Longitudinal evaluation of circulating tumor cells in operable locally advanced esophageal squamous cell carcinoma

Longitudinal evaluation of circulating tumor cells in operable locally advanced esophageal squamous cell carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Longitudinal evaluation of circulating tumor cells in operable locally advanced esophageal squamous cell carcinoma Jin Wang, Shuang Chen, Weidi Zhang, Yang Li, Lu Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4640180/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: The detection of circulating tumor cells (CTCs) at a single time point offers restricted insights into prognostic assessment. This study aims to longitudinally investigate alterations in CTCs status throughout the neoadjuvant chemotherapy, surgical, and postoperative chemotherapy phases within a homogeneous cohort. Further more, an assessment of the interrelation between patients' treatment response, survival prognosis, and CTCs status was conducted. Materials and methods: Thirty-one patients diagnosed with operable locally advanced esophageal squamous cell carcinoma were randomly allocated to either the surgical or neoadjuvant group. CTCs detection was systematically conducted at various time points throughout the treatment on individual patients. Associations between the presence of CTCs/CTM and therapeutic efficacy, as well as clinical outcomes, were subject to rigorous analysis. Results: We performed a total of 136 CTCs detections at the time points of pre-neoadjuvant chemotherapy, preoperative, postoperative day 2, post-operative 1 week, post-operative 3 months. The CTCs were detected in 6/12(50％), 14/31(45.16％), 22/31(70.97％), 16/31(51.6％) and 5/31(16.1％) of patients before neoadjuvant treatment, prior to surgery, on postoperative day 2, one week after surgery, and three months post-surgery, respectively. The CTC count exhibited an increase before and after surgery, whereas a decrease was observed before and after neoadjuvant chemotherapy. Among patients initially identified with CTCs+, those in the neoadjuvant group experienced extended progression-free survival (PFS) (p=0.05) and overall survival (OS) (p=0.04) compared to those in the surgical group. On postoperative day 2, 17 patients had CTCs＜4, while 14 patients had CTCs≥4. Individuals with CTCs＜4 demonstrated significantly prolonged PFS (p＜0.01, HR=6.26, 95% CI 1.96-19.96) and OS (p＜0.01, HR=6.63, 95% CI 2.08-21.13) compared to those with CTCs≥4. CTM was detected in 45.16% of patients at any given time. Survival analysis indicated that CTM- patients exhibited longer PFS (p=0.029, HR=2.92, 95% CI 1.05-8.07) and OS (p=0.03, HR=2.92, 95% CI 1.06-8.09) than CTM+ patients. Conclusions: Single-time detection offers limited information for prognostic evaluation, a dynamic fluctuation in the quantity of CTCs/CTM throughout the course of treatment, exhibiting a robust predictive impact on patient prognosis. Biological sciences/Cancer/Cancer therapy/Chemotherapy Biological sciences/Stem cells/Cancer stem cells circulating tumor cells operable locally advanced esophageal squamous cell carcinoma neoadjuvant chemotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Locally advanced esophageal squamous cell carcinoma (AJCC TNM staging system≥cT2 and/or cN1-3, M0, LAEC) is characterized by the invasion of local structures or involvement of regional lymph nodes without distant metastases. However, relying solely on surgery for the treatment of locally advanced cases is linked to a median survival ranging from 12 to 18 months and a five-year survival rate of 15–39% [ 1-3 ] . When surgical resection is the exclusive therapeutic approach, both local and systemic recurrence is prevalent, affecting 35–50% of patients [ 4 ] . A standard initial therapeutic strategy for these patients involves neoadjuvant chemotherapy (nCT) or chemoradiotherapy (nCRT) followed by surgery [ 5 , 6 ] . This approach serves to decrease surgical complexity by reducing tumor size, enhance the R0 resection rate, and improve patients' tolerance to chemotherapy compared to postoperative chemotherapy. Additionally, it facilitates the early treatment of micrometastases. Circulating tumor cells (CTCs) represent tumor cells that are released into the peripheral blood from primary tumors or metastatic lesions, either spontaneously or as a result of surgical intervention [ 6 ] . CTCs possess the potential to serve as precursors of metastases [ 7 ] , offering crucial guidance for the prognosis of diverse tumors [ 8-10 ] . In contrast to histology, CTCs present an advantage in repeatedly obtaining required specimens with low invasiveness. The clinical significance of tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma was previously examined [ 11 ] . It was determined that CTCs function as independent prognostic indicators of patient outcomes in ESCC. The consideration of CTCs has the potential to enhance the accuracy of preoperative staging in ESCC [ 12 ] . However, the appearance of CTCs undergoes dynamic changes during treatment, including alterations in quantity and molecular phenotype [ 13-15 ] . Single-time detection offers limited information for prognostic evaluation. This study aims to: 1) longitudinally investigate changes in CTCs status during neoadjuvant chemotherapy, surgery, and postoperative chemotherapy within the same cohort; 2) assess patients' response to treatment in real-time; 3) further analyze the prognostic significance of CTCs status. Methods Study design The flow chart of the experiment was shown in Figure 1, and its duration was from July 2017 to December 2018. All 31 participants granted written informed consent for their involvement, then a 5-year follow-up was performed. The 31 patients with LAEC were followed for at least 5 years. LAEC progression, and times of recurrence, death, and disease free survival were recorded. Progression-free survival (PFS) was defined as the time from the onset of CTCs testing to CT progression or patient death. If the patient was lost to follow-up, or at the end of follow-up LAEC had not progressed, then this was recorded as a delete value. When a patient was not rehospitalized after discharge and the telephone follow-up was not answered by patients or their relatives, the patient’s initial hospitalization time was also taken as a PFS deletion. Overall survival was analyzed when patients were followed for 5 years. Approval was granted by the Shandong Provincial Cancer Research Institute Ethics Committee approved this study(201702012). CTCs testing and follow-up methods were as our previous study[12]. Written informed consent was obtained from the participants. All methods in this study were in accordance with the Declaration of Helsinki. Patients Pathological confirmation of esophageal squamous cell carcinoma (ESCC) through endoscopic biopsy was conducted prior to surgery (Stage II-IIIb). Gastroscopy and endoscopic ultrasonography were employed for a comprehensive assessment of tumor location and depth of invasion. Enhanced CT scans of the chest and abdomen were utilized to evaluate the extent of external invasion and the presence of suspected lymph node metastasis. When necessary, endoscopic ultrasonography or B-ultrasonography was performed to ascertain the existence of liver or distant metastasis. The exclusion criteria are as follows: 1) a history of malignant tumors; TNM stage IIIc-Ⅳ (T4b or N3 or M1); 2) severe complications affecting the heart, liver, lung, brain, kidneys, and other organs; 3) tumor perforation into the lung leading to abscess formation; 4) cervical lymph node metastasis occurring in the lower thoracic segment of esophageal cancer; 5) paraperitoneal lymph node metastasis in cervical esophageal cancer. Surgery and chemotherapy Video-assisted thoracoscopic surgery (VATS) in conjunction with laparoscopic or open thoracic McKeown esophagectomy was employed for the procedure. Lymph node dissection encompassed: 1) chest: paraesophageal lymph nodes, subcarina lymph nodes, left and right pararyngeal recurrent nerve lymph nodes, trachea, and bronchial lymph nodes; 2) abdomen: lymph nodes of paracardia, lymph nodes of the lesser curvature of the stomach, especially lymph nodes of the left gastric vessel, lymph nodes of the common hepatic artery, and suspected lymph nodes of the splenic artery. For patients with middle and upper thoracic esophageal cancer, selective neck dissection is considered viable when cervical lymph node metastasis is suspected based on preoperative enhanced CT evaluation of the right recurrent nerve lymph node and ultrasound exploration of cervical lymph nodes. In the neoadjuvant arm, the DP regimen comprised 2 cycles of docetaxel (75 mg/m2 on Day 1) and cisplatin (25 mg/m2 on Day 1-3), administered once every 3 weeks. Two cycles of the DP regimen were continued 1 month after surgery. In the chemotherapy arm, 4 cycles of the DP regimen were continued 1 month after surgery. Postoperative adjuvant concurrent chemoradiotherapy was determined based on pathological type, lymph node metastasis, positive surgical margin, and postoperative stage. CTCs analysis A total of 136 CTCs detections were peformed. The time points selected for CTCs detection were as follows: 1) In the neoadjuvant arm(n=12, 60 CTCs detections): pre-neoadjuvant chemotherapy, preoperative, postoperative day 2, post-operative 1 week, post-operative 3 months. 2) In the surgery arm(n=19, 76 CTCs detections): preoperative, post-operative day 2, post-operative 1 week, post-operative 3 months. The ISET assay was conducted following the protocol outlined by Vona et al [ 16 ] . A total of 5 milliliters of whole blood were diluted to 8 mL using a buffer containing 0.2% formaldehyde and filtered through an 8mm pore size membrane. An automated testing platform was employed to process the samples. The collected CTCs and circulating tumor microemboli were stained with Romanowsky stain, air-dried, mounted, and independently reviewed by three senior cytopathologists without prior knowledge. Cells were identified as CTCs if they exhibited ≥4 of the following morphological characteristics: atypical nucleus (irregular shape or presence of a nodular, lobulated contour); nuclear-cytoplasmic ratio >0.8; nuclear long diameter >18mm; hyperchromatic nuclei and nonhomogeneous staining; thickened, sunken, wrinkled, and jagged nuclear membrane; presence of nuclear chromatin side-shift or a large nucleoli or presence of abnormal mitotic figures; and presence of tumor cell aggregations, or circulating tumor microemboli (Figure 2). Statistical analysis Survival analysis was conducted utilizing the "survival" and "survminer" packages in R version 4.3.0. Scatter plots were generated employing "ggplot2" and "RColorBrewer." The comparison of two datasets and the creation of receiver operating characteristic (ROC) curves were executed using the Statistical Package for Social Sciences software (SPSS Version 27). Additional statistical analyses were carried out through the utilization of EXCEL(Microsoft Excel 2016) software. Results Patients Enrolled were 31 patients diagnosed with stage I to III esophageal squamous cell carcinoma (neoadjuvant arm: 12, surgery arm: 19). The clinicopathological characteristics of the patients are illustrated in Table 1 and Figure 3. Longitudinal Evaluation of CTCs Detected in Patient Samples The longitudinal evolution of CTC numbers was depicted in Figure 4. Prior to neoadjuvant therapy, CTCs were found in 6 out of 12 patients (50%, median CTC count 0.5; range 0–5). Before surgery, CTCs were found in 14 out of 31 patients (45.16%, median CTC count 0; range 0–19). On postoperative day 2, CTCs were found in 22 out of 31 patie.0nts (70.97%, median CTC count 3; range 0–37). At one week postoperative, CTCs were found in 16 out of 31 patients (51.6%, median CTC count 1; range 0–14). Three months postoperative, CTCs were found in 5 out of 31 patients (16.1%, median CTC count 0; range 0–4). A comparative analysis of CTC numbers at various time points was conducted. Patients were categorized into three groups: all patients (31), the surgery arm (19), and the neoadjuvant arm (12). The results revealed statistical differences in CTC numbers between preoperative and postoperative day 2 (p=0.05), preoperative and postoperative 3 months (p=0), postoperative day 2 and postoperative 1 week (p=0.008), postoperative day 2 and postoperative 3 months (p=0), and postoperative 1 week and postoperative 3 months (p=0) among all 31 patients. In the surgery arm, statistical significance was observed in preoperative vs postoperative 3 months (p=0) and postoperative 1 week vs postoperative 3 months (p=0). In the neoadjuvant arm, with the exception of preoperative vs postoperative 1 week (p=0.231), pre-neoadjuvant chemotherapy vs preoperative (p=0.212), pre-neoadjuvant chemotherapy vs postoperative 1 week (p=0.832), all other comparative analyses exhibited statistical significance (Table 2). Number of CTCs change after neoadjuvant chemotherapy or surgery The number of circulating tumor cells (CTCs) was computed at two distinct time points for 31 patients through subtraction. The outcomes were categorized into two groups: a reduced or equal group and an increased group. The findings indicated that, in the comparison of CTCs numbers between patients undergoing neoadjuvant chemotherapy (pre-neoadjuvant minus preoperative), 66.70% exhibited a decrease or remained unchanged, while 33.3% showed an increase (Figure 5a). In the analysis of CTCs numbers for patients undergoing surgery (postoperative day 2 minus pre-treatment), 58% experienced an increase, whereas 42% demonstrated a reduction or remained unchanged (Figure 5b). Similarly, for the interval between post-operative 1 week and preoperative, 61.3% displayed a decrease or remained equal, and 38.7% manifested an increase (Figure 5c). Association between CTCs and survival time The median survival time for the cohort of 31 patients was 36 months. Among patients initially detected with circulating tumor cells (CTCs+, n=13), the neoadjuvant arm exhibited an extended progression-free survival (PFS) (p=0.05, Figure 6a) and overall survival (OS) (p=0.04, Figure 6b) compared to the surgical arm. The ROC curve (Figure 7) demonstrated that the number of CTCs on postoperative day 2 provided a superior diagnostic value for predicting patient prognosis (AUC=0.758, p=0.014, 95% CI 0.579-0.938). The determined cutoff value for the number of CTCs was 3.5. Consequently, a cutoff value of 4 was selected for subsequent analysis. On postoperative day 2, 17 patients had CTCs<4, while 14 patients had CTCs≥4. Patients with CTCs<4 exhibited significantly prolonged PFS (p<0.01, HR=6.26, 95% CI 1.96-19.96, Figure 6c) and OS (p<0.01, HR=6.63, 95% CI 2.08-21.13, Figure 6d) compared to those with CTCs≥4. Longitudinal Evaluation of CTCs and survival time In the comparison of CTCs numbers between postoperative day 2 and pre-treatment, 20 patients exhibited an increased quantity of CTCs. Among them, 10 patients experienced an increase exceeding 3, while the remaining 10 patients showed an increase of less than 3. Notably, patients with a CTCs increase of ≤3 demonstrated significantly prolonged progression-free survival (median, 59 months vs. 20 months, p=0.035, HR=3.29, 95% CI 1-10.9, Figure 6e) and overall survival (median, 61 months vs. 21 months, p=0.035, HR=3.78, 95% CI 1-14.3, Figure 6f) compared to those whose CTCs increased by >3. Association between CTM and survival time CTM was detected in 14 out of 31 patients (45.16%) at all time points, as previously indicated. The majority of CTM instances were identified post-initial treatment, with only 3 (21.4%) CTM+ patients detected pre-treatment. The CTM detection rates were 26.3% (5/19) and 75% (9/12) in the surgery arm and neoadjuvant arm, respectively. Survival analysis revealed that CTM-negative patients exhibited an extended progression-free survival (PFS) (p=0.029, HR=2.92, 95% CI 1.05-8.07, Figure 6g) and overall survival (OS) (p=0.03, HR=2.92, 95% CI 1.06-8.09, Figure 6h) compared to CTM-positive patients. Further analysis of CTM-positive patients indicated that, when comparing the difference in CTCs numbers between postoperative day 2 and preoperative, patients with a decreased or unchanged number of CTCs demonstrated superior survival (no statistical difference, Figure 6i-j) compared to those with an increased number. Discussion Random allocation was employed for the division of 31 patients diagnosed with operable LAEC into the operation and neoadjuvant groups. CTCs detection was conducted at various time points throughout the treatment on the same patient. Differential impacts on the alterations in the quantity of CTCs/CTM were observed between surgery and neoadjuvant chemotherapy. Neoadjuvant chemotherapy resulted in a reduction in the number of CTCs/CTM, while surgery led to an increase in the number of CTCs/CTM (Fig. 4 ). The study findings suggested that regardless of an increase or decrease, this alteration served as a predictive indicator for patient outcomes. Neoadjuvant interventions manifested both local and systemic effects, delivering a survival advantage through potential synergistic actions and stood as the standard treatment for LAEC [ 17 ] . In this investigation, a noticeable decline in CTCs number post pre-neoadjuvant chemotherapy was evident (66.70% reduction or equivalence, 33.30% increase, Fig. 5 a), signifying the efficacy of neoadjuvant therapy. Subsequent analysis disclosed that within the cohort of initially identified CTCs + patients, the neoadjuvant arm exhibited an extended progression-free survival (PFS) (p = 0.05) and overall survival (OS) (p = 0.04) compared to the surgery arm. In essence, first-time detected CTCs + patients derived more substantial survival benefits, strongly advocating the consideration of neoadjuvant therapy. In summary, this investigation highlights that neoadjuvant therapy can promptly diminish the number of CTCs in the bloodstream and, in the long term, effectively elongate the survival duration of patients. This bears significant ramifications for the management of LAEC. In the context of surgical procedures, the dissemination of tumor cells into the bloodstream was recognized. Hashimoto et al. [ 18 ] established a significant correlation between the increased count of lobar pulmonary vein CTCs during surgical manipulation and postoperative distant metastasis in NSCLC patients who underwent complete resection. Wei et al. [ 19 ] observed that prioritizing the ligation of effluent veins during surgery might mitigate tumor cell dissemination and enhance survival outcomes in non–small cell lung cancer patients. Postoperative day 2 served as a crucial temporal reference point in our study, revealing a statistical discrepancy in CTC numbers between preoperative and postoperative day 2 for all 31 patients (p = 0.05), as well as for 12 patients in the neoadjuvant arm (p = 0.02). Regarding survival analysis, patients with CTCs<4 exhibited superior survival compared to those with CTCs ≥ 4. When comparing the difference in CTC numbers between postoperative day 2 and pre-treatment, patients with an increase of ≤ 3 CTCs demonstrated significantly better survival outcomes than those with an increase of >3 CTCs. The ROC curve(Fig. 7 ) further underscored the heightened diagnostic efficacy of postoperative day 2 in comparison to other time points. Consequently, CTCs on postoperative day 2 emerged as a potent predictor of the survival prognosis for LAEC patients. Implementing diverse strategies to reduce the number of CTCs on postoperative day 2 could exert a profoundly positive influence on patient prognosis. Research findings have demonstrated a notable correlation between an immediate increase in CTCs count before being defined as PR and prolonged efficacy and survival. Ikeda et al. [ 20 ] reported that patients with a ≥ 1.5 times increase in CTCs count (n = 6; median, 732.5 days) exhibited significantly longer progression-free survival (PFS) compared to those with < 1.5 times increase (n = 3; median, 127 days). Likewise, a substantial elevation in the CTCs count was observed in our study on postoperative day 2. As indicated earlier, CTCs<4 or CTCs increased ≤ 3 signify improved patient survival. The CTCs clusters, often referred to as circulating tumor microemboli (CTM), composed of more than 2 CTCs, platelets, fibroblasts, etc., were suggested to possess 23- to 50-fold stronger metastatic ability [ 21 , 22 ] . Previous research has demonstrated that pancreatic ductal adenocarcinoma patients with CTM experienced poorer overall survival and disease-free survival compared to those without CTM [ 8 , 9 ] . Similarly, our survival analysis revealed that CTM- patients had a longer PFS (p = 0.029, HR = 2.92, 95% CI 1.05–8.07) and OS (p = 0.03, HR = 2.92, 95% CI 1.06–8.09) than CTM + patients. Moreover, it was observed that the majority of CTM were detected after initial treatment, with only 21.4% detected pre-treatment; the CTM detection rate in the surgery arm and neoadjuvant arm showed a significant difference of 26.3% (5/19) VS 75% (9/12). Further investigation is required to elucidate the reasons behind these results. Conclusions The research indicates that dynamic changes in the quantity of CTCs/CTMs do occur during the treatment process, and such variations play a significant predictive role in the prognosis of patients. Due to the relatively small sample size in this study, further validation is required in larger cohorts. Additionally, the relationship between the occurrence of CTMs and treatment modalities warrants further investigation. Declarations The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Lu Han , E-mail address: [email protected] Author Contribution WJ , CS ,and LHwrote the main manuscript text and LY and SZG prepared figures and tables.All authors have read this manuscript and would like to have it considered exclusively for publication in “scientific reports”. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.Lu Han E-mail address: [email protected] References Hulscher JB, van Sandick JW, de Boer AG, et al. Extended transthoracic resection compared with limited transhiatal resection for adenocarcinoma of the esophagus. N Engl J Med 2002;347(21):1662–69. Omloo JMT, Lagarde SM, Hulscher JBF, et al. Extended Transthoracic Resection Compared With Limited Transhiatal Resection for Adenocarcinoma of the Mid/Distal Esophagus. Annals of Surgery 2007;246:992-1001. Oppedijk V, van der Gaast A, van Lanschot JJB, et al. Patterns of Recurrence After Surgery Alone Versus Preoperative Chemoradiotherapy and Surgery in the CROSS Trials. Journal of Clinical Oncology 2014;32:385-91. Chen G, Wang Z, Liu Xy, Liu Fy. Recurrence Pattern of Squamous Cell Carcinoma in the Middle Thoracic Esophagus after Modified Ivor‐Lewis Esophagectomy. World Journal of Surgery 2007;31:1108-15. Shapiro J, van Lanschot JJB, Hulshof MCCM, et al. Neoadjuvant chemoradiotherapy plus surgery versus surgery alone for oesophageal or junctional cancer (CROSS): long-term results of a randomised controlled trial. The Lancet Oncology 2015;16:1090-98. Ando N, Kato H, Igaki H, et al. A Randomized Trial Comparing Postoperative Adjuvant Chemotherapy with Cisplatin and 5-Fluorouracil Versus Preoperative Chemotherapy for Localized Advanced Squamous Cell Carcinoma of the Thoracic Esophagus (JCOG9907). Annals of Surgical Oncology 2011;19:68-74. Cohen SJ, Punt CJA, Iannotti N, et al. Relationship of Circulating Tumor Cells to Tumor Response, Progression-Free Survival, and Overall Survival in Patients With Metastatic Colorectal Cancer. Journal of Clinical Oncology 2008;26:3213-21. Jin F, Zhu L, Shao J, et al. Circulating tumour cells in patients with lung cancer universally indicate poor prognosis. European Respiratory Review 2022;31:220151. Gao T, Mao J, Huang J, et al. Prognostic significance of circulating tumor cell measurement in the peripheral blood of patients with nasopharyngeal carcinoma. Clinics 2023;78:100179. Ko JMY, Lam KO, Kwong DLW, et al. Circulating Tumor Cell Enumeration for Serial Monitoring of Treatment Outcomes for Locally Advanced Esophageal Squamous Cell Carcinoma. Cancers 2023;15:832. Tang H, Wang H, Fang Y, et al. Neoadjuvant chemoradiotherapy versus neoadjuvant chemotherapy followed by minimally invasive esophagectomy for locally advanced esophageal squamous cell carcinoma: a prospective multicenter randomized clinical trial. Annals of Oncology 2023;34:163-72. Han L, Li Y-J, Zhang W-D, Song P-P, Li H, Li S. Clinical significance of tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma. Medicine 2019;98:e13921. Barsan V, Cristofanilli M, Mani SA. Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition. Breast Diseases: A Year Book Quarterly 2013;24:225-26. Tan K, Leong SM, Kee Z, et al. Longitudinal monitoring reveals dynamic changes in circulating tumor cells (CTCs) and CTC-associated miRNAs in response to chemotherapy in metastatic colorectal cancer patients. Cancer Letters 2018;423:1-8. Wang SQ, Shuai ZF, Zhang XJ, Wu T, Dong HY, Liu T, Wen QT, Yu XW. Detection of CTCs and CSCs in the staging and metastasis of non-small cell lung cancer based on microfluidic chip and the diagnostic significance. Eur Rev Med Pharmacol Sci. 2020 Sep;24(18):9487-96. Vona G, Sabile A, Louha M, et al. Isolation by Size of Epithelial Tumor Cells. The American Journal of Pathology 2000;156:57-63. Han J, Wang Z, Liu C. Survival and complications after neoadjuvant chemotherapy or chemoradiotherapy for esophageal cancer: a meta-analysis. Future Oncol. 2021 Jun;17(17):2257-74. Hashimoto M, Tanaka F, Yoneda K, et al. Positive correlation between postoperative tumor recurrence and changes in circulating tumor cell counts in pulmonary venous blood (pvCTC) during surgical manipulation in non-small cell lung cancer. Journal of Thoracic Disease 2018;10:298-306. Wei S, Guo C, He J, 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. JAMA Surgery 2019;154:e190972. Ikeda M, Koh Y, Teraoka S, et al. Longitudinal Evaluation of PD-L1 Expression on Circulating Tumor Cells in Non-Small Cell Lung Cancer Patients Treated with Nivolumab. Cancers 2021;13:2290. Diaz LA, Bardelli A. Liquid Biopsies: Genotyping Circulating Tumor DNA. Journal of Clinical Oncology 2014;32:579-86. Aceto N, Bardia A, Miyamoto David T, et al. Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis. Cell 2014;158:1110-22. Tables Table 1 Clinical pathological characteristics of the patients. Patietnts,No.(%) Characteristics Surgery group(n=19) Neoadjuvant group(n=12) Age,y ＜60 5（26.3） 5（41.7） ≥60 14（73.7） 7（58.3） Sex Male 15（78.9） 12（100） Female 4（21.1） 0 Alcohol drinking 6（31.6） 5（41.7） no drinking 13（68.4） 7（58.3） KPS ≥90 11（57.9） 9（75.0） ＜90 8（42.1） 3（25.0） Tumor location upper 2（10.5） 4（33.3） middle 10（52.6） 6（50.0） lower 7（36.8） 2（16.7） AJCC-UICC stage I 0 1（8.3） II 12（63.2） 4（33.3） III 7（36.8） 7（58.3） IV 0 0 Differentiation grade well 2（10.5） 5（41.7） mode 13（68.4） 4（33.3） poor 4（21.1） 2（16.7） other 0 1（8.3） Tumor depth, Tis-T1 2（10.5） 2（16.7） T2 5（26.3） 1（8.3） T3 11（57.9） 7（58.3） T4 2（10.5） 2（16.7） LNM yes 12（63.2） 8(66.7) no 7（36.8） 4(33.3) venous invasion positive 18（94.7） 19(100) negative 1（5.3） 0 CEA ＜3.4 16（84.2） 9（75.0） ≥3.4 3（15.8） 3（25.0） Platelet normal 11（57.9） 11(91.7) abnormal 8（42.1） 1（8.3） N/L ratio normal 1.5-3.5 9（47.4） 9（75.0） abnormal 10（52.6） 2（16.7） SCC normal 2.5 17（89.5） 10(83.3) abnormal 2（10.5） 2(15.4) Cyfra21-1 normal 3.3 14（73.7） 10(83.3) abnormal 5（26.3） 2(15.4) Table 2 Differences in the number of CTCs between surgery arm and neoadjuvant arm at different time points all p surgery arm p neoadjuvant arm p preoperative vs postoperative day 2 0.050 0.225 0.020 preoperative vs postoperative 1 week 0.312 0.396 0.231 preoperative vs postoperative 3 month 0.000 0.000 0.003 postoperative day 2 vs postoperative 1 week 0.008 0.083 0.003 postoperative day 2 vs postoperative 3 month 0.000 0.001 0.000 postoperative 1 week vs postoperative 3 month 0.000 0.000 0.002 pre-neoadjuvant chemotherapy vs preoperative NA NA 0.212 pre-neoadjuvant chemotherapy vs postoperative day 2 NA NA 0.003 pre-neoadjuvant chemotherapy vs postoperative 1 week NA NA 0.832 pre-neoadjuvant chemotherapy vs postoperative 3 month NA NA 0.022 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4640180","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329999322,"identity":"4bca696b-88a3-41cf-b76b-59009c7ca623","order_by":0,"name":"Jin Wang","email":"","orcid":"","institution":"JeonbukNational University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Wang","suffix":""},{"id":329999323,"identity":"7abfc1e7-42f5-4b2e-b6dc-ef8a5e21b2b3","order_by":1,"name":"Shuang Chen","email":"","orcid":"","institution":"The University of 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05:54:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4640180/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4640180/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61184159,"identity":"b4fd3269-6798-4866-b9ea-bd8a09c05b66","added_by":"auto","created_at":"2024-07-26 17:10:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":171553,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4640180/v1/622eb2162ba8fd48216d7231.png"},{"id":61184164,"identity":"a7385176-2c23-455a-a2bb-57062e8617a8","added_by":"auto","created_at":"2024-07-26 17:10:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1716091,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure 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legend\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4640180/v1/6ab3fd14d45d8de16aa8c164.png"},{"id":61184163,"identity":"894fca8f-282d-498d-bc2f-d16503add026","added_by":"auto","created_at":"2024-07-26 17:10:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":154365,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4640180/v1/60634f6f97bc63cf63441179.png"},{"id":61936750,"identity":"13ad7590-d7b7-4eab-b9ce-67081977245c","added_by":"auto","created_at":"2024-08-07 09:19:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6035484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4640180/v1/547ab6c5-eacf-404f-8975-9c0e722d970d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Longitudinal evaluation of circulating tumor cells in operable locally advanced esophageal squamous cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLocally advanced esophageal squamous cell carcinoma (AJCC TNM staging system≥cT2 and/or cN1-3, M0, LAEC) is characterized by the invasion of local structures or involvement of regional lymph nodes without distant metastases. However, relying solely on surgery for the treatment of locally advanced cases is linked to a median survival ranging from 12 to 18 months and a five-year survival rate of 15–39%\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e1-3\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. When surgical resection is the exclusive therapeutic approach, both local and systemic recurrence is prevalent, affecting 35–50% of patients\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e4\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. A standard initial therapeutic strategy for these patients involves neoadjuvant chemotherapy (nCT) or chemoradiotherapy (nCRT) followed by surgery\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e5\u003c/sup\u003e\u003csup\u003e, \u003c/sup\u003e\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. This approach serves to decrease surgical complexity by reducing tumor size, enhance the R0 resection rate, and improve patients' tolerance to chemotherapy compared to postoperative chemotherapy. Additionally, it facilitates the early treatment of micrometastases.\u003c/p\u003e\n\u003cp\u003eCirculating tumor cells (CTCs) represent tumor cells that are released into the peripheral blood from primary tumors or metastatic lesions, either spontaneously or as a result of surgical intervention\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. CTCs possess the potential to serve as precursors of metastases\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e7\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e, offering crucial guidance for the prognosis of diverse tumors\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e8-10\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. In contrast to histology, CTCs present an advantage in repeatedly obtaining required specimens with low invasiveness.\u003c/p\u003e\n\u003cp\u003eThe clinical significance of tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma was previously examined\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. It was determined that CTCs function as independent prognostic indicators of patient outcomes in ESCC. The consideration of CTCs has the potential to enhance the accuracy of preoperative staging in ESCC\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e12\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. However, the appearance of CTCs undergoes dynamic changes during treatment, including alterations in quantity and molecular phenotype\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e13-15\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. Single-time detection offers limited information for prognostic evaluation. This study aims to: 1) longitudinally investigate changes in CTCs status during neoadjuvant chemotherapy, surgery, and postoperative chemotherapy within the same cohort; 2) assess patients' response to treatment in real-time; 3) further analyze the prognostic significance of CTCs status.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe flow chart of the experiment was shown in Figure 1, and its duration was from July 2017 to December 2018. All 31 participants granted written informed consent for their involvement, then a 5-year follow-up was performed. The 31 patients with LAEC were followed for at least 5 years. LAEC progression, and times of recurrence, death, and disease free survival were recorded. Progression-free survival (PFS) was defined as the time from the onset of CTCs testing to CT progression or patient death. If the patient was lost to follow-up, or at the end of follow-up LAEC had not progressed, then this was recorded as a delete value. When a patient was not rehospitalized after discharge and the telephone follow-up was not answered by patients or their relatives, the patient’s initial hospitalization time was also taken as a PFS deletion. Overall survival was analyzed when patients were followed for 5 years. Approval was granted by the Shandong Provincial Cancer Research Institute Ethics Committee approved this study(201702012). CTCs testing and follow-up methods were as our previous study[12].\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the participants. All methods in this study were in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePathological confirmation of esophageal squamous cell carcinoma (ESCC) through endoscopic biopsy was conducted prior to surgery (Stage II-IIIb). Gastroscopy and endoscopic ultrasonography were employed for a comprehensive assessment of tumor location and depth of invasion. Enhanced CT scans of the chest and abdomen were utilized to evaluate the extent of external invasion and the presence of suspected lymph node metastasis. When necessary, endoscopic ultrasonography or B-ultrasonography was performed to ascertain the existence of liver or distant metastasis. The exclusion criteria are as follows: 1) a history of malignant tumors; TNM stage IIIc-Ⅳ\u0026nbsp;(T4b or N3 or M1); 2) severe complications affecting the heart, liver, lung, brain, kidneys, and other organs; 3) tumor perforation into the lung leading to abscess formation; 4) cervical lymph node metastasis occurring in the lower thoracic segment of esophageal cancer; 5) paraperitoneal lymph node metastasis in cervical esophageal cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurgery and chemotherapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVideo-assisted thoracoscopic surgery (VATS) in conjunction with laparoscopic or open thoracic McKeown esophagectomy was employed for the procedure. Lymph node dissection encompassed: 1) chest: paraesophageal lymph nodes, subcarina lymph nodes, left and right pararyngeal recurrent nerve lymph nodes, trachea, and bronchial lymph nodes; 2) abdomen: lymph nodes of paracardia, lymph nodes of the lesser curvature of the stomach, especially lymph nodes of the left gastric vessel, lymph nodes of the common hepatic artery, and suspected lymph nodes of the splenic artery. For patients with middle and upper thoracic esophageal cancer, selective neck dissection is considered viable when cervical lymph node metastasis is suspected based on preoperative enhanced CT evaluation of the right recurrent nerve lymph node and ultrasound exploration of cervical lymph nodes.\u003c/p\u003e\n\u003cp\u003eIn the neoadjuvant arm, the DP regimen comprised 2 cycles of docetaxel (75 mg/m2 on Day 1) and cisplatin (25 mg/m2 on Day 1-3), administered once every 3 weeks. Two cycles of the DP regimen were continued 1 month after surgery. In the chemotherapy arm, 4 cycles of the DP regimen were continued 1 month after surgery. Postoperative adjuvant concurrent chemoradiotherapy was determined based on pathological type, lymph node metastasis, positive surgical margin, and postoperative stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCTCs analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 136 CTCs detections were peformed. The time points selected for CTCs detection were as follows: 1) In the neoadjuvant arm(n=12, 60 CTCs detections): pre-neoadjuvant chemotherapy, preoperative, postoperative day 2, post-operative 1 week, post-operative 3 months. 2) In the surgery arm(n=19, 76 CTCs detections): preoperative, post-operative day 2, post-operative 1 week, post-operative 3 months. The ISET assay was conducted following the protocol outlined by Vona et al\u003csup\u003e[\u003c/sup\u003e\u003ca href=\"#_ENREF_16\" title=\"Vona, 2000 #380\"\u003e\u003csup\u003e16\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e]\u003c/sup\u003e. A total of 5 milliliters of whole blood were diluted to 8 mL using a buffer containing 0.2% formaldehyde and filtered through an 8mm pore size membrane. An automated testing platform was employed to process the samples. The collected CTCs and circulating tumor microemboli were stained with Romanowsky stain, air-dried, mounted, and independently reviewed by three senior cytopathologists without prior knowledge. Cells were identified as CTCs if they exhibited\u0026nbsp;≥4 of the following morphological characteristics: atypical nucleus (irregular shape or presence of a nodular, lobulated contour); nuclear-cytoplasmic ratio \u0026gt;0.8; nuclear long diameter \u0026gt;18mm; hyperchromatic nuclei and nonhomogeneous staining; thickened, sunken, wrinkled, and jagged nuclear membrane; presence of nuclear chromatin side-shift or a large nucleoli or presence of abnormal mitotic figures; and presence of tumor cell aggregations, or circulating tumor microemboli (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurvival analysis was conducted utilizing the \"survival\" and \"survminer\" packages in R version 4.3.0. Scatter plots were generated employing \"ggplot2\" and \"RColorBrewer.\" The comparison of two datasets and the creation of receiver operating characteristic (ROC) curves were executed using the Statistical Package for Social Sciences software (SPSS Version 27). Additional statistical analyses were carried out through the utilization of EXCEL(Microsoft Excel 2016) software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnrolled were 31 patients diagnosed with stage I to III esophageal squamous cell carcinoma (neoadjuvant arm: 12, surgery arm: 19). The clinicopathological characteristics of the patients are illustrated in Table 1 and Figure 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLongitudinal Evaluation of CTCs Detected in Patient Samples \u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eThe longitudinal evolution of CTC numbers was depicted in Figure 4. Prior to neoadjuvant therapy, CTCs were found in 6 out of 12 patients (50%, median CTC count 0.5; range 0–5). Before surgery, CTCs were found in 14 out of 31 patients (45.16%, median CTC count 0; range 0–19). On postoperative day 2, CTCs were found in 22 out of 31 patie.0nts (70.97%, median CTC count 3; range 0–37). At one week postoperative, CTCs were found in 16 out of 31 patients (51.6%, median CTC count 1; range 0–14). Three months postoperative, CTCs were found in 5 out of 31 patients (16.1%, median CTC count 0; range 0–4).\u003c/p\u003e\n\u003cp\u003eA comparative analysis of CTC numbers at various time points was conducted. Patients were categorized into three groups: all patients (31), the surgery arm (19), and the neoadjuvant arm (12). The results revealed statistical differences in CTC numbers between preoperative and postoperative day 2 (p=0.05), preoperative and postoperative 3 months (p=0), postoperative day 2 and postoperative 1 week (p=0.008), postoperative day 2 and postoperative 3 months (p=0), and postoperative 1 week and postoperative 3 months (p=0) among all 31 patients. In the surgery arm, statistical significance was observed in preoperative vs postoperative 3 months (p=0) and postoperative 1 week vs postoperative 3 months (p=0). In the neoadjuvant arm, with the exception of preoperative vs postoperative 1 week (p=0.231), pre-neoadjuvant chemotherapy vs preoperative (p=0.212), pre-neoadjuvant chemotherapy vs postoperative 1 week (p=0.832), all other comparative analyses exhibited statistical significance (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNumber of CTCs change after neoadjuvant chemotherapy or surgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe number of circulating tumor cells (CTCs) was computed at two distinct time points for 31 patients through subtraction. The outcomes were categorized into two groups: a reduced or equal group and an increased group. The findings indicated that, in the comparison of CTCs numbers between patients undergoing neoadjuvant chemotherapy (pre-neoadjuvant minus preoperative), 66.70% exhibited a decrease or remained unchanged, while 33.3% showed an increase (Figure 5a). In the analysis of CTCs numbers for patients undergoing surgery (postoperative day 2 minus pre-treatment), 58% experienced an increase, whereas 42% demonstrated a reduction or remained unchanged (Figure 5b). Similarly, for the interval between post-operative 1 week and preoperative, 61.3% displayed a decrease or remained equal, and 38.7% manifested an increase (Figure 5c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssociation between CTCs and survival time\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eThe median survival time for the cohort of 31 patients was 36 months. Among patients initially detected with circulating tumor cells (CTCs+, n=13), the neoadjuvant arm exhibited an extended progression-free survival (PFS) (p=0.05, Figure 6a) and overall survival (OS) (p=0.04, Figure 6b) compared to the surgical arm. The ROC curve (Figure 7) demonstrated that the number of CTCs on postoperative day 2 provided a superior diagnostic value for predicting patient prognosis (AUC=0.758, p=0.014, 95% CI 0.579-0.938).\u003c/p\u003e\n\u003cp\u003eThe determined cutoff value for the number of CTCs was 3.5. Consequently, a cutoff value of 4 was selected for subsequent analysis. On postoperative day 2, 17 patients had CTCs\u0026lt;4, while 14 patients had CTCs≥4. Patients with CTCs\u0026lt;4 exhibited significantly prolonged PFS (p\u0026lt;0.01, HR=6.26, 95% CI 1.96-19.96, Figure 6c) and OS (p\u0026lt;0.01, HR=6.63, 95% CI 2.08-21.13, Figure 6d) compared to those with CTCs≥4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLongitudinal Evaluation of CTCs and survival time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the comparison of CTCs numbers between postoperative day 2 and pre-treatment, 20 patients exhibited an increased quantity of CTCs. Among them, 10 patients experienced an increase exceeding 3, while the remaining 10 patients showed an increase of less than 3. Notably, patients with a CTCs increase of ≤3 demonstrated significantly prolonged progression-free survival (median, 59 months vs. 20 months, p=0.035, HR=3.29, 95% CI 1-10.9, Figure 6e) and overall survival (median, 61 months vs. 21 months, p=0.035, HR=3.78, 95% CI 1-14.3, Figure 6f) compared to those whose CTCs increased by \u0026gt;3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssociation between CTM and survival time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCTM was detected in 14 out of 31 patients (45.16%) at all time points, as previously indicated. The majority of CTM instances were identified post-initial treatment, with only 3 (21.4%) CTM+ patients detected pre-treatment. The CTM detection rates were 26.3% (5/19) and 75% (9/12) in the surgery arm and neoadjuvant arm, respectively. Survival analysis revealed that CTM-negative patients exhibited an extended progression-free survival (PFS) (p=0.029, HR=2.92, 95% CI 1.05-8.07, Figure 6g) and overall survival (OS) (p=0.03, HR=2.92, 95% CI 1.06-8.09, Figure 6h) compared to CTM-positive patients. Further analysis of CTM-positive patients indicated that, when comparing the difference in CTCs numbers between postoperative day 2 and preoperative, patients with a decreased or unchanged number of CTCs demonstrated superior survival (no statistical difference, Figure 6i-j) compared to those with an increased number.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRandom allocation was employed for the division of 31 patients diagnosed with operable LAEC into the operation and neoadjuvant groups. CTCs detection was conducted at various time points throughout the treatment on the same patient. Differential impacts on the alterations in the quantity of CTCs/CTM were observed between surgery and neoadjuvant chemotherapy. Neoadjuvant chemotherapy resulted in a reduction in the number of CTCs/CTM, while surgery led to an increase in the number of CTCs/CTM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The study findings suggested that regardless of an increase or decrease, this alteration served as a predictive indicator for patient outcomes.\u003c/p\u003e \u003cp\u003eNeoadjuvant interventions manifested both local and systemic effects, delivering a survival advantage through potential synergistic actions and stood as the standard treatment for LAEC \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In this investigation, a noticeable decline in CTCs number post pre-neoadjuvant chemotherapy was evident (66.70% reduction or equivalence, 33.30% increase, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), signifying the efficacy of neoadjuvant therapy. Subsequent analysis disclosed that within the cohort of initially identified CTCs\u0026thinsp;+\u0026thinsp;patients, the neoadjuvant arm exhibited an extended progression-free survival (PFS) (p\u0026thinsp;=\u0026thinsp;0.05) and overall survival (OS) (p\u0026thinsp;=\u0026thinsp;0.04) compared to the surgery arm. In essence, first-time detected CTCs\u0026thinsp;+\u0026thinsp;patients derived more substantial survival benefits, strongly advocating the consideration of neoadjuvant therapy. In summary, this investigation highlights that neoadjuvant therapy can promptly diminish the number of CTCs in the bloodstream and, in the long term, effectively elongate the survival duration of patients. This bears significant ramifications for the management of LAEC.\u003c/p\u003e \u003cp\u003eIn the context of surgical procedures, the dissemination of tumor cells into the bloodstream was recognized. Hashimoto et al.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e established a significant correlation between the increased count of lobar pulmonary vein CTCs during surgical manipulation and postoperative distant metastasis in NSCLC patients who underwent complete resection. Wei et al. \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e observed that prioritizing the ligation of effluent veins during surgery might mitigate tumor cell dissemination and enhance survival outcomes in non\u0026ndash;small cell lung cancer patients. Postoperative day 2 served as a crucial temporal reference point in our study, revealing a statistical discrepancy in CTC numbers between preoperative and postoperative day 2 for all 31 patients (p\u0026thinsp;=\u0026thinsp;0.05), as well as for 12 patients in the neoadjuvant arm (p\u0026thinsp;=\u0026thinsp;0.02). Regarding survival analysis, patients with CTCs\u0026lt;4 exhibited superior survival compared to those with CTCs\u0026thinsp;\u0026ge;\u0026thinsp;4. When comparing the difference in CTC numbers between postoperative day 2 and pre-treatment, patients with an increase of \u0026le;\u0026thinsp;3 CTCs demonstrated significantly better survival outcomes than those with an increase of \u0026gt;3 CTCs. The ROC curve(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e) further underscored the heightened diagnostic efficacy of postoperative day 2 in comparison to other time points. Consequently, CTCs on postoperative day 2 emerged as a potent predictor of the survival prognosis for LAEC patients. Implementing diverse strategies to reduce the number of CTCs on postoperative day 2 could exert a profoundly positive influence on patient prognosis.\u003c/p\u003e \u003cp\u003eResearch findings have demonstrated a notable correlation between an immediate increase in CTCs count before being defined as PR and prolonged efficacy and survival. Ikeda et al.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e reported that patients with a\u0026thinsp;\u0026ge;\u0026thinsp;1.5 times increase in CTCs count (n\u0026thinsp;=\u0026thinsp;6; median, 732.5 days) exhibited significantly longer progression-free survival (PFS) compared to those with \u0026lt;\u0026thinsp;1.5 times increase (n\u0026thinsp;=\u0026thinsp;3; median, 127 days). Likewise, a substantial elevation in the CTCs count was observed in our study on postoperative day 2. As indicated earlier, CTCs\u0026lt;4 or CTCs increased\u0026thinsp;\u0026le;\u0026thinsp;3 signify improved patient survival.\u003c/p\u003e \u003cp\u003eThe CTCs clusters, often referred to as circulating tumor microemboli (CTM), composed of more than 2 CTCs, platelets, fibroblasts, etc., were suggested to possess 23- to 50-fold stronger metastatic ability\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Previous research has demonstrated that pancreatic ductal adenocarcinoma patients with CTM experienced poorer overall survival and disease-free survival compared to those without CTM\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Similarly, our survival analysis revealed that CTM- patients had a longer PFS (p\u0026thinsp;=\u0026thinsp;0.029, HR\u0026thinsp;=\u0026thinsp;2.92, 95% CI 1.05\u0026ndash;8.07) and OS (p\u0026thinsp;=\u0026thinsp;0.03, HR\u0026thinsp;=\u0026thinsp;2.92, 95% CI 1.06\u0026ndash;8.09) than CTM\u0026thinsp;+\u0026thinsp;patients. Moreover, it was observed that the majority of CTM were detected after initial treatment, with only 21.4% detected pre-treatment; the CTM detection rate in the surgery arm and neoadjuvant arm showed a significant difference of 26.3% (5/19) VS 75% (9/12). Further investigation is required to elucidate the reasons behind these results.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe research indicates that dynamic changes in the quantity of CTCs/CTMs do occur during the treatment process, and such variations play a significant predictive role in the prognosis of patients. Due to the relatively small sample size in this study, further validation is required in larger cohorts. Additionally, the relationship between the occurrence of CTMs and treatment modalities warrants further investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLu Han , E-mail address:\u0026nbsp;\u003c/strong\u003e\u003cstrong\[email protected]\u003c/strong\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWJ , CS ,and LHwrote the main manuscript text and LY and SZG prepared figures and tables.All authors have read this manuscript and would like to have it considered exclusively for publication in \u0026ldquo;scientific reports\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.Lu Han E-mail address: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHulscher JB, van Sandick JW, de Boer AG, et al. 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Cell 2014;158:1110-22.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Clinical pathological characteristics of the patients.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"532\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.44360902255639%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.27067669172932%\" valign=\"bottom\"\u003e\n \u003cp\u003ePatietnts,No.(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.285714285714285%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eSurgery group(n=19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNeoadjuvant group(n=12)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAge,y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;＜60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5（26.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5（41.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026ge;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14（73.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7（58.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15（78.9）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12（100）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4（21.1）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAlcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; drinking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6（31.6）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5（41.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; no drinking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13（68.4）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7（58.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eKPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026ge;90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11（57.9）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9（75.0）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;＜90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8（42.1）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3（25.0）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTumor location\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; upper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（10.5）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4（33.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; middle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10（52.6）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6（50.0）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; lower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7（36.8）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（16.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAJCC-UICC stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1（8.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12（63.2）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4（33.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7（36.8）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7（58.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDifferentiation grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ewell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（10.5）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5（41.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003emode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13（68.4）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4（33.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003epoor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4（21.1）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（16.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eother\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1（8.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTumor depth,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTis-T1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（10.5）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（16.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5（26.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1（8.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11（57.9）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7（58.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eT4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（10.5）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（16.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLNM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12（63.2）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8(66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eno\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7（36.8）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4(33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003evenous invasion\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18（94.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19(100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1（5.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e＜3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16（84.2）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9（75.0）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026ge;3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3（15.8）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3（25.0）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePlatelet\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003enormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11（57.9）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11(91.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eabnormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8（42.1）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1（8.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eN/L ratio\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003enormal 1.5-3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9（47.4）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9（75.0）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eabnormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10（52.6）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（16.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSCC\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003enormal 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17（89.5）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10(83.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eabnormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2（10.5）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2(15.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCyfra21-1\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003enormal 3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14（73.7）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10(83.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eabnormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5（26.3）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2(15.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Differences in the number of CTCs between surgery arm and neoadjuvant arm at different time points\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable style=\"width: 4.3e+2pt;border: none;border-collapse:collapse;margin-left:6.75pt;margin-right:6.75pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;border-top:solid black 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;border-top:solid black 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eall\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003ep\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;border-top:solid black 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003esurgery arm\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003ep\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;border-top:solid black 1.0pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eneoadjuvant arm\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003ep\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epreoperative vs postoperative day 2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.050\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e0.225\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.020\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epreoperative vs postoperative 1 week\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e0.312\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e0.396\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e0.231\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epreoperative vs postoperative 3 month\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epostoperative day 2 vs postoperative 1 week\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.008\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e0.083\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epostoperative day 2 vs postoperative 3 month\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epostoperative 1 week vs postoperative 3 month\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epre-neoadjuvant chemotherapy vs preoperative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e0.212\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epre-neoadjuvant chemotherapy vs postoperative day 2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epre-neoadjuvant chemotherapy vs postoperative 1 week\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e0.832\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:238.75pt;border:none;border-bottom:solid black 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003epre-neoadjuvant chemotherapy vs postoperative 3 month\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.5pt;border:none;border-bottom:solid black 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:66.85pt;border:none;border-bottom:solid black 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eNA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:91.25pt;border:none;border-bottom:solid black 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:13.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:middle;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:red;'\u003e0.022\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"circulating tumor cells, operable locally advanced esophageal squamous cell carcinoma, neoadjuvant chemotherapy","lastPublishedDoi":"10.21203/rs.3.rs-4640180/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4640180/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eThe detection of circulating tumor cells (CTCs) at a single time point offers restricted insights into prognostic assessment. This study aims to longitudinally investigate alterations in CTCs status throughout the neoadjuvant chemotherapy, surgical, and postoperative chemotherapy phases within a homogeneous cohort. Further more, an assessment of the interrelation between patients' treatment response, survival prognosis, and CTCs status was conducted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods: \u003c/strong\u003eThirty-one patients diagnosed with operable locally advanced esophageal squamous cell carcinoma were randomly allocated to either the surgical or neoadjuvant group. CTCs detection was systematically conducted at various time points throughout the treatment on individual patients. Associations between the presence of CTCs/CTM and therapeutic efficacy, as well as clinical outcomes, were subject to rigorous analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe performed a total of 136 CTCs detections at the time points of pre-neoadjuvant chemotherapy, preoperative, postoperative day 2, post-operative 1 week, post-operative 3 months. The CTCs were detected in 6/12(50％), 14/31(45.16％), 22/31(70.97％), 16/31(51.6％) and 5/31(16.1％) of patients before neoadjuvant treatment, prior to surgery, on postoperative day 2, one week after surgery, and three months post-surgery, respectively. The CTC count exhibited an increase before and after surgery, whereas a decrease was observed before and after neoadjuvant chemotherapy. Among patients initially identified with CTCs+, those in the neoadjuvant group experienced extended progression-free survival (PFS) (p=0.05) and overall survival (OS) (p=0.04) compared to those in the surgical group. On postoperative day 2, 17 patients had CTCs＜4, while 14 patients had CTCs≥4. Individuals with CTCs＜4 demonstrated significantly prolonged PFS (p＜0.01, HR=6.26, 95% CI 1.96-19.96) and OS (p＜0.01, HR=6.63, 95% CI 2.08-21.13) compared to those with CTCs≥4. CTM was detected in 45.16% of patients at any given time. Survival analysis indicated that CTM- patients exhibited longer PFS (p=0.029, HR=2.92, 95% CI 1.05-8.07) and OS (p=0.03, HR=2.92, 95% CI 1.06-8.09) than CTM+ patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eSingle-time detection offers limited information for prognostic evaluation, a dynamic fluctuation in the quantity of CTCs/CTM throughout the course of treatment, exhibiting a robust predictive impact on patient prognosis.\u003c/p\u003e","manuscriptTitle":"Longitudinal evaluation of circulating tumor cells in operable locally advanced esophageal squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-26 17:10:40","doi":"10.21203/rs.3.rs-4640180/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a7456e70-9c1a-4829-851e-0529dded8e52","owner":[],"postedDate":"July 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34928996,"name":"Biological sciences/Cancer/Cancer therapy/Chemotherapy"},{"id":34928997,"name":"Biological sciences/Stem cells/Cancer stem cells"}],"tags":[],"updatedAt":"2024-08-07T09:19:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-26 17:10:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4640180","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4640180","identity":"rs-4640180","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}