing a significant difference (P = .004).

      Conclusions

      We provide the first evidence to show that early PV ligation can prevent PVCTCs from spreading into the circulation, offering an innovative surgical concept for the principle sequence of pulmonary vessel management.

      Graphical abstract

      Key Words

      Abbreviations and Acronyms:

      CT (computed tomography), CTC (circulating tumor cell), ELG (early ligation group), EpCAM (epithelial cell adhesion molecule), FISH (fluorescence in situ hybridization), IQR (interquartile range), LLG (late ligation group), NSCLC (non–small cell lung cancer), PA (pulmonary artery), Post-PVCTCs (postoperative circulating tumor cells in the pulmonary vein), Pre-PVCTCs (preoperative circulating tumor cells in the pulmonary vein), PV (pulmonary vein), PVB (pulmonary vein blood), SN (solid nodule), SSN (subsolid nodule), TBCD (telomerase reverse transcriptase-based circulating tumor cell detection), VATS-L (video-assisted thoracoscopic surgery lobectomy)
      Figure thumbnail fx2
      Early pulmonary vein ligation can intercept shedding circulating tumor cells at the stump.
      Early pulmonary vein ligation can prevent tumor cells from spreading intraoperatively.
      Early PV ligation prevents the spread of tumor cells via the hematogenous metastatic pathway in a simple and feasible way. It also offers an innovative surgical concept for the principle sequence of pulmonary vessel management.
      See Commentaries on pages 1636 and 1637.
      Video-assisted thoracoscopic surgery lobectomy (VATS-L) is the preferred means of surgical treatment for early-stage lung cancer.
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      Video-assisted thoracoscopic lobectomy for lung cancer.
      However, it has been reported that 20% to 50% of these patients develop distant metastasis and local recurrence within 5 years after surgery.
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      Updated perspectives have highlighted the fact that surgical manipulation might promote the dissemination of circulating tumor cells (CTCs) and contribute to subsequent metastasis.
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      Does the mobilization of circulating tumour cells during cancer therapy cause metastasis?.
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      Circulating tumor cells detected in the tumor-draining pulmonary vein are associated with disease recurrence after surgical resection of NSCLC.
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      Tumor cells are dislodged into the pulmonary vein during lobectomy.
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      Significant increase in circulating tumour cells in pulmonary venous blood during surgical manipulation in patients with primary lung cancer.
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      Therefore, it is of great importance to develop a new surgical technique to reduce CTC spread during surgery.
      As the unique effluent vein of the entire lobe region, the pulmonary vein (PV) is the first channel for CTC dissemination during surgery and is able to harbor vast numbers of CTCs released from the primary tumor.
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      Pulmonary venous blood sampling significantly increases the yield of circulating tumor cells in early-stage lung cancer.
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      (96.7%) revealed a higher CTC detection rate in the PV than in peripheral vessels (6.6%-91.3%), even for early-stage lung cancer. In our previous pilot study, we also found that surgical manipulation could unintentionally mobilize CTCs into the PV.
      • Duan X.
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      • Han Y.
      • et al.
      Circulating tumor cells in the pulmonary vein increase significantly after lobectomy: a prospective observational study.
      Thus, PV blood samples are ideal for assessing the effects of surgical manipulations on CTC mobilization at surgery.
      Ligation of the PV before the pulmonary artery (PA) is advised to prevent CTCs from spreading through the outflow channel.
      • Kurusu Y.
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      • Mita S.
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      The sequence of vessel ligation affects tumor release into the circulation.
      A large-population study was designed to compare the different sequences of vessel ligation (vein-first or artery-first), and accumulating evidence is strongly biased toward the advantage of the vein-first technique in reducing intraoperative tumor cell spread.
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      Effect of vein-first vs artery-first surgical technique on circulating tumor cells and survival in patients with non-small cell lung cancer: a randomized clinical trial and registry-based propensity score matching analysis.
      However, in actual clinical settings, following the principle of PV-PA surgical procedures, some surgeons are accustomed to ligating the PV immediately after dissecting it and then dissecting other hilar structures; others also dissect the PV at the beginning of lobectomy but choose to transect it later, after all bronchovascular pedicles are either partially or entirely dissected. The critical temporal window from completion of the PV dissection to the exposure of other hilar structures (arteries and bronchus) might provide the opportunity for CTCs to shed during the operation. Accordingly, we designed the current study to investigate whether the timing of PV ligation, namely, the early or late PV ligation technique, can affect CTC dissemination during VATS-L using telomerase reverse transcriptase-based CTC detection (TBCD), which we have established in previous studies.
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      • Duan X.
      • Zhang Z.
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      • Zhao C.
      • Liang C.
      • et al.
      Combination of CT and telomerase+ circulating tumor cells improves diagnosis of small pulmonary nodules.
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      • Bao L.
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      • Dong M.
      • Yin L.
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      Tumor-selective replication herpes simplex virus-based technology significantly improves clinical detection and prognostication of viable circulating tumor cells.

      Methods

      Study Design and Participants

      This study was a prospective observational clinical trial registered at the Chinese Clinical Trial Registry (ChiCTR1800016879). Between July 2018 and August 2019, 44 patients with non-small cell lung cancer (NSCLC) who underwent VATS-L at the Department of Thoracic Surgery at Beijing Chest Hospital affiliated with Capital Medical University were enrolled (Figure 1). All participants were required to meet the following inclusion criteria: (1) aged 18 to 80 years, (2) peripheral NSCLC with T1, (3) simple thoracoscopic lobectomy following oncosurgical principles (PV-PA procedures), and (4) the resected lung lobe was extracted smoothly from the thoracic cavity and the PV stump remained intact. Participants were excluded if they had: (1) multiple intralobar primary tumors; and (2) mediastinal lymph node metastasis. The Ethics Committee of Beijing Chest Hospital (number 2018-04-01; date, January 29, 2018) approved the protocol for this study. All participants provided written informed consent to participate in this study and for the subsequent publication of the study results.
      Figure thumbnail gr1
      Figure 1Consolidated Standards of Reporting Trials (CONSORT) diagram of the patient cohort. VATS-L, Video-assisted thoracoscopic surgery lobectomy; NSCLC, non–small cell lung cancer; PA, pulmonary artery; PV, pulmonary vein; ELG, early ligation group; LLG, late ligation group; PVCTCs, circulating tumor cells in pulmonary vein.
      The preoperative examination included regular blood tests and cardiopulmonary function tests. Preoperative staging studies including enhanced computed tomography (CT) scanning, brain magnetic resonance imaging, isotope bone scanning, and abdomen B-ultrasound were routinely performed. All surgeries in our trial were performed by 4 chief thoracic surgeons with uniform 2-port VATS-L consisting of an operation port (average diameter: 5.57 cm [range, 4.8-6.2 cm]) and an observation port (approximately 2 cm), and followed by systematic mediastinal lymphadenectomy. All operations were successfully performed, except 1, during which the patient had to be converted to an open thoracotomy because of unexpected severe adhesion of the hilar lymph node.

      Sample Collection, Time Partitioning, and Patient Grouping

      The detailed procedures of blood sample collection have been reported in a previous article.
      • Duan X.
      • Zhu Y.
      • Cui Y.
      • Yang Z.
      • Zhou S.
      • Han Y.
      • et al.
      Circulating tumor cells in the pulmonary vein increase significantly after lobectomy: a prospective observational study.
      A 4-mL pulmonary vein blood (PVB) sample was collected using a 24- or 25-gauge disposable vein infusion needle attached to a 10-mL syringe before PV ligation was performed. Another 4 mL of blood was obtained from the PV stump of the resected pulmonary lobe at the end of the operation. All blood samples were stored in 4 mL K2E (EDTA) tubes, maintained at 4 °C, and transported to the laboratory for analysis within 4 hours (Figure 2).
      Figure thumbnail gr2
      Figure 2Schematic showing pulmonary vein (PV) blood sample collection and the workflow of circulating tumor cell (CTC) detection and validation. FISH, Fluorescence in situ hybridization.
      After the operation port (4th intercostal space) and the observation port (7th intercostal space) were successfully placed, an endoscopic ring clamp was inserted into the thoracic cavity to grip the lung lobe. At this point, the lobectomy start time was recorded. The timing of PV ligation was recorded when the vessel cutting stapler transected the PV. When the target lung lobe was completely resected, the lobectomy end time was recorded.
      We defined patients who underwent early and late PV ligation techniques as the early ligation group (ELG; Figure 3, A) and late ligation group (LLG; Figure 3, B), respectively. In detail, the early PV ligation technique was performed as follows (Video 1): the PV was dissected and then transected immediately using an endoscopic vascular cutting stapler. Then, the artery branches and bronchus were sequentially dissected and transected. Conversely, the late PV ligation technique was performed as follows (Video 1): the PV was exposed at the beginning of the lobectomy but was not ligated until the PA branches, bronchus, and pulmonary fissure were exposed either partially or entirely (Figure 3, C).
      Figure thumbnail gr3
      Figure 3Schematic diagram showing how the early ligation group (ELG) and late ligation group (LLG) were divided. A, Early PV ligation technique: the upper PV was first dissected and then transected immediately. B, Late PV ligation technique: the upper PV was first dissected, but it was not transected until the right upper pulmonary artery branches, bronchus and pulmonary fissure were exposed either partially or entirely. A and B, right upper lobe resection. C, Schematic diagram showing the timing of PV ligation and the time point of hilar vessel ligation between the ELG and LLG groups. PV, Pulmonary vein; PA, pulmonary artery.

      Virus

      The oHSV1-hTERTp-GFP virus with an endogenous ICP4 promoter was replaced with the hTERT promoter, and genes encoding cell protein 34.5 (ICP34.5) were replaced with the GFP gene, as described in our previous work (Figure 2).
      • Zhang W.
      • Duan X.
      • Zhang Z.
      • Yang Z.
      • Zhao C.
      • Liang C.
      • et al.
      Combination of CT and telomerase+ circulating tumor cells improves diagnosis of small pulmonary nodules.
      ,
      • Zhang W.
      • Bao L.
      • Yang S.
      • Qian Z.
      • Dong M.
      • Yin L.
      • et al.
      Tumor-selective replication herpes simplex virus-based technology significantly improves clinical detection and prognostication of viable circulating tumor cells.
      The purified viruses were aliquoted and stored at −80 °C until use.

      CTCs in the PV Detection via TBCD

      Four milliliters of blood was collected in 4-mL EDTA tubes and centrifuged at 500g for 5 minutes. The plasma was removed, and red blood cell lysis buffer was used to lyse red blood cells in a 50-mL centrifuge tube until the liquid was transparent; the samples were centrifuged at 500g for 5 minutes, and all the liquid was discarded to collect the cell pellet. After resuspending and washing the cell pellet with phosphate buffered saline, oHSV1-hTERTp-GFP was used to infect the total cells at a multiplicity of infection of 1 (multiplicity of infection = virus titer × virus volume/number of cells = 1). Then, the treated cells were placed in an incubator for 24 hours at 5% CO2 and 37 °C. The transduced cells were collected and stained with CD45. Finally, flow cytometry was used to identify CTCs. The CTCs were defined as GFP+CD45− cells (Figure E1, A).

      Validation of CTCs via ImageStreamX (Amnis) and Fluorescence In Situ Hybridization

      Two tubes of blood were treated with a standard CTC detection process. For FlowSight imaging, 1 tube of treated blood cells was incubated with an eFluor 450-labeled anti-CD45 antibody and APC-labeled anti-EpCAM antibody and detected using the ImageStreamX Mark II system (Amnis). CD45−GFP+epithelial cell adhesion molecule (EpCAM)+ cells were recorded as CTCs (Figure E1, B). For fluorescence in situ hybridization (FISH), MACS magnetic beads (Miltenyi Biotec) were used to isolate CD45− cells. After fixation, the cells were labeled with a dual-color FISH probe set. The PTEN FISH probe set consists of a Spectrum Red-labeled probe specific for the PTEN gene on the chromosome 10q23.3 region and a Spectrum Green-labeled centromere 10 (CEP10) probe (Celnovte FISH system). The EGFR FISH probe set consists of a Spectrum Red-labeled probe specific for the EGFR gene on the chromosome 7q11.2 region and a Spectrum Green-labeled centromere 7 (CEP7) probe (Celnovte FISH system). DAPI was used to stain nuclei.

      Statistics

      IBM SPSS Statistics 26.0 and GraphPad Prism version 8.2 were used to analyze the data. The CTC count is presented as the median (interquartile range [IQR]), and other continuous data are presented as the mean ± SD. The nonparametric Mann–Whitney U test was used to test 2 patient groups (asymmetric and continuous data). Categorical data are reported as proportions and percentages and were analyzed using the χ2 test or Fisher exact test when data were sparse. All P values were 2-sided.

      Results

      Patient Characteristics

      The characteristics of the patients enrolled in this study are presented in Table 1. A total of 44 lung cancer patients were enrolled. Eighteen patients were assigned to the ELG, and 26 patients were assigned to the LLG. The mean pathological tumor sizes were 2.42 ± 0.64 cm and 2.32 ± 0.62 cm in the ELG and LLG, respectively; no difference was observed between the 2 groups (P = .702). There were also no statistical differences in sex, age, smoking history, position, histology, spicule sign, tumor density, pN, pTNM, CT biopsy, tumor markers (single carcinoembryonic antigen or single soluble fragment of cytokeratin-19 positive), or vessel/lymphatic invasion between the 2 groups (P > .05).
      Table 1Patient characteristics
      VariableELG (n = 18)LLG (n = 26)P value
      Sex.050
       Male5 (27.8)15 (57.7)
       Female13 (72.2)11 (42.3)
      Age, y56.5 ± 9.1062.35 ± 7.70.400
      Smoking history.632
       Yes5 (27.8)9 (34.6)
       No13 (72.2)17 (65.4)
      cT size, cm2.24 ± 0.612.44 ± 0.51.402
      pT size, cm2.42 ± 0.642.32 ± 0.62.702
      Position.827
       RUL5 (27.8)11 (42.3)
       RML1 (5.6)1 (3.9)
       RLL4 (22.2)3 (11.5)
       LUL4 (22.2)6 (23.1)
       LLL4 (22.2)5 (19.2)
      Histology.419
       Adenocarcinoma14 (77.8)23 (88.5)
       Non-adenocarcinoma4 (22.2)3 (11.5)
      Spicule sign.977
       Yes7 (38.9)10 (38.5)
       No11 (61.1)16 (61.5)
      Tumor density>.999
       SN16 (88.9)22 (84.6)
       SSN2 (11.1)4 (15.4)
      pN.489
       Positive3 (16.7)7 (26.9)
       Negative15 (83.3)19 (73.1)
      pTNM.489
       I15 (83.3)19 (73.1)
       II-IIIA3 (16.7)7 (26.9)
      CT-guided biopsy.614
       Yes9 (50.0)15 (57.7)
       No9 (50.0)11 (42.3)
      TMs>.999
       Positive4 (22.2)5 (19.2)
       Negative14 (77.8)21 (80.8)
      Vessel/lymphatic invasion.576
       Positive7 (38.9)8 (30.8)
       Negative11 (61.1)18 (69.2)
      Data are presented as n (%) or mean ± SD, except where otherwise noted. ELG, Early ligation group; LLG, late ligation group; RUL, right upper lobe; RML, right middle lobe; RLL, right lower lobe; LUL, left upper lobe; LLL, left lower lobe; SN, solid nodules; SSN, subsolid nodules; CT, computed tomography; TMs, tumor markers, single carcinoembryonic antigen positive or single soluble fragment of cytokeratin-19 positive.

      Distribution of PV CTCs and the Association With Clinical Characteristics

      To further compare the difference in PV CTCs in the ELG and LLG (Table 2), we studied the distribution of PV CTCs before and after the operation. As shown in Figure 4, A, the median postoperative PV CTC (Post-PVCTC) count was 9 (IQR, 6-18) per 4 mL of PVB, which was higher than the median preoperative PV CTC (Pre-PVCTC) count of 1 (IQR, 0-3; P < .001). This result suggested that VATS-L inevitably promotes the dissemination of CTCs during surgery.
      Table 2Associations of clinical characteristics of patients (n = 44) and PV CTCs
      Clinical factors (n = 44)Pre-PVCTCsPost-PVCTCs
      Median (IQR)P valueMedian (IQR)P value
      Sex.826.777
       Male (n = 20)1 (0-2.75)11 (5.25-25.5)
       Female (n = 24)1 (0-3.75)9 (6.25-16.5)
      Age, y.430.169
       ≤60 (n = 23)2 (0-4)10 (10-26)
       >60 (n = 21)1 (0-2)9 (5.5-16.5)
      Smoking history.133.910
       Yes (n = 14)0.5 (0-1)9.5 (5.75-22)
       No (n = 30)2 (0-4)9 (6-19)
      Position.065.236
       RUL + RML + LUL (n = 28)1.5 (0-4)9 (6,14.5)
       RLL + LLL (n = 16)0 (0-1.75)16 (6.5-36.25)
      Histology.241.254
       Adenocarcinoma (n = 37)1 (0-3.5)9 (6-17.5)
       Non-adenocarcinoma (n = 7)0 (0-1)15 (7-37)
      Spicule sign.755.913
       Yes (n = 17)1 (0-4)8 (6.5-20.5)
       No (n = 27)1 (0-3)10 (5-18.)
      Tumor density.140.043
       SN (n = 38)1 (0-4)10 (6.75-22.5)
       SSN (n = 6)0 (0-1.5)5 (0.75-9.75)
      pT.917.220
       <1 cm (n = 2)1 (0-2)12.5 (12-13)
       1-1.9 cm (n = 14)4.25 (1-6)8.5 (3.5-11.25)
       2-3.5 cm (n = 28)1 (0-2.75)10.5 (7-25.5)
      pN.002.751
       Positive (n = 10)4 (1.75-6)11 (7-17.25)
       Negative (n = 34)1 (0-2)8.5 (6-19)
      pTNM.002.751
       Stage I (n = 34)1 (0-2)8.5 (6-19)
       Stage II- IIIA (n = 10)4 (1.75-6)11 (7-17.25)
      CT-guided biopsy.525.555
       Yes (n = 20)1 (0-2.75)8 (6.25-16.5)
       No (n = 24)1 (0-3.75)9.5 (6-21)
      TMs.098.226
       Positive (n = 9)2 (0.5-5)15 (9-22)
       Negative (n = 35)1 (0-2)8 (6-18)
      Vessel/lymphatic invasion.063.030
       Positive (n = 15)2 (1-5)15 (8-26)
       Negative (n = 29)1 (0-2.5)8 (5-14)
      PV, Pulmonary vein; CTCs, circulating tumor cells; Pre-PVCTCs, preoperative circulating tumor cells in pulmonary vein; Post-PVCTCs, postoperative circulating tumor cells in pulmonary vein; IQR, interquartile range; RUL, right upper lobe; RML, right middle lobe; LUL, left upper lobe; RLL, right lower lobe; LLL, left lower lobe; SN, solid nodules; SSN, subsolid nodules; CT, computed tomography; TMs, tumor makers, single carcinoembryonic antigen positive or single soluble fragment of cytokeratin-19 positive.
      Figure thumbnail gr4
      Figure 4Distribution of circulating tumor cells in pulmonary vein (PVCTCs) in the preoperative and postoperative groups. A, Comparison of PVCTC numbers for the preoperation and postoperation groups (P < .001). B, Comparison of PVCTC numbers for the solid nodules (SN) and subsolid nodules (SSN) in the preoperation (P = .140) and postoperation groups (P = .043); C, Comparison of PVCTC numbers for the vessel/lymphatic invasion-positive patients and invasion-negative patients in the preoperation (P = .063) and postoperation groups (P = .030); D, Comparison of the PVCTC numbers for the pN-positive patients and pN-negative patients in the preoperation (P = .002) and postoperation groups (P = .751); E, Comparison of PVCTC numbers for the stage I patients and stage II-IIIA patients in the preoperation (P = .002) and postoperation groups (P = .751). Box-and-whiskers dot plots: median (horizontal line), 25th and 75th percentiles (box), range (whiskers), and observations (dots).
      Then, we further analyzed the factors affecting CTC dissemination among the patient clinical characteristics. As shown in Figure 4, B, there was no difference of the Pre-PVCTCs count among solid nodule (SN) tumors and subsolid nodule (SSN) tumors (1 [IQR, 0-4] vs 0 [IQR, 0-1.5] per 4 mL PVB) (P = .140; Table 2). Interestingly, the Post-PVCTC count among the SN tumors was higher than that among the SSN tumors (10 [IQR, 6.75-22.5] vs 5 [IQR, 0.75-9.75] per 4 mL PVB; P = .043; Figure 4, B; Table 2). These results indicate that SN tumors tend to release more CTCs into the PV than SSN tumors, and surgery might contribute to this difference in CTC release.
      Similarly, more PV CTCs were detected among tumors with vessel or lymphatic invasion in the postoperative group (15 [IQR, 8-6] vs 8 [IQR, 5-14] per 4 mL PVB; P = .030; Figure 4, C; Table 2). However, the difference was not found in the preoperative group (2 [IQR, 1-5] vs 1 [IQR, 0-2] per 4 mL PVB; P = .063; Figure 4C; Table 2). This result indicates that tumors with vessel or lymphatic invasion might contribute to CTC spread. As shown in Figure 4, D, we detected more Pre-PVCTCs in patients who were pN positive than in those who were pN negative (4 [IQR, 1.75-6] vs 1 [IQR, 0-2]; P = .002; Table 2), but there was no difference in the postoperative group (11 [IQR, 7-17.25] vs 8.5 [IQR, 6-19]; P = .751; Table 2). pTNM staging also showed a difference between stage II to IIIA and stage I patients (4 [IQR, 1.75-6] vs 1 [IQR, 0-2]; P = .002; Figure 4, E; Table 2) before the operation. Furthermore, sex, age, smoking history, position, histology, spicule sign, pathological tumor size, CT-guided tumor biopsy, and tumor markers were not associated with PV CTC number.

      Comparison of the Operation Time for the ELG and LLG

      Because of the differences in surgical techniques that were performed, we compared the operation time for the ELG and LLG. The total lobectomy time was not statistically different between the 2 groups (Figure 5, E; P = .063), whereas the PV ligation time in the ELG was significantly shorter than that in the LLG (24.44 ± 14.03 vs 73.77 ± 30.69 minutes; P < .001; Figure 5, A). We also observed that the ratio of PV ligation time to the total time was less than 0.5 (range, 0.11-0.46) in the ELG, whereas it was greater than 0.5 in the LLG (range, 0.50-0.97; P < .001; Figure 5, B).
      Figure thumbnail gr5
      Figure 5Comparison of (PV) ligation time and circulating tumor cells in pulmonary vein (PVCTC) count for the early ligation group (ELG) and late ligation group (LLG). A, Comparison of the PV ligation time for the ELG and the LLG (P < .001). B, Comparison of the ratio of PV ligation time to total time for the ELG and the LLG (P < .001). C, Comparison of the circulating tumor cell (CTC) interception time for the ELG and LLG (P = .001). D, Comparison of the ratio of the CTC interception time to total time for the ELG and LLG (P < .001). E, Comparison of the total lobectomy time for the ELG and LLG (P = .063). F, Comparison of preoperative circulating tumor cells in pulmonary vein (pre-PVCTC) counts for the ELG and LLG (P = .729). G, Comparison of postoperative circulating tumor cells in pulmonary vein (post-PVCTC) counts for the ELG and LLG (P = .004). H, Comparison of ΔPVCTC numbers for the ELG and LLG after the lung lobe was resected (P = .006). Box-and-whiskers dot plots: median (horizontal line), 25th and 75th percentiles (box), range (whiskers), and observations (dots).
      Conversely, the CTC interception time (total lobectomy time minus PV ligation time) in the ELG was longer than that in the LLG (P = .001; Figure 5, C), and the ratio of CTC interception time to total time was >0.5 (range, 0.54-0.89) in the ELG but <0.5 (range, 0.03-0.50) in the LLG (P < .001; Figure 5, D). These results indicate that early PV ligation is usually completed during the first half of the total lobectomy time; thus, the CTC interception time is longer.

      Comparison of PV CTCs for the ELG and LLG

      As shown, surgical manipulation can cause CTC shedding, and ELG has a longer CTC interception time. Thus, we wondered whether there were differences in PV CTCs between the 2 groups. As shown in Figure 5, F-H and Table 3, there were no differences between patients in the ELG and LLG before the surgical operation (1 [IQR, 0-3.25] vs 1 [IQR, 0-3.25] per 4 mL PVB; P = .729; Figure 5, F; Table 3). Interestingly, the number of PV CTCs in the ELG was higher than that in the LLG after lobectomy (16 [IQR, 9.5-36.75] vs 8 [IQR, 4.75-12.25] per 4 mL PVB; P = .004; Figure 5, G; Table 3). To further compare the differences in Post-PVCTCs between the ELG and LLG, we used the Post-PVCTC count minus the Pre-PVCTC count to adjust values to ΔPV CTCs. As shown in Figure 5, H and Table 3, the ΔPV CTC count in the ELG was also higher than that in the LLG (16 [IQR, 6.75-35.5] vs 6 [IQR, 4-9] per 4 mL PVB; P = .006). These results suggest that many CTCs enter the circulation through the PV during surgical manipulations when the PV is not ligated early.
      Table 3Comparison of PV CTCs for ELG and LLG
      ELG (n = 18)LLG (n = 26)P value
      Pre-PVCTCs1 (0-3.25)1 (0-3.25).729
      Post-PVCTCs16 (9.5-36.75)8 (4.75-12.25).004
      ΔPV CTCs16 (6.75-35.5)6 (4-9).006
      Data are presented as median (interquartile range) except where otherwise noted. PV, Pulmonary vein; CTCs, circulating tumor cells; ELG, early ligation group; LLG, late ligation group; Pre-PVCTCs, preoperative circulating tumor cells in pulmonary vein; Post-PVCTCs, postoperative circulating tumor cells in pulmonary vein; ΔPV CTCs, postoperative circulating tumor cells in pulmonary vein minus preoperative circulating tumor cells in pulmonary vein.

      Validation of PV CTCs

      We performed FlowSight imaging and FISH to confirm that PV CTCs were derived from the primary lung cancer site. We first introduced EpCAMs to identify CTCs using FlowSight images. The typical results of 3 patients are shown in Figure 6, A. CTCs were larger than white blood cells and highly expressed GFP and EpCAM but not CD45 (CD45−/GFP+/EpCAM+). Moreover, white blood cells expressed only CD45 (CD45+/GFP−/EpCAM−).
      Figure thumbnail gr6
      Figure 6Validation of circulating tumor cells (CTCs) via FlowSight and fluorescence in situ hybridization (FISH). A, Images of CTCs from 3 representative lung cancer patients acquired using FlowSight imaging. CD45-eFluor 450 (purple), hTERTp-GFP (green), EpCAM (red), and bright-field digital images are shown for CTCs and white blood cells (WBCs). CTCs were defined as CD45-/GFP+/EpCAM+. WBCs were only marked with CD45. B, Images of CTCs from the same patients acquired using FISH. Dual-color FISH results for heterozygous PTEN (10q23) deletion and EGFR amplification (7q11.2) in CTCs.
      As shown in Figure 6, B, FISH images (magnification 60 × ) were captured for 2 patients. PTEN FISH showed that CTCs exhibited the typical signal configuration for heterozygous deletion of the PTEN gene with 1 red signal; moreover, chromosome 10, where the deleted gene for PTEN is located, had 2 standard green fluorescence signals. EGFR FISH showed that CTCs exhibited an amplified signal with more than 2 red signals, and chromosome 7 had 2 standard green fluorescence signals. These results indicated that PV CTCs were derived from primary lung cancer.

      Discussion

      We conducted a prospective clinical trial to investigate, for the first time, the association between CTC dissemination and the timing of PV ligation on the basis of whether early or late PV ligation techniques were used. Our results revealed that early PV ligation during lobectomy better prevents CTC shedding into the PV stump than late PV ligation. We provide an innovative surgical concept for the principle sequence of pulmonary vessel management (PV-PA procedure), which highlights the significant advantage of early PV ligation in terms of preventing tumor cells from spreading.
      Several studies have revealed that surgical manipulation can inadvertently mobilize PV CTCs into the PV.
      • Yao X.
      • Williamson C.
      • Adalsteinsson V.A.
      • D’Agostino R.S.
      • Fitton T.
      • Smaroff G.G.
      • et al.
      Tumor cells are dislodged into the pulmonary vein during lobectomy.
      ,
      • Hashimoto M.
      • Tanaka F.
      • Yoneda K.
      • Takuwa T.
      • Matsumoto S.
      • Okumura Y.
      • et al.
      Significant increase in circulating tumour cells in pulmonary venous blood during surgical manipulation in patients with primary lung cancer.
      ,
      • Duan X.
      • Zhu Y.
      • Cui Y.
      • Yang Z.
      • Zhou S.
      • Han Y.
      • et al.
      Circulating tumor cells in the pulmonary vein increase significantly after lobectomy: a prospective observational study.
      In this regard, we also found a higher number of PV CTCs postoperatively than preoperatively, which aligned with the our perspective. Yao and colleagues
      • Yao X.
      • Williamson C.
      • Adalsteinsson V.A.
      • D’Agostino R.S.
      • Fitton T.
      • Smaroff G.G.
      • et al.
      Tumor cells are dislodged into the pulmonary vein during lobectomy.
      raised the critical issue that the order of pulmonary vessel ligation and the timing of PV ligation might influence the number of PV CTCs spreading. Regarding VATS-L, the process of extracting resected lung lobes from the chest cavity might occasionally squeeze specimens or disturb the tumor itself, which is a significant challenge. We therefore formulated strict inclusion/exclusion criteria, as described previously, and included only patients who had peripheral NSCLC tumors with a diameter ≤3 cm. With these objective quality control approaches, we believe that the results of the study are reliable. Moreover, because blood flow stops when the PA branches are completely transected, tumor cells will be less able to shed and circulate into the PV at that time.
      Our results revealed that Pre-PVCTCs are correlated with lymph node metastasis and pTNM stage, particularly in patients who are pN positive. However, there was no relationship between the number of Post-PVCTCs and these same factors; the exact reason needs to be further clarified in the future. Reddy and colleagues
      • Reddy R.M.
      • Murlidhar V.
      • Zhao L.
      • Grabauskiene S.
      • Zhang Z.
      • Ramnath N.
      • et al.
      Pulmonary venous blood sampling significantly increases the yield of circulating tumor cells in early-stage lung cancer.
      reported that the Pre-PVCTC count is significantly higher with preoperative bronchoscopic biopsy than with CT-guided biopsy. Our study did not obtain the same result because the peripheral lung cancer cases enrolled in our trial did not require evaluation using bronchoscopic biopsy. Furthermore, a significant change in PV CTCs was not found between patients with CT-guided biopsy and those with non–CT-guided biopsy in our study. Accordingly, CT-guided biopsy might only slightly or transiently affect CTC shedding from the primary tumor into the bloodstream.
      Another intriguing finding in our study is that the Post-PVCTC count was increased in patients with SNs compared with those with SSNs. A possible explanation is that SSNs, which have a more indolent biological behavior, might be less likely to shed CTCs to promote invasion and metastasis.
      • Xing X.
      • Yang F.
      • Huang Q.
      • Guo H.
      • Li J.
      • Qiu M.
      • et al.
      Decoding the multicellular ecosystem of lung adenocarcinoma manifested as pulmonary subsolid nodules by single-cell RNA sequencing.
      We also found that Post-PVCTCs were more numerous in patients with positive vessel/lymphatic invasion, which was similar to the findings of Hashimoto and colleagues.
      • Hashimoto M.
      • Tanaka F.
      • Yoneda K.
      • Takuwa T.
      • Matsumoto S.
      • Okumura Y.
      • et al.
      Significant increase in circulating tumour cells in pulmonary venous blood during surgical manipulation in patients with primary lung cancer.
      In contrast, another study revealed no relationship between the PV CTC count and any clinical or histological factors.
      • Yao X.
      • Williamson C.
      • Adalsteinsson V.A.
      • D’Agostino R.S.
      • Fitton T.
      • Smaroff G.G.
      • et al.
      Tumor cells are dislodged into the pulmonary vein during lobectomy.
      A possible reason might be the different proportions of enrolled patients on the basis of pathological staging and the different surgical treatment modalities used (thoracoscopic or thoracotomic lobectomy).
      Some surgical techniques might cause tumor cells to be released into the bloodstream because of repeated manipulation of the tumor-bearing organ and the unintentional squeezing of the tumor during the operation.
      • Yamashita J.I.
      • Kurusu Y.
      • Fujino N.
      • Saisyoji T.
      • Ogawa M.
      Detection of circulating tumor cells in patients with non-small cell lung cancer undergoing lobectomy by video-assisted thoracic surgery: a potential hazard for intraoperative hematogenous tumor cell dissemination.
      ,
      • Weitz J.
      • Kienle P.
      • Lacroix J.
      • Willeke F.
      • Benner A.
      • Lehnert T.
      • et al.
      Dissemination of tumor cells in patients undergoing surgery for colorectal cancer.
      A no-touch isolation technique has been proposed previously and is broadly used during the resection of colorectal cancer,
      • Turnbull R.J.
      • Kyle K.
      • Watson F.R.
      • Spratt J.
      Cancer of the colon: the influence of the no-touch isolation technic on survival rates.
      ,
      • Sales J.P.
      • Wind P.
      • Douard R.
      • Cugnenc P.H.
      • Loric S.
      Blood dissemination of colonic epithelial cells during no-touch surgery for rectosigmoid cancer.
      pancreatic cancer,
      • Gall T.M.
      • Jacob J.
      • Frampton A.E.
      • Krell J.
      • Kyriakides C.
      • Castellano L.
      • et al.
      Reduced dissemination of circulating tumor cells with no-touch isolation surgical technique in patients with pancreatic cancer.
      hepatocellular carcinoma,
      • Lee J.M.
      • Lee K.W.
      • Kim H.C.
      • Yi N.J.
      • Suh K.S.
      No touch isolation technique for the prevention of postoperative recurrence of hepatocellular carcinoma after liver transplantation-combined with trans-arterial radioembolization.
      ,
      • Li Y.
      • Xu K.S.
      • Li J.S.
      • Jia W.D.
      • Liu W.B.
      • He X.D.
      • et al.
      The research of no-touch isolation technique on the prevention of postoperative recurrence and metastasis of hepatocellular carcinoma after hepatectomy.
      epithelial ovarian carcinoma,
      • Sznurkowski J.J.
      En bloc pelvic resection for advanced ovarian cancer preceded by central ligation of vessels supplying the tumor bed: a description of surgical technique and a feasibility study.
      and peripheral lung cancer,
      • Yasukawa M.
      • Sawabata N.
      • Kawaguchi T.
      • Taniguchi S.
      Wedge resection of tumor before lobectomy for lung cancer could be a no-touch isolation technique.
      with the goal of controlling viable CTC dissemination by preligating the vessels near the tumor. It has been deeply embodied by Kitagawa and colleagues
      • Kitagawa H.
      • Tajima H.
      • Nakagawara H.
      • Makino I.
      • Miyashita T.
      • Terakawa H.
      • et al.
      A modification of radical antegrade modular pancreatosplenectomy for adenocarcinoma of the left pancreas: significance of en bloc resection including the anterior renal fascia.
      in their “early vascular control technique” to ligate the short gastric vessels and the splenic artery earlier in the procedure during the surgical treatment of distal pancreatic cancer.
      Regarding thoracoscopic lobectomy, several studies have shown that PV CTC dissemination at surgery is responsible for subsequent tumor recurrence.
      • Crosbie P.A.
      • Shah R.
      • Krysiak P.
      • Zhou C.
      • Morris K.
      • Tugwood J.
      • et al.
      Circulating tumor cells detected in the tumor-draining pulmonary vein are associated with disease recurrence after surgical resection of NSCLC.
      ,
      • Chemi F.
      • Rothwell D.G.
      • McGranahan N.
      • Gulati S.
      • Abbosh C.
      • Pearce S.P.
      • et al.
      Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.
      ,
      • Murlidhar V.
      • Reddy R.M.
      • Fouladdel S.
      • Zhao L.
      • Ishikawa M.K.
      • Grabauskiene S.
      • et al.
      Poor prognosis indicated by venous circulating tumor cell clusters in early-stage lung cancers.
      The single-direction thoracoscopic lobectomy proposed by Liu and colleagues
      • Liu L.
      • Che G.
      • Pu Q.
      • Ma L.
      • Wu Y.
      • Kan Q.
      • et al.
      A new concept of endoscopic lung cancer resection: single-direction thoracoscopic lobectomy.
      complies with the surgical principle of the PV-PA procedure, which has an active role in preventing CTC dissemination.
      • Wei S.
      • Guo C.
      • He J.
      • Tan Q.
      • Mei J.
      • Yang Z.
      • et al.
      Effect of vein-first vs artery-first surgical technique on circulating tumor cells and survival in patients with non-small cell lung cancer: a randomized clinical trial and registry-based propensity score matching analysis.
      Additionally, such a surgical technique can diminish repeated surgical manipulations, even for lobectomy with hypoplastic lung fissures. However, some thoracic surgeons misinterpret the true value of these implications and neglect the importance of the timing of PV ligation (early or late), which is inconsistent with oncosurgical principles. Collectively, the results of our trial show that the application of an early PV ligation technique could better prevent hematogenous dissemination of CTCs, which provides new insight into the surgical treatment of lung cancer (Figure 7). Furthermore, early PV ligation techniques should be seriously considered, especially for lung cancer patients with pure solid tumor components and larger-sized, central-type, and more aggressive tumor characteristics. In our trial, 2 patients with small cell lung cancer underwent early PV ligation, and we also isolated abundant Post-PVCTCs; however, these 2 cases failed to meet our inclusion criteria because of the pathologic results (Figure E2).
      Figure thumbnail gr7
      Figure 7Schematic summary of the study flow and key findings. Total patients were divided into the early ligation group (ELG) and late ligation group (LLG). We collected 4 mL preoperative and postoperative pulmonary venous blood and detected circulating tumor cells (CTCs) via telomerase reverse transcriptase-based method (TBCD). The number of postoperative circulating tumor cells in the PV (Post-PVCTCs) and ΔPVCTCs in the ELG was higher than in the LLG. PV, Pulmonary vein; Pre-PVCTCs, preoperative circulating tumor cells in pulmonary vein; Post-PVCTCs, postoperative circulating tumor cells in pulmonary vein.
      In this study, we used the TERT-based CTC detection method (TBCD) for PV CTC detection. Telomerase is a pan tumor hallmark, and it is highly expressed in approximately 80% of malignant tumors. Previous studies confirmed that telomerase-based CTC detection is a promising method.
      • Soria E.
      • Vallejo M.
      • Saenz L.
      • Ramírez N.
      Telomerase-specific attenuated viruses, a definitive strategy or just one more in circulating tumor cells detection anthology?.
      Accordingly, TBCD has shown high CTC detection efficiency in our previous studies, even for small lung nodules.
      • Zhang W.
      • Duan X.
      • Zhang Z.
      • Yang Z.
      • Zhao C.
      • Liang C.
      • et al.
      Combination of CT and telomerase+ circulating tumor cells improves diagnosis of small pulmonary nodules.
      ,
      • Zhang W.
      • Bao L.
      • Yang S.
      • Qian Z.
      • Dong M.
      • Yin L.
      • et al.
      Tumor-selective replication herpes simplex virus-based technology significantly improves clinical detection and prognostication of viable circulating tumor cells.
      ,
      • Gao H.
      • Liu W.
      • Yang S.
      • Zhang W.
      • Li X.
      • Qin H.
      • et al.
      Detection of circulating tumor cells using oHSV1-hTERT-GFP in lung cancer.
      In addition, we tested 178 healthy donors and proved that the average number of CD45−/GFP+ cells was 0.63 per 4 mL peripheral blood in healthy donors in the previous study, which means that other cells are not contaminating the CTCs.
      • Zhang W.
      • Bao L.
      • Yang S.
      • Qian Z.
      • Dong M.
      • Yin L.
      • et al.
      Tumor-selective replication herpes simplex virus-based technology significantly improves clinical detection and prognostication of viable circulating tumor cells.
      Some studies have indicated that higher telomerase activity is associated with a higher degree of malignancy in tumors.
      • Yuan X.
      • Larsson C.
      • Xu D.
      Mechanisms underlying the activation of TERT transcription and telomerase activity in human cancer: old actors and new players.
      Chemi and colleagues
      • Chemi F.
      • Rothwell D.G.
      • McGranahan N.
      • Gulati S.
      • Abbosh C.
      • Pearce S.P.
      • et al.
      Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.
      have proven that increasing PV CTC count was associated with poor tumor prognosis, and showed the overlap of somatic mutations (91%) detected between single PV CTCs and metastatic tumor. Thus, we believe that the PV CTCs captured with our method had higher cell viability and might strengthen the significance of our research. Yet, it has not been clear whether the shed PV CTCs are viable in circulation, and further investigation is still needed to study the mechanisms of tumor metastasis through PV CTC dissemination.
      There are several potential limitations in this study. First, although our study illustrates the significance of the early PV ligation technique in preventing the spread of tumor cells, it might increase the probability of pulmonary congestion in complex lobectomy. Second, we did not include the clinical outcome in our study. To reduce the possible negative effects of squeezing the tumor during extraction of the resected lung lobe after surgery and to ensure the authenticity of the results, we formulated strict inclusion/exclusion criteria and enrolled only patients with peripheral and smaller tumor sizes (≤3 cm). Thus, a longer follow-up time was needed to obtain disease-free survival and overall survival outcomes. Third, the sample size was relatively small. In the future, we will conduct further prospective, randomized trials with larger sample sizes to investigate the benefits of the early PV ligation technique.

      Conclusions

      Based on the results of our reasearch, we provide the first evidence to show that early PV ligation can prevent PV CTCs from spreading into the circulation, offering an innovative surgical concept for the principle sequence of pulmonary vessel management.

      Conflict of Interest Statement

      The authors reported no conflicts of interest.
      The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.
      The authors thank Drs Zhidong Liu, Shijie Zhou, Daping Yu, and Yi Han for providing the assistance of drawing PV blood samples intraoperatively.

      Supplementary Data

      Appendix E1

      Figure thumbnail fx4
      Figure E1A, Flow cytometry analysis process for circulating tumor cells in pulmonary vein (PVCTCs) on the basis of the telomerase reverse transcriptase-based method (TBCD). Circulating tumor cells (CTCs) were defined as GFP+ CD45-. B, ImageStreamX (Amnis) system analysis process for PVCTC validation on the basis of the TBCD. The CTCs were defined as GFP+ CD45- EpCAM+.
      Figure thumbnail fx5
      Figure E2A, The results of computed tomography (CT) images, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) staining in patient 1 with small cell lung cancer (SCLC). The IHC results of this patient were TTF-1+, CD56+, syn+, and Ki67 (80%+) (magnification 20 ×). B, The results of CT images, HE staining, and IHC staining in patient 2 with SCLC. The IHC results of this patient were TTF-1+, CD56+, syn+, and Ki67 (80%+). C, Schematic diagram showing the pulmonary vein (PV) ligation time and the total lobectomy time of 2 patients. Because the early PV ligation technique was performed in them, we can observe that the PV ligation time was located at the first half of the total lobectomy time. D, The circulating tumor cell (CTC) number of SCLC patient 1 and patient 2 before (Pre) and after (Post) surgery. SCLC patient 1: preoperative circulating tumor cells in pulmonary vein (Pre-PVCTC) versus postoperative circulating tumor cells in pulmonary vein (Post-PVCTC) = 9 versus 106 per 4 mL blood. SCLC patient 2: Pre-PVCTC versus Post-PVCTC = 7 versus 1263 per 4 mL blood.

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      Linked Article

      • Commentary: Catch me if you can: Does reducing circulating tumor cells with early pulmonary vein ligation improve survival after lobectomy?
        The Journal of Thoracic and Cardiovascular SurgeryVol. 164Issue 6
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          In this issue of the Journal, Duan and colleagues1 evaluated early versus late pulmonary vein ligation and concluded that early ligation can prevent circulating tumor cells from entering the circulation. The potential benefit of decreasing circulating tumor cells on survival is appealing, since 90% of cancer-related deaths are due to metastatic disease,2 and the rate of recurrence for surgically resected lung cancers is 30% to 77%.3
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      • Commentary: A surgical shotgun?
        The Journal of Thoracic and Cardiovascular SurgeryVol. 164Issue 6
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          It has been hypothesized in patients and shown in preclinical studies that local treatment of cancer, whether surgery or radiation therapy, can lead to mechanical disruption of the tumor, shedding of tumor cells, suppression of antitumor immunity, and enhancement of the metastatic process.1,2 Specific to lung cancer, previous studies have shown increases in pulmonary venous and peripheral blood circulating tumor cells (CTCs) following surgery, raising the question of whether ligating the pulmonary vein and cessation of effluent flow early during lobectomy might reduce shedding of CTCs.
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