Porphyromonas gingivalis levels correlate with Toll-like receptor 4 expression status and prognosis after esophagectomy for advanced thoracic esophageal squamous cell carcinoma

Porphyromonas gingivalis levels correlate with Toll-like receptor 4 expression status and prognosis after esophagectomy for advanced thoracic 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 Research Article Porphyromonas gingivalis levels correlate with Toll-like receptor 4 expression status and prognosis after esophagectomy for advanced thoracic esophageal squamous cell carcinoma Takatoshi Yoneya, Yusuke Sato, Kyoko Nomura, Akiyuki Wakita, Yushi Nagaki, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5874217/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 Background. Poor oral health is an independent risk factor for upper-aerodigestive tract cancers, including esophageal squamous cell carcinoma (ESCC). Porphyromonas gingivalis (P. gingivalis) , the most studied periodontal pathogen, is a Gram-negative bacterium with walls composed of lipopolysaccharide (LPS). Toll-like receptor 4 (TLR4) recognizes LPS, and we previously reported that ESCC patients exhibiting high TLR4 expression also show significantly poorer survival than patients expressing lower levels of TLR4. We therefore hypothesized that P. gingivalis affects disease progression and prognosis in patients with ESCC. Patients and Methods. We used an ESCC tissue microarray to assess the P. gingivalis status of ESCC patients and to determine whether P. gingivalis levels correlate with prognosis after curative esophagectomy. We also tested whether the combined P. gingivalis and TLR4 statuses correlate with prognosis in these patients. Results . Clinical ESCC samples from 175 patients were tested for P. gingivalis . High P. gingivalis levels (3+ and 2+) correlated with significantly poorer 5-year overall survival (OS) and disease-specific survival (DSS) after esophagectomy than lower P. gingivalis levels (1+ and 0). Univariate and multivariate analyses showed that the P. gingivalis status is an independent prognostic factor affecting 5-year OS and DSS. Moreover, the combined P. gingivalis and TLR4 statuses are also an independent prognostic factor affecting 5-year OS and DSS. Conclusion . P. gingivalis infection leads to TLR4 expression and a poorer prognosis in ESCC patients after esophagectomy. Preventing P. gingivalis infection may be an effective strategy for improving the long-term outcomes of patients with ESCC. Porphyromonas gingivalis Toll-like receptor TLR4 esophageal squamous cell carcinoma ESCC prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Esophageal cancer is divided into two main histological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Well-known risk factors for ESCC are cigarette smoking, heavy alcohol consumption, poor dietary habits, and inactivation of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) [1,2]. In addition, recent reports suggest that poor oral health is also an independent risk factor for upper-aerodigestive tract cancers, including ESCC [3–6]. Consistent with that finding, we recently reported that the severity of periodontitis is an independent risk factor contributing to a poorer long-term prognosis after esophagectomy [7]. Toll-like receptors (TLRs) are a family of pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs) released from various viruses, bacteria, fungi and parasites [8,9]. These receptors play vital roles in mediating innate and antigen-specific adaptive immunity. A total of 10 TLRs, recognizing different PAMPs, have been identified in human beings [10–13]. It has been reported that about 700 species of bacteria reside in the mouths of human beings [14,15]. Among the oral bacterial flora are species classified as “beneficial” or “opportunistic” as well as “bad bacteria,” which are periodontal pathogens. These periodontal pathogens are classified as “red complex,” “orange complex” or “yellow complex,” among others, based on their relation to periodontitis [16–18]. All three species classified as “red complex,” including the most well-studied Porphyromonas gingivalis , and most species classified as “orange complex” are Gram-negative bacteria with cell walls composed of lipopolysaccharide (LPS). TLR4 recognizes LPS, and we previously reported that ESCC patients exhibiting high TLR4 expression exhibit significantly poorer survival than those expressing lower levels of TLR4 [19]. Moreover, we recently reported that LPS promotes ESCC cell proliferation and migration in vitro by enhancing CCL2 production via TLR4 signaling [20]. These findings suggest that there may be a relationship between P. gingivalis infection and TLR4 expression status in clinical ESCC specimens. In the present study, therefore, we examined the association between P. gingivalis levels and expression of TLR4 in clinical specimens from ESCC patients and their correlation with long-term survival. MATERIALS AND METHODS Patients This study was approved by the Ethics Committee of Akita University School of Medicine on 20 November 2019. All experiments adhered to the principles of the Helsinki Declaration, and all study participants provided informed written consent. Between January 2000 and December 2011, 507 patients received esophagectomy to treat esophageal cancer at Akita University Hospital. Among them, 177 patients with pT2-pT4 thoracic ESCC who had received curative esophagectomy with no preoperative treatment were enrolled in this study. The clinical cancer stages and treatment strategies were discussed and decided by a cancer board composed of radiologists, oncologists, gastroenterologists and surgeons. Pathological stages were determined according to the UICC International Union Against Cancer Tumor-Node-Metastasis (TNM) Classification of Malignant Tumors (8th edition). Esophagectomy Our standard operative procedure was right thoracoscopic or robot-assisted thoracoscopic esophagectomy with extended three-field lymph node dissection. The three fields included: (1) mediastinal fields, involving the periesophageal region and areas around the trachea and bilateral main bronchus; (2) abdominal fields, involving the perigastric region and areas around the celiac axis; and (3) cervical fields involving the bilateral periesophageal and supraclavicular regions. Reconstruction often required insertion of a gastric tube via the posterior mediastinal route or the retrosternal route [21,22]. ESCC Tissue Microarray An ESCC tissue microarray (TMA) was constructed at the Pathology Institute, Toyama, Japan, as previously described [19–23]. Previous evaluations of TMAs showed that use of double cores measuring 0.6 mm in diameter adequately reflected the entire section and were sufficient to overcome cancer tissue heterogeneity [24]. We therefore employed triplicate cores measuring 0.6 mm in diameter to enhance the reliability further. Triplicate cores were randomly collected from separate carcinoma areas and transferred to the TMA. The total TMA block contained 531 cores (3 cores each from 177 paraffin main tumor blocks from the enrolled ESCC patients). However, six cores from two patients were lost from the microarray because of the thickness of the transplanted tissue. The evaluation was therefore based on 175 cases. Immunohistochemistry (IHC) Sections (4 µm) from the TMA were immunohistochemically examined using standard procedures [19–23]. Briefly, sections were incubated in citrate buffer (pH 6.0) for 10 min at 121 ℃ in an autoclave for antigen retrieval. Endogenous peroxidase activity was inactivated by incubation for 5 min in 3.0% H 2 O 2 , and internal biotin was blocked using a biotin-blocking system (Dako, Denmark). The sections were then incubated with an anti- P. gingivalis monoclonal antibody (1 µg/mL; D376-3, Medical Biological Laboratories Co., LTD., Tokyo, Japan) overnight at 4 ℃, after which the antigen was detected using a catalyzed signal amplification system (Dako) with a DAB peroxidase substrate. Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted. IHC staining was imaged and viewed using a NanoZoomer Digital Pathology C9600 slide scanner and Virtual Slide Viewer software (NDP.view2 version 2.9.29) (Hamamatsu Photonics, Hamamatsu, Japan). Using the HER2 IHC scoring system (ASCO and CAP guidelines), staining scores were then assigned by three physicians blinded to the clinical and prognostic data [25]. A sample was assigned an IHC score of 3 + if there was intense P. gingivalis staining in the cytoplasm or nuclei in more than 30% of cells, 2 + if there was moderate staining in the cytoplasm or nuclei in > 10% of cells, 1 + if there was weak staining, or 0 if there was no staining. High P. gingivalis levels were defined as an IHC score of 3 + or 2+; low levels were defined as a score of 1 + or 0. In cases where there was disagreement, the score assigned by 2 of the 3 physicians was used. Statistical Analysis Continuous variables are presented as the median (range: minimum–maximum). To assess differences between the P. gingivalis -high and P. gingivalis -low groups, the Mann-Whitney-Wilcoxon test (for continuous variables) or χ 2 and Fisher’s exact tests (for categorical variables) was used. The clinical end-points of this study were overall survival (OS) and disease-specific survival (DSS). Length of survival (censored at 60 months for longer survivors) was calculated from the date of esophagectomy to the patient’s death or the date of the last clinical follow-up. Kaplan-Meier curves were constructed to assess the association between OS and DSS and P. gingivalis status. Curves were compared using the log-rank test. Cox’s proportional hazards model was used to assess prognostic factors associated with OS and DSS in ESCC patients after curative esophagectomy. The variables adjusted for multivariate analysis were age, gender, pT, pN, pStage and tumor differentiation. All statistical analyses were performed using JMP Pro 17.2.0. Two-sided values of p < 0.05 were considered statistically significant. RESULTS Immunohistochemical Analysis of P. gingivalis levels Three representative cores assigned an IHC score of 3+, 2+, 1 + or 0 (Fig. 1 ) . IHC using the monoclonal antibody for P. gingivalis showed the presence of antigen in the cytoplasm of ESCC cells. Scored 3+, 2+, 1 + or 0 were obtained from 15 patients (8.6%), 89 patients (50.9%), 65 patients (37.1%) and 6 patients (3.4%), respectively. Thus, 104 patients were considered P. gingivalis -high (scores of 3 + or 2+), while 71 patients were considered P. gingivalis -low (scores of 1 + or 0). The clinicopathological characteristics of the two groups are summarized (Table 1 ). The P. gingivalis -high group exhibited significantly stronger TLR4 expression, whereas there were no significant differences between the groups with respect to the other factors shown in the table. Table 1. The clinicopathological characteristics of the ESCC patients (n=175) Characteristics P. gingivalis -high n= 104 ( 59.4 %) P. gingivalis -low n= 71 ( 40.6 %) P Gender Female Male 12 ( 16.9 %) 59 ( 83.1 %) 0. 311 12 ( 11.5 %) 92 ( 88.5 %) Age at surgery 66( 38-82 ) 66( 40-78 ) 0.546 Habitual smoking Current Past Never 57 ( 54.8 %) 25 ( 24.0 %) 22 ( 21.2 %) 42 ( 59.2 %) 13 ( 18.3 %) 16 ( 22.5% ) 0. 665 Habitual alcohol consumption Current Past Never 76 ( 73.1 %) 16 ( 15.4 %) 12 ( 11.5 %) 55 ( 77.5 %) 6 ( 8.4 %) 10 (14.1 %) 0. 379 Tumor location Upper Middle Lower 4 ( 3.8 %) 65 ( 62.5 %) 35 ( 33.7 %) 2 ( 2.8 %) 50 ( 70.4 %) 19 ( 26.8 %) 0. 554 Depth of invasion (pT) pT2 pT3 pT4a 21 ( 20.2 %) 75 ( 72.1 %) 8 ( 7.7 %) 10 ( 14.1 %) 60 ( 84.5 %) 1 ( 1.4 %) 0.0 83 Lymph node metastasis (pN UICC) pN0 pN1 pN2 pN3 M1 Lymph (supraclavicular) 27 ( 26.0 %) 33 ( 31.7 %) 19 ( 18.3 %) 13 ( 12.5 %) 1 2 (11.5%) 22 ( 31.0 %) 22 ( 31.0 %) 15 (21.1 %) 7 ( 9.9 %) 5 ( 7.0 %) 0. 791 Pathological stage pStage ⅡA pStage ⅡB pStage ⅢA pStage ⅢB pStage ⅣA pStage ⅣB (M1 Lymph) 13 ( 12.5 %) 13 ( 12.5 %) 3 ( 2.9%) 47 ( 45.2 %) 16 ( 15.4 %) 1 2 (11.5%) 7 ( 9.9 %) 14 (19.7%) 4 ( 5.6 %) 34 ( 47.9 %) 7 ( 9.9 %) 5 ( 7.0 %) 0. 493 Tumor differentiation Well Moderate Poor 16 ( 15.4 %) 53 ( 51.0 %) 35 ( 33.6 %) 13 ( 18.3 %) 38 (53.5 %) 20 ( 28.2 %) 0.714 TLR4 expression TLR4 1+ TLR4 2+ TLR4 3+ 17 ( 16.3 %) 58 ( 55.8 %) 29 ( 27.9 %) 28 ( 39.4 %) 34 ( 47.9 %) 9 ( 12.7 %) 0.001* Adjuvant chemotherapy Positive Negative 64 ( 61.5 %) 40 ( 38.5 %) 41 ( 57.8 %) 30 ( 42.2 %) 0. 615 Recurrence of ESCC Positive Negative 52 ( 50.0 %) 52 (5 0.0 %) 27 ( 38.0 %) 44 ( 62.0 %) 0. 118 Prognosis Alive Deceased from ESCC Deceased from other cancer Deceased from other diseases 39 ( 37.5 %) 47 ( 45.2 %) 4 (3.8%) 14 ( 13.5 %) 37 ( 52.1 %) 19 ( 26.8 %) 1 (1.4%) 14 ( 19.7 %) 0.0 51 Pneumonia (C-D ≧ GradeII) Positive Negative 23 (22.1%) 81 (77.9%) 10 (14.1%) 61 (85.9%) 0.182 Anastomotic Leakage (C-D ≧ GradeI) Positive Negative 22 (21.6%) 82 (78.9%) 8 (11.3%) 63 (88.7%) 0.088 P. gingivalis Levels, TLR4 Expression and 5-Year OS and DSS in 175 ESCC Patients The 89 censored cases were followed for 60 months. Kaplan-Meier curves illustrating the association between P. gingivalis levels and survival revealed that high P. gingivalis expression was associated with significantly poorer 5-year OS and DSS (p = 0.0195 and p = 0.0314, respectively, Fig. 2 ). In addition, TLR4 expression status significantly (p = 0.0145) reflected the P. gingivalis levels (Fig. 3 ). Combined P. gingivalis and TLR4 Status and 5-Year OS and DSS in 175 ESCC Patients The Kaplan-Meier curves relating 5-year OS and DSS to the four profiles combining P. gingivalis and TLR4 levels are shown (Fig. 4 ). Evaluation of TLR4 expression status was performed using same methods in our previous study [19]. Patients exhibiting combined P. gingivalis -high and TLR4-high had much poorer 5-year OS and DSS than any of the other combinations of P. gingivalis and TLR4 levels (p = 0.0222, 0.0348, respectively). P. gingivalis Levels are a Prognostic Factor Affecting 5-Year OS and DSS In univariate analyses, significant prognostic factors affecting 5-year OS were P. gingivalis levels (high vs. low), the combination of P. gingivalis and TLR4 levels (High-high vs. others), age (70 and older vs. younger), lymph node metastasis (N + vs. N-), pStage (Ⅲ-Ⅳ vs. Ⅰ-Ⅱ) and tumor differentiation (poorly vs. not poorly differentiated). Significant prognostic factors affecting 5-year DSS were P. gingivalis levels (high vs. low), the combination of P. gingivalis and TLR4 levels (High-high vs. others), lymph node metastasis (N + vs. N-), pStage (Ⅲ-Ⅳ vs. Ⅰ-Ⅱ) and tumor differentiation (poorly vs. not poorly differentiated) (Table 2 ). In addition, multivariate analysis showed that in every combination with age, gender, depth of invasion, lymph node metastasis, pStage and tumor differentiation, P. gingivalis levels are an independent prognostic factor affecting 5-year OS and DSS. Similarly, in every combination with age, gender, depth of invasion, lymph node metastasis, pStage and tumor differentiation, the combination of P. gingivalis and TLR4 levels is an independent prognostic factor affecting 5-year OS and DSS (Table 3 ). Table 2 Univariate analysis of survival in ESCC (Cox hazard models) OS DSS Variables P HR 95% CI P HR 95% CI P. gingivalis levels high (n = 104) vs. low (n = 71) 0.021* 1.710 1.084–2.698 0.034* 1.791 1.045–3.070 Combination of P. gingivalis and TLR4 High-high vs. others 0.002* 1.962 1.269–3.033 0.005* 2.095 1.253–3.503 Age 70 and older (n = 66) vs. younger (n = 109) 0.034* 1.575 1.035–2.396 0.206 1.378 0.839–2.263 Gender Male (n = 151) vs. Female (n = 24) 0.105 1.825 0.882–3.776 0.319 1.491 0.680–3.269 Depth of invasion T3-4 (n = 144) vs. T2 (n = 31) 0.433 1.257 0.710–2.227 0.183 1.655 0.788–3.472 Lymph node metastasis N+ (n = 126) vs. N- (n = 49) < 0.001* 5.770 2.785–11.953 < 0.001* 8.570 3.111–23.610 Pathological stage Ⅲ-Ⅳ (n = 121) vs. Ⅰ-Ⅱ (n = 54) < 0.001* 5.162 2.667–9.992 < 0.001* 9.821 3.564–27.062 Tumor differentiation poorly (n = 55) vs. not poorly (n = 120) 0.003* 1.900 1.240–2.911 0.006* 2.004 1.218–3.297 *Statistically significant Table 3 Multivariate analysis of survival in ESCC (Cox hazard models) OS DSS Variables P HR 95% CI P HR 95% CI P. gingivalis levels Crude (P. gingivalis expression) High vs. low 0.021* 1.710 1.084–2.698 0.034* 1.791 1.045–3.070 Adjusted for age and gender 0.016* 1.762 1.110–2.798 0.029* 1.841 1.066–3.178 Adjusted for age, gender, depth of invasion, lymph node metastasis, pathological stage and tumor differentiation 0.020* 1.747 1.094–2.788 0.043* 1.762 1.018–3.050 Combination between P. gingivalis and TLR4 Crude (combination between P. gingivalis and TLR4) High-high vs. others 0.002* 1.962 1.269–3.033 0.005* 2.095 1.253–3.503 Adjusted for age and gender 0.001* 2.108 1.346–3.302 0.003* 2.237 1.318–3.797 Adjusted for age, gender, depth of invasion, lymph node metastasis, pathological stage and tumor differentiation 0.004* 1.967 1.247–3.104 0.010* 2.010 1.178–3.431 *Statistically significant DISCUSSION We have shown that ESCC patients with high P. gingivalis levels after curative esophagectomy have significantly poorer 5-year OS and DSS than patients with low levels. Moreover, patients exhibiting a P. gingivalis -high/TLR4-high profile had much poorer 5-year OS and DSS than other P. gingivalis /TLR4 profiles. The P. gingivalis -high/TLR4-high profile emerged as an independent prognostic factor significantly affecting 5-year OS and DSS. We also found a strong correlation between high P. gingivalis levels and high TLR4 expression. These findings could have a profound impact on the understanding and treatment of ESCC. Several earlier reports have also shown that P. gingivalis infection leads to a poorer prognosis in ESCC. Miao-Fen Chen et al. reported that more than half of their ESCC patients were infected with P. gingivalis , which was associated with poor differentiation and disease progression [26]. Shegan Gao et al. reported that patients infected with P. gingivalis had a significantly shorter median OS than patients without the infection [27]. Our present findings are consistent with that earlier clinical evidence. With respect to the mechanism by which P. gingivalis exerts its adverse effect, it has been shown that the bacterium induces IL-6 production but that esophageal cancer develops and progresses in an IL-6-dependent manner [26]. P. gingivalis infection also suppresses ESCC cell apoptosis and promotes ESCC cell viability via the transcriptional signal transduction activator 3 (STAT3) pathway [27]. LPS released by P. gingivalis may affect ESCC cell proliferation and migration via TLR4 signaling and selectin ligand-induced adhesion [28]. Moreover, LPS reportedly activates the NF-κB pathway, leading to the release of inflammatory cytokines and induction of carcinogenesis [29]. Meng et al. showed that P. gingivalis upregulates expression of key molecules involved in the NF-κB signaling pathway in vitro, including cyclin D1, matrix metalloproteinase 2 (MMP2) and C-MYC, thereby promoting proliferation, migration and invasion by ESCC cells [30]. In addition, Liang et al. showed that miR-194 expression is significantly upregulated and expression of its direct target, GRHL3, is suppressed after P. gingivalis infection. They also found that expression of the tumor suppressor gene PTEN, a direct regulatory target of GRHL3, is also diminished by activation of the phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) pathway, suggesting that P. gingivalis infection promotes ESCC growth and migration [31]. In addition to esophageal cancer, P. gingivalis infection is significantly associated with colorectal, pancreatic, prostate, breast, uterine, lung and oropharyngeal cancers as well as non-Hodgkin's lymphoma and leukemia [32–39]. In colorectal cancer, IFNGR1 is reportedly degraded through palmitoylation in lysosomes, enabling tumor immune escape [40]. This is consistent with the findings of Shen et al., who reported that decreased expression of IFNGR1 and its palmitoylation increased the proliferative, migratory and invasive capacity of ESCC cells [41]. In addition, Chattopadhyay et al. reported that P. gingivalis promotes oral squamous cell carcinoma by activating the anti-apoptotic Janus kinase 1 (JAK1)/STAT3 and PI3K/Akt pathways in oral epithelial cells [42]. Liang et al. also reported a similar mechanism of cancer promotion in ESCC [31]. On the other hand, a meta-analysis conducted by Michaud et al. found a negative association between periodontal disease and risk of colorectal cancer [43]. However, we could find no other study reporting that P. gingivalis inhibits the development or progression of a cancer, including ESCC. Thus, P. gingivalis is implicated in numerous malignancies and is an important prognostic factor. By contrast, lactic acid bacteria are known to exert immunomodulatory, antagonistic and pathogen-suppressive effects and to exhibit strong inhibitory effects on periodontal pathogens [44]. Based on these reports and our earlier findings [17,20], we initiated a clinical trial: Improvement of oral environment with toothpaste and mouthwash containing Lactobacillus and the incident rate of pneumonia after esophagectomy for esophageal cancer patients (LacPEC study, jRCTs021230010) in June 2023. Toothpaste and mouthwash containing Lactobacillus, “beneficial” Gram-positive bacteria, are already commercially available in Japan and have been shown to significantly reduce periodontal Gram-negative bacteria and candida albicans without affecting normal components of the oral flora, including Streptococcus mitis and Streptococcus oralis . The results of this clinical trial suggest predominance of “beneficial” Gram-positive bacteria in the oral environment before esophagectomy is beneficial for preventing postoperative complications and, consequently, for better long-term survival. In the present study, IHC using monoclonal antibody for P. gingivalis showed the presence of antigen in the cytoplasm of ESCC cells. Same results were proved in more earlier reports [45]. These results mean that part of the structures of P. gingivalis have invaded or have been taken into ESCC cells. How those part of the structures of P. gingivalis affect ESCC cells and those mechanisms are subjects for the further research. Our previous study proved that LPS released from Gram-negative bacteria promotes ESCC proliferation in vitro and in vivo, and the influences of LPS can be inhibited by using a TLR4 blocker [20]. Therefore, LPS released from Gram-negative bacteria increases the proliferation of ESCC cells through TLR4 signaling. This phenomenon seems to be one of the molecular oncological mechanisms of the results of the present study. There are limitations to this study. The most important limitation is the lack of data on the status of the patients’ oral cavity environment and an assessment of their oral bacterial flora before esophagectomy. Because we started preoperative assessment of the oral cavity environment by dentists before esophagectomy in 2009 [46], those data were not available for most patients in the cohort used for this study. While we have demonstrated that P. gingivalis is associated with TLR4 expression status and patient prognosis, we have not determined whether other red complexes, Treponema denticola and Tannerella forsythia , are also prognostic factors. Another limitation is that the present study is a single-institute, retrospective study. To solve this limitation, we need to validate specimens from other institute in the further study. CONCLUSION We have shown that infection of ESCC tissue by P. gingivalis increases expression of TLR4, which leads to a poor prognosis in ESCC patients after curative esophagectomy. To improve the long-term outcomes of ESCC patients, it would be valuable to focus on improving the oral environment and preventing P. gingivalis infection. Abbreviations P. gingivalis : Porphyromonas gingivalis TLRs: Toll-like receptors ESCC: Esophageal squamous cell carcinoma PAMPs: Pathogen-associated molecular patterns LPS: Lipopolysaccharide Declarations Acknowledgements The authors would like to express our deepest gratitude to the patients who contributed tumor samples for this study. Authors' contributions Conceptualization, Y.S. and T.Y.; methodology and investigation, Y.S., T.Y., A.W. and Y.N. validation, R.S., T.Y., T.T. M.Y. and M.T.; formal analysis, K.N.; writing—original draft preparation, T.Y. and Y.S.; writing, review and editing, T.Y. and Y.S., and K.T.; project administration, K. I. All authors have read and agreed to the published version of the manuscript. Funding Not applicable. Data availability The datasets used or analyzed during the current study are available from the corresponding author on reasonable request. Conflict of interest The authors have no conflict of interest to declare. Ethics approval and consent to participate This study was approved by the Ethics Committee of Akita University School of Medicine on 20 November 2019. All experiments adhered to the principles of the Helsinki Declaration, and all study participants provided informed written consent. Consent for publication Not applicable. References V A McCormack, D Menya, M O Munishi, et al. Informing etiologic research priorities for squamous cell esophageal cancer in Africa: a review of setting-specific exposures to known and putative risk factors. Int J Cancer. 2017 Jan 15;140(2):259-271. https://doi.org/10.1002/ijc.30292 Chien-Hung Lee, Deng-Chyang Wu, I-Chen Wu, et al. 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Ann Surg Oncol. 2016 Mar;23(3):936-42. https://doi.org/10.1245/s10434-015-4909-1 Yusuke Sato, Satoru Motoyama, Akiyuki Wakita, et al. TLR3 expression status predicts prognosis in patients with advanced thoracic esophageal squamous cell carcinoma after esophagectomy. Am J Surg. 2018 Aug;216(2):319-325. https://doi.org/10.1016/j.amjsurg.2018.01.038 Robert L Camp, Veronique Neumeister, David L Rimm. A decade of tissue microarrays: Progress in the discovery and validation of cancer biomarkers. J Clin Oncol. 2008 Dec 1;26(34):5630-7. https://doi.org/10.1200/JCO.2008.17.3567 Antonio C Wolff, M Elizabeth H Hammond, Jared N Schwartz, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131(1):18-43. https://doi.org/10.5858/2007-131-18-ASOCCO Miao-Fen Chen, Ming-Shian Lu, Ching-Chuan Hsieh, Wen-Cheng Chen. Porphyromonas gingivalis promotes tumor progression in esophageal squamous cell carcinoma. Cell Oncol (Dordr). 2021 Apr;44(2):373-384. https://doi.org/10.1007/s13402-020-00573-x Shegan Gao, Yiwen Liu, Xiaoxian Duan, et al. Porphyromonas gingivalis infection exacerbates oesophageal cancer and promotes resistance to neoadjuvant chemotherapy. Br J Cancer. 2021 Aug;125(3):433-444. https://doi.org/10.1038/s41416-021-01419-5 Mathieu C Rousseau, Rich Y C Hsu, Jonathan D Spicer, et al. Lipopolysaccharide-induced toll-like receptor 4 signaling enhances the migratory ability of human esophageal cancer cells in a selectin-dependent manner. Surgery. 2013 Jul;154(1):69-77. https://doi.org/10.1016/j.surg.2013.03.006 Atsuo Ochi, Andrew H Nguyen, Andrea S Bedrosian, et al. MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells. J Exp Med. 2012 Aug 27;209(9):1671-87. https://doi.org/10.1084/jem.20111706 Meng F, Li R, Ma L, et al. Porphyromonas gingivalis promotes the motility of esophageal squamous cell carcinoma by activating NF-κB signaling pathway. Microbes Infect. 2019 Aug-Sep;21(7):296-304. https://doi.org/10.1016/j.micinf.2019.01.005 Gaofeng Liang, Haojie Wang, Hao Shi, et al. Porphyromonas gingivalis promotes the proliferation and migration of esophageal squamous cell carcinoma through the miR-194/GRHL3/ PTEN/Akt Axis. ACS Infect Dis. 2020 May 8;6(5):871-881. https://doi.org/10.1021/acsinfecdis.0c00007 T J COOK. Blood dyscrasias as related to periodontal disease; with special reference to leukemia. J Periodontol (1930). 1947 Oct;18(4):159-65. https://doi.org/10.1902/jop.1947.18.4.159 F M WENTZ. Oral Manifestations of the Blood Diseases. J Am Dent Assoc. 1952 Jun;44(6):698-701. https://doi.org/10.14219/jada.archive.1952.0114 Stefano Corbella, Paolo Veronesi, Viviana Galimberti, Roberto Weinstein, Massimo Del Fabbro, Luca Francetti. Is periodontitis a risk indicator for cancer? A meta-analysis. PLoS One. 2018 Apr 17;13(4):e0195683. https://doi.org/10.1371/journal.pone.0195683 Paige M Bracci. Oral Health and the Oral Microbiome in Pancreatic Cancer: An Overview of Epidemiological Studies. Cancer J. 2017 Nov/Dec;23(6):310-314. https://doi.org/10.1097/PPO.0000000000000287 P Maisonneuve, S Amar, A B Lowenfels. Periodontal disease, edentulism, and pancreatic cancer: a meta-analysis. Ann Oncol. 2017 May 1;28(5):985-995. https://doi.org/10.1093/annonc/mdx019 Dominique S Michaud, Zhuxuan Fu, Jian Shi, Mei Chung. Periodontal Disease, Tooth Loss, and Cancer Risk. Epidemiol Rev. 2017 Jan 1;39(1):49-58. https://doi.org/10.1093/epirev/mxx006 Li Xiao, Qianyu Zhang, Yanshuang Peng, Daqing Wang, Ying Liu. The effect of periodontal bacteria infection on incidence and prognosis of cancer: A systematic review and meta-analysis. Medicine (Baltimore). 2020 Apr;99(15):e19698. https://doi.org/10.1097/MD.0000000000019698 Tingting Shi, Min Min, Chenyu Sun, Yun Zhang, Mingming Liang, Yehuan Sun. Periodontal disease and susceptibility to breast cancer: A meta-analysis of observational studies. J Clin Periodontol. 2018 Sep;45(9):1025-1033. https://doi.org/10.1111/jcpe.12982 Wan Du, Fang Hua, Xiong Li, et al. Loss of Optineurin Drives Cancer Immune Evasion via Palmitoylation-Dependent IFNGR1 Lysosomal Sorting and Degradation. Cancer Discov. 2021 Jul;11(7):1826-1843. https://doi.org/10.1158/2159-8290.CD-20-1571 L Shen, D Zhang, S Gao. Effect of Porphyromonas gingivalis infection on IFNGR1 palmitoylation in esophageal cancer cells. Nan Fang Yi Ke Da Xue Xue Bao. 2023 Jul 20;43(7):1155-1163. https://doi.org/10.12122/j.issn.1673-4254.2023.07.12 Indranil Chattopadhyay, Mukesh Verma, Madhusmita Panda. Role of Oral Microbiome Signatures in Diagnosis and Prognosis of Oral Cancer. Technol Cancer Res Treat. 2019 Jan 1:18:1533033819867354. https://doi.org/10.1177/1533033819867354 Dominique S Michaud, Zhuxuan Fu, Jian Shi, Mei Chung. Periodontal Disease, Tooth Loss, and Cancer Risk. Epidemiol Rev 2017 Jan 1;39(1):49-58. https://doi.org/10.1093/epirev/mxx006 Wei-Kuang Lai, Ying-Chen Lu, Chun-Ren Hsieh, et al. Developing Lactic Acid Bacteria as an Oral Healthy Food. Life (Basel). 2021 Mar 24;11(4):268. https://doi.org/10.3390/life11040268 G Amodini Rajakaruna 1, Mariko Negi, Keisuke Uchida, Masaki Sekine, Asuka Furukawa, Takashi Ito, Daisuke Kobayashi, Yoshimi Suzuki, Takumi Akashi, Makoto Umeda, Walter Meinzer, Yuichi Izumi, Yoshinobu Eishi. Localization and density of Porphyromonas gingivalis and Tannerella forsythia in gingival and subgingival granulation tissues affected by chronic or aggressive periodontitis. Sci Rep. 2018 Jun 22;8(1):9507. https://doi.org/10.1038/s41598-018-27766-7 Yusuke Sato, Satoru Motoyama, Hiroshi Takano, et al. Esophageal Cancer Patients Have a High Incidence of Severe Periodontitis and Preoperative Dental Care Reduces the Likelihood of Severe Pneumonia after Esophagectomy. Dig Surg. 2016;33(6):495-502. https://doi.org/10.1159/000446927 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5874217","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":408236008,"identity":"64ad6430-efed-4e44-b0a1-3724995dfc5e","order_by":0,"name":"Takatoshi 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14:10:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5874217/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5874217/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75188338,"identity":"42fc45fd-94fb-4949-b255-3eda3b4718b7","added_by":"auto","created_at":"2025-01-31 18:01:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14480877,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining for \u003cem\u003eP. gingivalis\u003c/em\u003e in an ESCC tissue microarray. The representative photomicrographs show triplicate specimens scored 3+, 2+, 1+ or 0. The triplicate cores are shown at 100x magnification, along with high-magnification images (500x)\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5874217/v1/6b25b2ec535e74c4aea22df7.png"},{"id":75188337,"identity":"43d5b614-df4f-4084-9cae-8cbf25fbb376","added_by":"auto","created_at":"2025-01-31 18:01:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":605704,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan–Meier survival curves illustrating the association between \u003cem\u003eP. gingivalis\u003c/em\u003e status (high or low) and 5-year OS (\u003cstrong\u003ea\u003c/strong\u003e) and DSS (\u003cstrong\u003eb\u003c/strong\u003e) in ESCC patients after curative esophagectomy. The log-rank test was used to evaluate differences between the two groups (5-year OS: p=0.0195 DSS: p=0.0314). \u003cem\u003eP. g\u003c/em\u003e, \u003cem\u003eP. gingivalis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5874217/v1/a8f78a5be8ff7cb88c240840.png"},{"id":75188342,"identity":"b699bf77-01a1-4f90-ab25-e78720cab699","added_by":"auto","created_at":"2025-01-31 18:01:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":224382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eP. gingivalis\u003c/em\u003elevels and TLR4 expression status. \u003cem\u003eP. gingivalis\u003c/em\u003elevels significantly reflect TLR4 expression status (p=0.0145)\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5874217/v1/dde8db9af1153b57c339d5e2.png"},{"id":75188356,"identity":"178db9aa-64fd-4a42-b1ac-003df92b37a6","added_by":"auto","created_at":"2025-01-31 18:01:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":837295,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan–Meier survival curves illustrating the association between the combined \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4 expression status (\u003cem\u003eP. gingivalis\u003c/em\u003e-high TLR4-high, \u003cem\u003eP. gingivalis\u003c/em\u003e-low TLR4-high, \u003cem\u003eP. gingivalis\u003c/em\u003e-high TLR4-low, \u003cem\u003eP. gingivalis\u003c/em\u003e-low TLR4-low) and 5-year OS (\u003cstrong\u003ea\u003c/strong\u003e) and DSS (\u003cstrong\u003eb\u003c/strong\u003e) in ESCC patients after curative esophagectomy. The log-rank test was used to evaluate differences among the four groups (5-year OS: p=0.0222 DSS: p=0.0348). \u003cem\u003eP. g\u003c/em\u003e, \u003cem\u003eP. gingivalis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5874217/v1/26de223d41d6acf5563f6341.png"},{"id":77264056,"identity":"c1172ccd-e74a-40c4-a89f-c98035b8a2fe","added_by":"auto","created_at":"2025-02-26 20:48:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27186229,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5874217/v1/012a4ab9-30c2-4998-95c4-693552523fd6.pdf"}],"financialInterests":"","formattedTitle":"Porphyromonas gingivalis levels correlate with Toll-like receptor 4 expression status and prognosis after esophagectomy for advanced thoracic esophageal squamous cell carcinoma","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEsophageal cancer is divided into two main histological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Well-known risk factors for ESCC are cigarette smoking, heavy alcohol consumption, poor dietary habits, and inactivation of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) [1,2]. In addition, recent reports suggest that poor oral health is also an independent risk factor for upper-aerodigestive tract cancers, including ESCC [3\u0026ndash;6]. Consistent with that finding, we recently reported that the severity of periodontitis is an independent risk factor contributing to a poorer long-term prognosis after esophagectomy [7].\u003c/p\u003e \u003cp\u003eToll-like receptors (TLRs) are a family of pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs) released from various viruses, bacteria, fungi and parasites [8,9]. These receptors play vital roles in mediating innate and antigen-specific adaptive immunity. A total of 10 TLRs, recognizing different PAMPs, have been identified in human beings [10\u0026ndash;13].\u003c/p\u003e \u003cp\u003eIt has been reported that about 700 species of bacteria reside in the mouths of human beings [14,15]. Among the oral bacterial flora are species classified as \u0026ldquo;beneficial\u0026rdquo; or \u0026ldquo;opportunistic\u0026rdquo; as well as \u0026ldquo;bad bacteria,\u0026rdquo; which are periodontal pathogens. These periodontal pathogens are classified as \u0026ldquo;red complex,\u0026rdquo; \u0026ldquo;orange complex\u0026rdquo; or \u0026ldquo;yellow complex,\u0026rdquo; among others, based on their relation to periodontitis [16\u0026ndash;18]. All three species classified as \u0026ldquo;red complex,\u0026rdquo; including the most well-studied \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, and most species classified as \u0026ldquo;orange complex\u0026rdquo; are Gram-negative bacteria with cell walls composed of lipopolysaccharide (LPS). TLR4 recognizes LPS, and we previously reported that ESCC patients exhibiting high TLR4 expression exhibit significantly poorer survival than those expressing lower levels of TLR4 [19]. Moreover, we recently reported that LPS promotes ESCC cell proliferation and migration in vitro by enhancing CCL2 production via TLR4 signaling [20]. These findings suggest that there may be a relationship between \u003cem\u003eP. gingivalis\u003c/em\u003e infection and TLR4 expression status in clinical ESCC specimens. In the present study, therefore, we examined the association between \u003cem\u003eP. gingivalis\u003c/em\u003e levels and expression of TLR4 in clinical specimens from ESCC patients and their correlation with long-term survival.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003e This study was approved by the Ethics Committee of Akita University School of Medicine on 20 November 2019. All experiments adhered to the principles of the Helsinki Declaration, and all study participants provided informed written consent. Between January 2000 and December 2011, 507 patients received esophagectomy to treat esophageal cancer at Akita University Hospital. Among them, 177 patients with pT2-pT4 thoracic ESCC who had received curative esophagectomy with no preoperative treatment were enrolled in this study. The clinical cancer stages and treatment strategies were discussed and decided by a cancer board composed of radiologists, oncologists, gastroenterologists and surgeons. Pathological stages were determined according to the UICC International Union Against Cancer Tumor-Node-Metastasis (TNM) Classification of Malignant Tumors (8th edition).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEsophagectomy\u003c/h3\u003e\n\u003cp\u003eOur standard operative procedure was right thoracoscopic or robot-assisted thoracoscopic esophagectomy with extended three-field lymph node dissection. The three fields included: (1) mediastinal fields, involving the periesophageal region and areas around the trachea and bilateral main bronchus; (2) abdominal fields, involving the perigastric region and areas around the celiac axis; and (3) cervical fields involving the bilateral periesophageal and supraclavicular regions. Reconstruction often required insertion of a gastric tube via the posterior mediastinal route or the retrosternal route [21,22].\u003c/p\u003e\n\u003ch3\u003eESCC Tissue Microarray\u003c/h3\u003e\n\u003cp\u003eAn ESCC tissue microarray (TMA) was constructed at the Pathology Institute, Toyama, Japan, as previously described [19\u0026ndash;23]. Previous evaluations of TMAs showed that use of double cores measuring 0.6 mm in diameter adequately reflected the entire section and were sufficient to overcome cancer tissue heterogeneity [24]. We therefore employed triplicate cores measuring 0.6 mm in diameter to enhance the reliability further. Triplicate cores were randomly collected from separate carcinoma areas and transferred to the TMA. The total TMA block contained 531 cores (3 cores each from 177 paraffin main tumor blocks from the enrolled ESCC patients). However, six cores from two patients were lost from the microarray because of the thickness of the transplanted tissue. The evaluation was therefore based on 175 cases.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eSections (4 \u0026micro;m) from the TMA were immunohistochemically examined using standard procedures [19\u0026ndash;23]. Briefly, sections were incubated in citrate buffer (pH 6.0) for 10 min at 121 ℃ in an autoclave for antigen retrieval. Endogenous peroxidase activity was inactivated by incubation for 5 min in 3.0% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and internal biotin was blocked using a biotin-blocking system (Dako, Denmark). The sections were then incubated with an anti-\u003cem\u003eP. gingivalis\u003c/em\u003e monoclonal antibody (1 \u0026micro;g/mL; D376-3, Medical Biological Laboratories Co., LTD., Tokyo, Japan) overnight at 4 ℃, after which the antigen was detected using a catalyzed signal amplification system (Dako) with a DAB peroxidase substrate. Finally, the sections were counterstained with Mayer\u0026rsquo;s hematoxylin, dehydrated and mounted. IHC staining was imaged and viewed using a NanoZoomer Digital Pathology C9600 slide scanner and Virtual Slide Viewer software (NDP.view2 version 2.9.29) (Hamamatsu Photonics, Hamamatsu, Japan). Using the HER2 IHC scoring system (ASCO and CAP guidelines), staining scores were then assigned by three physicians blinded to the clinical and prognostic data [25]. A sample was assigned an IHC score of 3\u0026thinsp;+\u0026thinsp;if there was intense \u003cem\u003eP. gingivalis\u003c/em\u003e staining in the cytoplasm or nuclei in more than 30% of cells, 2\u0026thinsp;+\u0026thinsp;if there was moderate staining in the cytoplasm or nuclei in \u0026gt;\u0026thinsp;10% of cells, 1\u0026thinsp;+\u0026thinsp;if there was weak staining, or 0 if there was no staining. High \u003cem\u003eP. gingivalis\u003c/em\u003e levels were defined as an IHC score of 3\u0026thinsp;+\u0026thinsp;or 2+; low levels were defined as a score of 1\u0026thinsp;+\u0026thinsp;or 0. In cases where there was disagreement, the score assigned by 2 of the 3 physicians was used.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eContinuous variables are presented as the median (range: minimum\u0026ndash;maximum). To assess differences between the \u003cem\u003eP. gingivalis\u003c/em\u003e-high and \u003cem\u003eP. gingivalis\u003c/em\u003e-low groups, the Mann-Whitney-Wilcoxon test (for continuous variables) or χ 2 and Fisher\u0026rsquo;s exact tests (for categorical variables) was used. The clinical end-points of this study were overall survival (OS) and disease-specific survival (DSS). Length of survival (censored at 60 months for longer survivors) was calculated from the date of esophagectomy to the patient\u0026rsquo;s death or the date of the last clinical follow-up. Kaplan-Meier curves were constructed to assess the association between OS and DSS and \u003cem\u003eP. gingivalis\u003c/em\u003e status. Curves were compared using the log-rank test. Cox\u0026rsquo;s proportional hazards model was used to assess prognostic factors associated with OS and DSS in ESCC patients after curative esophagectomy. The variables adjusted for multivariate analysis were age, gender, pT, pN, pStage and tumor differentiation. All statistical analyses were performed using JMP Pro 17.2.0. Two-sided values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical Analysis of P. gingivalis levels\u003c/h2\u003e \u003cp\u003eThree representative cores assigned an IHC score of 3+, 2+, 1\u0026thinsp;+\u0026thinsp;or 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. IHC using the monoclonal antibody for \u003cem\u003eP. gingivalis\u003c/em\u003e showed the presence of antigen in the cytoplasm of ESCC cells. Scored 3+, 2+, 1\u0026thinsp;+\u0026thinsp;or 0 were obtained from 15 patients (8.6%), 89 patients (50.9%), 65 patients (37.1%) and 6 patients (3.4%), respectively. Thus, 104 patients were considered \u003cem\u003eP. gingivalis\u003c/em\u003e-high (scores of 3\u0026thinsp;+\u0026thinsp;or 2+), while 71 patients were considered \u003cem\u003eP. gingivalis\u003c/em\u003e-low (scores of 1\u0026thinsp;+\u0026thinsp;or 0). The clinicopathological characteristics of the two groups are summarized (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The \u003cem\u003eP. gingivalis\u003c/em\u003e-high group exhibited significantly stronger TLR4 expression, whereas there were no significant differences between the groups with respect to the other factors shown in the table.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe clinicopathological characteristics of the ESCC patients (n=175)\u003c/p\u003e\n \u003ctable style=\"border-collapse: collapse;border: none;width: 576px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCharacteristics\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eP. gingivalis\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-high\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003en=\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e104 (\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e59.4\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eP. gingivalis\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-low\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003en=\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e71 (\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e40.6\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;P\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eGender\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e \u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eFemale\u003c/span\u003e\u003c/p\u003e\n \u003cp 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style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAge at surgery\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;height:5.15pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e66(\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e38-82\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;height:5.15pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e66(\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e40-78\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;height:5.15pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.546\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;height:26.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eHabitual smoking\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eCurrent\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePast\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New 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style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e21 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e20.2\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e75 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e72.1\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e8 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e7.7\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e10 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e14.1\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e60 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e84.5\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e1.4\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e83\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eLymph node metastasis (pN UICC)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epN0\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epN1\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epN2\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epN3\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eM1 Lymph (supraclavicular)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e27 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e26.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e33 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e31.7\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e19 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e18.3\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e13 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e12.5\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;(11.5%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e22 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e31.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e22 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e31.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e15\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e(21.1\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e7 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e9.9\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e5 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e7.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e791\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:justify;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePathological stage\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epStage ⅡA\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epStage ⅡB\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epStage ⅢA\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epStage ⅢB\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epStage ⅣA\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003epStage ⅣB (M1 Lymph)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e13 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e12.5\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e13 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e12.5\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e3 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e2.9%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e47 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e45.2\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e16 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e15.4\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;(11.5%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e7 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style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eNegative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e64 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e61.5\u003c/span\u003e\u003cspan style='font-family: \"Times New 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style='font-family:\"Times New Roman\",serif;'\u003e41 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e57.8\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e30 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e42.2\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e615\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eRecurrence of ESCC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePositive\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eNegative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e52 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e50.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e52 (5\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e0.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e27 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e38.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e44 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e62.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e118\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePrognosis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAlive\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eDeceased from ESCC\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eDeceased from other cancer\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;text-indent:5.0pt;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eDeceased from other diseases\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e39 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e37.5\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e47 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e45.2\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e4 (3.8%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e14 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e13.5\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e37 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e52.1\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e19 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e26.8\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e1 (1.4%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e14 (\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e19.7\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;'\u003e%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.0\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e51\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePneumonia (C-D\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Yu Mincho\",serif;'\u003e≧\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eGradeII)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e \u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePositive\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;Negative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e23 (22.1%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e81 (77.9%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e10 (14.1%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e61 (85.9%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.182\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:170.1pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eAnastomotic Leakage (C-D\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Yu Mincho\",serif;'\u003e≧\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eGradeI)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e \u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ePositive\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e \u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eNegative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e22 (21.6%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e82 (78.9%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:99.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e8 (11.3%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e63 (88.7%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:70.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e0.088\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:left;line-height:13.0pt;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eP. gingivalis Levels, TLR4 Expression and 5-Year OS and DSS in 175 ESCC Patients\u003c/h3\u003e\n\u003cp\u003eThe 89 censored cases were followed for 60 months. Kaplan-Meier curves illustrating the association between \u003cem\u003eP. gingivalis\u003c/em\u003e levels and survival revealed that high \u003cem\u003eP. gingivalis\u003c/em\u003e expression was associated with significantly poorer 5-year OS and DSS (p\u0026thinsp;=\u0026thinsp;0.0195 and p\u0026thinsp;=\u0026thinsp;0.0314, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, TLR4 expression status significantly (p\u0026thinsp;=\u0026thinsp;0.0145) reflected the \u003cem\u003eP. gingivalis\u003c/em\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCombined P. gingivalis and TLR4 Status and 5-Year OS and DSS in 175 ESCC Patients\u003c/h2\u003e \u003cp\u003eThe Kaplan-Meier curves relating 5-year OS and DSS to the four profiles combining \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4 levels are shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Evaluation of TLR4 expression status was performed using same methods in our previous study [19]. Patients exhibiting combined \u003cem\u003eP. gingivalis\u003c/em\u003e-high and TLR4-high had much poorer 5-year OS and DSS than any of the other combinations of \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4 levels (p\u0026thinsp;=\u0026thinsp;0.0222, 0.0348, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eP. gingivalis Levels are a Prognostic Factor Affecting 5-Year OS and DSS\u003c/h2\u003e \u003cp\u003eIn univariate analyses, significant prognostic factors affecting 5-year OS were \u003cem\u003eP. gingivalis\u003c/em\u003e levels (high vs. low), the combination of \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4 levels (High-high vs. others), age (70 and older vs. younger), lymph node metastasis (N\u0026thinsp;+\u0026thinsp;vs. N-), pStage (Ⅲ-Ⅳ vs. Ⅰ-Ⅱ) and tumor differentiation (poorly vs. not poorly differentiated). Significant prognostic factors affecting 5-year DSS were \u003cem\u003eP. gingivalis\u003c/em\u003e levels (high vs. low), the combination of \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4 levels (High-high vs. others), lymph node metastasis (N\u0026thinsp;+\u0026thinsp;vs. N-), pStage (Ⅲ-Ⅳ vs. Ⅰ-Ⅱ) and tumor differentiation (poorly vs. not poorly differentiated) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, multivariate analysis showed that in every combination with age, gender, depth of invasion, lymph node metastasis, pStage and tumor differentiation, \u003cem\u003eP. gingivalis\u003c/em\u003e levels are an independent prognostic factor affecting 5-year OS and DSS. Similarly, in every combination with age, gender, depth of invasion, lymph node metastasis, pStage and tumor differentiation, the combination of \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4 levels is an independent prognostic factor affecting 5-year OS and DSS (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariate analysis of survival in ESCC (Cox hazard models)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDSS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariables\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP. gingivalis\u003c/b\u003e \u003cb\u003elevels\u003c/b\u003e\u003c/p\u003e \u003cp\u003ehigh (n\u0026thinsp;=\u0026thinsp;104) vs. low (n\u0026thinsp;=\u0026thinsp;71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.021*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.084\u0026ndash;2.698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.034*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.045\u0026ndash;3.070\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCombination of\u003c/b\u003e \u003cb\u003eP. gingivalis\u003c/b\u003e \u003cb\u003eand TLR4\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHigh-high vs. others\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.269\u0026ndash;3.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.005*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.253\u0026ndash;3.503\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003cp\u003e70 and older (n\u0026thinsp;=\u0026thinsp;66) vs. younger (n\u0026thinsp;=\u0026thinsp;109)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.034*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.035\u0026ndash;2.396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.839\u0026ndash;2.263\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMale (n\u0026thinsp;=\u0026thinsp;151) vs. Female (n\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.825\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.882\u0026ndash;3.776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.680\u0026ndash;3.269\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDepth of invasion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eT3-4 (n\u0026thinsp;=\u0026thinsp;144) vs. T2 (n\u0026thinsp;=\u0026thinsp;31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.710\u0026ndash;2.227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.788\u0026ndash;3.472\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLymph node metastasis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eN+ (n\u0026thinsp;=\u0026thinsp;126) vs. N- (n\u0026thinsp;=\u0026thinsp;49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.785\u0026ndash;11.953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.111\u0026ndash;23.610\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePathological stage\u003c/b\u003e\u003c/p\u003e \u003cp\u003eⅢ-Ⅳ (n\u0026thinsp;=\u0026thinsp;121) vs. Ⅰ-Ⅱ (n\u0026thinsp;=\u0026thinsp;54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.667\u0026ndash;9.992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.564\u0026ndash;27.062\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor differentiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003epoorly (n\u0026thinsp;=\u0026thinsp;55) vs. not poorly (n\u0026thinsp;=\u0026thinsp;120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.240\u0026ndash;2.911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.006*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.218\u0026ndash;3.297\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e*Statistically significant\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultivariate analysis of survival in ESCC (Cox hazard models)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDSS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariables\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP. gingivalis\u003c/b\u003e \u003cb\u003elevels\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude (P. gingivalis expression)\u003c/p\u003e \u003cp\u003eHigh vs. low\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.021*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.084\u0026ndash;2.698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.034*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.045\u0026ndash;3.070\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdjusted for age and gender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.016*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.110\u0026ndash;2.798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.029*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.066\u0026ndash;3.178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdjusted for age, gender, depth of invasion, lymph node metastasis, pathological stage and tumor differentiation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.020*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.747\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.094\u0026ndash;2.788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.043*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.018\u0026ndash;3.050\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCombination between\u003c/b\u003e \u003cb\u003eP. gingivalis\u003c/b\u003e \u003cb\u003eand TLR4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude (combination between \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4)\u003c/p\u003e \u003cp\u003eHigh-high vs. others\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.269\u0026ndash;3.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.005*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.253\u0026ndash;3.503\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdjusted for age and gender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.346\u0026ndash;3.302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.003*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.318\u0026ndash;3.797\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdjusted for age, gender, depth of invasion, lymph node metastasis, pathological stage and tumor differentiation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.004*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.247\u0026ndash;3.104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.010*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.178\u0026ndash;3.431\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e*Statistically significant\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe have shown that ESCC patients with high \u003cem\u003eP. gingivalis\u003c/em\u003e levels after curative esophagectomy have significantly poorer 5-year OS and DSS than patients with low levels. Moreover, patients exhibiting a \u003cem\u003eP. gingivalis\u003c/em\u003e-high/TLR4-high profile had much poorer 5-year OS and DSS than other \u003cem\u003eP. gingivalis\u003c/em\u003e/TLR4 profiles. The \u003cem\u003eP. gingivalis\u003c/em\u003e-high/TLR4-high profile emerged as an independent prognostic factor significantly affecting 5-year OS and DSS. We also found a strong correlation between high \u003cem\u003eP. gingivalis\u003c/em\u003e levels and high TLR4 expression. These findings could have a profound impact on the understanding and treatment of ESCC.\u003c/p\u003e \u003cp\u003eSeveral earlier reports have also shown that \u003cem\u003eP. gingivalis\u003c/em\u003e infection leads to a poorer prognosis in ESCC. Miao-Fen Chen et al. reported that more than half of their ESCC patients were infected with \u003cem\u003eP. gingivalis\u003c/em\u003e, which was associated with poor differentiation and disease progression [26]. Shegan Gao et al. reported that patients infected with \u003cem\u003eP. gingivalis\u003c/em\u003e had a significantly shorter median OS than patients without the infection [27]. Our present findings are consistent with that earlier clinical evidence. With respect to the mechanism by which \u003cem\u003eP. gingivalis\u003c/em\u003e exerts its adverse effect, it has been shown that the bacterium induces IL-6 production but that esophageal cancer develops and progresses in an IL-6-dependent manner [26]. \u003cem\u003eP. gingivalis\u003c/em\u003e infection also suppresses ESCC cell apoptosis and promotes ESCC cell viability via the transcriptional signal transduction activator 3 (STAT3) pathway [27]. LPS released by \u003cem\u003eP. gingivalis\u003c/em\u003e may affect ESCC cell proliferation and migration via TLR4 signaling and selectin ligand-induced adhesion [28]. Moreover, LPS reportedly activates the NF-κB pathway, leading to the release of inflammatory cytokines and induction of carcinogenesis [29]. Meng et al. showed that \u003cem\u003eP. gingivalis\u003c/em\u003e upregulates expression of key molecules involved in the NF-κB signaling pathway in vitro, including cyclin D1, matrix metalloproteinase 2 (MMP2) and C-MYC, thereby promoting proliferation, migration and invasion by ESCC cells [30]. In addition, Liang et al. showed that miR-194 expression is significantly upregulated and expression of its direct target, GRHL3, is suppressed after \u003cem\u003eP. gingivalis\u003c/em\u003e infection. They also found that expression of the tumor suppressor gene PTEN, a direct regulatory target of GRHL3, is also diminished by activation of the phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) pathway, suggesting that \u003cem\u003eP. gingivalis\u003c/em\u003e infection promotes ESCC growth and migration [31].\u003c/p\u003e \u003cp\u003eIn addition to esophageal cancer, \u003cem\u003eP. gingivalis\u003c/em\u003e infection is significantly associated with colorectal, pancreatic, prostate, breast, uterine, lung and oropharyngeal cancers as well as non-Hodgkin's lymphoma and leukemia [32\u0026ndash;39]. In colorectal cancer, IFNGR1 is reportedly degraded through palmitoylation in lysosomes, enabling tumor immune escape [40]. This is consistent with the findings of Shen et al., who reported that decreased expression of IFNGR1 and its palmitoylation increased the proliferative, migratory and invasive capacity of ESCC cells [41]. In addition, Chattopadhyay et al. reported that \u003cem\u003eP. gingivalis\u003c/em\u003e promotes oral squamous cell carcinoma by activating the anti-apoptotic Janus kinase 1 (JAK1)/STAT3 and PI3K/Akt pathways in oral epithelial cells [42]. Liang et al. also reported a similar mechanism of cancer promotion in ESCC [31]. On the other hand, a meta-analysis conducted by Michaud et al. found a negative association between periodontal disease and risk of colorectal cancer [43]. However, we could find no other study reporting that \u003cem\u003eP. gingivalis\u003c/em\u003e inhibits the development or progression of a cancer, including ESCC. Thus, \u003cem\u003eP. gingivalis\u003c/em\u003e is implicated in numerous malignancies and is an important prognostic factor.\u003c/p\u003e \u003cp\u003eBy contrast, lactic acid bacteria are known to exert immunomodulatory, antagonistic and pathogen-suppressive effects and to exhibit strong inhibitory effects on periodontal pathogens [44]. Based on these reports and our earlier findings [17,20], we initiated a clinical trial: Improvement of oral environment with toothpaste and mouthwash containing Lactobacillus and the incident rate of pneumonia after esophagectomy for esophageal cancer patients (LacPEC study, jRCTs021230010) in June 2023. Toothpaste and mouthwash containing Lactobacillus, \u0026ldquo;beneficial\u0026rdquo; Gram-positive bacteria, are already commercially available in Japan and have been shown to significantly reduce periodontal Gram-negative bacteria and candida albicans without affecting normal components of the oral flora, including \u003cem\u003eStreptococcus mitis\u003c/em\u003e and \u003cem\u003eStreptococcus oralis\u003c/em\u003e. The results of this clinical trial suggest predominance of \u0026ldquo;beneficial\u0026rdquo; Gram-positive bacteria in the oral environment before esophagectomy is beneficial for preventing postoperative complications and, consequently, for better long-term survival.\u003c/p\u003e \u003cp\u003eIn the present study, IHC using monoclonal antibody for \u003cem\u003eP. gingivalis\u003c/em\u003e showed the presence of antigen in the cytoplasm of ESCC cells. Same results were proved in more earlier reports [45]. These results mean that part of the structures of \u003cem\u003eP. gingivalis\u003c/em\u003e have invaded or have been taken into ESCC cells. How those part of the structures of \u003cem\u003eP. gingivalis\u003c/em\u003e affect ESCC cells and those mechanisms are subjects for the further research.\u003c/p\u003e \u003cp\u003eOur previous study proved that LPS released from Gram-negative bacteria promotes ESCC proliferation in vitro and in vivo, and the influences of LPS can be inhibited by using a TLR4 blocker [20]. Therefore, LPS released from Gram-negative bacteria increases the proliferation of ESCC cells through TLR4 signaling. This phenomenon seems to be one of the molecular oncological mechanisms of the results of the present study.\u003c/p\u003e \u003cp\u003eThere are limitations to this study. The most important limitation is the lack of data on the status of the patients\u0026rsquo; oral cavity environment and an assessment of their oral bacterial flora before esophagectomy. Because we started preoperative assessment of the oral cavity environment by dentists before esophagectomy in 2009 [46], those data were not available for most patients in the cohort used for this study. While we have demonstrated that \u003cem\u003eP. gingivalis\u003c/em\u003e is associated with TLR4 expression status and patient prognosis, we have not determined whether other red complexes, \u003cem\u003eTreponema denticola\u003c/em\u003e and \u003cem\u003eTannerella forsythia\u003c/em\u003e, are also prognostic factors. Another limitation is that the present study is a single-institute, retrospective study. To solve this limitation, we need to validate specimens from other institute in the further study.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eWe have shown that infection of ESCC tissue by \u003cem\u003eP. gingivalis\u003c/em\u003e increases expression of TLR4, which leads to a poor prognosis in ESCC patients after curative esophagectomy. To improve the long-term outcomes of ESCC patients, it would be valuable to focus on improving the oral environment and preventing \u003cem\u003eP. gingivalis\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eP. gingivalis\u003c/em\u003e: \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTLRs: Toll-like receptors\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eESCC: Esophageal squamous cell carcinoma\u003c/p\u003e\n\u003cp\u003ePAMPs: Pathogen-associated molecular patterns\u003c/p\u003e\n\u003cp\u003eLPS: Lipopolysaccharide\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express our deepest gratitude to the patients who contributed tumor samples for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Y.S. and T.Y.; methodology and investigation, Y.S., T.Y., A.W. and Y.N. validation, R.S., T.Y., T.T. M.Y. and M.T.; formal analysis, K.N.; writing\u0026mdash;original draft preparation, T.Y. and Y.S.; writing, review and editing, T.Y. and Y.S., and K.T.; project administration, K. I. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Akita University School of Medicine on 20 November 2019. All experiments adhered to the principles of the Helsinki Declaration, and all study participants provided informed written consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eV A McCormack, D Menya, M O Munishi, et al. Informing etiologic research priorities for squamous cell esophageal cancer in Africa: a review of setting-specific exposures to known and putative risk factors. Int J Cancer. 2017 Jan 15;140(2):259-271. https://doi.org/10.1002/ijc.30292\u003c/li\u003e\n\u003cli\u003eChien-Hung Lee, Deng-Chyang Wu, I-Chen Wu, et al. Genetic modulation of ADH1B and ALDH2 polymorphisms with regard to alcohol and tobacco consumption for younger aged esophageal squamous cell carcinoma diagnosis. 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Effect of Porphyromonas gingivalis infection on IFNGR1 palmitoylation in esophageal cancer cells. Nan Fang Yi Ke Da Xue Xue Bao. 2023 Jul 20;43(7):1155-1163. https://doi.org/10.12122/j.issn.1673-4254.2023.07.12\u003c/li\u003e\n\u003cli\u003eIndranil Chattopadhyay, Mukesh Verma, Madhusmita Panda. Role of Oral Microbiome Signatures in Diagnosis and Prognosis of Oral Cancer. Technol Cancer Res Treat. 2019 Jan 1:18:1533033819867354. https://doi.org/10.1177/1533033819867354\u003c/li\u003e\n\u003cli\u003eDominique S Michaud, Zhuxuan Fu, Jian Shi, Mei Chung. Periodontal Disease, Tooth Loss, and Cancer Risk. Epidemiol Rev 2017 Jan 1;39(1):49-58. https://doi.org/10.1093/epirev/mxx006\u003c/li\u003e\n\u003cli\u003eWei-Kuang Lai, Ying-Chen Lu, Chun-Ren Hsieh, et al. Developing Lactic Acid Bacteria as an Oral Healthy Food. Life (Basel). 2021 Mar 24;11(4):268. https://doi.org/10.3390/life11040268\u003c/li\u003e\n\u003cli\u003eG Amodini Rajakaruna 1, Mariko Negi, Keisuke Uchida, Masaki Sekine, Asuka Furukawa, Takashi Ito, Daisuke Kobayashi, Yoshimi Suzuki, Takumi Akashi, Makoto Umeda, Walter Meinzer, Yuichi Izumi, Yoshinobu Eishi. Localization and density of Porphyromonas gingivalis and Tannerella forsythia in gingival and subgingival granulation tissues affected by chronic or aggressive periodontitis. Sci Rep. 2018 Jun 22;8(1):9507. https://doi.org/10.1038/s41598-018-27766-7\u003c/li\u003e\n\u003cli\u003eYusuke Sato, Satoru Motoyama, Hiroshi Takano, et al. Esophageal Cancer Patients Have a High Incidence of Severe Periodontitis and Preoperative Dental Care Reduces the Likelihood of Severe Pneumonia after Esophagectomy. Dig Surg. 2016;33(6):495-502. https://doi.org/10.1159/000446927\u003c/li\u003e\n\u003c/ol\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":"Porphyromonas gingivalis, Toll-like receptor, TLR4, esophageal squamous cell carcinoma, ESCC, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-5874217/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5874217/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground.\u003c/strong\u003e Poor oral health is an independent risk factor for upper-aerodigestive tract cancers, including esophageal squamous cell carcinoma (ESCC). \u003cem\u003ePorphyromonas gingivalis (P. gingivalis)\u003c/em\u003e, the most studied periodontal pathogen, is a Gram-negative bacterium with walls composed of lipopolysaccharide (LPS). Toll-like receptor 4 (TLR4) recognizes LPS, and we previously reported that ESCC patients exhibiting high TLR4 expression also show significantly poorer survival than patients expressing lower levels of TLR4. We therefore hypothesized that \u003cem\u003eP. gingivalis\u003c/em\u003e affects disease progression and prognosis in patients with ESCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients and Methods.\u003c/strong\u003e We used an ESCC tissue microarray to assess the \u003cem\u003eP. gingivalis\u003c/em\u003e status of ESCC patients and to determine whether \u003cem\u003eP. gingivalis\u003c/em\u003elevels correlate with prognosis after curative esophagectomy. We also tested whether the combined \u003cem\u003eP. gingivalis\u003c/em\u003eand TLR4 statuses correlate with prognosis in these patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e. Clinical ESCC samples from 175 patients were tested for \u003cem\u003eP. gingivalis\u003c/em\u003e. High \u003cem\u003eP. gingivalis\u003c/em\u003e levels (3+ and 2+) correlated with significantly poorer 5-year overall survival (OS) and disease-specific survival (DSS) after esophagectomy than lower \u003cem\u003eP. gingivalis\u003c/em\u003e levels (1+ and 0). Univariate and multivariate analyses showed that the \u003cem\u003eP. gingivalis\u003c/em\u003e status is an independent prognostic factor affecting 5-year OS and DSS. Moreover, the combined \u003cem\u003eP. gingivalis\u003c/em\u003e and TLR4 statuses are also an independent prognostic factor affecting 5-year OS and DSS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e. \u003cem\u003eP. gingivalis\u003c/em\u003e infection leads to TLR4 expression and a poorer prognosis in ESCC patients after esophagectomy. Preventing \u003cem\u003eP. gingivalis\u003c/em\u003e infection may be an effective strategy for improving the long-term outcomes of patients with ESCC.\u003c/p\u003e","manuscriptTitle":"Porphyromonas gingivalis levels correlate with Toll-like receptor 4 expression status and prognosis after esophagectomy for advanced thoracic esophageal squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 18:01:22","doi":"10.21203/rs.3.rs-5874217/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":"6ba0dce3-86b7-44c6-9a05-ab25b29b20a9","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-26T20:39:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-31 18:01:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5874217","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5874217","identity":"rs-5874217","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}