Low expression of PRDM5 predicts poor prognosis of esophageal squamous cell carcinoma

Low expression of PRDM5 predicts poor prognosis of 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 Low expression of PRDM5 predicts poor prognosis of esophageal squamous cell carcinoma Jing Guo, Qiuxin Yang, Sheng Wei, Jingjing Shao, Tianye Zhao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1362257/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 2 You are reading this latest preprint version Abstract Background: The role of the transcription factor PRDM5 in esophageal squamous cell carcinoma (ESCC) has not been revealed. This study investigated the relationship between PRDM5 expression and survival outcome in esophageal squamous cell carcinoma and explored the mechanism in tumor development. Methods: In present study, expression of PRDM5 mRNA in esophageal squamous cell carcinoma patients was conducted using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. The expression of PRDM5 was assessed by immunohistochemical staining. Kaplan-Meier curve and Cox regression analysis was performed to analyze the survival outcome and independent predictive factors. qRT-PCR and Methylation-specific PCR were performed to identify the mRNA level of PRDM5 and Methylation rate. Cibersort algorithm to analyze the relationship between PRDM5 expression and immune cell invasion. Western-blot was performed to confirm the expression of esophageal tumor tissues and adjacent tissues. Results: The TCGA database and GEO database show that PRDM5 mRNA level in esophageal squamous cell carcinoma adjacent tissues was higher than that of cancer tissues,and ESCC patients with high expression of PRDM5 mRNA had better overall survival.Tissue microarray showed that the protein level of PRDM5 in the adjacent tissues of patients with ESCC was higher than that in cancer tissues, and the expression level of PRDM5 was significantly correlated with the grade of clinicopathological characteristics (p<0.001). Patients with high expression of PRDM5 displayed a better OS and DFS. Cox regression analysis showed that PRDM5 was an independent risk factor and prognostic factor for ESCC patients(HR: 2.626, 95%CI: 1.824-3.781; p<0.001). The protein level of PRDM5 matched with the transcriptional level, whereas the DNA methylation affected the transcriptional level. Cibersort showed that T cells CD4 memory resting, mast cells resting, eosinophils, M2 macrophages and mast cells activated were significantly positively correlated with PRDM5 expression (P<0.05), while regulatory T cells, monocytes and dendritic cells negatively correlated with PRDM5 expression (P <0.05). Conclusion: PRDM5 can be used as a biomarker to predict the survival of ESCC patients. Furthermore, PRDM5 expression in ESCC cells may affect WNT/β-catenin signaling pathways, thus further affect the ESCC cell proliferation, migration, and invasion capacity. PRDM5 esophageal squamous cell carcinoma prognosis WNT signaling pathway immunohistochemical DNA methylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Esophageal cancer is one of the most common cancers in the world, which seriously threatens human life and health. The International Agency for Research on Cancer announced that the incidence and mortality of esophageal cancer are growing in recent years 1 . In China, the incidence and mortality of esophageal cancer rank third and fourth among all malignant tumors respectively 2 . Therefore, esophageal cancer has always been the main malignant tumor threatening the health of Chinese residents. Due to the obvious regional differences in the incidence of esophageal cancer, the incidence of ESCC is higher than esophageal adenocarcinoma. Although we have made great progress in the diagnosis and treatment of esophageal cancer in recent years, due to the high recurrence rate after treatment and the limitations of drugs and treatment strategies after metastasis. Therefore, the overall survival rate of ESCC is still disappointing in china. There is still a lack of reliability markers to guide and predict the prognosis of esophageal cancer. Most patients with esophageal cancer are ESCC in china. Therefore, there is an urgent need for effective and independent markers to predict clinical prognosis. This study aimed to investigate whether PRDM5 might be a biomarker for the prognosis of ESCC and explore the mechanism in tumor development. PRDM5 belongs to PRDM family which is a PR range zinc finger domain and quantity structure (except PRDM11) 3 . The PRDM protein N at the end of the PR domain is 20–30% similar to SET and belongs to the SET protein subtype 4 . SET has histone lysine protein transferase activity, which can change the chromosome structure and affect the form of epigenetic reprogramming, therefore may affect gene expression 5 . According to the mode of action, Zinc finger protein can bind to DNA specifically, regulate the transcription factor, and have transcriptional activity. Zinc finger protein can also mediate the interaction between proteins and participate in the post-transcriptional regulation process such as the ripening, clipping and degradation of mRNA, showing RNA binding properties 6 , 7 . The PRDM family of proteins has an unequal number of C2H2 zinc finger protein structures, while PRDM5 has 1 PR domain and 16 zinc finger structures 8 . PRDM5 has a PR/SET domain at the N-terminal, but non-histone methyltransferase (HMTase) activity. In contrast, PRDM5 acts as an epigenetic modifier by recruiting histone-modifying enzymes such as HMTase G9A and HDAC1 to the target gene 9 . It was a stress response gene that exists as a protective factor when it is not silenced by methylated 10 . Silencing PRDM5 by methylation in tumor cell lines leads to changes in various tumors including nasopharyngeal, esophageal, gastric, hepatocyte, and cervical cancer. Whereas PRDM5 methylation was seldom detected in lung, colon, ovarian, and bladder cancer cell lines 10 . Immunostaining showed nuclear expression of infected cells indicating that PRDM5 is a nuclear protein 11 . PRDM5 also plays an essential role in many aspects such as cell cycle and cell function. Besides, PRDM5 also regulates the G2/M phase cell cycle 9 , 11 , 12 . Silencing PRDM5 alone can reduce the apoptosis of glioma cells and intestinal epithelial cells 12 , 13 . Ectopic PRDM5 expression can significantly reduce the colony formation efficiency of all cancer cells in monolayer culture 10 , 12 . PRDM5 overexpression significantly inhibits the activity of the TOP flash reporter molecule, which further inhibits WNT/B-catenin signal conversion, thereby inhibiting the activity of its downstream target gene CCND1 4, 10, 14 . CHIP analysis found that PRDM5 directly binds to CDK4 and TWIST1 promoters. In addition, PRDM5 expresses the CDK4 and TWIST1 promoters of H3K4me3 and the levels of acetyl histone H4 are significantly reduced 10 . CHIP sequencing of colorectal cancer cell lines found that the response genes Adamts9, Col1a1, Mmp13 and Mgll in PRDM5 have been confirmed strongly regulated by PRDM5 15 . Further exploration of the downstream pathways can reveal that PRDM5 can be regulated by WNT/β-catenin signal, and as an epigenetic modifier of the expression of a variety of cancer genes to exert its tumor suppressor effect. Furthermore, PRDM5 also served as an early diagnostic and prognostic marker, while detection of PRDM5 DNA methylation in gastric secretion may be used as a diagnose method in early gastric cancer 16 . PRDM5 methylation is also an early event in the occurrence and development of BRAF mutant colorectal cancer. In addition, PRDM5 protein levels were significantly reduced in both mutant and wild-type intestinal polyps, especially in BRAF mutant and wild-type cancers. This indicates that PRDM5 down-regulation may start in the early stages of tumor development and continue with the development of the disease 17 . In another research, PRDM5 expression levels in glioma specimens were low and correlated with clinicopathological parameters and poor prognosis 12 . However, the protein level of PRDM5 in ESCC and the correlation between survival has not been studied so far. All the above evidence suggests that PRDM5 plays a key role in the carcinogenesis and development of tumors, and guides us to further explore whether PRDM5 acts as a tumor suppressor gene in ESCC and the prognostic significance of PRDM5. Methods Bioinformation analysis of public datasets TCGA&GEO PRDM5 mRNA levels and corresponding information of samples were downloaded from TCGA (https://www.cancer.gov/) and GEO database (http://www. ncbi.nlm.nih.gov /GEO/). The database was collected under the following conditions: (1) containing at least 10 cases of normal tissue; (2) PRDM5 expression data were extracted comprehensively. In addition, two ESCC data sets were downloaded from database: GSE53624 and GSE53622. Both the TCGA and GEO databases contain information on the clinical characteristics and prognosis information of patients with ESCC. Human esophageal squamous cell carcinoma specimens and clinical data collection Tissue microarrays were obtained from 2 groups of independent samples of patients with esophageal squamous cell carcinoma from 2 medical centers. The discovery sets included 169 patients underwent curative esophagectomy obtained from the First People's Hospital of Nantong City from 2010 to 2014. Due to the poor quality of tissue microarrays and the lost of patient's clinical information, and 154 cases were used for research. Among them, there were 147 paired tissues between cancer and adjacent cancer. The validation group included 279 patients underwent curative esophagectomy from 2013 to 2014 in the Affiliated Hospital of Nantong University. Due to the poor quality of tissue microarrays and the lost of patient's clinical information, there were 255 cases for research. Clinical baseline data were collected for each patient retrospectively. None of the patients received any of the preoperative anticancer treatment such as preoperative chemotherapy, radiotherapy and immunotherapy before surgery, and concurrent radical Esophageal squamous cell carcinoma surgery. All the information of patients including age, gender, tumor size, depth of invasion (T), lymph node status (N), metastasis (M), and overall survival (OS) were recorded from computerized medical records. Using of human tissues with informed consent was confirmed by each patient. This research was approved by the Clinical Research Ethics Committee of each hospital. Immunohistochemistry (IHC) staining and scoring Formalin-fixed paraffin-embedded surgical specimens were performed for construction of tissue microarray (TMA). Tissue blocks were sectioned at 2mm and prepared on glass slides. A subsequent immunohistochemistry study was conducted to identify expression of PRDM5. The immunohistochemical protocols were performed as below. TMA was firstly fixed by formaldehyde and repaired by EDTA, then hydrogen peroxide was used to extinguish endogenous HRP. After rinsing, the primary PRDM5 antibody (purchased from abcam) was diluted (1:1000) with antibody diluent. The primary antibody was taken out the next day and washed three times and then incubated with a secondary antibody (rabbit 1:2000 Proteintech) for 20 minutes. All specimens were independently reassessed by 2 pathologists according to the International Union against Cancer TNM classification system. The intensity grades of staining are described as follows: negative (0), weak (1), medium (2), and strong (3), while the degree of staining was scored according to the percentage of positive cells：0（0%）; 1（1%-25%）; 2(26%-50%); 3(51%- 75%); and 4(76%-100%). The combined score was calculated by multiplying the intensity and density of staining eventually. Low expression of PRDM5 is defined as a score of 1-6, and high expression of PRDM5 is defined as a score of ≥7. Quantitative real-time PCR (qRT-PCR) A total of 15 pairs of esophageal tumor and para-tumor tissues were collected from surgical specimens of patients underwent radical surgery of esophageal tumor in Nantong Tumor Hospital. Total RNA was extracted using Trizol (Sigma) according to the manufacturer’s instructions, and then 1ug of obtaining cDNA was extracted for reverse transcription and detect the expression level of endogenous PRDM5 mRNA. Accurate quantification was achieved using the standard curve, which was produced by continuously diluting a known amount of RNA from the in vitro transcription response and using the dilution series for TaqManqPCR with the patient sample. Quantitative analysis of mRNA expression was conducted using the StepOnePlus™ Real-Time PCR System. The primers and TaqMan probes for analysis were designed using the manufacturer's software, PrimerExpress, and the PRDM5 primers were supplied by and. The reference gene GAPDH is used as an internal control for RNA quality. All quantitative analyses were repeated to assess the consistency of the results. Relatively standardization of the target relative to the GAPDH gene expression level calculation for Δ Ct = Ct (target) - Ct (GAPDH). The relative quantification values of PRDM5 were calculated using the 2 - △△ CT method. Methylation-specific polymerase chain reaction (MSP) By methylation-specific polymerase chain reaction (methylation-specific polymerase chain reaction, MSP) method, collection of esophageal squamous cell carcinoma patients who resection of carcinoma tissue and paired normal tissue adjacent to carcinoma, using tissue genomic DNA extraction kit (QIAGEN GmbH) to extract DNA, the DNA methylation kit (ZYMO RESEARCH.) for DNA denaturation and bisulfite conversion. The transformed DNA was used as a template for PCR amplification. PRDM5 methylation-specific primers and non-methylation-specific primers (Servicebio) were designed. The methylation-specific primers were upstream 5-TTGTTTCGGGTTTCGCGTTC-3 and downstream 5-ATTCCTACTACGAAAACGCG-3. PRDM5 geneno-methylation-specific primers were upstream 5-TAGTTTTGTTTTGGGTTTTGT-3 and downstream 5- CCATTCCTACTACAAAAACACA-3. PCR reaction system: DNA template 1 L, 5 × PCR buffer 10 L, upstream and downstream primers 0.5 L each, with ribozyme free water added up to 20 L. PCR reaction conditions: pre-denaturation at 95 ℃ for 120 s, denaturation at 95 ℃ for 60 s, annealing at 55 ℃ for 60 s, elongation at 72 ℃ for 60 s, 40 cycles. PCR products were analyzed by the gelatinization imaging system. For example, the target band amplified by gene methylation-specific primers indicated positive methylation of the PRDM5 gene, and the gene methylation rate was calculated. Western blot analysis A total of 15 pairs of esophageal tumor and para-tumor tissues were collected from surgical specimens of patients underwent radical surgery of esophageal tumor in Nantong Tumor Hospital. Extract tissue protein: Take out fresh esophageal cancer and adjacent tissues from the refrigerator at -80℃, rewarm on ice, and then weigh 0.1 g of each sample into a 1.5 mL EP tube, add 1 mL Mix the protein lysate and lyse it on ice for 10 minutes, put it in a biological sample homogenizer, fully homogenize and ice bath for 30 minutes, then transfer to a low-temperature centrifuge, set at 4℃, 12000 rpm, and centrifuge for 20 minutes. After centrifugation, transfer the supernatant to a new EP tube. Prepare the protein loading solution according to 5× loading buffer: protein lysate=1:4, pipette and mix well, dry at 100℃ Boil in a thermostat for 10 minutes, and centrifuge again at 4°C and 12000 rpm for 10 minutes. Finally, determine the protein concentration in each sample and record it. Store it at -20°C. When used, take it out and boil for 3 minutes. Centrifuge at 4°C and 12000 rpm for 3 minutes before loading. Sample Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (microporous). At room temperature, the membrane was enclosed in 5% bovine serum albumin for 1 hour. The membrane was incubated with a primary antibody at 4℃ overnight, GAPDH (1:500, #66009-1-1g; Proteintech), PRDM5(1:1000, # ab7609; Abcam) were washed three times with TBST and incubated at room temperature with horseradish peroxidase-conjugated secondary antibodies for 1 hour. TBST was used three times and ECL (bio-rad) visualized. Immune Cell Infiltration Analysis A deconvolution algorithm based on CIBERSORT gene expression (http://cibersort.stanford.edu/) 18 was used to evaluate the immune cell infiltration in 179 patients obtained from the GEO database (http://www. ncbi.nlm.nih.gov /GEO/). The samples were divided into two groups according to the average expression of PRDM5, Wilcoxon Signed Rank test was used to compare immune cell content between groups with high and low expression of PRDM5 mRNA. Heatmaps and violin plots were analyzed and plotted using the packages“pheatmap”and“vioplot”, respectively. Statistical analysis All statistical analyses were performed using the SPSS19.0 software (SPSS, Chicago, Illinois) and GraphPad Prism 5 software (GraphPad Software, Inc.) R software (http://www. r-project.org/) was used to extract and standardize mRNA expression data from these databases, and then GraphPad Prism Version 8.0 (GraphPad Software, Inc.) was used for statistical analysis. The correlation between PRDM5 gene expression and copy number variation and DNA methylation was analyzed using online database cBioPortal (http://www.cbioportal.org/) and MEXPRESS (http://mexpress.be/). The paired or unpaired Student's t-test was performed to analyze the statistical significance between two groups. Cox regression model was used to determine the risk factors of OS through univariate and multivariate analysis. The association between expression levels and progression-free survival in Esophageal squamous cell carcinoma patients were analyzed by Kaplan–Meier survival curves, using Kaplan-Meier Plotter. Log-rank test was used for statistical analysis. A p-value<0.05 was considered statistically significant. Results Esophageal tumor tissues exhibit decreased PRDM5 mRNA expression PRDM5 mRNA levels and corresponding information of samples were downloaded from TCGA and GEO database. From TCGA database and GSE53622, GSE53624 two data sets, it was found that PRDM5 mRNA level in esophageal tumor tissue was significantly lower than para-tumor tissue (Fig. 1 A). Correlation of PRDM5 mRNA levels with overall survival (OS) in esophageal tumor patients Based on the TCGA database, patients of Esophageal squamous cell carcinoma with high PRDM5 mRNA levels displayed longer OS than those with low expression ( p =0.0254) (Fig. 1 b). Whereas, although there is no statistical significance between the high mRNA levels of PRDM5 expression with the OS in GEO data sets, we did observe the trend of a prolonged OS from the point of view of survival analysis (Fig. 1 B). Patients’ characteristics and immunohistochemical in TMA Patient characteristics and pathological and clinical features are shown in Table 1 . To investigate the relationship between PRDM5 and prognosis of Esophageal squamous cell carcinoma, immunohistochemistry was performed to evaluate the expression level of PRDM5 in tissue microarray (TMA) of 409 patients (discovery data set 154, validation data set 255). Esophageal squamous cell carcinoma tissues and adjacent tissues were paired in the discovery set. As indicated before, the combined score was calculated by multiplying the intensity and density of staining eventually. Low expression of PRDM5 was defined as a score of 0-6, while high expression of PRDM5 was defined as a score of ≥7. It was found that the expression of PRDM5 in adjacent tissues was higher than in tumor tissues (Fig. 2 D). Furthermore, it was also shown that PRDM5 expression was lower in poorly differentiated Esophageal squamous cell carcinoma and on the contrary in highly differentiated Esophageal squamous cell carcinoma (Fig. 2 A-C. The immunohistochemical staining showed a high expression of PRDM5 displayed 29.2% in the discovery data set and 30.1% in the validation data set (Fig. 2 E). Detailed characteristics are summarized in Table 1 ,2. The correlation of PRDM5 exprssion and DFS, OS In the discovery data set, we analyzed the relationship between the expression of PRDM5 and the DFS and OS of patients. As indicated in Fig. 3 A, patients with high expression of PRDM5 had prolonged DFS and OS comparing with patients with low expression. It was either shown in the validation set and the combined data set (Fig. 3 B-C). Besides, the expression levels of PRDM5 were related to TNM staging, tumor differentiation, and vascular invasion in the combined data set (Table 3 ). Through univariate analysis, we found that T stage (HR: 0.573, 95%CI: 0.43-0.764; p<0.001), N stage(HR: 0.526, 95%CI: 0.401-0.69; p<0.001), TNM staging(HR: 0.509, 95%CI: 0.388-0.667;p<0.001), PRDM5 expression level (HR: 2.986, 95%CI: 2.084-4.278; p<0.001), differentiation degree (HR: 0.724, 95%CI: 0.537-0.976; p=0.034), nerve infiltration (HR: 0.643, 95%CI: 0.429-0.964; p=0.0.033), and vascular invasion (HR: 0.497, 95%CL: 0.359-0.688; p<0.001) were risk factors of ESCC patients. Multivariate analysis revealed the independent prognostic factors for ESCC patients were expression of PRDM5 (HR: 2.626, 95%CI: 1.824-3.781; p<0.001), T stage (HR: 0.719, 95%CI: 0.533-0.969; p=0.0.03), TNM stages (HR: 0.657, 95%CI: 0.49-0.881; p<0.001), and vascular invasion (HR: 0.694, 95%CL: 0.493-0.976; p=0.0.036) (Table 4 ). Stratified analysis of OS in esophageal tumor patients Detailed research was conducted to investigate the correlation of several factors related to overall survival in esophageal tumor patients. Patients were stratified into several groups according to factors as below: postoperative radiotherapy, postoperative chemoradiotherapy, postoperative chemotherapy, no treatment. As shown in Fig. 4 A, postoperative radiotherapy could prolong the OS in total group, while the postoperative chemoradiotherapy didn’t affect the OS. In Fig. 4 B, postoperative therapy didn’t affect the OS in stage Ⅰ-Ⅱ Esophageal squamous cell carcinoma patients. Whereas, in stage Ⅲ-Ⅳ Esophageal squamous cell carcinoma patients, postoperative treatment could extend the OS especially in patients who underwent postoperative radiotherapy (Fig. 4 B-C). Moreover, the high expression level of PRDM5 was associate with longer OS in patients who underwent postoperative therapy(Fig. 4 D-E). The analysis based on the relapse-free survival (RFS) showed there are significant differences among the low expression PRDM5 and the high group (Fig. 4 F). Prognostic nomogram and calibration plots of Esophageal squamous cell carcinoma Based on the results of univariate and multivariate analysis, we constructed a nomogram to predict the operating system at 3-year, 5-year, and 10-year after surgery (Supplementary Figure 1). According to preliminary analysis, predictive factors include T status, vascular invasion, tumor grade, T stage, TNM staging, and PRDM5 staining, all of which were important prognostic indicators of OS. The transcriptional level of PRDM5 was consistent with the protein level and was affected by methylation Next, the expression of PRDM5 of esophageal tumor tissues and paired para-tumor tissues were analyzed. It was found that the expression of PRDM5 was higher in 15 specimens of esophageal tumor than para-tumor tissues (Fig. 5 A). We selected 10 pairs of tissues with high expression of PRDM5-mRNA level in para-tumor tissues, Western-blot was performed using 10 pairs of cancer and adjacent tissues to detect protein expression levels and found the protein level of PRDM5 in adjacent tissues was higher than that tumor tissues, which proved that PRDM5 had consistency in both the transcription level and protein level changes (Fig. 5 B). We then performed methylation PCR on eight pairs of Esophageal squamous cell carcinoma tissue and found that PRDM5 DNA was highly methylated in cancer tissues, and absent in adjacent tissues (Fig. 5 C). Therefore, we further investigated the relationship between PRDM5 DNA methylation and copy number in ESCC in cBioPortal. There was no significant difference between PPRDM5 DNA methylation and copy number (Supplementary Fig. 6A). However, there was a significant negative correlation between PRDM5-mRNA expression and DNA methylation, which was confirmed by Pearson correlation coefficient (Spearman: -0.61, p =1.02E-19) (Supplementary Fig. 6B). We also found in the MEXPRESS ( http://mexpress.be/ ) database that methylation at CPG position 120921881 was negatively correlated with PRDM5-mRNA expression (Supplementary Fig. 6C). Therefore, all these data suggested that methylation at CPG Island 120921881(the promoter of PRDM5) leads to down-regulation of PRDM5 mRNA expression, which in turn leads to a decrease in PRDM5 protein level. PRDM5 is associated with immune infiltration of ESCC We also used the Cibersort algorithm to analyze the expression levels of 22 immune cell subgroups and evaluate their correlation with PRDM5 expression. The results showed that T cells CD4 memory resting, Mast cells resting, Eosinophils, M2 macrophages and Mast cells activated were significantly positively correlated with PRDM5expression (P < 0.05). T cells regulatory (Tregs), Monocytes, and Dendritic cells resting were negatively correlated with PRDM5 expression(Fig. 7 A). In addition, we evaluated possible associations between 22 immune cells, and heat maps showed weak to moderate correlations in the rates of different tumor-infiltrating immune cell subgroups(Fig. 7 B). Discussion In this study, we did research on the clinical role of PRDM5 ,Based on this research, we found that the expression of PRDM5 in esophageal para-tumor tissue was significantly higher than in tumor tissues, regardless of mRNA level or protein level, and Esophageal squamous cell carcinoma patients with high expression of PRDM5 had a better prognosis. Recent studies indicated that high levels of PRDM5 are associated with better outcomes for glioma and colorectal cancer 12 , 17 . Our research showed that postoperative chemotherapy is necessary for esophageal patients with stage III-IV after surgery, and postoperative radiotherapy significantly prolonged the patient's OS. However, postoperative adjuvant treatment for patients with stage Ⅰ-Ⅱ Esophageal squamous cell carcinoma after surgery has little benefit on OS, the results also suggested that postoperative adjuvant treatment for patients with high expression of PRDM5 can help to prolong DFS and OS. This might indicate that PRDM5 could affect the therapeutic effect. Previous studies have shown that ectopic expression of PRDM5 can inhibit the proliferation of gastric cancer, nasopharyngeal cancer, and brain glioma cells 10 , 12 . Our study showed that the expression of PRDM5 was correlated with pathological status T stage. It also demonstrated that over-expression of PRDM5 inhibited proliferation activity in esophageal carcinoma cell. This aroused our thinking, indicating that the patient's pathological information such as the high expression of PRDM5 should be considered comprehensively when choosing postoperative adjuvant treatment and patients treatment strategies. However, since it is difficult to obtain dynamic monitoring only by immunohistochemical methods, we suggest that it might be more helpful to detect PRDM5 expression in patients' serum. This experiment proved that PRDM5 transcriptional level and protein level were consistent, and the transcriptional level was affected by methylation. Previous studies have found that PRDM5 expression is lower in multiple cancer species than in adjacent tissues, including breast, ovarian, liver, lung, and colon cancers, and cervical cancers 10 , and that silencing or downregulation of PRDM5 is often caused by DNA methylation 10 , 15 , 17 , 19 . And then extended to epigenetics, studies have shown that long-term heavy drinking can leading to homocysteine and S-adenosine homocysteine (SAH) increment, resulting in histone modifications and changes in gene expression. Eventually affect the small RNA family which serve as epigenetic regulators causing DNA promoter methylation 20 . In this study, Esophageal squamous cell carcinoma patients with drinking history were relatively small proportion, so the history of drinking was not statistically significant. However, we could find that the PRDM5 expression was often lower in patients with a long-term history of drinking, which may be related to DNA methylation. Studies have shown that PRDM5 is a stress response gene 10 . It may provide protection when the esophagus is physically or chemically stimulated, but once methylation is silenced or its expression is reduced, it may lead to the occurrence and development of Esophageal squamous cell carcinoma. We also found in database that methylation at CPG position 120921881 was negatively correlated with PRDM5 mRNA expression, suggesting that methylation is negatively correlated with PRDM5 mRNA expression. In future studies, we would confirm whether this site causes the silencing or reduction of PRDM5 expression. It is worth considering whether blocking the methylation at certain site should be considered in clinical applications for patients with low PRDM5 expression. The methylation of PRDM5 may be involved in the early events of esophageal squamous cell carcinoma, such as esophagitis. Then this may also be used as a biomarker for the early diagnosis of esophageal squamous cell carcinoma. Future research should expand the sample size of esophageal squamous cell carcinoma and add patient samples from patients with esophagitis for further research.,We believe that in the future, PRDM5 methylation may also become part of cancer screening, and there is a blood-based screening for DNA methylation markers in colorectal cancer 21 , we showed that epigenetic therapy of PRDM5 is achievable. In the future, we anticipate targeted therapies will also include epigenetically inactivated tumor inhibitors, such as the CRISPRDCAS9 technology and the viral application of the epigenetic editor to reactivate. our results showed that T cells CD4 memory resting activated were significantly positively correlated with PRDM5expression ,Recent studies have shown that CD4+ T cells are not a pure cell lineage with a single function, but a diverse cell population with complex functions. In addition, CD4+ T cells can be used not only as helper cells, but also as effective effector cells or partners with macrophages and eosinophils to eliminate various tumors, and Patients with increased CD4+ T cell ratio in serum samples of patients with esophageal squamous cell carcinoma undergoing radiotherapy have better survival 22 . Whether the better prognosis of patients with esophageal squamous cell carcinoma with high expression of PRDM5 is related to the recruitment of memory CD4+ T cells by PRDM5 to enhance its killing effect on tumor cells. The effect of PRDM5 on immune function will be worthy of our further study. Previous studies have shown that the inhibition of WNT signaling by ectopic expression of PRDM5 weakens the transcriptional activity of CCND1 gene, which in turn affects the changes of CDK4. 10 . Studies have shown that CDK4 can phosphorylate Smad3, which is very important in response to TGF-β signal transcription activity. Phosphorylation of Smad3 by CDK4 inhibits its anti-proliferation function 23 . CDK4 also directly phosphorylates FOXM1 at multiple sites to positively regulate its activity, possibly protecting cancer cells from senescence by inhibiting reactive oxygen species (ROS) levels, and promoting the entry of G1/S phase by regulating the expression of several genes in cancer cells (such as cyclin E2, MYB and MCM2) 24 . CDK4 can phosphorylate MEP50 and increase methyltransferase activity and cell survival function 25 . Our previous results confirmed that the expression level of PRDM5 is related to the pathological characteristics of tumor size, then we speculate whether the expression of PRDM5 affected CDK4 and thus affected the proliferation ability of esophageal squamous cell carcinoma cells. We further speculate that PRDM5 may be involved in the CCND1-CDK4/CDK6 pathway, and that patients with low expression of PRDM5 may increased the expression levels of CCND1 and CDK4. It is worth exploring whether CDK4/CDK6 inhibitors (Palbociclib) can be considered for this type of patients, We will further verify its relationship with CCND1 and CDK4 in future cell function experiments, which is of great significance for clinical treatments. The previous results indicate that the expression of PRDM5 is related to the clinical pathological parameter lymph node metastasis status, and we speculate that it is related to the WNT signaling pathway and TWIST1.Studies have shown that β-catenin was found in the form of continuous protein methylation after activation and accumulated in the silent PRDM5 cell line. This leads to the activation of the Wnt/β-catenin signaling pathway, downstream LEF/TCF transcriptional activity and the start of EMT process 10 . Many studies have indicated that the WNT signaling pathway can impinge the development of Esophageal squamous cell carcinoma through the EMT pathway 26 – 29 . Studies have also shown that PRDM5 can bind to the promoter of TWIST1 to affect its transcriptional activity 10 . Studies have shown that patients with esophageal squamous cell carcinoma overexpressing TWIST1 had a poor prognosis, and the increase of TWIST1 can affected the invasion and migration ability of esophageal squamous cell carcinoma 30 – 32 . Relevant studies have shown that silence of TWIST1 in esophageal squamous cell carcinoma, cervical cancer and non-small cell lung cancer can enhance its drug sensitivity to cisplatin 33 – 35 , and studies have also shown that increased expression of TWIST1 can enhance the radiotherapy resistance of esophageal squamous cell carcinoma cells 36 . We suspect that this may be related to the longer overall survival of patients with high expression of PRDM5 that we have observed after adjuvant treatment after surgery. Patients with high expression of PRDM5 may have lower levels of TWIST1. In future studies, we should further verify the correlation between PRDM5 and TWIST1 at the cellular level. It is best to monitor the blood dynamics of clinical samples of esophageal squamous cell carcinoma to assess their impact on the treatment effect. Taken together, this work uncovers PRDM5 is a negative prognostic factor in esophageal tumor cell. Future researches should focus on how to conduct clinical detection of methylation sites to predict the prognosis of Esophageal squamous cell carcinoma patients, In the future, more detailed studies are still needed to describe and reveal the underlying mechanism of PRDM5 multiple pathological functions in esophageal squamous cell carcinoma. A comprehensive understanding of the multiple roles of PRDM5 in esophageal squamous cell carcinoma will provide more important clinical value for PRDM5 as a diagnostic indicator, prognostic marker and treatment target for clinical treatment. Conclusions As a tumor suppressor gene in Esophageal squamous cell carcinoma, PRDM5 can be used as a biomarker to predict the survival of patients with Esophageal squamous cell carcinoma, and the high expression level of PRDM5 is associate with longer OS in patients who underwent postoperative therapy. Furthermore, PRDM5 expression in Esophageal squamous cell carcinoma cells may affects WNT/β-catenin signaling pathways, thus further affect the Esophageal squamous cell carcinoma cell proliferation, migration, and invasion capacity. Abbreviations ESCC: Esophageal squamous cell carcinoma; IARC: The International Agency for Research on Cancer; EMT: Epithelial-Mesenchymal Transition; IHC: Immunohistochemistry; TCGA: The Cancer Genome Atlas; GEO: Gene Expression DataSets; qRT-PCR: quantitative Real-time Polymerase Chain Reaction; MSP: Methylation-specific polymerase chain reaction; TMA: Tissue microarray Confidence intervals; CI: Confidence intervals; HR: Hazard ratio; OS: Overall survival; DFS: Disease-free survival; PBS: Phosphate buffer saline; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PRDM5: PR domain zinc finger protein 5; Si-RNA: Small interfering RNA; TCF/LEF: T-cell Factor/Lymphoid Enhancing Factor; BRAF: B-Raf proto-oncogene, serine/threonine kinase; CHIP: Chromatin Immunoprecipitation; HDAC1: Histone deacetylase 1. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of The Affiliated Hospital of Nantong University. All patients signed an informed consent approved by the institutional review board. The authors have declared that no competing interest. Consent to publish Not applicable. Availability of data and materials All the data generated or analyzed during this study are included in this published article. The GSE53622 dataset is available at (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53622) and GSE53624 dataset is available at (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53624) respectively. Competing Interests The authors have declared that no competing interest exists. Funding This research was supported by the funding of “Postgraduate Research&Practice Innovation Program of Jiangsu Province”, Scientific Research Subject of the Health commission of Nantong (WKZL2018048) and the Scientific Research Subject of Nantong Science and Technology Bureau (MSZ19233). Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JG, TYZ, QXY, and JL. SW, JJS, and LYG performed statistical analysis. The first draft of the manuscript was written by JG, JC and GRW, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank the Department of Radiotherapy, Department of Oncology & Cancer Research Center Nantong of Affiliated Tumor Hospital of Nantong University for providing the patient information used in this article. References Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018, 68(6):394–424. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J: Cancer statistics in China, 2015. CA Cancer J Clin 2016, 66(2):115–132. Cathrine K. 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Chunbao Zang1, Xujie Liu1,* Wu1, Bingqian Zhang1, Bing Li2,*, Shuhong Ma1, YH, Weiwei Dai1, SJ, Shaolin Li1, Yujia He1, Wenli, Zhiping Peng1: IL-6/STAT3/TWIST inhibition reverses ionizing radiationinduced EMT and radioresistance in esophageal squamous carcinoma. Oncotarget 2017, 8(pp):11228-11238. Tables Table 1 Relationship between PRDM5 expression and clinicopathological characteristics in Discovery Data Set PRDM5 LOW PRDM5 HIGH P-value Patient characteristics N=109 N=45 Gender 0.872 female 28 11 male 81 34 Age 0.864 ≧60 79 32 < 60 30 13 Tumorinformation Location 0.866 uper 7 2 Middle 50 20 Lower 52 23 Tobacco 0.812 N0 89 36 YES 20 9 Alcohol 0.331 N0 93 41 YES 16 4 T stage 0.105 I-II 38 22 III-IV 71 23 N stage 0.091 N0 59 31 YES 50 14 M stage 0.360 N0 107 45 YES 2 0 TNM stage 0.122 I-II 63 32 III-IV 46 13 Histological grade <0.001 Well 3 7 Moderately 65 35 Poorly 41 3 Accompanying diseases 0.933 N0 89 37 YES 20 8 Positive margin N0 101 41 0.744 YES 8 4 Nerve invasion 0.178 N0 87 40 YES 22 5 Vascular invasion 0.013 N0 83 42 YES 26 3 Table 2 Relationship between PRDM5 expression and clinicopathological characteristics in Validation Data Set Patient characteristics N=178 N=77 Gender 0.177 female 54 17 male 124 60 Age 0.496 ≧60 134 61 < 60 44 16 T stage 0.012 I-II 67 35 III-IV 111 42 N stage 0.061 N0 93 50 YES 85 27 M stage 0.540 N0 177 76 YES 1 1 TNM stage 0.013 I-II 95 54 III-IV 83 23 Histological grade 0.030 Well 19 14 Moderately 89 45 Poorly 70 18 Nerve invasion 0.232 N0 167 75 YES 11 2 Vascular invasion 0.045 N0 147 71 YES 31 6 Table 3 Relationship between PRDM5 expression and clinicopathological characteristics in Combined Data Set Patient characteristics N=287 N=122 Gender 0.241 female 82 28 male 205 94 Age 0.668 ≧60 74 93 < 60 213 29 T stage 0.003 I-II 105 57 III-IV 182 65 N stage 0.012 N0 152 81 YES 135 41 M stage 0.832 N0 284 121 YES 3 1 TNM stage 0.004 I-II 158 86 III-IV 129 36 Histological grade <0.001 Well 22 21 Moderately 154 80 Poorly 111 21 Nerve invasion 0.073 N0 254 115 YES 33 7 Vascular invasion 0.002 N0 230 113 YES 57 9 Table 4 Univariate and multivariate analysis of characteristics associated with overall survival Variables Univariate analysis multivariate analysis Hazard Ratio 95%CI P-value Hazard Ratio 95%CI P-value Age 1.041 0.764 1.418 0.798 gender 0.889 0.653 1.212 0.458 T stage 0.573 0.43 0.764 <0.001 0.719 0.533 0.969 0.03 N stage 0.526 0.401 0.69 <0.001 M stage 0.385 0.143 1.037 0.059 TNM stage 0.509 0.388 0.667 <0.001 0.657 0.49 0.881 <0.001 Histological grade 0.724 0.537 0.976 0.034 PRDM5 2.986 2.084 4.278 <0.001 2.626 1.824 3.781 <0.001 Vascular invasion 0.497 0.359 0.688 <0.001 0.694 0.493 0.976 0.036 Nerve invasion 0.643 0.429 0.964 0.033 Supplementary Files SupplementaryFig1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 Feb, 2022 First submitted to journal 29 Nov, 2021 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. 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(B) PRDM5 mRNA level and OS in esophageal cancer.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/b06dafb33272c33e5e954768.png"},{"id":18370892,"identity":"82500482-4f7f-4bef-bccd-e9bb1a4f449d","added_by":"auto","created_at":"2022-02-18 16:19:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":818496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative pictures of PRDM5 expression in ESCC tissues \u003c/strong\u003e(left50×, middle 100×,right20×). (A) Poorly differentiated carcinoma and para-cancer tissue (B)Moderately differentiated carcinoma and para-cancer tissue (C) Highly differentiated carcinoma and para-cancer tissue (D) Protein level of PRDM5 carcinoma and para-cancer tissue. (E)The expression of PRDM5 in esophageal carcinoma with well, middle and poor differentiation.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/bc6d9c6b0c9a0f17d670b14c.png"},{"id":18370610,"identity":"326866b1-a9b8-4b83-97d4-fb7fb34d7064","added_by":"auto","created_at":"2022-02-18 16:16:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":143133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between PRDM5 expression and OS/DFS in esophageal cancer patients.\u003c/strong\u003e (A) Discovery Data Set. (B) Validation Data Set. (C) Combined Data Set.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/042b8aa2fcdad31e4dc080db.png"},{"id":18370613,"identity":"2a8ac838-0244-4d4e-8ea3-cc8cb20ac32e","added_by":"auto","created_at":"2022-02-18 16:16:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":170263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of PRDMA5 and postoperative treatment on overall survival (OS). \u003c/strong\u003e(A) postoperative treatment and OS. (B) Postoperative treatment and OS in stage Ⅰ-Ⅱ. (C) Postoperative treatment and OS in stage Ⅲ-Ⅳ. (D) PRDM5 expression and OS in patients underwent postoperative radiotherapy. (E) PRDM5 expression and OS in patients underwent postoperative chemotherapy. (F) PRDM5 expression and RFS.\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/685ca4633441e3fd671119dc.png"},{"id":18370614,"identity":"c771f7a8-50d7-4878-9d6d-4c99297b5137","added_by":"auto","created_at":"2022-02-18 16:16:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":146776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRDM5 expression in adjacent tissues and tumor tissues.\u003c/strong\u003e (A) mRNA level in adjacent tissues and tumor tissues. (B) Protein level of PRDM5 in adjacent tissues and tumor tissues. (C) Methylation-specific PCR in adjacent tissues and tumor tissues.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/6d3099b546d94a554395963d.png"},{"id":18370612,"identity":"b742ebf7-b178-449d-99db-1c31d661e036","added_by":"auto","created_at":"2022-02-18 16:16:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":207511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between PRDM5 in online databases and methylation. \u003c/strong\u003e(A) The relationship between PRDM5 methylation and DNA copy number. (B) The relationship between PRDM5 methylation and mRNA. (C) PRDM5 methylation site prediction.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/4c2a9713e9d2c7af85d05812.png"},{"id":18370611,"identity":"011f7d66-b00b-43f8-872b-0fff4238b6c9","added_by":"auto","created_at":"2022-02-18 16:16:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":354447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relationship between the expression of PRDM5 and immune infiltration in patients with esophageal squamous cell carcinoma. \u003c/strong\u003e(A) The ratio of 22 subtypes of immune cells in the high and low PRDM5 expression groups in esophageal squamous cell carcinoma samples is different. (B) Heat map of 22 immune infiltrating cells in esophageal squamous cell carcinoma samples.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/6257fa66f6ff529b1a87c0c6.png"},{"id":18370893,"identity":"ab307b01-1ad2-41bd-8c83-fcb842693b6d","added_by":"auto","created_at":"2022-02-18 16:19:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1256824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/7afe81af-a9cf-47b9-88c7-e264392679ac.pdf"},{"id":18370608,"identity":"886f33d3-80ad-44fb-8ec8-b1ce7e7a09bc","added_by":"auto","created_at":"2022-02-18 16:16:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":187832,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1362257/v1/6c8728df8e0b4e86df7e2387.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eLow expression of PRDM5 predicts poor prognosis of esophageal squamous cell carcinoma\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eEsophageal cancer is one of the most common cancers in the world, which seriously threatens human life and health. The International Agency for Research on Cancer announced that the incidence and mortality of esophageal cancer are growing in recent years\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In China, the incidence and mortality of esophageal cancer rank third and fourth among all malignant tumors respectively\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Therefore, esophageal cancer has always been the main malignant tumor threatening the health of Chinese residents. Due to the obvious regional differences in the incidence of esophageal cancer, the incidence of ESCC is higher than esophageal adenocarcinoma. Although we have made great progress in the diagnosis and treatment of esophageal cancer in recent years, due to the high recurrence rate after treatment and the limitations of drugs and treatment strategies after metastasis. Therefore, the overall survival rate of ESCC is still disappointing in china. There is still a lack of reliability markers to guide and predict the prognosis of esophageal cancer. Most patients with esophageal cancer are ESCC in china. Therefore, there is an urgent need for effective and independent markers to predict clinical prognosis. This study aimed to investigate whether PRDM5 might be a biomarker for the prognosis of ESCC and explore the mechanism in tumor development.\u003c/p\u003e \u003cp\u003ePRDM5 belongs to PRDM family which is a PR range zinc finger domain and quantity structure (except PRDM11)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The PRDM protein N at the end of the PR domain is 20\u0026ndash;30% similar to SET and belongs to the SET protein subtype\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. SET has histone lysine protein transferase activity, which can change the chromosome structure and affect the form of epigenetic reprogramming, therefore may affect gene expression\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. According to the mode of action, Zinc finger protein can bind to DNA specifically, regulate the transcription factor, and have transcriptional activity. Zinc finger protein can also mediate the interaction between proteins and participate in the post-transcriptional regulation process such as the ripening, clipping and degradation of mRNA, showing RNA binding properties\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The PRDM family of proteins has an unequal number of C2H2 zinc finger protein structures, while PRDM5 has 1 PR domain and 16 zinc finger structures\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePRDM5 has a PR/SET domain at the N-terminal, but non-histone methyltransferase (HMTase) activity. In contrast, PRDM5 acts as an epigenetic modifier by recruiting histone-modifying enzymes such as HMTase G9A and HDAC1 to the target gene\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. It was a stress response gene that exists as a protective factor when it is not silenced by methylated\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Silencing PRDM5 by methylation in tumor cell lines leads to changes in various tumors including nasopharyngeal, esophageal, gastric, hepatocyte, and cervical cancer. Whereas PRDM5 methylation was seldom detected in lung, colon, ovarian, and bladder cancer cell lines\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Immunostaining showed nuclear expression of infected cells indicating that PRDM5 is a nuclear protein\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. PRDM5 also plays an essential role in many aspects such as cell cycle and cell function. Besides, PRDM5 also regulates the G2/M phase cell cycle\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Silencing PRDM5 alone can reduce the apoptosis of glioma cells and intestinal epithelial cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Ectopic PRDM5 expression can significantly reduce the colony formation efficiency of all cancer cells in monolayer culture\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. PRDM5 overexpression significantly inhibits the activity of the TOP flash reporter molecule, which further inhibits WNT/B-catenin signal conversion, thereby inhibiting the activity of its downstream target gene CCND1\u003csup\u003e4, 10, 14\u003c/sup\u003e. CHIP analysis found that PRDM5 directly binds to CDK4 and TWIST1 promoters. In addition, PRDM5 expresses the CDK4 and TWIST1 promoters of H3K4me3 and the levels of acetyl histone H4 are significantly reduced\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. CHIP sequencing of colorectal cancer cell lines found that the response genes Adamts9, Col1a1, Mmp13 and Mgll in PRDM5 have been confirmed strongly regulated by PRDM5\u003csup\u003e15\u003c/sup\u003e. Further exploration of the downstream pathways can reveal that PRDM5 can be regulated by WNT/β-catenin signal, and as an epigenetic modifier of the expression of a variety of cancer genes to exert its tumor suppressor effect.\u003c/p\u003e \u003cp\u003eFurthermore, PRDM5 also served as an early diagnostic and prognostic marker, while detection of PRDM5 DNA methylation in gastric secretion may be used as a diagnose method in early gastric cancer\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. PRDM5 methylation is also an early event in the occurrence and development of BRAF mutant colorectal cancer. In addition, PRDM5 protein levels were significantly reduced in both mutant and wild-type intestinal polyps, especially in BRAF mutant and wild-type cancers. This indicates that PRDM5 down-regulation may start in the early stages of tumor development and continue with the development of the disease\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In another research, PRDM5 expression levels in glioma specimens were low and correlated with clinicopathological parameters and poor prognosis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the protein level of PRDM5 in ESCC and the correlation between survival has not been studied so far. All the above evidence suggests that PRDM5 plays a key role in the carcinogenesis and development of tumors, and guides us to further explore whether PRDM5 acts as a tumor suppressor gene in ESCC and the prognostic significance of PRDM5.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eBioinformation analysis of public datasets TCGA\u0026amp;GEO\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePRDM5 mRNA levels and corresponding information of samples were downloaded from TCGA (https://www.cancer.gov/) and GEO database (http://www. ncbi.nlm.nih.gov /GEO/). The database was collected under the following conditions: (1) containing at least 10 cases of normal tissue; (2) PRDM5 expression data were extracted comprehensively. In addition, two ESCC data sets were downloaded from database: GSE53624 and GSE53622. Both the TCGA and GEO databases contain information on the clinical characteristics and prognosis information of patients with ESCC. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman esophageal squamous cell carcinoma specimens and clinical data collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue microarrays were obtained from 2 groups of independent samples of patients with esophageal squamous cell carcinoma from 2 medical centers. The discovery sets included 169 patients underwent curative esophagectomy obtained from the First People\u0026apos;s Hospital of Nantong City from 2010 to 2014. Due to the poor quality of tissue microarrays and the lost of patient\u0026apos;s clinical information, and 154 cases were used for research. Among them, there were 147 paired tissues between cancer and adjacent cancer. The validation group included 279 patients underwent curative esophagectomy from 2013 to 2014 in the Affiliated Hospital of Nantong University. Due to the poor quality of tissue microarrays and the lost of patient\u0026apos;s clinical information, there were 255 cases for research. Clinical baseline data were collected for each patient retrospectively. None of the patients received any of the preoperative anticancer treatment such as preoperative chemotherapy, radiotherapy and immunotherapy before surgery, and concurrent radical Esophageal squamous cell carcinoma surgery. All the information of patients including age, gender, tumor size, depth of invasion (T), lymph node status (N), metastasis (M), and overall survival (OS) were recorded from computerized medical records. Using of human tissues with informed consent was confirmed by each patient. This research was approved by the Clinical Research Ethics Committee of each hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) staining and scoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded surgical specimens were performed for construction of tissue microarray (TMA). Tissue blocks were sectioned at 2mm and prepared on glass slides. A subsequent immunohistochemistry study was conducted to identify expression of PRDM5. The immunohistochemical protocols were performed as below. TMA was firstly fixed by formaldehyde and repaired by EDTA, then hydrogen peroxide was used to extinguish endogenous HRP. After rinsing, the primary PRDM5 antibody (purchased from abcam) was diluted (1:1000) with antibody diluent. The primary antibody was taken out the next day and washed three times and then incubated with a secondary antibody (rabbit 1:2000 Proteintech) for 20 minutes.\u003c/p\u003e\n\u003cp\u003eAll specimens were independently reassessed by 2 pathologists according to the International Union against Cancer TNM classification system. The intensity grades of staining are described as follows: negative (0), weak (1), medium (2), and strong (3), while the degree of staining was scored according to the percentage of positive cells：0（0%）; 1（1%-25%）; 2(26%-50%); 3(51%- 75%); and 4(76%-100%). The combined score was calculated by multiplying the intensity and density of staining eventually. Low expression of PRDM5 is defined as a score of 1-6, and high expression of PRDM5 is defined as a score of \u0026ge;7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qRT-PCR) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 15 pairs of esophageal tumor and para-tumor tissues were collected from surgical specimens of patients underwent radical surgery of esophageal tumor in Nantong Tumor Hospital. Total RNA was extracted using Trizol (Sigma) according to the manufacturer\u0026rsquo;s instructions, and then 1ug of obtaining cDNA was extracted for reverse transcription and detect the expression level of endogenous PRDM5 mRNA. Accurate quantification was achieved using the standard curve, which was produced by continuously diluting a known amount of RNA from the in vitro transcription response and using the dilution series for TaqManqPCR with the patient sample. Quantitative analysis of mRNA expression was conducted using the StepOnePlus\u0026trade; Real-Time PCR System. The primers and TaqMan probes for analysis were designed using the manufacturer\u0026apos;s software, PrimerExpress, and the PRDM5 primers were supplied by and. The reference gene GAPDH is used as an internal control for RNA quality. All quantitative analyses were repeated to assess the consistency of the results. Relatively standardization of the target relative to the GAPDH gene expression level calculation for \u0026Delta; Ct = Ct (target) - Ct (GAPDH). The relative quantification values of PRDM5 were calculated using the 2 \u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCT\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethylation-specific polymerase chain reaction (MSP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy methylation-specific polymerase chain reaction (methylation-specific polymerase chain reaction, MSP) method, collection of esophageal squamous cell carcinoma patients who resection of carcinoma tissue and paired normal tissue adjacent to carcinoma, using tissue genomic DNA extraction kit (QIAGEN GmbH) to extract DNA, the DNA methylation kit (ZYMO RESEARCH.) for DNA denaturation and bisulfite conversion. The transformed DNA was used as a template for PCR amplification. PRDM5 methylation-specific primers and non-methylation-specific primers (Servicebio) were designed. The methylation-specific primers were upstream 5-TTGTTTCGGGTTTCGCGTTC-3 and downstream 5-ATTCCTACTACGAAAACGCG-3. PRDM5 geneno-methylation-specific primers were upstream 5-TAGTTTTGTTTTGGGTTTTGT-3 and downstream 5- CCATTCCTACTACAAAAACACA-3. PCR reaction system: DNA template 1 L, 5 \u0026times; PCR buffer 10 L, upstream and downstream primers 0.5 L each, with ribozyme free water added up to 20 L. PCR reaction conditions: pre-denaturation at 95 ℃ for 120 s, denaturation at 95 ℃ for 60 s, annealing at 55 ℃ for 60 s, elongation at 72 ℃ for 60 s, 40 cycles. PCR products were analyzed by the gelatinization imaging system. For example, the target band amplified by gene methylation-specific primers indicated positive methylation of the PRDM5 gene, and the gene methylation rate was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 15 pairs of esophageal tumor and para-tumor tissues were collected from surgical specimens of patients underwent radical surgery of esophageal tumor in Nantong Tumor Hospital. Extract tissue protein: Take out fresh esophageal cancer and adjacent tissues from the refrigerator at -80℃, rewarm on ice, and then weigh 0.1 g of each sample into a 1.5 mL EP tube, add 1 mL Mix the protein lysate and lyse it on ice for 10 minutes, put it in a biological sample homogenizer, fully homogenize and ice bath for 30 minutes, then transfer to a low-temperature centrifuge, set at 4℃, 12000 rpm, and centrifuge for 20 minutes. After centrifugation, transfer the supernatant to a new EP tube. Prepare the protein loading solution according to 5\u0026times; loading buffer: protein lysate=1:4, pipette and mix well, dry at 100℃ Boil in a thermostat for 10 minutes, and centrifuge again at 4\u0026deg;C and 12000 rpm for 10 minutes. Finally, determine the protein concentration in each sample and record it. Store it at -20\u0026deg;C. When used, take it out and boil for 3 minutes. Centrifuge at 4\u0026deg;C and 12000 rpm for 3 minutes before loading. Sample Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (microporous). At room temperature, the membrane was enclosed in 5% bovine serum albumin for 1 hour. The membrane was incubated with a primary antibody at 4℃ overnight, GAPDH (1:500, #66009-1-1g; Proteintech), PRDM5(1:1000, # ab7609; Abcam) were washed three times with TBST and incubated at room temperature with horseradish peroxidase-conjugated secondary antibodies for 1 hour. TBST was used three times and ECL (bio-rad) visualized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune Cell Infiltration Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA deconvolution algorithm based on CIBERSORT gene expression (http://cibersort.stanford.edu/) \u003csup\u003e18\u003c/sup\u003ewas used to evaluate the immune cell infiltration in 179 patients obtained from the GEO database (http://www. ncbi.nlm.nih.gov /GEO/). The samples were divided into two groups according to the average expression of PRDM5, Wilcoxon Signed Rank test was used to compare immune cell content between groups with high and low expression of PRDM5 mRNA. Heatmaps and violin plots were analyzed and plotted using the packages\u0026ldquo;pheatmap\u0026rdquo;and\u0026ldquo;vioplot\u0026rdquo;, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using the SPSS19.0 software (SPSS, Chicago, Illinois) and GraphPad Prism 5 software (GraphPad Software, Inc.) R software (http://www. r-project.org/) was used to extract and standardize mRNA expression data from these databases, and then GraphPad Prism Version 8.0 (GraphPad Software, Inc.) was used for statistical analysis. The correlation between PRDM5 gene expression and copy number variation and DNA methylation was analyzed using online database cBioPortal (http://www.cbioportal.org/) and MEXPRESS (http://mexpress.be/). The paired or unpaired Student\u0026apos;s t-test was performed to analyze the statistical significance between two groups. Cox regression model was used to determine the risk factors of OS through univariate and multivariate analysis. The association between expression levels and progression-free survival in Esophageal squamous cell carcinoma patients were analyzed by Kaplan\u0026ndash;Meier survival curves, using Kaplan-Meier Plotter. Log-rank test was used for statistical analysis. A p-value\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003cp\u003e\u003cstrong\u003eEsophageal tumor tissues exhibit decreased PRDM5 mRNA expression\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePRDM5 mRNA levels and corresponding information of samples were downloaded from TCGA and GEO database. From TCGA database and GSE53622, GSE53624 two data sets, it was found that PRDM5 mRNA level in esophageal tumor tissue was significantly lower than para-tumor tissue (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCorrelation of PRDM5 mRNA levels with overall survival (OS) in esophageal tumor patients\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBased on the TCGA database, patients of Esophageal squamous cell carcinoma with high PRDM5 mRNA levels displayed longer OS than those with low expression (\u003cem\u003ep\u003c/em\u003e=0.0254) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Whereas, although there is no statistical significance between the high mRNA levels of PRDM5 expression with the OS in GEO data sets, we did observe the trend of a prolonged OS from the point of view of survival analysis (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePatients\u0026rsquo; characteristics and immunohistochemical in TMA\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003ePatient characteristics and pathological and clinical features are shown in Table\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. To investigate the relationship between PRDM5 and prognosis of Esophageal squamous cell carcinoma, immunohistochemistry was performed to evaluate the expression level of PRDM5 in tissue microarray (TMA) of 409 patients (discovery data set 154, validation data set 255). Esophageal squamous cell carcinoma tissues and adjacent tissues were paired in the discovery set. As indicated before, the combined score was calculated by multiplying the intensity and density of staining eventually. Low expression of PRDM5 was defined as a score of 0-6, while high expression of PRDM5 was defined as a score of \u0026ge;7. It was found that the expression of PRDM5 in adjacent tissues was higher than in tumor tissues (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Furthermore, it was also shown that PRDM5 expression was lower in poorly differentiated Esophageal squamous cell carcinoma and on the contrary in highly differentiated Esophageal squamous cell carcinoma (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-C. The immunohistochemical staining showed a high expression of PRDM5 displayed 29.2% in the discovery data set and 30.1% in the validation data set (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). Detailed characteristics are summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e,2.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003c/div\u003e\n\u003cp\u003e\u003cstrong id=\"isPasted\"\u003eThe correlation of PRDM5 exprssion and DFS, OS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the discovery data set, we analyzed the relationship between the expression of PRDM5 and the DFS and OS of patients. As indicated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, patients with high expression of PRDM5 had prolonged DFS and OS comparing with patients with low expression. It was either shown in the validation set and the combined data set (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Besides, the expression levels of PRDM5 were related to TNM staging, tumor differentiation, and vascular invasion in the combined data set (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Through univariate analysis, we found that T stage (HR: 0.573, 95%CI: 0.43-0.764; p\u0026lt;0.001), N stage(HR: 0.526, 95%CI: 0.401-0.69; p\u0026lt;0.001), TNM staging(HR: 0.509, 95%CI: 0.388-0.667;p\u0026lt;0.001), PRDM5 expression level (HR: 2.986, 95%CI: 2.084-4.278; p\u0026lt;0.001), differentiation degree (HR: 0.724, 95%CI: 0.537-0.976; p=0.034), nerve infiltration (HR: 0.643, 95%CI: 0.429-0.964; p=0.0.033), and vascular invasion (HR: 0.497, 95%CL: 0.359-0.688; p\u0026lt;0.001) were risk factors of ESCC patients. Multivariate analysis revealed the independent prognostic factors for ESCC patients were expression of PRDM5 (HR: 2.626, 95%CI: 1.824-3.781; p\u0026lt;0.001), T stage (HR: 0.719, 95%CI: 0.533-0.969; p=0.0.03), TNM stages (HR: 0.657, 95%CI: 0.49-0.881; p\u0026lt;0.001), and vascular invasion (HR: 0.694, 95%CL: 0.493-0.976; p=0.0.036) (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong id=\"isPasted\"\u003eStratified analysis of OS in esophageal tumor patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetailed research was conducted to investigate the correlation of several factors related to overall survival in esophageal tumor patients. Patients were stratified into several groups according to factors as below: postoperative radiotherapy, postoperative chemoradiotherapy, postoperative chemotherapy, no treatment. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, postoperative radiotherapy could prolong the OS in total group, while the postoperative chemoradiotherapy didn\u0026rsquo;t affect the OS. In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, postoperative therapy didn\u0026rsquo;t affect the OS in stage Ⅰ-Ⅱ Esophageal squamous cell carcinoma patients. Whereas, in stage Ⅲ-Ⅳ Esophageal squamous cell carcinoma patients, postoperative treatment could extend the OS especially in patients who underwent postoperative radiotherapy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Moreover, the high expression level of PRDM5 was associate with longer OS in patients who underwent postoperative therapy(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). The analysis based on the relapse-free survival (RFS) showed there are significant differences among the low expression PRDM5 and the high group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrognostic nomogram and calibration plots of Esophageal squamous cell carcinoma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the results of univariate and multivariate analysis, we constructed a nomogram to predict the operating system at 3-year, 5-year, and 10-year after surgery (Supplementary Figure 1). According to preliminary analysis, predictive factors include T status, vascular invasion, tumor grade, T stage, TNM staging, and PRDM5 staining, all of which were important prognostic indicators of OS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe transcriptional level of PRDM5 was consistent with the protein level and was affected by methylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, the expression of PRDM5 of esophageal tumor tissues and paired para-tumor tissues were analyzed. It was found that the expression of PRDM5 was higher in 15 specimens of esophageal tumor than para-tumor tissues (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). We selected 10 pairs of tissues with high expression of PRDM5-mRNA level in para-tumor tissues, Western-blot was performed using 10 pairs of cancer and adjacent tissues to detect protein expression levels and found the protein level of PRDM5 in adjacent tissues was higher than that tumor tissues, which proved that PRDM5 had consistency in both the transcription level and protein level changes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). We then performed methylation PCR on eight pairs of Esophageal squamous cell carcinoma tissue and found that PRDM5 DNA was highly methylated in cancer tissues, and absent in adjacent tissues (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Therefore, we further investigated the relationship between PRDM5 DNA methylation and copy number in ESCC in cBioPortal. There was no significant difference between PPRDM5 DNA methylation and copy number (Supplementary Fig. 6A). However, there was a significant negative correlation between PRDM5-mRNA expression and DNA methylation, which was confirmed by Pearson correlation coefficient (Spearman: -0.61, \u003cem\u003ep\u003c/em\u003e=1.02E-19) (Supplementary Fig.\u0026nbsp;6B). We also found in the MEXPRESS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mexpress.be/\u003c/span\u003e\u003c/span\u003e) database that methylation at CPG position 120921881 was negatively correlated with PRDM5-mRNA expression (Supplementary Fig.\u0026nbsp;6C). Therefore, all these data suggested that methylation at CPG Island 120921881(the promoter of PRDM5) leads to down-regulation of PRDM5 mRNA expression, which in turn leads to a decrease in PRDM5 protein level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong id=\"isPasted\"\u003ePRDM5 is associated with immune infiltration of ESCC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also used the Cibersort algorithm to analyze the expression levels of 22 immune cell subgroups and evaluate their correlation with PRDM5 expression. The results showed that T cells CD4 memory resting, Mast cells resting, Eosinophils, M2 macrophages and Mast cells activated were significantly positively correlated with PRDM5expression (P \u0026lt; 0.05). T cells regulatory (Tregs), Monocytes, and Dendritic cells resting were negatively correlated with PRDM5 expression(Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). In addition, we evaluated possible associations between 22 immune cells, and heat maps showed weak to moderate correlations in the rates of different tumor-infiltrating immune cell subgroups(Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we did research on the clinical role of PRDM5 ,Based on this research, we found that the expression of PRDM5 in esophageal para-tumor tissue was significantly higher than in tumor tissues, regardless of mRNA level or protein level, and Esophageal squamous cell carcinoma patients with high expression of PRDM5 had a better prognosis. Recent studies indicated that high levels of PRDM5 are associated with better outcomes for glioma and colorectal cancer\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our research showed that postoperative chemotherapy is necessary for esophageal patients with stage III-IV after surgery, and postoperative radiotherapy significantly prolonged the patient's OS. However, postoperative adjuvant treatment for patients with stage Ⅰ-Ⅱ Esophageal squamous cell carcinoma after surgery has little benefit on OS, the results also suggested that postoperative adjuvant treatment for patients with high expression of PRDM5 can help to prolong DFS and OS. This might indicate that PRDM5 could affect the therapeutic effect.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that ectopic expression of PRDM5 can inhibit the proliferation of gastric cancer, nasopharyngeal cancer, and brain glioma cells\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our study showed that the expression of PRDM5 was correlated with pathological status T stage. It also demonstrated that over-expression of PRDM5 inhibited proliferation activity in esophageal carcinoma cell. This aroused our thinking, indicating that the patient's pathological information such as the high expression of PRDM5 should be considered comprehensively when choosing postoperative adjuvant treatment and patients treatment strategies. However, since it is difficult to obtain dynamic monitoring only by immunohistochemical methods, we suggest that it might be more helpful to detect PRDM5 expression in patients' serum.\u003c/p\u003e \u003cp\u003eThis experiment proved that PRDM5 transcriptional level and protein level were consistent, and the transcriptional level was affected by methylation. Previous studies have found that PRDM5 expression is lower in multiple cancer species than in adjacent tissues, including breast, ovarian, liver, lung, and colon cancers, and cervical cancers\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and that silencing or downregulation of PRDM5 is often caused by DNA methylation\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. And then extended to epigenetics, studies have shown that long-term heavy drinking can leading to homocysteine and S-adenosine homocysteine (SAH) increment, resulting in histone modifications and changes in gene expression. Eventually affect the small RNA family which serve as epigenetic regulators causing DNA promoter methylation \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In this study, Esophageal squamous cell carcinoma patients with drinking history were relatively small proportion, so the history of drinking was not statistically significant. However, we could find that the PRDM5 expression was often lower in patients with a long-term history of drinking, which may be related to DNA methylation. Studies have shown that PRDM5 is a stress response gene\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It may provide protection when the esophagus is physically or chemically stimulated, but once methylation is silenced or its expression is reduced, it may lead to the occurrence and development of Esophageal squamous cell carcinoma. We also found in database that methylation at CPG position 120921881 was negatively correlated with PRDM5 mRNA expression, suggesting that methylation is negatively correlated with PRDM5 mRNA expression. In future studies, we would confirm whether this site causes the silencing or reduction of PRDM5 expression. It is worth considering whether blocking the methylation at certain site should be considered in clinical applications for patients with low PRDM5 expression. The methylation of PRDM5 may be involved in the early events of esophageal squamous cell carcinoma, such as esophagitis. Then this may also be used as a biomarker for the early diagnosis of esophageal squamous cell carcinoma. Future research should expand the sample size of esophageal squamous cell carcinoma and add patient samples from patients with esophagitis for further research.,We believe that in the future, PRDM5 methylation may also become part of cancer screening, and there is a blood-based screening for DNA methylation markers in colorectal cancer \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, we showed that epigenetic therapy of PRDM5 is achievable. In the future, we anticipate targeted therapies will also include epigenetically inactivated tumor inhibitors, such as the CRISPRDCAS9 technology and the viral application of the epigenetic editor to reactivate. our results showed that T cells CD4 memory resting activated were significantly positively correlated with PRDM5expression ,Recent studies have shown that CD4+ T cells are not a pure cell lineage with a single function, but a diverse cell population with complex functions. In addition, CD4+ T cells can be used not only as helper cells, but also as effective effector cells or partners with macrophages and eosinophils to eliminate various tumors, and Patients with increased CD4+ T cell ratio in serum samples of patients with esophageal squamous cell carcinoma undergoing radiotherapy have better survival\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Whether the better prognosis of patients with esophageal squamous cell carcinoma with high expression of PRDM5 is related to the recruitment of memory CD4+ T cells by PRDM5 to enhance its killing effect on tumor cells. The effect of PRDM5 on immune function will be worthy of our further study.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that the inhibition of WNT signaling by ectopic expression of PRDM5 weakens the transcriptional activity of CCND1 gene, which in turn affects the changes of CDK4.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Studies have shown that CDK4 can phosphorylate Smad3, which is very important in response to TGF-β signal transcription activity. Phosphorylation of Smad3 by CDK4 inhibits its anti-proliferation function\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. CDK4 also directly phosphorylates FOXM1 at multiple sites to positively regulate its activity, possibly protecting cancer cells from senescence by inhibiting reactive oxygen species (ROS) levels, and promoting the entry of G1/S phase by regulating the expression of several genes in cancer cells (such as cyclin E2, MYB and MCM2) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. CDK4 can phosphorylate MEP50 and increase methyltransferase activity and cell survival function\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Our previous results confirmed that the expression level of PRDM5 is related to the pathological characteristics of tumor size, then we speculate whether the expression of PRDM5 affected CDK4 and thus affected the proliferation ability of esophageal squamous cell carcinoma cells. We further speculate that PRDM5 may be involved in the CCND1-CDK4/CDK6 pathway, and that patients with low expression of PRDM5 may increased the expression levels of CCND1 and CDK4. It is worth exploring whether CDK4/CDK6 inhibitors (Palbociclib) can be considered for this type of patients, We will further verify its relationship with CCND1 and CDK4 in future cell function experiments, which is of great significance for clinical treatments.\u003c/p\u003e \u003cp\u003eThe previous results indicate that the expression of PRDM5 is related to the clinical pathological parameter lymph node metastasis status, and we speculate that it is related to the WNT signaling pathway and TWIST1.Studies have shown that β-catenin was found in the form of continuous protein methylation after activation and accumulated in the silent PRDM5 cell line. This leads to the activation of the Wnt/β-catenin signaling pathway, downstream LEF/TCF transcriptional activity and the start of EMT process\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Many studies have indicated that the WNT signaling pathway can impinge the development of Esophageal squamous cell carcinoma through the EMT pathway\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Studies have also shown that PRDM5 can bind to the promoter of TWIST1 to affect its transcriptional activity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Studies have shown that patients with esophageal squamous cell carcinoma overexpressing TWIST1 had a poor prognosis, and the increase of TWIST1 can affected the invasion and migration ability of esophageal squamous cell carcinoma\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Relevant studies have shown that silence of TWIST1 in esophageal squamous cell carcinoma, cervical cancer and non-small cell lung cancer can enhance its drug sensitivity to cisplatin\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and studies have also shown that increased expression of TWIST1 can enhance the radiotherapy resistance of esophageal squamous cell carcinoma cells\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We suspect that this may be related to the longer overall survival of patients with high expression of PRDM5 that we have observed after adjuvant treatment after surgery. Patients with high expression of PRDM5 may have lower levels of TWIST1. In future studies, we should further verify the correlation between PRDM5 and TWIST1 at the cellular level. It is best to monitor the blood dynamics of clinical samples of esophageal squamous cell carcinoma to assess their impact on the treatment effect.\u003c/p\u003e \u003cp\u003eTaken together, this work uncovers PRDM5 is a negative prognostic factor in esophageal tumor cell. Future researches should focus on how to conduct clinical detection of methylation sites to predict the prognosis of Esophageal squamous cell carcinoma patients, In the future, more detailed studies are still needed to describe and reveal the underlying mechanism of PRDM5 multiple pathological functions in esophageal squamous cell carcinoma. A comprehensive understanding of the multiple roles of PRDM5 in esophageal squamous cell carcinoma will provide more important clinical value for PRDM5 as a diagnostic indicator, prognostic marker and treatment target for clinical treatment.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAs a tumor suppressor gene in Esophageal squamous cell carcinoma, PRDM5 can be used as a biomarker to predict the survival of patients with Esophageal squamous cell carcinoma, and the high expression level of PRDM5 is associate with longer OS in patients who underwent postoperative therapy. Furthermore, PRDM5 expression in Esophageal squamous cell carcinoma cells may affects WNT/β-catenin signaling pathways, thus further affect the Esophageal squamous cell carcinoma cell proliferation, migration, and invasion capacity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eESCC: Esophageal squamous cell carcinoma; IARC: The International Agency for Research on Cancer; EMT: Epithelial-Mesenchymal Transition; IHC: Immunohistochemistry; TCGA: The Cancer Genome Atlas; GEO: Gene Expression DataSets; qRT-PCR: quantitative Real-time Polymerase Chain Reaction; MSP: Methylation-specific polymerase chain reaction; TMA: Tissue microarray Confidence intervals; CI: Confidence intervals; HR: Hazard ratio; OS: Overall survival; DFS: Disease-free survival; PBS: Phosphate buffer saline; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PRDM5: PR domain zinc finger protein 5; Si-RNA: Small interfering RNA; TCF/LEF: T-cell Factor/Lymphoid Enhancing Factor; BRAF: B-Raf proto-oncogene, serine/threonine kinase; CHIP: Chromatin Immunoprecipitation; HDAC1: Histone deacetylase 1.\u003c/p\u003e"},{"header":"Declarations","content":"\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 The Affiliated Hospital of Nantong University. All patients signed an informed consent approved by the institutional review board.\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated or analyzed during this study are included in this published article. The GSE53622 dataset is available at (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53622) and GSE53624 dataset is available at (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53624) respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the funding of \u0026ldquo;Postgraduate Research\u0026amp;Practice Innovation Program of Jiangsu Province\u0026rdquo;, Scientific Research Subject of the Health commission of Nantong (WKZL2018048) and the Scientific Research Subject of Nantong Science and Technology Bureau (MSZ19233).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by JG, TYZ, QXY, and JL. SW, JJS, and LYG performed statistical analysis. The first draft of the manuscript was written by JG, JC and GRW, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Department of Radiotherapy, Department of Oncology \u0026amp; Cancer Research Center Nantong of Affiliated Tumor Hospital of Nantong University for providing the patient information used in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Cancer Cell Int 2020, 20:20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe J, Zhou M, Chen X, Yue D, Yang L, Qin G, Zhang Z, Gao Q, Wang D, Zhang C \u003cem\u003eet al\u003c/em\u003e: Inhibition of SALL4 reduces tumorigenicity involving epithelial-mesenchymal transition via Wnt/beta-catenin pathway in esophageal squamous cell carcinoma. J Exp Clin Cancer Res 2016, 35(1):98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui Y, Zhang L, Wang W, Ma S, Liu H, Zang X, Zhang Y, Guan F: Downregulation of nicotinamide N-methyltransferase inhibits migration and epithelial-mesenchymal transition of esophageal squamous cell carcinoma via Wnt/beta-catenin pathway. Mol Cell Biochem 2019, 460(1-2):93\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForghanifard MM, Moaven O, Farshchian M, Montazer M, Raeisossadati R, Abdollahi A, Moghbeli M, Nejadsattari T, Parivar K, Abbaszadegan MR: Expression analysis elucidates the roles of MAML1 and Twist1 in esophageal squamous cell carcinoma aggressiveness and metastasis. Ann Surg Oncol 2012, 19(3):743\u0026ndash;749.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee KW, Sung CO, Kim JH, Kang M, Yoo HY, Kim HH, Um SH, Kim SH: CD10 expression is enhanced by Twist1 and associated with poor prognosis in esophageal squamous cell carcinoma with facilitating tumorigenicity in vitro and in vivo. Int J Cancer 2015, 136(2):310\u0026ndash;321.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzadpanah MH, Abbaszadegan MR, Fahim Y, Forghanifard MM: Ectopic expression of TWIST1 upregulates the stemness marker OCT4 in the esophageal squamous cell carcinoma cell line KYSE30. Cell Mol Biol Lett 2017, 22:33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Y, Jia Y, Wang S, Liu N, Gao D, Zhang L, Lin Z, Wang S, Kong F, Peng C \u003cem\u003eet al\u003c/em\u003e: Downregulation of MUTYH contributes to cisplatinresistance of esophageal squamous cell carcinoma cells by promoting Twistmediated EMT. Oncol Rep 2019, 42(6):2716\u0026ndash;2727.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu K, Chen L, Han X, Wang J, Wang J: Short hairpin RNA targeting Twist1 suppresses cell proliferation and improves chemosensitivity to cisplatin in HeLa human cervical cancer cells. Oncol Rep 2012, 27(4):1027\u0026ndash;1034.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin HO, Hong SE, Woo SH, Lee JH, Choe TB, Kim EK, Noh WC, Lee JK, Hong SI, Kim JI \u003cem\u003eet al\u003c/em\u003e: Silencing of Twist1 sensitizes NSCLC cells to cisplatin via AMPK-activated mTOR inhibition. Cell Death Dis 2012, 3:e319.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChunbao Zang1, Xujie Liu1,* Wu1, Bingqian Zhang1, Bing Li2,*, Shuhong Ma1, YH, Weiwei Dai1, SJ, Shaolin Li1, Yujia He1, Wenli, Zhiping Peng1: IL-6/STAT3/TWIST inhibition reverses ionizing radiationinduced EMT and radioresistance in esophageal squamous carcinoma. Oncotarget 2017, 8(pp):11228-11238.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRelationship between PRDM5 expression and clinicopathological characteristics in Discovery Data Set\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePRDM5 LOW\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePRDM5 HIGH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePatient characteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN=109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN=45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.872\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.864\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e≧60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumorinformation Location\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.866\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003euper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTobacco\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.812\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.331\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIII-IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.091\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.360\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTNM stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.122\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI-II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIII-IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHistological grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModerately\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePoorly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAccompanying diseases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.933\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive margin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.744\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.178\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTable 2 Relationship between PRDM5 expression and clinicopathological characteristics in Validation Data Set\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"96%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003ePatient characteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003eN=178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003eN=77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eGender\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.177\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003emale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.496\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003e≧60 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003e\u0026lt; 60\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eT stage \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eI-II \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eIII-IV \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eN stage\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.061\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eN0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eM stage \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.540\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eN0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eTNM stage \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eI-II \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eIII-IV \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eHistological grade\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eWell\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eModerately\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003ePoorly\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eN0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eVascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.571428571428573%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.46938775510204%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.428571428571427%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.53061224489796%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 3 Relationship between PRDM5 expression and clinicopathological characteristics in Combined Data Set\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"93%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003ePatient characteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003eN=287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003eN=122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eGender\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.241\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003emale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.668\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003e≧60 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003e\u0026lt; 60\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eT stage \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eI-II \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eIII-IV \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eN stage\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eN0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eM stage \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.832\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eN0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eTNM stage \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eI-II \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eIII-IV \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eHistological grade\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eWell\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eModerately\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e154\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003ePoorly\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.073\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eN0\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eVascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eN0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.551020408163264%\"\u003e\n \u003cp\u003eYES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.408163265306122%\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.448979591836736%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.591836734693878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 4 \u003cstrong\u003eUnivariate and multivariate analysis of characteristics associated with overall survival\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" id=\"isPasted\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.05351170568562%\"\u003e\n \u003cp\u003eUnivariate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003emultivariate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.05351170568562%\"\u003e\n \u003cp\u003eHazard Ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e95%CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003eHazard Ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e95%CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.05351170568562%\"\u003e\n \u003cp\u003e1.041\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e0.764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e1.418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003e0.798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n 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width=\"9.03010033444816%\"\u003e\n \u003cp\u003e0.388\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e0.667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003e0.657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e0.881\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n \u003cp\u003eHistological grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.05351170568562%\"\u003e\n \u003cp\u003e0.724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e0.537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e0.976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003e0.034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n \u003cp\u003ePRDM5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.05351170568562%\"\u003e\n \u003cp\u003e2.986\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e2.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e4.278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003e2.626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e1.824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e3.781\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n \u003cp\u003eVascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.05351170568562%\"\u003e\n \u003cp\u003e0.497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e0.359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e0.688\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003e0.694\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e0.493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e0.976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003e0.036\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.39464882943144%\"\u003e\n \u003cp\u003eNerve invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.05351170568562%\"\u003e\n \u003cp\u003e0.643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e0.429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.862876254180602%\"\u003e\n \u003cp\u003e0.964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.528428093645484%\"\u003e\n \u003cp\u003e0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.050167224080267%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.03010033444816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.190635451505017%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.8595317725752505%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PRDM5, esophageal squamous cell carcinoma, prognosis, WNT signaling pathway, immunohistochemical, DNA methylation","lastPublishedDoi":"10.21203/rs.3.rs-1362257/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1362257/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe role of the transcription factor PRDM5 in esophageal squamous cell carcinoma (ESCC) has not been revealed. This study investigated the relationship between PRDM5 expression and survival outcome in esophageal squamous cell carcinoma and explored the mechanism in tumor development.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn present study, expression of PRDM5 mRNA in esophageal squamous cell carcinoma patients was conducted using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. The expression of PRDM5 was assessed by immunohistochemical staining. Kaplan-Meier curve and Cox regression analysis was performed to analyze the survival outcome and independent predictive factors. qRT-PCR and Methylation-specific PCR were performed to identify the mRNA level of PRDM5 and Methylation rate. Cibersort algorithm to analyze the relationship between PRDM5 expression and immune cell invasion. Western-blot was performed to confirm the expression of esophageal tumor tissues and adjacent tissues. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe TCGA database and GEO database show that PRDM5 mRNA level in esophageal squamous cell carcinoma adjacent tissues was higher than that of cancer tissues,and ESCC patients with high expression of PRDM5 mRNA had better overall survival.Tissue microarray showed that the protein level of PRDM5 in the adjacent tissues of patients with ESCC was higher than that in cancer tissues, and the expression level of PRDM5 was significantly correlated with the grade of clinicopathological characteristics (p\u0026lt;0.001). Patients with high expression of PRDM5 displayed a better OS and DFS. Cox regression analysis showed that PRDM5 was an independent risk factor and prognostic factor for ESCC patients(HR: 2.626, 95%CI: 1.824-3.781; p\u0026lt;0.001). The protein level of PRDM5 matched with the transcriptional level, whereas the DNA methylation affected the transcriptional level. Cibersort showed that T cells CD4 memory resting, mast cells resting, eosinophils, M2 macrophages and mast cells activated were significantly positively correlated with PRDM5 expression (P\u0026lt;0.05), while regulatory T cells, monocytes and dendritic cells negatively correlated with PRDM5 expression (P \u0026lt;0.05).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003ePRDM5 can be used as a biomarker to predict the survival of ESCC patients. Furthermore, PRDM5 expression in ESCC cells may affect WNT/β-catenin signaling pathways, thus further affect the ESCC cell proliferation, migration, and invasion capacity.\u003c/p\u003e","manuscriptTitle":"Low expression of PRDM5 predicts poor prognosis of esophageal squamous cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-18 16:16:42","doi":"10.21203/rs.3.rs-1362257/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2022-02-15T14:11:34+00:00","index":0,"fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2021-11-29T09:01:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fecde46c-592a-4a47-b3b9-11686e9c8399","owner":[],"postedDate":"February 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-06-16T12:11:31+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-18 16:16:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1362257","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1362257","identity":"rs-1362257","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}