Serum proteinase-3 levels as a predictor of progression-free survival of first-line chemotherapy in metastatic colorectal cancer

Serum proteinase-3 levels as a predictor of progression-free survival of first-line chemotherapy in metastatic colorectal cancer | 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 Serum proteinase-3 levels as a predictor of progression-free survival of first-line chemotherapy in metastatic colorectal cancer Kei Furuya, Masao Nakajima, Ryouichi Tsunedomi, Yuki Nakagami, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3393984/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Feb, 2024 Read the published version in BMC Cancer → Version 1 posted 8 You are reading this latest preprint version Abstract Background: To improve the prognosis of patients with metastatic colorectal cancer (mCRC), investigating predictive biomarkers for prognosis and chemotherapeutic responsiveness is necessary. Therefore, this study aimed to analyze the clinical significance of serum proteinase-3 (PRTN3) as a predictor for prognosis and chemosensitivity, especially to bevacizumab, in mCRC. Methods: This single-center retrospective observational study enrolled 79 patients with mCRC in our hospital and 353 patients with colorectal cancer from the TCGA database. Preoperative serum PRTN3 levels were measured using enzyme-linked immunosorbent assay. The clinicopathological characteristics and prognosis according to serum PRTN3 levels were then evaluated. PRTN3 expression in tumor and stromal cells was evaluated immunohistochemically. The impact of PRTN3 levels on angiogenesis and bevacizumab sensitivity was evaluated using the tube formation assay. Results : Serum PRTN3 level was an independent poor prognostic factor for progression-free survival (PFS) (hazard ratio: 2.082; 95% confidence interval: 1.118–3.647; P =0.010) in patients with mCRC. Similarly, prognostic analysis with TCGA data set showed poorer overall survival in patients with PRTN3 expression compared to those without PRTN3 expression, especially in patients with stage IV. Immunohistochemical analysis of resected specimens revealed that stromal neutrophils expressed PRTN3, and their expression level was significantly correlated with serum PRTN3 levels. Interestingly, the effectiveness of first line chemotherapy was significantly poorer in the high serum PRTN3 level group. High serum PRTN3 was significantly associated with poorer PFS (hazard ratio, 3.027; 95% confidence interval, 1.175–7.793; P =0.0161) in patients treated with bevacizumab, an anti-angiogenic inhibitor. Tube formation assay revealed that PRTN3 administration notably augmented angiogenesis while simultaneously attenuating the anti-angiogenic influence exerted by bevacizumab. Conclusions: Serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy especially for bevacizumab in patients with mCRC; however, future studies are warranted to confirm our results. biomarker bevacizumab chemotherapy metastatic colorectal cancer proteinase-3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Colorectal cancer (CRC) is the second leading cause of cancer-specific death worldwide [ 1 ]. The standard treatment for metastatic CRC (mCRC) is a multidisciplinary approach involving chemotherapy, immunotherapy, and surgery [ 2 – 4 ]. However, the therapeutic efficacy of chemotherapy needs to be enhanced to improve mCRC survival. On the other hand, the additive or synergistic effects of the combination of anti-vascular endothelial growth factor (VEGF) monoclonal antibodies (mAbs), such as bevacizumab (Bev), and cytotoxic agents have been shown to overcome resistance to chemotherapy [ 5 ]. One possible mechanism for this effect is the direct anti-vascular effects of cytotoxic agents that intensify the pro-apoptotic effects of anti-VEGF mAbs on the vascular endothelium [ 6 ]. Another possibility is that normalization of tumor vessels by anti-VEGF mAbs increases the uptake of cytotoxic agents and antibodies [ 5 , 7 ]. Moreover, anti-VEGF mAbs, especially Bev, are currently the standard of care for mCRC; however, not all patients benefit. Additionally, adverse effects, such as hypertension, proteinuria, gastrointestinal perforation, and stroke, are often observed [ 2 , 8 ]. Thus, an alternative approach is to optimize the benefit of chemotherapy regimens using biomarkers that predict chemosensitivity. However, only a few biomarkers (e.g., RAS mutations, status, and UGT1A1 polymorphism) have been identified to guide chemotherapy selection in clinical practice [ 2 , 9 ] and some of these markers require invasive measurement. Moreover, no clinical and biological factors have been identified in response to Bev, despite extensive research to explore the predictive biomarkers in response to Bev therapy [ 10 ]. Therefore, more predictive and less invasive biomarkers need to be further explored [ 11 – 14 ]. We have previously identified prognostic biomarkers of CRC by comprehensive proteomic analysis [ 15 – 17 ]. Proteinase-3 (PRTN3) belongs to the neutrophil-derived protease family and is stored within azurophil granules [ 18 ]. It is involved in neutrophil differentiation and proliferation, inflammation, and vasculitis [ 19 , 20 ], as well as in the invasion of tumor and endothelial cells through the activation of matrix metalloproteinases (MMP)-2 [ 21 , 22 ]. In cancer, PRTN3 expression in tumor tissue is associated with poor prognosis in some carcinomas [ 23 – 25 ]. However, the relationship between serum PRTN3 levels and CRC remains unclear. Therefore, this study aimed to analyze the clinical significance of serum PRTN3 as a predictor of chemosensitivity and prognosis in mCRC. Methods Study design and patients This was a single-center, retrospective, observational study conducted between January 2008 to December 2019. In total, 79 serum samples were obtained from consecutive patients with mCRC at our hospital and stored at − 80°C until use. Of them, 48 patients who underwent surgery before chemotherapy, 48 primary tumors and five metastatic liver tumors were surgically obtained. Additionally, peripheral whole blood samples for flow cytometric analysis were obtained from 12 patients with mCRC (Fig. 1 ). Computed tomography was performed every 2–3 months to evaluate treatment efficacy Thusing the remaining target lesions in patients who did not undergo radical resection. Otherwise the efficacy was evaluated during preoperative chemotherapy in patients with radical resection according to RECIST criteria version 1.1. This study was approved by the Ethics Committee of Yamaguchi University Hospital (H20-102, H23-135, and H28-074) and was performed in accordance with the Declaration of Helsinki. Comprehensive proteomics analysis of serum and tumor tissue Comprehensive analysis of protein levels in the pretreatment serum and tumor tissue lysates of 24 patients with mCRC was performed using SOMAscan (Soma Logic, Boulder, CO) following the manufacturer’s protocol (Soma Logic) [ 15 , 16 ]. Briefly, total protein in frozen CRC samples was quantified by adding a Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Kanagawa, Japan) using a Qiagen Tissue Lyser (Qiagen Scientific, Tokyo, Japan). Samples were sent to Soma Logic and analyzed using the SOMAscan assay. The SOMAscan readings are displayed as relative fluorescence units. Analysis of relationship between PRTN3 gene expression and mCRC prognosis using The Cancer Genome Atlas database Transcriptional and corresponding clinical information of 276 patients with CRC was obtained from The Cancer Genome Atlas (TCGA, PanCancer Atlas, https://portal.gdc.cancer.gov/ Accessed 3 Jun 2021). Survival curve analysis was performed for the PRTN3-expression group (those with mRNA expression quantification of > 0 fragments per kilobase of exon per million mapped reads [FPKM]) and the non-expression group (those with mRNA expression quantification of 0 FPKM). Measurement of serum PRTN3 levels Serum PRTN3 concentration was measured using the human PRTN3 enzyme-linked immunosorbent assay (ELISA) kit (ab226902; Abcam, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, after adding 50 µl of a standard or sample solution to appropriate wells, 50 µl of the antibody cocktail (capture antibody and detector antibody) was added, and the samples were incubated at room temperature for 1 h. After washing, 100 µl of TMB Development Solution was added to each well and incubated for 10 min. The absorbance was read at a wavelength of 450 nm after adding 100 µl of a stop solution. Absorbance was measured using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). A standard curve was prepared for stock standards. The sensitivity of this assay was 150 pg/ml. All standards, controls, and samples were measured in duplicates. Immunohistochemical staining Immunohistochemistry (IHC) was performed using 4-µm-thick formalin-fixed paraffin-embedded (FFPE) sections. The sections were deparaffinized with xylene and alcohol, followed by antibody activation using 10 mM sodium citrate buffer (Agilent Technologies, Tokyo, Japan) at pH = 6.0 for 20 min at 95°C. Endogenous peroxidase activity was blocked with a blocking solution (S2023; Agilent Technologies) for 5 min, followed by blocking of non-specific reactions for 10 min at room temperature (X0909; Agilent Technologies) and incubation with rabbit anti-human PRTN3 antibody (EPR6277, Abcam; dilution 1:200) at 4°C overnight. After washing, the sections were incubated with an appropriate secondary antibody (K4003; Agilent Technologies; undiluted) for 30 min at room temperature. DAB (Agilent Technologies) was used according to the manufacturer’s protocol and allowed to react with the sections for 3 min. Contrast staining was performed with hematoxylin, and the samples were sealed after dehydration. Images were obtained using an all-in-one fluorescence microscope (BZ-X710, KEYENCE, Osaka, Japan). PRTN3 expression in tissue samples was evaluated by a pathologist and an investigator who were blinded to patient outcomes. Focusing on the tumor cells, a semi-quantitative evaluation of PRTN3 expression was performed using the composite expression score (CES) as described previously [ 26 ]. CES = 4 × (intensity score − 1) + frequency score. Intensity scores: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The frequency scores were as follows: 1 (5–24%), 2 (25–49%), 3 (50–74%), and 4 (75–100%). To analyze PRTN3 expression in stromal cells, five areas on each slide that were most abundant in positive cells were photographed under ×400 high-power fields, and the number of positive stromal cells was counted. Immunofluorescence staining Immunofluorescence staining was performed with 4-µm-thick FFPE sections. After deparaffinization and antigen activation (pH 6.0, 95°C, 20 min), non-specific reactions were blocked. The sections were then incubated with the anti-human PRTN3 antibody (at the same dilution rate for IHC), anti-CD3 antibody (17A2; Thermo Fisher Scientific, dilution 1:100), anti-CD68 antibody (PG-M1, Abcam; dilution 1:400), and anti-CD66b antibody (G10F5, Novus Biologicals, Centennial, CO; dilution 1:200) at 4°C overnight. The sections were incubated with secondary antibody mixtures (anti-mouse Alexa Fluor 488; Thermo Fisher Scientific; dilution 1:1000 and anti-rabbit Alexa Fluor 555; Thermo Fisher Scientific; dilution 1:1000) for 40 min at room temperature. Thereafter, they were stained with DAPI for nuclear contrast staining. Images were obtained using an all-in-one fluorescence microscope (BZ-X710, KEYENCE). Flow cytometry Fresh peripheral blood (100 µl) was collected in EDTA-anticoagulation tubes and reacted at room temperature for 30 min in the dark with anti-PRTN3 antibody (PR3G-2, Abcam; undiluted) and anti-CD66b antibody (G10F5, BioLegend, San Diego, CA; undiluted) according to the manufacturer’s protocol. Erythrocytes were lysed in lysis solution (BD Biosciences, San Jose, CA, USA) at room temperature for 10 min in the dark and centrifuged at 1500 rpm for 10 min. The cells were then washed with PBS. Finally, the cells were resuspended in 500 µl cell FIX (BD Biosciences; dilution, 1:10). Flow cytometric data were acquired using a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA) with the NovoExpress software (version 1.3.0, ACEA Biosciences) and analyzed using the FlowJo software (Tree Star, Ashland, OR, USA). Tube formation assay Tube formation assay was performed using the endothelial tube formation kit (Cell Biolabs, San Diego, USA) according to the manufacturer’s protocol. Briefly, pre-chilled 96-well plates were coated with ECM gel (50 µL/well) and incubated at 37℃ for 1 h. Followed by addition of 2.5×10 4 cells of human umbilical vein endothelial cells (Takara, Tokyo, Japan) and 150 µL culture medium per well. Additionally, PRTN3 (RPB434Hu02, Cloud Clone Corp., USA) was added at a final concentration of 0–1000 ng/mL, and Bev (Chugai Pharmaceutical Co., Tokyo, Japan) was added at a final concentration of 0–2.5 mg/mL to each well. After incubation at 37℃ and 5% CO 2 for 2 h, the number of branches and tube area were evaluated using an all-in-one fluorescence microscope (BZ-X710, KEYENCE). Statistical analysis The cutoff value of serum PRTN3 was determined using time-dependent receiver operating characteristic (ROC) curve analysis. Between-group differences were estimated using the Mann–Whitney U-test. Categorical variables were compared using the chi-squared test. Overall survival (OS) was defined from the date of initial diagnosis to the date of mortality. Survival curves were generated using the Kaplan–Meier method and compared using the log-rank test. Cox’s proportional hazards model was used to estimate the hazard ratios (HRs) and perform multivariate analysis. Multivariate analysis corrected for tendency factors ( P < 0.10) in the univariate analysis. Progression-free survival (PFS) for first-line chemotherapy was defined as the interval from the date of the first chemotherapy to the date of progression or death. All statistical analyses were performed using JMP Pro16 (SAS Institute, Cary, NC, USA). Statistical significance was set at P < 0.05. Results Candidate proteins predictive of mCRC prognosis The good (OS ≥ 3 years) and poor (OS < 2 years) prognosis groups included nine and 11 patients, respectively. The candidate proteins ranked according to their Fisher ratio are presented in Table S1 . Myeloperoxidase was the top candidate protein in serum, but is already reported to be associated with CRC prognosis [ 27 ]. Therefore, it may be difficult to identify its novel roles for mCRC and was thus excluded. The expression of PRTN3, the second candidate protein in serum, was significantly higher in the poor prognosis group than in the good prognosis group ( P < 0.001). The other candidate proteins were known prognostic markers in patients with mCRC and were therefore also excluded from the analysis [ 28 – 30 ]. In addition, PRTN3 was also one of the top 10 proteins in the tissue samples. Figure 2 illustrates the relationship between PRTN3 expression in tumor tissues and CRC prognosis in the TCGA database analysis. OS was significantly worse in the expression group than in the non-expression group ( P = 0.0328) but only in patients with stage IV CRC. Meanwhile, there were no significant differences among other disease stages. These results suggest that PRTN3 may be associated with the prognosis of mCRC. Therefore, we focused on PRTN3 as a predictive marker for mCRC prognosis. Clinicopathological characteristics and relationship between serum PRTN3 levels and mCRC The high (≥ 21.6 ng/ml) and low (< 21.6 ng/ml) PRTN3 expression groups involved 45 and 34 patients, respectively. There were no significant between-group differences in terms of tumor markers, TNM category, histology, or therapeutic interventions including chemotherapy regimen and surgical procedures (Table 1 ). PRTN3 is a neutrophil-associated protein. However, there was also no difference in neutrophil counts between the two groups. PFS was significantly worse in the high expression group (HR: 2.173; 95% CI: 1.269–3.723; P = 0.037, Fig. 3 a). In the multivariate analysis, high serum PRTN3 expression (HR: 2.082; 95% CI: 1.188–3.647; P = 0.010) was an independent risk factor for progression (Table 2 ). Table 1 Clinicopathological patient characteristics mCRC (n = 79) Characteristics Serum PRTN3 low (n = 34) Serum PRTN3 high (n = 45) P -value Age (years) 67.5 [57.25, 76.25] 66 [55.5, 74.0] 0.593 Sex (male/female) 15/19 26/19 0.229 WBC (/µl) 6395 [4740, 7925] 7290 [5480, 8410] 0.160 Neutrophil count (/µl) 4133 [2803, 5279] 5011 [3593, 6682] 0.089 CEA level (ng/ml) 35.35 [12.95, 90.7] 51.8 [11.4, 331.7] 0.392 CA19-9 level (U/ml) 49.85 [10.6, 485.0] 66.2 [17.9, 260.8] 0.628 Tumor location (right/left) 13/21 21/24 0.454 T category (1/2/3/4) 0/1/11/22 0/1/22/22 0.337 N category (0/1/2/3) 6/15/8/5 11/13/12/9 0.559 Metastasis M1 (a/ b/c) 21/5/2 20/10/7 0.181 H (0/1/2/3) 11/11/9/3 14/13/11/7 0.847 PUL (0/1/2/3) 25/5/4/0 30/7/7/1 0.778 P (0/1/2/3) 26/2/3/3 30/3/6/6 0.808 Histological grade (un-/differentiated) 5/29 5/40 0.634 RAS (mutant/wild) 12/22 23/21 0.270 Chemotherapy 1st line (singlet/doublet/triplet) 7/27/0 9/34/2 0.310 Fluorouracil (+/-) 34/0 45/0 - Oxaliplatin (+/-) 28/6 34/11 0.467 Irinotecan (+/-) 0/34 4/41 0.074 Anti-VEGF antibody (Bev) (+/-) 10/24 19/26 0.240 Anti-EGFR antibody (+/-) 14/20 11/34 0.114 Number of regimens (1/2/3-) 13/10/11 21/13/11 0.685 Operation Primary resection (+/-) 29/5 38/7 0.917 Metastasis resection (+/-) 20/14 24/21 0.626 Radical resection (R0, 1/R2, unresected-) 17/17 19/26 0.492 Data are presented as n or as the median [interquartile range]. Bev, bevacizumab; CA19-9, carbohydrate antigen19-9; CEA, carcinoembryonic antigen; EGFR, Epidermal growth factor receptor; H, hepatic metastasis; P, peritoneal metastasis; PUL, pulmonary metastasis; VEGF, vascular endothelial growth factor; WBC, white blood cell Table 2 Univariate and multivariate analyses of risk factors of progression-free survival Factor Cut-off Univariate analysis Multivariate analysis HR 95% CI P -value HR 95% CI P -value Lower Upper Lower Upper Serum PRTN3 (ng/ml) > 21.6 2.173 1.269 3.723 0.005 2.082 1.188 3.647 0.010 Age (years) > 65 0.921 0.545 1.555 0.757 Sex Male/female 1.162 1.162 1.940 0.565 WBC count (/µl) > 8600 1.707 0.922 3.162 0.089 1.807 0.964 3.388 0.065 Neutrophil count (/µl) > 4920 1.477 0.884 2.470 0.137 CEA level (ng/ml) > 6.0 0.893 0.354 2.254 0.812 CA19-9 level (U/ml) > 37 1.517 0.889 2.588 0.127 Tumor location Right/left 1.263 0.756 2.109 0.372 T category 1.039 0.678 1.638 0.865 N category 1.360 1.050 1.756 0.020 1.222 0.931 1.604 0.148 Metastasis a/b/c 1.200 0.808 1.725 0.355 Operation (radical resection) R0, 1/R2, unresected 0.619 0.369 1.037 0.068 0.589 0.341 1.018 0.058 95% CI, 95% confidence interval; CA19-9, carbohydrate antigen19-9; CEA, carcinoembryonic antigen; WBC, white blood cell Although there was no significant between-group difference in OS, the high expression group had a significantly poorer 2-year survival than the low expression group (50.4% vs. 78.7%; HR: 3.095; 95% CI: 1.331–7.197; P = 0.009, Fig. 3 b). In the multivariate analysis, high serum PRTN3 expression (HR: 4.531; 95% CI: 1.569–13.085; P = 0.005), high N category (HR: 1.572; 95% CI: 1.056–2.339; P = 0.026), and radical resection (HR: 0.133; 95% CI: 0.046–0.383; P < 0.001) were independent risk factors for poor 2-year survival (Table 3 ). These findings indicated that high serum PRTN3 levels may be associated with poor PFS, which may be related to poor 2-year survival in patients with mCRC. Table 3 Univariate and multivariate analyses of risk factors of 2-year survival Factor Cut-off Univariate analysis Multivariate analysis HR 95% CI P -value HR 95% CI P -value Lower Upper Lower Upper Serum PRTN3 (ng/ml) > 21.6 3.095 1.331 7.197 0.009 4.531 1.569 13.085 0.005 Age (years) > 65 0.786 0.387 1.595 0.505 Sex Male/female 1.918 0.919 4.005 0.083 1.890 0.745 4.792 0.180 WBC count (/µl) > 8600 1.018 0.418 2.483 0.968 Neutrophil count (/µl) > 4920 1.520 0.751 3.075 0.245 CEA level (ng/ml) > 6.0 4.093 0.557 30.07 0.166 CA19-9 level (U/ml) > 37 1.146 0.693 3.062 0.321 Tumor location Right/left 1.228 0.401 1.653 0.570 T category 1.001 0.546 1.925 0.997 N category 1.663 1.160 2.406 0.006 1.572 1.056 2.339 0.026 Metastasis a/b/c 1.724 1.016 2.814 0.044 1.633 0.903 2.955 0.105 Operation (radical resection) R0, 1/R2, unresected 0.241 0.103 0.561 0.001 0.133 0.046 0.383 < 0.001 95% CI, 95% confidence interval; CA19-9, carbohydrate antigen19-9; CEA, carcinoembryonic antigen; WBC, white blood cell Relationship between serum PRTN3 levels and PRTN3 levels in CRC tissues IHC analysis (Fig. 4 ) showed that in the primary tumor, PRTN3 was expressed in both cancer and stromal cells. However, serum PRTN3 levels were not correlated with the degree of PRTN3 expression in cancer cells (Fig. 4 b). In contrast, the number of PRTN3-positive cells in the stroma was significantly higher in the high serum PRTN3 group than in the low serum PRTN3 group ( P = 0.0051, Fig. 4 d). Similar results were observed for liver metastases ( P = 0.035, Fig. 4 f). In the analysis according to the number of PRTN3-positive cells in the stroma, OS was not significantly different between the high and low groups. However, a high number of PRTN3-positive cells in the stroma was associated with poorer PFS (HR: 2.020; 95% CI: 0.962–4.243; P = 0.063, data not shown). Moreover, PRTN3 was mostly expressed in neutrophils (CD66b) and rarely in lymphocytes (CD3) and macrophages (CD68) (Fig. 5 ). PRTN3 expression in peripheral blood cells In the flow cytometric analysis of peripheral blood cells, almost all PRTN3-positive cells expressed CD66b (Fig. S1 b); median of 30.6% (range, 9.81–51.1%) of CD66b-positive cells (Fig. S1 c). These results suggest that PRTN3 is expressed in neutrophils, but not in all neutrophils. However, there was no significant correlation between the number of PRTN3-positive neutrophils and the serum PRTN3 levels (Fig. S1 d). Relationship between chemotherapy efficacy and serum PRTN3 levels With respect to the association between serum PRTN3 level and chemosensitivity, the results showed that the effectiveness of chemotherapy was lower in patients with high serum PRTN3 levels than in those with low serum PRTN3 levels (Table 4 ). Furthermore, subgroup analyses according to the use of Bev showed that effectiveness of chemotherapy was significantly different regardless of Bev usage (Table 4 ). On the other hand, the PFS was significantly different between patients with high and with low PRTN3 expression only in the Bev subgroup (HR: 3.027; 95% CI: 1.175–7.793; P = 0.0161, Fig. 6 ). Although more frequent RAS mutations (P < 0.001) and higher level of CA19-9 expression (P = 0.008) were observed in the Bev subgroup, these factors were not significantly associated with PFS in the Bev and no Bev subgroups, respectively (P = 0.864 and P = 0.123) (data not shown). These results indicate that serum PRTN3 levels may be associated with the effectiveness of chemotherapy, especially Bev chemotherapy. Table 4 Best overall response by Response Evaluation Criteria in Solid Tumors All specimens With bevacizumab treatment Without bevacizumab treatment Serum PRTN3 low (n = 34) Serum PRTN3 high (n = 45) P -value Serum PRTN3 low (n = 10) Serum PRTN3 high (n = 19) P -value Serum PRTN3 low (n = 24) Serum PRTN3 high (n = 26) P -value Best overall response < 0.001 < 0.001 0.004 Partial response 21 (61.8) 5 (11.1) 8 (80.0) 2 (10.5) 13 (54.2) 3 (11.5) Stable disease 10 (29.4) 27 (60.0) 2 (20.0) 13 (68.4) 8 (33.3) 14 (53.9) Progressive disease 3 (8.8) 13 (28.9) 0 4 (21.1) 3 (12.5) 9 (34.6) Data are presented as n (%). Abbreviations: PRTN3, proteinase-3 Association of PRTN3 expression with angiogenesis and bevacizumab resistance A tube formation assay was performed to support the hypothesis that PRTN3 promotes angiogenesis and that PRTN3 is involved in Bev resistance. PRTN3 administration gradually increased the number of branches and tube area in a dose-dependent manner (Fig. 7 ). Conversely, the number of branches and tube area were decreased by Bev administration. Moreover, PRTN3 administration increased the number of branches and tube area in a dose-dependent manner regardless of Bev administration. These results suggest that PRTN3 could be involved in resistance to Bev chemotherapy. Discussion This is the first study to report the importance of serum PRTN3 levels in patients with mCRC. We found that high serum PRTN3 levels were associated with worse PFS (Fig. 3 , Table 2 ) and response to chemotherapy, especially to Bev chemotherapy (Fig. 6 , Table 4 ). Additionally, a tube formation assay confirmed the relationship between PRTN3 expression and sensitivity to Bev (Fig. 7 ). These results support the idea that PRTN3 could be a predictive biomarker of PFS by reflecting the response to Bev therapy in patients with mCRC. This could be partly attributed to the various factors influencing tumor angiogenesis as well as host factors. TCGA database analysis in the current study showed a difference in prognosis according to the status of PRTN3 expression only in stage IV patients (Fig. 2 ). This result could potentially be attributed to the well-known fact that Bev was preferably administered to stage IV CRC patients and also supports our hypothesis that PRTN3 level is associated with Bev response. Furthermore, we found that PRTN3 is expressed in peripheral and stromal neutrophils in patients with mCRC (Figs. 4 and 5 , Fig. S1 ). PRTN3 promotes the activation of (MMP)-2, which is involved in angiogenesis and tumor invasion [ 21 , 22 ]. PRTN3 is also activated by IL-32, which induces protease-activated receptor 2 (PAR2) signaling [ 31 , 32 ]. One of the downstream pathways of the IL-32-PRTN3-PAR2 axis is the Ras-Raf pathway, which is involved in angiogenesis [ 31 , 33 , 34 ]. Moreover, the Ras-Raf pathway downstream of VEGF and its receptor signaling is implicated in tumor angiogenesis, therefore, therapies targeting this pathway are commonly used in mCRC [ 5 , 35 ]. Schiffmann et al. reported that a high number of CD177-positive neutrophils in the stroma is associated with poor response to Bev chemotherapy for mCRC, thus leading to poor prognosis [ 36 ]. These reports support our findings that PRTN3 is an important regulator of tumor angiogenesis and can thus be a predictive biomarker of Bev response. Our study showed that serum PRTN3 levels were significantly associated with the number of PRTN3-positive cells in stroma, and almost all PRTN3-positive cells were neutrophils (Figs. 4 and 5 ). Therefore, we hypothesized that PRTN3 may play a role in stromal neutrophil function. Tumor-associated neutrophils are involved in tumor angiogenesis [ 37 ]. Therefore, the possibility of a relationship between neutrophil-associated angiogenesis and Bev sensitivity should be investigated in the future. Currently, Bev chemotherapy is the standard treatment for mCRC. However, resistance to Bev is also a major obstacle to successful treatment [ 10 ]. Therefore, predictive biomarkers of response to Bev and novel strategies for overcoming resistance to Bev are required. Our study found that PRTN3 may influence the therapeutic effect of Bev (Table 4 , Figs. 6 and 7 ). Serum PRTN3 levels may potentially be beneficial in forecasting the efficacy of Bev treatment, thus facilitating the selection of optimal chemotherapeutic regimen. PRTN3 has been reported to promote angiogenesis through MMPs [ 21 , 22 ]. As the serum PRTN3 level before Bev initiation is an independent predictive factor for response, angiogenesis by PRTN3 may exceed the inhibitory effect of Bev on angiogenesis, resulting in chemotherapy resistance. In addition, even when VEGF is inhibited, the activation of other pathways, stimulated by PRTN3, may result in chemotherapy resistance. Our tube formation assay results support this hypothesis. Thus, identification of PRTN3 and its signals and their inhibition may overcome resistance to Bev. This study had some limitations, namely, its retrospective, single-center design; small sample size; and profound advances in chemotherapy during the study period. Therefore, further studies, such as experiments analyzing the molecular and cellular mechanisms underlying the effects of PRTN3 on tumor angiogenesis and the relationship between PRTN3 and stromal neutrophils, are warranted to confirm our results. Conclusions In conclusion, serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy in patients with mCRC. In Addition, PRTN3 might predict the efficacy of bevacizumab. Abbreviations CRC: Colorectal cancer mCRC: Metastatic colorectal cancer VEGF: vascular endothelial growth factor mAbs: monoclonal antibodies Bev: bevacizumab PRTN3: Protenase-3 TCGA: The Cancer Genome Atlas FPKM: fragments per kilobase of exon per million ELISA: enzyme-linked immunosorbent assay IHC: Immunohistochemistry FFPE: formalin-fixed paraffin-embedded CES: composite expression score ROC: receiver operating characteristic OS: Overall survival HR: hazard ratio PFS: Progression-free survival MMPs: matrix metalloproteinases PAR2: protease-activated receptor 2 Declarations Ethics approval and consent to participate Ethics approval and consent to participate Ethical, legal, and social implications were approved by the Ethics Committee of Yamaguchi University Hospital (H20-102, H23-135, and H28-074). This study was conducted per the Helsinki Declaration for experimentation on human subjects. All the samples were obtained with informed consent from the patients. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflict of interest The authors have no conflict of interest. Funding This study was performed as part of a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT; 11039020) and The Japan Agency for Medical Research and Development (AMED; 15cm0106085h0005). This study was supported in part by a grant from the Japan Agency for Medical Research and Development for Leading Advanced Projects for Medical Innovation (LEAP; 16am0001006h0003). Contributions KF, MN, RT and SH conceived and designed the study. KF, MN, RT, HM, YS, YT, YW, ST, NM, MI, NS, ST, TI, SH and HN were involved in the collection and assembly of data. KF, MN, RT, MX and YH performed the experiments. KF, NM, RT and YN analyzed the data. KF and HN wrote the manuscript. All authors have read and approved the manuscript. Acknowledgements We would like to thank the patients and staff of Yamaguchi University Hospital for participating in this study. Author information Authors and Affiliations Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan Kei Furuya, Masao Nakajima, Ryouichi Tsunedomi, Yuki Nakagami, Ming Xu, Hiroto Matsui, Yukio Tokumitsu, Yoshitaro Shindo, Yusaku Watanabe, Shinobu Tomochika, Noriko Maeda, Michihisa Iida, Nobuaki Suzuki, Shigeru Takeda, Shoichi Hazama & Hiroaki Nagano Oncology Center, Yamaguchi University Hospital, Ube, Yamaguchi 755-8505, Japan Tatsuya Ioka Department of Diagnostic Pathology, Yamaguchi University Hospital, Ube, Yamaguchi 755-8505, Japan Yoshinobu Hoshii Department of Digestive Surgery, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan Tomio Ueno References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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IL-32-PAR2 axis is an innate immunity sensor providing alternative signaling for LPS-TRIF axis. Sci Rep. 2013;3:2960. Novick D, Rubinstein M, Azam T, Rabinkov A, Dinarello CA, Kim SH. Proteinase 3 is an IL-32 binding protein. Proc Natl Acad Sci U S A. 2016;103:3316–21. Kawaguchi M, Yamamoto K, Kataoka H, Izumi A, Yamashita F, Kiwaki T, et al. Protease-activated receptor-2 accelerates intestinal tumor formation through activation of nuclear factor-κB signaling and tumor angiogenesis in ApcMin/+ mice. Cancer Sci. 2020;111:1193–202. Pawar NR, Buzza MS, Antalis TM. Membrane-anchored serine proteases and protease-activated Receptor-2-Mediated signaling: Co-conspirators in cancer progression. Cancer Res. 2019;79:301–10. Ferrara N, Adamis AP. Ten years of anti-vascular endothelial growth factor therapy. Nat Rev Drug Discov. 2016;15:385–403. Schiffmann LM, Fritsch M, Gebauer F, Günther SD, Stair NR, Seeger JM, et al. Tumour-infiltrating neutrophils counteract anti-VEGF therapy in metastatic colorectal cancer. Br J Cancer. 2019;120:69–78. Jaillon S, Ponzetta A, Di Mitri D, Santoni A, Bonecchi R, Mantovani A. Neutrophil diversity and plasticity in tumour progression and therapy. Nat Rev Cancer. 2020;20:485–503. Additional Declarations No competing interests reported. Supplementary Files Additionaltable1.docx Additional file 1: Table S1. Protein comprehensive proteomics analysis AdditionalFigure1.pdf Additional file 2: Fig. S1. PRTN3 expression in peripheral blood cells of patients with mCRC (a) Representative data of CD66b/PRTN3 staining of living cells. (b) Percentage of CD66b expression according to PRTN3 expression (n=12). (c) Percentage of PRTN3 expression according to CD66b expression (n=12). (d) Correlation between the serum PRTN3 level and the PRTN3-positive neutrophil count (n=12). R=0.3163. P=0.3166. *P<0.05 Abbreviations: mCRC, metastatic colorectal cancer; PRTN3, proteinase-3 Cite Share Download PDF Status: Published Journal Publication published 02 Feb, 2024 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 06 Dec, 2023 Reviews received at journal 28 Nov, 2023 Reviewers agreed at journal 14 Nov, 2023 Reviewers invited by journal 31 Oct, 2023 Editor assigned by journal 31 Oct, 2023 Editor invited by journal 04 Oct, 2023 Submission checks completed at journal 04 Oct, 2023 First submitted to journal 27 Sep, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3393984","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":237813374,"identity":"61d2bda4-9d22-4c82-8c51-c2e761d873e1","order_by":0,"name":"Kei Furuya","email":"","orcid":"","institution":"Yamaguchi University Graduate School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kei","middleName":"","lastName":"Furuya","suffix":""},{"id":237813375,"identity":"559c6c7c-2aaa-48b1-87bb-6876d88ce3ab","order_by":1,"name":"Masao Nakajima","email":"","orcid":"","institution":"Yamaguchi University 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03:44:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3393984/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3393984/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-024-11924-4","type":"published","date":"2024-02-02T15:01:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44378624,"identity":"7e7ecdb0-0d1d-40fe-ac61-8444060e5aed","added_by":"auto","created_at":"2023-10-10 18:07:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConsort diagram of this study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn total, 79 patients with mCRC were enrolled in the study. Patient sera were used to measure PRTN3 levels with ELISA. Overall, 48 primary tumor and 5 liver tumor samples obtained surgically from the 79 patients were subjected to IHC staining. The prognostic impact and association with the efficacy of first-line chemotherapy of PRTN3 levels were examined. Flow cytometric analysis was performed using peripheral whole blood samples from 12 patients with mCRC\u003c/p\u003e\n\u003cp\u003eAbbreviations: IHC, immunohistochemistry; mCRC, metastatic colorectal cancer; PRTN3, proteinase-3\u003c/p\u003e","description":"","filename":"Binder21.png","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/7543741818ae5f33f30e7b09.png"},{"id":44379970,"identity":"bb2a51eb-b617-4d4b-9cd5-490efb176fe9","added_by":"auto","created_at":"2023-10-10 18:15:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":239153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival analysis of patients with CRC according to PRTN3-expression from The Cancer Genome Atlas database\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patients were divided into two groups according to PRTN3 expression/non-expression. (a) All patients. (b) Stage I. (c) Stage II. (d) Stage III. (e) Stage IV\u003c/p\u003e\n\u003cp\u003eAbbreviations: mCRC, metastatic colorectal cancer; PRTN3, proteinase-3\u003c/p\u003e","description":"","filename":"Binder22.png","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/dc54aacc212fd58e265f5f70.png"},{"id":44379968,"identity":"b8a557b1-75f7-414a-8cb2-c40160e6b678","added_by":"auto","created_at":"2023-10-10 18:15:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":129656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival analysis according to serum PRTN3 levels in patients with mCRC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patients were divided into two groups according to serum PRTN3 levels based on a cutoff value of 21.6 ng/ml identified using time-dependent receiver operating characteristic curve analysis: high expression group (≥ 21.6 ng/ml) and low expression group (\u0026lt; 21.6 ng/ml). (a) Progression-free survival. (b) Overall survival and overall survival rates\u003c/p\u003e\n\u003cp\u003eAbbreviations: mCRC, metastatic colorectal cancer; PRTN3, proteinase-3\u003c/p\u003e","description":"","filename":"Binder23.png","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/130a49991690aa7ab8689375.png"},{"id":44378632,"identity":"a2eeccd8-6321-4dc3-bfa0-cbde5e67d2b4","added_by":"auto","created_at":"2023-10-10 18:07:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":789309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRTN3 expression in resected specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical (IHC) analysis was performed using primary and metastatic specimens. Scale bar: 100 µm. (a) Representative images of PRTN3 expression in primary tumors. CES=4 × (intensity score – 1) + frequency score. The intensity scores are 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The frequency scores are 1 (5–24%), 2 (25–49%), 3 (50–74%), and 4 (75–100%). (b) Correlation between serum PRTN3 and CES levels. (c) and € Representative cases of PRTN3 expression in stromal cells of primary colorectal and metastatic liver specimens. The upper row shows the case with a few positive cells in the stroma. The lower row shows the case with many positive cells in the stroma. (d) and (f) Correlation between serum PRTN3 levels and number of PRTN3-positive cells in the stroma of primary and metastatic tumors, respectively. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003eAbbreviations: CES, composite expression score; HPF, high-power field; PRTN3, proteinase-3\u003c/p\u003e","description":"","filename":"Binder24.png","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/b712e12785d5a3d9d83d4d74.png"},{"id":44379969,"identity":"79e293c2-6b0c-4ed2-b6a9-abf23da29f38","added_by":"auto","created_at":"2023-10-10 18:15:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":239886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRTN3 expression in immune cells in the tumor microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative immunofluorescence images of PRTN3 (green), CD66b (red), CD68 (red), CD3 (red), nuclei (blue), and merge (yellow) in the primary tissue. Scale bar: 100 µm\u003c/p\u003e\n\u003cp\u003eAbbreviations: PRTN3, proteinase-3\u003c/p\u003e","description":"","filename":"Binder25.png","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/3132bfe1cd3c2ac61a494da6.png"},{"id":44378626,"identity":"70a7eaad-2f5e-42ed-bf78-069b884100e1","added_by":"auto","created_at":"2023-10-10 18:07:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":122930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProgression-free survival according to PRTN3 expression in the anti-VEGF antibody treatment subgroups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Progression-free survival of patients treated with bevacizumab. (b) Progression-free survival of patients treated without bevacizumab. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Binder26.png","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/92d3e58495b0dad2f652b545.png"},{"id":44378627,"identity":"18e02c0f-d67c-4731-b4c8-761f78f5ab51","added_by":"auto","created_at":"2023-10-10 18:07:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":320087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAngiogenic effects of PRTN3 and bevacizumab sensitivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTube formation assay was performed using the endothelial tube formation kit. PRTN3 was added at a final concentration of 0-1000 ng/mL, and Bev was added at a final concentration of 0-2.5 mg/mL to 2.5×10\u003csup\u003e4\u003c/sup\u003e cells of human umbilical vein endothelial cells per each well. After incubation at 37℃ and 5% CO\u003csup\u003e2\u003c/sup\u003e for 2 h, the number of branches and tube area were evaluated. (a) Representative images of tube formation assay. Scale bar: 100 µm. (b) Number of branching points. The number of branches is increased in a PRTN3 dose-dependent manner. The number of branches is decreased with the administration of bevacizumab. PRTN3 administration increased the number of branches in a dose-dependent manner regardless of bevacizumab administration. (c) Tube area. The results are the same as those for the number of branches. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05\u003c/p\u003e","description":"","filename":"Binder27.png","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/80177ea77c68f8c666701e2b.png"},{"id":50674234,"identity":"caacb9a3-ef24-4785-8a96-4afa20d7ba48","added_by":"auto","created_at":"2024-02-05 15:09:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2612391,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/82d92b53-057b-4d04-816e-991ed5a60247.pdf"},{"id":44378625,"identity":"41435d54-bb94-4a52-a64f-ff6cb878289f","added_by":"auto","created_at":"2023-10-10 18:07:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1. \u003c/strong\u003eProtein comprehensive proteomics analysis\u003c/p\u003e","description":"","filename":"Additionaltable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/a7cc9ce6967048048f6705cf.docx"},{"id":44378630,"identity":"ac241f24-6bb9-46c4-9ed4-e70e66a0c916","added_by":"auto","created_at":"2023-10-10 18:07:05","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2296319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1. \u003c/strong\u003ePRTN3 expression in peripheral blood cells of patients with mCRC\u003c/p\u003e\n\u003cp\u003e(a) Representative data of CD66b/PRTN3 staining of living cells. (b) Percentage of CD66b expression according to PRTN3 expression (n=12). (c) Percentage of PRTN3 expression according to CD66b expression (n=12). (d) Correlation between the serum PRTN3 level and the PRTN3-positive neutrophil count (n=12). R=0.3163. P=0.3166. *P\u0026lt;0.05\u003c/p\u003e\n\u003cp\u003eAbbreviations: mCRC, metastatic colorectal cancer; PRTN3, proteinase-3\u003c/p\u003e","description":"","filename":"AdditionalFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3393984/v1/6be9a133d5cc173e0a896f0d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Serum proteinase-3 levels as a predictor of progression-free survival of first-line chemotherapy in metastatic colorectal cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eColorectal cancer (CRC) is the second leading cause of cancer-specific death worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The standard treatment for metastatic CRC (mCRC) is a multidisciplinary approach involving chemotherapy, immunotherapy, and surgery [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the therapeutic efficacy of chemotherapy needs to be enhanced to improve mCRC survival. On the other hand, the additive or synergistic effects of the combination of anti-vascular endothelial growth factor (VEGF) monoclonal antibodies (mAbs), such as bevacizumab (Bev), and cytotoxic agents have been shown to overcome resistance to chemotherapy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. One possible mechanism for this effect is the direct anti-vascular effects of cytotoxic agents that intensify the pro-apoptotic effects of anti-VEGF mAbs on the vascular endothelium [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Another possibility is that normalization of tumor vessels by anti-VEGF mAbs increases the uptake of cytotoxic agents and antibodies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, anti-VEGF mAbs, especially Bev, are currently the standard of care for mCRC; however, not all patients benefit. Additionally, adverse effects, such as hypertension, proteinuria, gastrointestinal perforation, and stroke, are often observed [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, an alternative approach is to optimize the benefit of chemotherapy regimens using biomarkers that predict chemosensitivity. However, only a few biomarkers (e.g., RAS mutations, status, and UGT1A1 polymorphism) have been identified to guide chemotherapy selection in clinical practice [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and some of these markers require invasive measurement. Moreover, no clinical and biological factors have been identified in response to Bev, despite extensive research to explore the predictive biomarkers in response to Bev therapy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, more predictive and less invasive biomarkers need to be further explored [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe have previously identified prognostic biomarkers of CRC by comprehensive proteomic analysis [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Proteinase-3 (PRTN3) belongs to the neutrophil-derived protease family and is stored within azurophil granules [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is involved in neutrophil differentiation and proliferation, inflammation, and vasculitis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], as well as in the invasion of tumor and endothelial cells through the activation of matrix metalloproteinases (MMP)-2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In cancer, PRTN3 expression in tumor tissue is associated with poor prognosis in some carcinomas [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the relationship between serum PRTN3 levels and CRC remains unclear. Therefore, this study aimed to analyze the clinical significance of serum PRTN3 as a predictor of chemosensitivity and prognosis in mCRC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and patients\u003c/h2\u003e \u003cp\u003eThis was a single-center, retrospective, observational study conducted between January 2008 to December 2019. In total, 79 serum samples were obtained from consecutive patients with mCRC at our hospital and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use. Of them, 48 patients who underwent surgery before chemotherapy, 48 primary tumors and five metastatic liver tumors were surgically obtained. Additionally, peripheral whole blood samples for flow cytometric analysis were obtained from 12 patients with mCRC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Computed tomography was performed every 2\u0026ndash;3 months to evaluate treatment efficacy Thusing the remaining target lesions in patients who did not undergo radical resection. Otherwise the efficacy was evaluated during preoperative chemotherapy in patients with radical resection according to RECIST criteria version 1.1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e This study was approved by the Ethics Committee of Yamaguchi University Hospital (H20-102, H23-135, and H28-074) and was performed in accordance with the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eComprehensive proteomics analysis of serum and tumor tissue\u003c/h2\u003e \u003cp\u003eComprehensive analysis of protein levels in the pretreatment serum and tumor tissue lysates of 24 patients with mCRC was performed using SOMAscan (Soma Logic, Boulder, CO) following the manufacturer\u0026rsquo;s protocol (Soma Logic) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Briefly, total protein in frozen CRC samples was quantified by adding a Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Kanagawa, Japan) using a Qiagen Tissue Lyser (Qiagen Scientific, Tokyo, Japan). Samples were sent to Soma Logic and analyzed using the SOMAscan assay. The SOMAscan readings are displayed as relative fluorescence units.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of relationship between PRTN3 gene expression and mCRC prognosis using The Cancer Genome Atlas database\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTranscriptional and corresponding clinical information of 276 patients with CRC was obtained from The Cancer Genome Atlas (TCGA, PanCancer Atlas, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Accessed 3 Jun 2021). Survival curve analysis was performed for the PRTN3-expression group (those with mRNA expression quantification of \u0026gt;\u0026thinsp;0 fragments per kilobase of exon per million mapped reads [FPKM]) and the non-expression group (those with mRNA expression quantification of 0 FPKM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of serum PRTN3 levels\u003c/h2\u003e \u003cp\u003eSerum PRTN3 concentration was measured using the human PRTN3 enzyme-linked immunosorbent assay (ELISA) kit (ab226902; Abcam, Tokyo, Japan) according to the manufacturer\u0026rsquo;s instructions. Briefly, after adding 50 \u0026micro;l of a standard or sample solution to appropriate wells, 50 \u0026micro;l of the antibody cocktail (capture antibody and detector antibody) was added, and the samples were incubated at room temperature for 1 h. After washing, 100 \u0026micro;l of TMB Development Solution was added to each well and incubated for 10 min. The absorbance was read at a wavelength of 450 nm after adding 100 \u0026micro;l of a stop solution. Absorbance was measured using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). A standard curve was prepared for stock standards. The sensitivity of this assay was 150 pg/ml. All standards, controls, and samples were measured in duplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eImmunohistochemistry (IHC) was performed using 4-\u0026micro;m-thick formalin-fixed paraffin-embedded (FFPE) sections. The sections were deparaffinized with xylene and alcohol, followed by antibody activation using 10 mM sodium citrate buffer (Agilent Technologies, Tokyo, Japan) at pH\u0026thinsp;=\u0026thinsp;6.0 for 20 min at 95\u0026deg;C. Endogenous peroxidase activity was blocked with a blocking solution (S2023; Agilent Technologies) for 5 min, followed by blocking of non-specific reactions for 10 min at room temperature (X0909; Agilent Technologies) and incubation with rabbit anti-human PRTN3 antibody (EPR6277, Abcam; dilution 1:200) at 4\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003eAfter washing, the sections were incubated with an appropriate secondary antibody (K4003; Agilent Technologies; undiluted) for 30 min at room temperature. DAB (Agilent Technologies) was used according to the manufacturer\u0026rsquo;s protocol and allowed to react with the sections for 3 min. Contrast staining was performed with hematoxylin, and the samples were sealed after dehydration. Images were obtained using an all-in-one fluorescence microscope (BZ-X710, KEYENCE, Osaka, Japan). PRTN3 expression in tissue samples was evaluated by a pathologist and an investigator who were blinded to patient outcomes. Focusing on the tumor cells, a semi-quantitative evaluation of PRTN3 expression was performed using the composite expression score (CES) as described previously [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCES\u0026thinsp;=\u0026thinsp;4 \u0026times; (intensity score\u0026thinsp;\u0026minus;\u0026thinsp;1)\u0026thinsp;+\u0026thinsp;frequency score. Intensity scores: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The frequency scores were as follows: 1 (5\u0026ndash;24%), 2 (25\u0026ndash;49%), 3 (50\u0026ndash;74%), and 4 (75\u0026ndash;100%).\u003c/p\u003e \u003cp\u003eTo analyze PRTN3 expression in stromal cells, five areas on each slide that were most abundant in positive cells were photographed under \u0026times;400 high-power fields, and the number of positive stromal cells was counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed with 4-\u0026micro;m-thick FFPE sections. After deparaffinization and antigen activation (pH 6.0, 95\u0026deg;C, 20 min), non-specific reactions were blocked. The sections were then incubated with the anti-human PRTN3 antibody (at the same dilution rate for IHC), anti-CD3 antibody (17A2; Thermo Fisher Scientific, dilution 1:100), anti-CD68 antibody (PG-M1, Abcam; dilution 1:400), and anti-CD66b antibody (G10F5, Novus Biologicals, Centennial, CO; dilution 1:200) at 4\u0026deg;C overnight. The sections were incubated with secondary antibody mixtures (anti-mouse Alexa Fluor 488; Thermo Fisher Scientific; dilution 1:1000 and anti-rabbit Alexa Fluor 555; Thermo Fisher Scientific; dilution 1:1000) for 40 min at room temperature. Thereafter, they were stained with DAPI for nuclear contrast staining. Images were obtained using an all-in-one fluorescence microscope (BZ-X710, KEYENCE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFresh peripheral blood (100 \u0026micro;l) was collected in EDTA-anticoagulation tubes and reacted at room temperature for 30 min in the dark with anti-PRTN3 antibody (PR3G-2, Abcam; undiluted) and anti-CD66b antibody (G10F5, BioLegend, San Diego, CA; undiluted) according to the manufacturer\u0026rsquo;s protocol. Erythrocytes were lysed in lysis solution (BD Biosciences, San Jose, CA, USA) at room temperature for 10 min in the dark and centrifuged at 1500 rpm for 10 min. The cells were then washed with PBS. Finally, the cells were resuspended in 500 \u0026micro;l cell FIX (BD Biosciences; dilution, 1:10). Flow cytometric data were acquired using a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA) with the NovoExpress software (version 1.3.0, ACEA Biosciences) and analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTube formation assay\u003c/h2\u003e \u003cp\u003eTube formation assay was performed using the endothelial tube formation kit (Cell Biolabs, San Diego, USA) according to the manufacturer\u0026rsquo;s protocol. Briefly, pre-chilled 96-well plates were coated with ECM gel (50 \u0026micro;L/well) and incubated at 37℃ for 1 h. Followed by addition of 2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells of human umbilical vein endothelial cells (Takara, Tokyo, Japan) and 150 \u0026micro;L culture medium per well. Additionally, PRTN3 (RPB434Hu02, Cloud Clone Corp., USA) was added at a final concentration of 0\u0026ndash;1000 ng/mL, and Bev (Chugai Pharmaceutical Co., Tokyo, Japan) was added at a final concentration of 0\u0026ndash;2.5 mg/mL to each well. After incubation at 37℃ and 5% CO\u003csup\u003e2\u003c/sup\u003e for 2 h, the number of branches and tube area were evaluated using an all-in-one fluorescence microscope (BZ-X710, KEYENCE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe cutoff value of serum PRTN3 was determined using time-dependent receiver operating characteristic (ROC) curve analysis. Between-group differences were estimated using the Mann\u0026ndash;Whitney U-test. Categorical variables were compared using the chi-squared test. Overall survival (OS) was defined from the date of initial diagnosis to the date of mortality. Survival curves were generated using the Kaplan\u0026ndash;Meier method and compared using the log-rank test. Cox\u0026rsquo;s proportional hazards model was used to estimate the hazard ratios (HRs) and perform multivariate analysis. Multivariate analysis corrected for tendency factors (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.10) in the univariate analysis. Progression-free survival (PFS) for first-line chemotherapy was defined as the interval from the date of the first chemotherapy to the date of progression or death. All statistical analyses were performed using JMP Pro16 (SAS Institute, Cary, NC, USA). Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eCandidate proteins predictive of mCRC prognosis\u003c/h2\u003e\n \u003cp\u003eThe good (OS\u0026thinsp;\u0026ge;\u0026thinsp;3 years) and poor (OS\u0026thinsp;\u0026lt;\u0026thinsp;2 years) prognosis groups included nine and 11 patients, respectively. The candidate proteins ranked according to their Fisher ratio are presented in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e. Myeloperoxidase was the top candidate protein in serum, but is already reported to be associated with CRC prognosis [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, it may be difficult to identify its novel roles for mCRC and was thus excluded. The expression of PRTN3, the second candidate protein in serum, was significantly higher in the poor prognosis group than in the good prognosis group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The other candidate proteins were known prognostic markers in patients with mCRC and were therefore also excluded from the analysis [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, PRTN3 was also one of the top 10 proteins in the tissue samples.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the relationship between PRTN3 expression in tumor tissues and CRC prognosis in the TCGA database analysis. OS was significantly worse in the expression group than in the non-expression group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0328) but only in patients with stage IV CRC. Meanwhile, there were no significant differences among other disease stages. These results suggest that PRTN3 may be associated with the prognosis of mCRC. Therefore, we focused on PRTN3 as a predictive marker for mCRC prognosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eClinicopathological characteristics and relationship between serum PRTN3 levels and mCRC\u003c/h2\u003e\n \u003cp\u003eThe high (\u0026ge;\u0026thinsp;21.6 ng/ml) and low (\u0026lt;\u0026thinsp;21.6 ng/ml) PRTN3 expression groups involved 45 and 34 patients, respectively. There were no significant between-group differences in terms of tumor markers, TNM category, histology, or therapeutic interventions including chemotherapy regimen and surgical procedures (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). PRTN3 is a neutrophil-associated protein. However, there was also no difference in neutrophil counts between the two groups. PFS was significantly worse in the high expression group (HR: 2.173; 95% CI: 1.269\u0026ndash;3.723; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the multivariate analysis, high serum PRTN3 expression (HR: 2.082; 95% CI: 1.188\u0026ndash;3.647; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) was an independent risk factor for progression (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eClinicopathological patient characteristics mCRC (n\u0026thinsp;=\u0026thinsp;79)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3 low\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3 high\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;45)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e -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\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.5 [57.25, 76.25]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66 [55.5, 74.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.593\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex (male/female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15/19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26/19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.229\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWBC (/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6395 [4740, 7925]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7290 [5480, 8410]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.160\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophil count (/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4133 [2803, 5279]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5011 [3593, 6682]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.089\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCEA level (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.35 [12.95, 90.7]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.8 [11.4, 331.7]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.392\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA19-9 level (U/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.85 [10.6, 485.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.2 [17.9, 260.8]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.628\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor location (right/left)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13/21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21/24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.454\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT category (1/2/3/4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/11/22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/1/22/22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.337\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN category (0/1/2/3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6/15/8/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11/13/12/9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.559\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMetastasis\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\u003eM1 (a/ b/c)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21/5/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/10/7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.181\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH (0/1/2/3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11/11/9/3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14/13/11/7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.847\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePUL (0/1/2/3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25/5/4/0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30/7/7/1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.778\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP (0/1/2/3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26/2/3/3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30/3/6/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.808\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHistological grade (un-/differentiated)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5/29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5/40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.634\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRAS (mutant/wild)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12/22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23/21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.270\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChemotherapy\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\u003e1st line (singlet/doublet/triplet)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7/27/0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9/34/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.310\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFluorouracil (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34/0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45/0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOxaliplatin (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34/11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.467\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIrinotecan (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0/34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4/41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnti-VEGF antibody (Bev) (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10/24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.240\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnti-EGFR antibody (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14/20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11/34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.114\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of regimens (1/2/3-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13/10/11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21/13/11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.685\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOperation\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\u003ePrimary resection (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29/5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38/7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.917\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMetastasis resection (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24/21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.626\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadical resection (R0, 1/R2, unresected-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17/17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.492\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eData are presented as n or as the median [interquartile range].\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eBev, bevacizumab; CA19-9, carbohydrate antigen19-9; CEA, carcinoembryonic antigen; EGFR, Epidermal growth factor receptor; H, hepatic metastasis; P, peritoneal metastasis; PUL, pulmonary metastasis; VEGF, vascular endothelial growth factor; WBC, white blood cell\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eUnivariate and multivariate analyses of risk factors of progression-free survival\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eFactor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eCut-off\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eUnivariate analysis\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eMultivariate analysis\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\" rowspan=\"2\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e -value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 12.8049%;\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 8.2927%;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e -value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 8.2927%;\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\" style=\"width: 8.2927%;\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \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\u003eUpper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 6.2195%;\"\u003e\n \u003cp\u003eUpper\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3 (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;21.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.188\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e3.647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.921\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.757\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.940\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.565\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWBC count (/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;8600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.922\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.162\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.807\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e3.388\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophil count (/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;4920\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.137\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCEA level (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.354\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.254\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.812\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA19-9 level (U/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.588\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.127\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor location\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRight/left\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.756\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.372\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT category\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.678\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.865\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN category\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.360\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.756\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e1.604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e0.148\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMetastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ea/b/c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.355\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\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOperation (radical resection)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR0, 1/R2, unresected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.619\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.369\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.341\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e1.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.7317%;\"\u003e\n \u003cp\u003e0.058\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e95% CI, 95% confidence interval; CA19-9, carbohydrate antigen19-9; CEA, carcinoembryonic antigen; WBC, white blood cell\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAlthough there was no significant between-group difference in OS, the high expression group had a significantly poorer 2-year survival than the low expression group (50.4% vs. 78.7%; HR: 3.095; 95% CI: 1.331\u0026ndash;7.197; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009, Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). In the multivariate analysis, high serum PRTN3 expression (HR: 4.531; 95% CI: 1.569\u0026ndash;13.085; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), high N category (HR: 1.572; 95% CI: 1.056\u0026ndash;2.339; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026), and radical resection (HR: 0.133; 95% CI: 0.046\u0026ndash;0.383; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were independent risk factors for poor 2-year survival (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings indicated that high serum PRTN3 levels may be associated with poor PFS, which may be related to poor 2-year survival in patients with mCRC.\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eUnivariate and multivariate analyses of risk factors of 2-year survival\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eFactor\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eCut-off\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eUnivariate analysis\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eMultivariate analysis\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\" rowspan=\"2\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e -value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e -value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \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\u003eUpper\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLower\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUpper\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3 (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;21.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.569\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.505\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 \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale/female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.918\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.745\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.180\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWBC count (/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;8600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.418\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.968\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 \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeutrophil count (/\u0026micro;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;4920\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.520\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.245\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 \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCEA level (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.166\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 \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCA19-9 level (U/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.693\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.321\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 \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor location\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRight/left\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.653\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.570\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 \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT category\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.546\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.925\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.997\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 \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN category\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.572\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.056\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMetastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ea/b/c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.814\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.955\u003c/p\u003e\n \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\u003eOperation (radical resection)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR0, 1/R2, unresected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.561\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.383\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e95% CI, 95% confidence interval; CA19-9, carbohydrate antigen19-9; CEA, carcinoembryonic antigen; WBC, white blood cell\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eRelationship between serum PRTN3 levels and PRTN3 levels in CRC tissues\u003c/h2\u003e\n \u003cp\u003eIHC analysis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) showed that in the primary tumor, PRTN3 was expressed in both cancer and stromal cells. However, serum PRTN3 levels were not correlated with the degree of PRTN3 expression in cancer cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). In contrast, the number of PRTN3-positive cells in the stroma was significantly higher in the high serum PRTN3 group than in the low serum PRTN3 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0051, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Similar results were observed for liver metastases (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). In the analysis according to the number of PRTN3-positive cells in the stroma, OS was not significantly different between the high and low groups. However, a high number of PRTN3-positive cells in the stroma was associated with poorer PFS (HR: 2.020; 95% CI: 0.962\u0026ndash;4.243; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.063, data not shown). Moreover, PRTN3 was mostly expressed in neutrophils (CD66b) and rarely in lymphocytes (CD3) and macrophages (CD68) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003ePRTN3 expression in peripheral blood cells\u003c/h2\u003e\n \u003cp\u003eIn the flow cytometric analysis of peripheral blood cells, almost all PRTN3-positive cells expressed CD66b (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eb); median of 30.6% (range, 9.81\u0026ndash;51.1%) of CD66b-positive cells (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ec). These results suggest that PRTN3 is expressed in neutrophils, but not in all neutrophils. However, there was no significant correlation between the number of PRTN3-positive neutrophils and the serum PRTN3 levels (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003ed).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eRelationship between chemotherapy efficacy and serum PRTN3 levels\u003c/h2\u003e\n \u003cp\u003eWith respect to the association between serum PRTN3 level and chemosensitivity, the results showed that the effectiveness of chemotherapy was lower in patients with high serum PRTN3 levels than in those with low serum PRTN3 levels (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, subgroup analyses according to the use of Bev showed that effectiveness of chemotherapy was significantly different regardless of Bev usage (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). On the other hand, the PFS was significantly different between patients with high and with low PRTN3 expression only in the Bev subgroup (HR: 3.027; 95% CI: 1.175\u0026ndash;7.793; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0161, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Although more frequent RAS mutations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and higher level of CA19-9 expression (P\u0026thinsp;=\u0026thinsp;0.008) were observed in the Bev subgroup, these factors were not significantly associated with PFS in the Bev and no Bev subgroups, respectively (P\u0026thinsp;=\u0026thinsp;0.864 and P\u0026thinsp;=\u0026thinsp;0.123) (data not shown). These results indicate that serum PRTN3 levels may be associated with the effectiveness of chemotherapy, especially Bev chemotherapy.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBest overall response by Response Evaluation Criteria in Solid Tumors\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\" colspan=\"3\"\u003e\n \u003cp\u003eAll specimens\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eWith bevacizumab treatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eWithout bevacizumab treatment\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\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3 low\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3 high\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3\u003c/p\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3\u003c/p\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3\u003c/p\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum PRTN3\u003c/p\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBest overall response\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\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\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\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\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\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePartial response\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (61.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (11.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (80.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (10.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (54.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (11.5)\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\u003eStable disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10 (29.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 (60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (68.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 (53.9)\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\u003eProgressive disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (8.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13 (28.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4 (21.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (34.6)\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 \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003eData are presented as n (%).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003eAbbreviations: PRTN3, proteinase-3\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eAssociation of PRTN3 expression with angiogenesis and bevacizumab resistance\u003c/h2\u003e\n \u003cp\u003eA tube formation assay was performed to support the hypothesis that PRTN3 promotes angiogenesis and that PRTN3 is involved in Bev resistance. PRTN3 administration gradually increased the number of branches and tube area in a dose-dependent manner (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Conversely, the number of branches and tube area were decreased by Bev administration. Moreover, PRTN3 administration increased the number of branches and tube area in a dose-dependent manner regardless of Bev administration. These results suggest that PRTN3 could be involved in resistance to Bev chemotherapy.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first study to report the importance of serum PRTN3 levels in patients with mCRC. We found that high serum PRTN3 levels were associated with worse PFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and response to chemotherapy, especially to Bev chemotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Additionally, a tube formation assay confirmed the relationship between PRTN3 expression and sensitivity to Bev (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results support the idea that PRTN3 could be a predictive biomarker of PFS by reflecting the response to Bev therapy in patients with mCRC. This could be partly attributed to the various factors influencing tumor angiogenesis as well as host factors. TCGA database analysis in the current study showed a difference in prognosis according to the status of PRTN3 expression only in stage IV patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This result could potentially be attributed to the well-known fact that Bev was preferably administered to stage IV CRC patients and also supports our hypothesis that PRTN3 level is associated with Bev response.\u003c/p\u003e \u003cp\u003eFurthermore, we found that PRTN3 is expressed in peripheral and stromal neutrophils in patients with mCRC (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PRTN3 promotes the activation of (MMP)-2, which is involved in angiogenesis and tumor invasion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. PRTN3 is also activated by IL-32, which induces protease-activated receptor 2 (PAR2) signaling [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. One of the downstream pathways of the IL-32-PRTN3-PAR2 axis is the Ras-Raf pathway, which is involved in angiogenesis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, the Ras-Raf pathway downstream of VEGF and its receptor signaling is implicated in tumor angiogenesis, therefore, therapies targeting this pathway are commonly used in mCRC [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Schiffmann et al. reported that a high number of CD177-positive neutrophils in the stroma is associated with poor response to Bev chemotherapy for mCRC, thus leading to poor prognosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These reports support our findings that PRTN3 is an important regulator of tumor angiogenesis and can thus be a predictive biomarker of Bev response.\u003c/p\u003e \u003cp\u003eOur study showed that serum PRTN3 levels were significantly associated with the number of PRTN3-positive cells in stroma, and almost all PRTN3-positive cells were neutrophils (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore, we hypothesized that PRTN3 may play a role in stromal neutrophil function. Tumor-associated neutrophils are involved in tumor angiogenesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, the possibility of a relationship between neutrophil-associated angiogenesis and Bev sensitivity should be investigated in the future.\u003c/p\u003e \u003cp\u003eCurrently, Bev chemotherapy is the standard treatment for mCRC. However, resistance to Bev is also a major obstacle to successful treatment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, predictive biomarkers of response to Bev and novel strategies for overcoming resistance to Bev are required. Our study found that PRTN3 may influence the therapeutic effect of Bev (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Serum PRTN3 levels may potentially be beneficial in forecasting the efficacy of Bev treatment, thus facilitating the selection of optimal chemotherapeutic regimen. PRTN3 has been reported to promote angiogenesis through MMPs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As the serum PRTN3 level before Bev initiation is an independent predictive factor for response, angiogenesis by PRTN3 may exceed the inhibitory effect of Bev on angiogenesis, resulting in chemotherapy resistance. In addition, even when VEGF is inhibited, the activation of other pathways, stimulated by PRTN3, may result in chemotherapy resistance. Our tube formation assay results support this hypothesis. Thus, identification of PRTN3 and its signals and their inhibition may overcome resistance to Bev.\u003c/p\u003e \u003cp\u003eThis study had some limitations, namely, its retrospective, single-center design; small sample size; and profound advances in chemotherapy during the study period. Therefore, further studies, such as experiments analyzing the molecular and cellular mechanisms underlying the effects of PRTN3 on tumor angiogenesis and the relationship between PRTN3 and stromal neutrophils, are warranted to confirm our results.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy in patients with mCRC. In Addition, PRTN3 might predict the efficacy of bevacizumab.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eCRC:\u0026nbsp;\u003c/strong\u003eColorectal cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emCRC:\u003c/strong\u003e Metastatic colorectal cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVEGF:\u003c/strong\u003e vascular endothelial growth factor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emAbs:\u0026nbsp;\u003c/strong\u003emonoclonal antibodies\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBev:\u003c/strong\u003e bevacizumab\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePRTN3:\u0026nbsp;\u003c/strong\u003eProtenase-3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTCGA:\u0026nbsp;\u003c/strong\u003eThe Cancer Genome Atlas\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFPKM:\u0026nbsp;\u003c/strong\u003efragments per kilobase of exon per million\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA:\u003c/strong\u003e enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC:\u003c/strong\u003e Immunohistochemistry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFFPE:\u003c/strong\u003e formalin-fixed paraffin-embedded\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCES:\u003c/strong\u003e composite expression score\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROC:\u0026nbsp;\u003c/strong\u003ereceiver operating characteristic\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOS:\u0026nbsp;\u003c/strong\u003eOverall survival\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHR:\u003c/strong\u003e hazard ratio\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePFS:\u003c/strong\u003e Progression-free survival\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMMPs:\u003c/strong\u003e matrix metalloproteinases\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePAR2:\u003c/strong\u003e protease-activated receptor 2\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eEthical, legal, and social implications were approved by the Ethics Committee of Yamaguchi University Hospital (H20-102, H23-135, and H28-074). This study was conducted per the Helsinki Declaration for experimentation on human subjects. All the samples were obtained with informed consent from the patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\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\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed as part of a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT; 11039020) and The Japan Agency for Medical Research and Development (AMED; 15cm0106085h0005). This study was supported in part by a grant from the Japan Agency for Medical Research and Development for Leading Advanced Projects for Medical Innovation (LEAP; 16am0001006h0003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKF, MN, RT and SH conceived and designed the study. KF, MN, RT, HM, YS, YT, YW, ST, NM, MI, NS, ST, TI, SH and HN were involved in the collection and assembly of data. KF, MN, RT, MX and YH performed the experiments. KF, NM, RT and YN analyzed the data. KF and HN wrote the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the patients and staff of Yamaguchi University Hospital for participating in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eDepartment of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan\u003c/p\u003e\n\u003cp\u003eKei Furuya, Masao Nakajima, Ryouichi Tsunedomi, Yuki Nakagami, Ming Xu, Hiroto Matsui, Yukio Tokumitsu, Yoshitaro Shindo, Yusaku Watanabe, Shinobu Tomochika, Noriko Maeda, Michihisa Iida, Nobuaki Suzuki, Shigeru Takeda, Shoichi Hazama \u0026amp; Hiroaki Nagano\u003c/p\u003e\n\u003cp\u003eOncology Center, Yamaguchi University Hospital, Ube, Yamaguchi 755-8505, Japan\u003c/p\u003e\n\u003cp\u003eTatsuya Ioka\u003c/p\u003e\n\u003cp\u003eDepartment of Diagnostic Pathology, Yamaguchi University Hospital, Ube, Yamaguchi 755-8505, Japan\u003c/p\u003e\n\u003cp\u003eYoshinobu Hoshii\u003c/p\u003e\n\u003cp\u003eDepartment of Digestive Surgery, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan\u003c/p\u003e\n\u003cp\u003eTomio Ueno\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Nat Rev Cancer. 2020;20:485\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\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":"biomarker, bevacizumab, chemotherapy, metastatic colorectal cancer, proteinase-3","lastPublishedDoi":"10.21203/rs.3.rs-3393984/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3393984/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eTo improve the prognosis of patients with metastatic colorectal cancer (mCRC), investigating predictive biomarkers for prognosis and chemotherapeutic responsiveness is necessary. Therefore, this study aimed to analyze the clinical significance of serum proteinase-3 (PRTN3) as a predictor for prognosis and chemosensitivity, especially to bevacizumab, in mCRC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThis single-center retrospective observational study enrolled 79 patients with mCRC in our hospital and 353 patients with colorectal cancer from the TCGA database. Preoperative serum PRTN3 levels were measured using enzyme-linked immunosorbent assay. The clinicopathological characteristics and prognosis according to serum PRTN3 levels were then evaluated. PRTN3 expression in tumor and stromal cells was evaluated immunohistochemically. The impact of PRTN3 levels on angiogenesis and bevacizumab sensitivity was evaluated using the tube formation assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Serum PRTN3 level was an independent poor prognostic factor for progression-free survival (PFS) (hazard ratio: 2.082; 95% confidence interval: 1.118–3.647; \u003cem\u003eP\u003c/em\u003e=0.010) in patients with mCRC. Similarly, prognostic analysis with TCGA data set showed poorer overall survival in patients with PRTN3 expression compared to those without PRTN3 expression, especially in patients with stage IV. Immunohistochemical analysis of resected specimens revealed that stromal neutrophils expressed PRTN3, and their expression level was significantly correlated with serum PRTN3 levels. Interestingly, the effectiveness of first line chemotherapy was significantly poorer in the high serum PRTN3 level group. High serum PRTN3 was significantly associated with poorer PFS (hazard ratio, 3.027; 95% confidence interval, 1.175–7.793; \u003cem\u003eP\u003c/em\u003e=0.0161) in patients treated with bevacizumab, an anti-angiogenic inhibitor. Tube formation assay revealed that PRTN3 administration notably augmented angiogenesis while simultaneously attenuating the anti-angiogenic influence exerted by bevacizumab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eSerum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy especially for bevacizumab in patients with mCRC; however, future studies are warranted to confirm our results.\u003c/p\u003e","manuscriptTitle":"Serum proteinase-3 levels as a predictor of progression-free survival of first-line chemotherapy in metastatic colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-10 18:07:00","doi":"10.21203/rs.3.rs-3393984/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2023-12-06T07:37:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-11-28T22:06:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"dc283437-6792-41a6-8b77-7039ae5718c2","date":"2023-11-14T20:54:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-10-31T11:04:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-31T08:25:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-10-04T10:44:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-10-04T10:19:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2023-09-28T03:32:04+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":"60208147-31f3-439a-b7e3-a74a81afcd76","owner":[],"postedDate":"October 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-02-05T15:06:05+00:00","versionOfRecord":{"articleIdentity":"rs-3393984","link":"https://doi.org/10.1186/s12885-024-11924-4","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2024-02-02 15:01:58","publishedOnDateReadable":"February 2nd, 2024"},"versionCreatedAt":"2023-10-10 18:07:00","video":"","vorDoi":"10.1186/s12885-024-11924-4","vorDoiUrl":"https://doi.org/10.1186/s12885-024-11924-4","workflowStages":[]},"version":"v1","identity":"rs-3393984","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3393984","identity":"rs-3393984","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}