PRDX4 Can Potentially Serve as an Independent Prognostic Marker for Early Recurrence of Oral Squamous Cell Carcinoma

PRDX4 Can Potentially Serve as an Independent Prognostic Marker for Early Recurrence of Oral Squamous Cell Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PRDX4 Can Potentially Serve as an Independent Prognostic Marker for Early Recurrence of Oral Squamous Cell Carcinoma Taijiro Hashimoto, Takeru Oyama, Yangxion Zhang, Yukinobu Ito, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7744178/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oral squamous cell carcinoma (OSCC), a main histological subtype of oral cavity cancer, remains one of the most prevailing tumors worldwide with the increasing incidence and mortality. Since a high rate of early local recurrence is one of the major risk factors for poor outcome of OSCC, its prognostic biomarkers are urgently needed. Peroxiredoxin 4 (PRDX4), a member of PRDXs, which are involved in the antioxidant defense mechanism, is a unique secreted subtype of PRDXs. A certain number of previous studies have that the overexpression of the PRDX4 protein has a clear relationship with tumor initiation and progression in many cancers. Furthermore, recent studies have revealed that PRDX4 promotes tumor development through the Wnt/β-catenin signaling pathway. In the present study, to assess the status of the PRDX4/β-catenin expression and its association with clinical outcomes, including early local recurrence in OSCC, we immunohistochemically examined PRDX4 expression levels and cytoplasmic β-catenin protein accumulation levels in a total of 72 postoperative OSCC samples. The high expression of PRDX4 was significantly correlated with smoking and alcohol intake and poorer early phase 2-year recurrence-free survival (RFS). In addition, a multivariate Cox regression analysis revealed that PRDX4 was an independent prognostic factor for 2-year-RFS. In addition, high β-catenin accumulation is significantly associated with distant metastasis. A comparison of the combination of a high expression of PRDX4 and high β-catenin protein accumulation groups with the other groups only showed a significant predominance among males. In conclusion, the increased expression of PRDX4 may be a useful independent prognostic biomarker for recurrence of OSCC, especially in the early postoperative phase. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Oral squamous cell carcinoma (OSCC) is the main histological subtype of oral cavity cancer, accounting for approximately 90% of all cancer cases [ 1 ]. Globally, the incidence of OSCC in 2022 was 389,846, with 188,438 associated deaths (Global Cancer Observatory), and the rate is constantly increasing [ 2 ]. Despite easy access to clinical examinations, the majority of OSCCs are diagnosed at an advanced stage because the early stages of the tumor are frequently asymptomatic [ 3 , 4 ]. OSCC is primarily treated with radical surgery with postoperative radiation and/or chemotherapy [ 5 ]. Approximately one-third of patients experience postoperative recurrence, with the most common pattern being local recurrence [ 5 ], which is a major risk factor for poor outcomes [ 6 ]. Recurrence usually occurs within the first 18 months after surgery, leading to poor overall survival (OS) [ 7 , 8 ]. Moreover, patients with early recurrence have a significantly worse prognosis than those with late recurrence [ 5 , 9 – 11 ]. However, the identification of early recurrence in the postoperative state can be a clinical challenge until large and symptomatic tumors emerge [ 5 ]. Because of the detrimental properties of early recurrence, prognostic and predictive biomarkers are urgently needed. We focused on peroxiredoxins (PRDXs) as candidate biomarkers for the early recurrence of OSCC. PRDXs protect organisms against oxidative stress caused by the accumulation of reactive oxygen species (ROS) by catalyzing peroxide reduction to remove excessive hydroxyperoxide [ 12 ]. PRDX4 is the only secreted subtype of PRDX localized in the endoplasmic reticulum (ER) and contributes to oxidative protein folding and the antioxidant function [ 12 – 14 ]. In the ER, PRDX4, in collaboration with endoplasmic reticulum oxidoreductin 1 (ERO1) via protein disulfide isomerase (PDI), acts as a sulfhydryl oxidase to introduce disulfide bonds into nascent proteins that are transferred to the Golgi body for secretion [ 12 ]. Decreased capacity for oxidative protein folding by PRDX4 leads to a large amount of ER stress, resulting in a number of diseases, including wound healing [ 15 ], liver disease [ 16 – 18 ], atherosclerosis [ 19 ], diabetes [ 20 , 21 ] and metabolic syndrome [ 13 , 21 ]. Moreover, aberrations in the expression of PRDX4 have been detected in a large number of solid and hematopoietic neoplasms, and the majority of them were significantly upregulated, including lung cancer (lung adenocarcinoma) [ 22 – 25 ], liver cancer (hepatocellular carcinoma and hepatoblastoma) [ 26 – 28 ], pancreatic cancer [ 29 ], gastric cancer [ 30 ], colorectal cancer [ 31 , 32 ], renal cancer (renal papillary carcinoma) [ 33 ], prostate cancer [ 31 , 34 , 35 ], breast cancer [ 31 , 36 ], ovarian cancer [ 31 ], brain tumor (glioma) [ 37 , 38 ], multiple myeloma [ 39 ], and acute myeloid and promyelocytic leukemia [ 31 , 40 ]. Certain cancers with high PRDX4 expression levels showed a significantly close association with tumor initiation and progression, resulting in a poor prognosis. More recently, the overexpression of PRDX4 predicted shortened survival in hepatoblastoma [ 27 ], and the strong immunohistochemical expression of PRDX4 predicted poorer survival in pancreatic ductal adenocarcinoma [ 29 ]. Aberrations in the expression of PRDX4 have also been reported in squamous cell carcinomas including OSCC [ 41 , 42 ]. The overexpression of PRDX4 has been reported in human OSCC tissues and cell lines, based on an immunohistochemical analysis and a meta-analysis, and is associated with increased cell proliferation, migration, and invasion through the reduced expression of adhesion molecules in vitro [ 41 , 43 ]. Furthermore, elevated expression levels of PRDX4 mRNA were detected using the Gene Expression Array based on quantitative reverse transcription PCR (qRT-PCR) in human OSCC tissue [ 44 ]. In the lung, the high expression of PRDX4 was observed in squamous cell carcinoma by immunostaining, which was significantly correlated with higher recurrence rates and shorter disease-free survival (DFS) [ 42 ]. These studies revealed that PRDX4 is significantly overexpressed in squamous cell carcinomas from various organ types. One of the major mechanisms by which the overexpression of PRDX4 contributes to malignancy is activation of the Wnt/β-catenin signaling pathway [ 45 , 46 ]. In fact, knockdown of PRDX4 by short hairpin RNA (shRNA) inhibited the Wnt/β-catenin signaling pathway in two renal cell carcinoma cell lines [ 45 ]. The degradation of PRDX4 inactivated the Wnt/β-catenin signaling pathway in hepatocellular carcinoma cells [ 46 ]. These studies have shown that PRDX4 can promote cell growth, invasion, and migration through the Wnt/β-catenin signaling pathway, which plays a crucial role in the initiation and progression of malignant neoplasms. However, to our knowledge, no previous studies have disclosed the relationship between increased PRDX4 expression and recurrence, especially early recurrence, after surgical treatment of OSCC and its underlying mechanisms, including the Wnt/β-catenin signaling pathway. The detection of increased PRDX4 expression levels in postsurgical lesions might indicate the need for immediate therapeutic interventions for recurrent OSCC. Our aim was to assess the potential application of the expression of PRDX4 and β-catenin as a biomarker, especially for the early recurrence after surgical treatment of OSCC. We investigated PRDX4/β-catenin expression levels and their association with the clinical characteristics and outcomes, including early local recurrence after surgical treatment of OSCC in 72 patients, based on an immunohistochemical analysis of PRDX4 and β-catenin. Materials and Methods Samples This study was approved by the Medical Ethics Committee of Kanazawa Medical University [C133]. A total of 85 cases of formalin-fixed paraffin-embedded (FFPE) tissue blocks were retrieved from archives of the Pathological Department of Kanazawa Medical University Hospital. Corresponding clinicopathological information was also collected from hospital electronic records. Thirteen cases were excluded from the analysis because of a lack of either FFPE tissue blocks or clinicopathological information. Immunohistochemistry Immunohistochemical staining was performed using the antibody-linked dextran polymer method for antibody-bridge labelling with hematoxylin counterstaining (EnVision; Dako Cytomation Corporation, Glostrup, Denmark). Antibodies against PRDX4 (Life Technologies Corporation, Carlsbad, CA, USA), β-catenin (Life Technologies Corporation), and S-100 protein (Dako Cytomation Corporation, Glostrup, Denmark) were used at 1:200, 1:1000, and 1:100 dilutions, respectively. Human PRDX4 transgenic mouse pancreatic cells were used as positive controls for PRDX4 [ 19 – 24 ], and well-differentiated tubular colorectal adenocarcinoma cells for β-catenin [ 47 ]. The 3-µm-thick unstained sections were deparaffinized with xylene, hydrated using a graded ethanol series (100, 90, and 80%) for 1 min each, and washed with PBS three times. The sections were unmasked in citrate buffer using Pascal (Agilent Technologies Japan, Ltd., Tokyo, Japan), incubated in 10% H 2 O 2 in methanol for 5 min to block endogenous peroxidase activity, and washed with PBS. The slides were then incubated with 1% serum for 1 h. First, the antibodies were applied and incubated overnight at 4°C. The second antibody-peroxidaselinked polymers were then applied and the sections were incubated with a solution consisting of 20 mg of 3.3’-diaminobenzidine tetrahydrochloride, 65 mg of sodium azide, and 20 mL of 30% H 2 O 2 in 100 mL of Tris-HCl (50 mM, pH 7.6). After counterstaining with Meyer’s hematoxylin, sections were observed under a light microscope. The sections were first scanned at low power for all fields (original magnification: ×40) with tumor and non-tumor tissues to account for the heterogeneity of distribution. Immunohistochemical analysis In each case, PRDX4 and β-catenin immunoreactivity was assessed semi-quantitatively by evaluating the proportion of cells with clearly higher expression levels in the total neoplastic OSCC cells in comparison to the adjacent non-tumor squamous cells, which presented very weak or negative expression levels of both proteins. The cutoff values for immunohistochemical staining of PRDX4 (10%) and β-catenin (10%) were selected and validated based on a receiver operating characteristic (ROC) curve analysis ( Fig. 1 ) . All patients were divided into two groups based on the expression of PRDX4 as follows: high when PRDX4 staining was ≥ 10% and low when staining was < 10%. All histological and immunohistochemical slides were evaluated by two independent observers (certified surgical pathologists in our department, A.S. and T.O.) using a blinded protocol design (observers were blinded to the clinicopathological data). The interobserver agreement, as measured by the interclass correlation coefficient, was excellent (> 99%) for all antibodies investigated. For the few (< 1%) instances of disagreement, a consensus score was determined by a third board-certified pathologist (S.Y.) in our department. Statistical analysis The correlations between the PRDX4 and/or β-catenin expression status and clinicopathological characteristics were analyzed using the chi-squared test. Statistical significance was set at P < 0.05. Survival curves were generated using the Kaplan-Meier method. Log-rank and generalized Wilcoxon tests were used for the statistical comparison of RFS between a cohort with high PRDX4 expression levels and a cohort with low expression levels, using GraphPad Prism 10 (GraphPad software, Tokyo, Japan). Statistical significance was set at P < 0.05. Results Clinicopathological characteristics of the cases A total of 72 patients who underwent surgery at Kanazawa Medical University Hospital were included in the present study. Table 1 described the detailed clinicopathological characteristics of the patients. Subsequently, the cases were dichotomized into two groups according to clinicopathological characteristics (e.g., sex, age, tumor differentiation, maximum diameter, T-category of TNM staging system, and lymph node or distant metastasis) (Table 2). The patients included 43 males and 25 females. The average age of the patients was 62.5 years (range, 26 to 93 years). Fourteen of 72 (19%) of the tumors were ≤ 30 mm in maximal diameter. Lymphatic, venous, and perineural invasion were observed in 19% (14/72), 22% (16/72), and 19% (14/72) of the tumors, respectively. The majority of tumors (96%; 69/72) were classified as well-to-moderately differentiated, while the remainder were poorly differentiated squamous cell carcinomas. Based on the TNM classification, 44% (32/72) of cases were classified as pathological T category ≥ 2 (pT2 or higher). Lymph node metastasis (pN1 or higher) was observed in 29% (21/72) of the tumors. Distant metastasis (M1) was observed in 7% (5/72) of the tumors; metastases were detected in the lung (3/5), liver (2/5), and spine (2/5). Local recurrence developed in 21% (15/72) of the patients. A smoking history was present in 71% (51/72) of the cases, and a drinking history was present in 64% (46/72). Association of the overexpression of PRDX4 and β-catenin accumulation with clinicopathological characteristics Figure 2 shows representative images of OSCC in the high-PRDX4 and high-β-catenin protein accumulation groups ( Fig. 2 A,B,C ) , and the low-PRDX4 and low-β-catenin protein accumulation groups ( Fig. 2 D,E,F ) . The expression of PRDX4 and β-catenin protein accumulation were both higher, especially in the tumor cell nests of the invasive fronts ( Fig. 2 B,C ) , in comparison to the low expression group ( Fig. 2 E,F ) . Moreover, in the invasive front, tumors with the high PRDX4 expression levels and β-catenin protein accumulation formed smaller tumor cell nests, suggesting malignant properties, compared to those with low PRDX4 expression levels and β-catenin protein accumulation. Table 3 shows that 51 (71%) samples showed high PRDX4 expression levels and 41 (57%) samples showed high β-catenin protein accumulation. The high expression of PRDX4 was significantly associated with a history of smoking (p = 0.011) and drinking (p = 0.009). All patients with distant metastasis were in the high β-catenin expression group (p = 0.044), and the group showed a significant male predominance (p = 0.016). Survival analysis Figure 3 shows 2-year-RFS in the PRDX4-high and PRDX4-low groups, β-catenin-high and β-catenin-low groups, and the combination of PRDX4-high and β-catenin-high vs. others. These analyses were performed using the Kaplan-Meier method. Figure 3 A shows that the high expression of PRDX4 was significantly associated with poorer 2-year-RFS (P = 0.043). However, Figs. 3 B and 3 C showed no significant differences in 2-year RFS according to the β-catenin expression status (p = 0.931) or the combined high expression of PRDX4 and β-catenin (p = 0.221). The median RFS of the PRDX4-high and PRDX4-low groups was 102 and 670 days, respectively, that of the β-catenin-high and β-catenin-low groups was 68 and 208 days, and that of the PRDX4-high-β-catenin-high-and other groups was 68 and 208 days, respectively. Cox regression analysis Multivariate Cox regression analyses were performed with sex, age, maximal tumor diameter, histological differentiation of SCC, pT category, lymph node and distant metastasis, perineural, lymphatic and venous invasion, and smoking and drinking history as covariates. Table 4 shows that the high expression of PRDX4 (P = 0.047), maximal tumor size > 30 mm (P = 0.029), and perineural invasion (P = 0.023) were independent prognostic factors for 2-year-RFS. Although perineural invasion was an independent prognostic factor for 2-year-RFS and was not correlated with the high expression of PRDX4, we additionally investigated the tumor microenvironment around the peripheral nerves by immunohistochemical staining of S-100 protein. Figure 4 shows that tiny PRDX4-positive tumor nests were frequently observed around S-100 protein-positive peripheral nerves. Discussion The present study revealed that the high expression of PRDX4 in OSCC is a significant prognostic factor, particularly for early recurrence. Furthermore, univariate and multivariate analyses of the Cox proportional regression model revealed that the high expression of PRDX4 predicted significantly worse 2-year-RFS in comparison to the low expression of PRDX4 (P = 0.043), indicating that an aberrantly increased PRDX4 expression level could be a candidate independent biomarker for early recurrence of OSCC. The early detection of PRDX4 in postoperative specimens might indicate the possibility of therapeutic intervention to improve patient survival. Moreover, PRDX4 could be a candidate serum biomarker to detect early recurrence of OSCC after surgical treatment, since the protein is known to be the only soluble PRDX subtype [ 12 , 13 ]. Indeed, the serum expression of PRDX4 can be detected in some tissues (e.g., pancreatic tissue), wherein protein secretion is critical for both exocrine and endocrine secretion [ 12 , 13 , 20 ]. The present study had some limitations. This was a retrospective study conducted within a single institution with a relatively small study population. Furthermore, we only evaluated histopathological sections (but not serum) and could not comprehensively analyze the role of PRDX4 in the prognosis of OSCC. Additionally, identical studies using other antibodies may be necessary to validate the results of this study in the future. Finally, the pivotal effects of the overexpression of PRDX4 on OSCC were determined using human OSCC cell lines and/or OSCC animal models in human PRDX4 transgenic mice. Despite these limitations, the univariate and multivariate Cox regression analyses in the present study revealed that perineural invasion was an independent prognostic factor for 2-year-RFS, although it was not associated with the increased expression of PRDX4 (Table 4) . Perineural invasion has been recognized as one of the critical histopathological characteristics in various malignancies, including those of the breast, pancreas, colorectum, and prostate, and it correlates with higher tumor grade, increased risk of locoregional recurrence, and a potentially worse prognosis [ 48 ]. As shown in Fig. 4 , tiny OSCC nests surrounding the peripheral nerves in the tumor microenvironment showed high PRDX4 expression levels. Accordingly, the localized overexpression of PRDX4 surrounding the peripheral nerves, instead of the overall expression of the tumor, might be closely associated with poor outcomes, including early recurrence. Further studies are needed to investigate the association between microenvironmentally localized high PRDX4 expression levels (e.g., perineural invasion) and early recurrence. In this study, we revealed the association between high PRDX4 expression levels and early recurrence of OSCC; however, the underlying mechanisms could not be elucidated. Since PRDX4 in the ER lumen mainly catalyzes oxidative protein folding, its decreased expression results in the accumulation of unfolded proteins, which causes cell apoptosis [ 12 , 13 , 18 ]. In contrast, malignant cells such as OSCC cells, as shown in Fig. 2 B and Fig. 4 B, significantly upregulate PRDX4 to protect themselves from apoptosis caused by the accumulation of unfolded protein, which promotes tumor initiation and development, possibly through the Wnt/β-catenin signaling pathway [ 45 , 46 ]. Thus, inhibiting the overexpression of PRDX4, together with the Wnt/β-catenin signaling pathway, could be an attractive therapeutic target for OSCC. Furthermore, we speculate that high PRDX4 levels could affect malignant characteristics by maintaining stem cell-like properties mainly activated by the Wnt/β-catenin signaling pathway [ 49 – 51 ]. Such stem cell-like tumor cells have been reported to express splicing variant isoforms of CD44 (CD44v)[ 52 , 53 ], leading to increased production of the antioxidant glutathione, which removes excessive ROS in the tumor microenvironment and may enhance malignant features [ 51 ]. Similarly, the antioxidative PRDX4 expression can be upregulated, especially in stem cell-like tumor cell populations within malignant neoplasms, including OSCC. Therefore, PRDX4 may be a candidate biomarker for stem cell-like tumor cells, which aggressively enhance the occurrence and growth of malignant neoplasms. Further in-depth studies will be required in the near future. In conclusion, we demonstrated that high PRDX4 expression in OSCC is a significant and independent prognostic factor, particularly for early recurrence, and may serve as a useful biomarker for detecting early recurrence in postoperative histopathological specimens and potentially in serum samples. The underlying mechanisms of the effects of PRDX4 on early recurrence of OSCC remain to be elucidated; however, our future study will partly focus on the prevention of unfolded protein accumulation in the ER by PRDX4 and the undifferentiated state of stem-like tumor cells induced likely by the Wnt/β-catenin signaling pathway. Declarations Ethics approval and consent to participate All intended procedures in the present study, including the use of specimens obtained from human subjects, were approved by the Medical Ethics Committee of Kanazawa Medical University (permission number: C133). All patients provided informed written consent according to the guidelines of the Japanese Society of Pathology. Consent for publication All patients provided informed written consent for publication. Availability of data and materials The datasets used and analyzed during the study are available from the corresponding author upon reasonable request. Competing interests All authors have no competing financial and/or non-financial interests. Funding This work was supported in part by Japan Society for the Promotion of Science (20K07454 to S. Y. and 23K06474 to T. O.), by Hokkoku Cancer Foundation (to S. Y.) and by Grant for Promoted Research from Kanazawa Medical University (S2023-A4 to T.O.) Authors' contributions Taijiro Hashimoto, Yangxion Zhang, Takeru Oyama, Akihiro Shioya and Sohsuke Yamada conceptualized and designed the experiments. Mitsuaki Yoshida, Taijiro Hashimoto, Yangxion Zhang and Jia Han performed experiments. Taijiro Hashimoto, Jia Han, Yukinobu Ito and Takeru Oyama analyzed the data and drafted the manuscript. Masashi Okuro, Morimasa Kitamura and Sohsuke Yamada have reviewed and edited the manuscript. Sohsuke Yamada supervised the study. All authors have read and approved the final version of the manuscript. Acknowledgements We would like to thank Brian Quinn for editing the manuscript. We thank the members of the Department of Pathology, Kanazawa Medical University Hospital, for providing expert technical assistance. References Muralidharan S, Nikalje M, Subramaniam T, Koshy JA, Koshy AV, Bangera D. A Narrative Review on Oral Squamous Cell Carcinoma. J Pharm Bioallied Sci. 2025; 17: S204-S6. https://doi.org/10.4103/jpbs.jpbs_593_25. Esperouz F, Ciavarella D, Lorusso M, Santarelli A, Lo Muzio L, Campisi G, et al. Critical review of OCT in clinical practice for the assessment of oral lesions. Front Oncol. 2025; 15: 1569197. https://doi.org/10.3389/fonc.2025.1569197. Jagadeesan D, Sathasivam KV, Fuloria NK, Balakrishnan V, Khor GH, Ravichandran M, et al. Comprehensive insights into oral squamous cell carcinoma: Diagnosis, pathogenesis, and therapeutic advances. Pathol Res Pract. 2024; 261: 155489. https://doi.org/10.1016/j.prp.2024.155489. Bagan J, Sarrion G, Jimenez Y. Oral cancer: clinical features. Oral Oncol. 2010; 46: 414-7. https://doi.org/10.1016/j.oraloncology.2010.03.009. Hosni A, Huang SH, Chiu K, Xu W, Su J, Bayley A, et al. Predictors of Early Recurrence Prior to Planned Postoperative Radiation Therapy for Oral Cavity Squamous Cell Carcinoma and Outcomes Following Salvage Intensified Radiation Therapy. Int J Radiat Oncol Biol Phys. 2019; 103: 363-73. https://doi.org/10.1016/j.ijrobp.2018.09.013. Montero PH, Patel SG. Cancer of the oral cavity. Surg Oncol Clin N Am. 2015; 24: 491-508. https://doi.org/10.1016/j.soc.2015.03.006. Lyu WN, Lin MC, Shen CY, Chen LH, Lee YH, Chen SK, et al. An Oral Microbial Biomarker for Early Detection of Recurrence of Oral Squamous Cell Carcinoma. ACS Infect Dis. 2023; 9: 1783-92. https://doi.org/10.1021/acsinfecdis.3c00269. Wang Y, Yang T, Gan C, Wang K, Sun B, Wang M, et al. Temporal and spatial patterns of recurrence in oral squamous cell carcinoma, a single-center retrospective cohort study in China. BMC Oral Health. 2023; 23: 679. https://doi.org/10.1186/s12903-023-03204-7. Liao CT, Chang JT, Wang HM, Ng SH, Hsueh C, Lee LY, et al. Salvage therapy in relapsed squamous cell carcinoma of the oral cavity: how and when? Cancer. 2008; 112: 94-103. https://doi.org/10.1002/cncr.23142. Kernohan MD, Clark JR, Gao K, Ebrahimi A, Milross CG. Predicting the prognosis of oral squamous cell carcinoma after first recurrence. Arch Otolaryngol Head Neck Surg. 2010; 136: 1235-9. https://doi.org/10.1001/archoto.2010.214. Liu SA, Wong YK, Lin JC, Poon CK, Tung KC, Tsai WC. Impact of recurrence interval on survival of oral cavity squamous cell carcinoma patients after local relapse. Otolaryngol Head Neck Surg. 2007; 136: 112-8. https://doi.org/10.1016/j.otohns.2006.07.002. Fujii J, Ochi H, Yamada S. A comprehensive review of peroxiredoxin 4, a redox protein evolved in oxidative protein folding coupled with hydrogen peroxide detoxification. Free Radic Biol Med. 2025; 227: 336-54. https://doi.org/10.1016/j.freeradbiomed.2024.12.015. Yamada S, Guo X. Peroxiredoxin 4 (PRDX4): Its critical in vivo roles in animal models of metabolic syndrome ranging from atherosclerosis to nonalcoholic fatty liver disease. Pathol Int. 2018; 68: 91-101. https://doi.org/10.1111/pin.12634. Hanaka T, Kido T, Noguchi S, Yamada S, Noguchi H, Guo X, et al. The overexpression of peroxiredoxin-4 affects the progression of idiopathic pulmonary fibrosis. BMC Pulm Med. 2019; 19: 265. https://doi.org/10.1186/s12890-019-1032-2. Yamaguchi R, Guo X, Zheng J, Zhang J, Han J, Shioya A, et al. PRDX4 Improved Aging-Related Delayed Wound Healing in Mice. J Invest Dermatol. 2021; 141: 2720-9. https://doi.org/10.1016/j.jid.2021.04.015. Zhang J, Guo X, Hamada T, Yokoyama S, Nakamura Y, Zheng J, et al. Protective Effects of Peroxiredoxin 4 (PRDX4) on Cholestatic Liver Injury. Int J Mol Sci. 2018; 19. https://doi.org/10.3390/ijms19092509. Homma T, Kurahashi T, Lee J, Nabeshima A, Yamada S, Fujii J. Double Knockout of Peroxiredoxin 4 (Prdx4) and Superoxide Dismutase 1 (Sod1) in Mice Results in Severe Liver Failure. Oxid Med Cell Longev. 2018; 2018: 2812904. https://doi.org/10.1155/2018/2812904. Nawata A, Noguchi H, Mazaki Y, Kurahashi T, Izumi H, Wang KY, et al. Overexpression of Peroxiredoxin 4 Affects Intestinal Function in a Dietary Mouse Model of Nonalcoholic Fatty Liver Disease. PLoS One. 2016; 11: e0152549. https://doi.org/10.1371/journal.pone.0152549. Guo X, Yamada S, Tanimoto A, Ding Y, Wang KY, Shimajiri S, et al. Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice. Antioxid Redox Signal. 2012; 17: 1362-75. https://doi.org/10.1089/ars.2012.4549. Ding Y, Yamada S, Wang KY, Shimajiri S, Guo X, Tanimoto A, et al. Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice. Antioxid Redox Signal. 2010; 13: 1477-90. https://doi.org/10.1089/ars.2010.3137. Nabeshima A, Yamada S, Guo X, Tanimoto A, Wang KY, Shimajiri S, et al. Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model. Antioxid Redox Signal. 2013; 19: 1983-98. https://doi.org/10.1089/ars.2012.4946. Shioya A, Guo X, Motono N, Mizuguchi S, Kurose N, Nakada S, et al. The Combination Of Weak Expression Of PRDX4 And Very High MIB-1 Labelling Index Independently Predicts Shorter Disease-free Survival In Stage I Lung Adenocarcinoma. Int J Med Sci. 2018; 15: 1025-34. https://doi.org/10.7150/ijms.25734. Zheng J, Guo X, Nakamura Y, Zhou X, Yamaguchi R, Zhang J, et al. Overexpression of PRDX4 Modulates Tumor Microenvironment and Promotes Urethane-Induced Lung Tumorigenesis. Oxid Med Cell Longev. 2020; 2020: 8262730. https://doi.org/10.1155/2020/8262730. Mizutani K, Guo X, Shioya A, Zhang J, Zheng J, Kurose N, et al. The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma. Int J Med Sci. 2019; 16: 1199-206. https://doi.org/10.7150/ijms.36071. Iwai S, Motono N, Oyama T, Shioya A, Yamada S, Uramoto H. The Clinical Relevance of the Expression of SGLT2 in Lung Adenocarcinoma. Oncology. 2024; 102: 710-9. https://doi.org/10.1159/000536060. Guo X, Noguchi H, Ishii N, Homma T, Hamada T, Hiraki T, et al. The Association of Peroxiredoxin 4 with the Initiation and Progression of Hepatocellular Carcinoma. Antioxid Redox Signal. 2019; 30: 1271-84. https://doi.org/10.1089/ars.2017.7426. Liu Y, Han J, Shioya A, Zhang YX, Dung VA, Oyama T, et al. The immunohistochemical combination of low SGLT2 expression and high PRDX4 expression independently predicts shortened survival in patients undergoing surgical resection for hepatoblastoma. Diagn Pathol. 2025; 20: 2. https://doi.org/10.1186/s13000-025-01596-4. Zheng J, Guo X, Shioya A, Yoshioka T, Matsumoto K, Hiraki T, et al. Peroxiredoxin 4 promotes embryonal hepatoblastoma cell migration but induces fetal cell differentiation. Am J Transl Res. 2020; 12: 2726-37. Han J, Itoh T, Shioya A, Sakurai M, Oyama T, Kumagai M, et al. The combination of the low immunohistochemical expression of peroxiredoxin 4 and perilipin 2 predicts longer survival in pancreatic ductal adenocarcinoma with peroxiredoxin 4 possibly playing a main role. Histol Histopathol. 2023; 38: 1415-27. https://doi.org/10.14670/HH-18-666. Park SY, Lee YJ, Park J, Kim TH, Hong SC, Jung EJ, et al. PRDX4 overexpression is associated with poor prognosis in gastric cancer. Oncol Lett. 2020; 19: 3522-30. https://doi.org/10.3892/ol.2020.11468. Jia W, Chen P, Cheng Y. PRDX4 and Its Roles in Various Cancers. Technol Cancer Res Treat. 2019; 18: 1533033819864313. https://doi.org/10.1177/1533033819864313. Zhou H, Li L, Chen J, Hou S, Zhou T, Xiong Y. Expression and prognostic value of PRDX family in colon adenocarcinoma by integrating comprehensive analysis and in vitro and in vivo validation. Front Oncol. 2023; 13: 1136738. https://doi.org/10.3389/fonc.2023.1136738. Kocaturk B. In silico analysis reveals PRDX4 as a prognostic and oncogenic marker in renal papillary cell carcinoma. Gene. 2023; 859: 147201. https://doi.org/10.1016/j.gene.2023.147201. Basu A, Banerjee H, Rojas H, Martinez SR, Roy S, Jia Z, et al. Differential expression of peroxiredoxins in prostate cancer: consistent upregulation of PRDX3 and PRDX4. Prostate. 2011; 71: 755-65. https://doi.org/10.1002/pros.21292. Ummanni R, Barreto F, Venz S, Scharf C, Barett C, Mannsperger HA, et al. Peroxiredoxins 3 and 4 are overexpressed in prostate cancer tissue and affect the proliferation of prostate cancer cells in vitro. J Proteome Res. 2012; 11: 2452-66. https://doi.org/10.1021/pr201172n. Jiang W, Wang M, Chen Q, Yu X, Liu G, He X, et al. Immune infiltration related PRDX4 facilitates the malignant features and drug resistance of breast cancer. Sci Rep. 2025; 15: 27507. https://doi.org/10.1038/s41598-025-13361-0. Szeliga M. Comprehensive analysis of the expression levels and prognostic values of PRDX family genes in glioma. Neurochem Int. 2022; 153: 105256. https://doi.org/10.1016/j.neuint.2021.105256. Kim TH, Song J, Alcantara Llaguno SR, Murnan E, Liyanarachchi S, Palanichamy K, et al. Suppression of peroxiredoxin 4 in glioblastoma cells increases apoptosis and reduces tumor growth. PLoS One. 2012; 7: e42818. https://doi.org/10.1371/journal.pone.0042818. Demasi AP, Martinez EF, Napimoga MH, Freitas LL, Vassallo J, Duarte AS, et al. Expression of peroxiredoxins I and IV in multiple myeloma: association with immunoglobulin accumulation. Virchows Arch. 2013; 463: 47-55. https://doi.org/10.1007/s00428-013-1433-1. Palande KK, Beekman R, van der Meeren LE, Beverloo HB, Valk PJ, Touw IP. The antioxidant protein peroxiredoxin 4 is epigenetically down regulated in acute promyelocytic leukemia. PLoS One. 2011; 6: e16340. https://doi.org/10.1371/journal.pone.0016340. Cao R, Zhang W, Zhang H, Wang L, Chen X, Ren X, et al. Comprehensive Analysis of the PRDXs Family in Head and Neck Squamous Cell Carcinoma. Front Oncol. 2022; 12: 798483. https://doi.org/10.3389/fonc.2022.798483. Hwang JA, Song JS, Yu DY, Kim HR, Park HJ, Park YS, et al. Peroxiredoxin 4 as an independent prognostic marker for survival in patients with early-stage lung squamous cell carcinoma. Int J Clin Exp Pathol. 2015; 8: 6627-35. Chang KP, Yu JS, Chien KY, Lee CW, Liang Y, Liao CT, et al. Identification of PRDX4 and P4HA2 as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using iTRAQ technology. J Proteome Res. 2011; 10: 4935-47. https://doi.org/10.1021/pr200311p. Pedro NF, Biselli JM, Maniglia JV, Santi-Neto D, Pavarino EC, Goloni-Bertollo EM, et al. Candidate Biomarkers for Oral Squamous Cell Carcinoma: Differential Expression of Oxidative Stress-Related Genes. Asian Pac J Cancer Prev. 2018; 19: 1343-9. https://doi.org/10.22034/APJCP.2018.19.5.1343. Li H, Wang Z, Chen X, Li S, Zhang F. Resveratrol Downregulated PRDX4 Expression to Inhibit the Progression of Renal Cell Carcinoma via Wnt/beta-Catenin Pathway. Food Sci Nutr. 2025; 13: e70352. https://doi.org/10.1002/fsn3.70352. Chen B, Lan J, Xiao Y, Liu P, Guo D, Gu Y, et al. Long noncoding RNA TP53TG1 suppresses the growth and metastasis of hepatocellular carcinoma by regulating the PRDX4/beta-catenin pathway. Cancer Lett. 2021; 513: 75-89. https://doi.org/10.1016/j.canlet.2021.04.022. Hugh TJ, Dillon SA, O'Dowd G, Getty B, Pignatelli M, Poston GJ, et al. beta-catenin expression in primary and metastatic colorectal carcinoma. Int J Cancer. 1999; 82: 504-11. https://doi.org/10.1002/(sici)1097-0215(19990812)82:43.0.co;2-6. Bahmad HF, Wegner C, Nuraj J, Avellan R, Gonzalez J, Mendez T, et al. Perineural Invasion in Breast Cancer: A Comprehensive Review. Cancers (Basel). 2025; 17. https://doi.org/10.3390/cancers17121900. Bhal S, Das B, Sinha S, Das C, Acharya SS, Maji J, et al. Resveratrol nanoparticles induce apoptosis in oral cancer stem cells by disrupting the interaction between beta-catenin and GLI-1 through p53-independent activation of p21. Med Oncol. 2024; 41: 167. https://doi.org/10.1007/s12032-024-02405-6. Esteban-Roman NF, Taddei E, Castro-Velazquez E, Villafuentes-Vidal L, Velez-Herrera A, Rubio-Osornio M, et al. Redox-Regulated Pathways in Glioblastoma Stem-like Cells: Mechanistic Insights and Therapeutic Implications. Brain Sci. 2025; 15. https://doi.org/10.3390/brainsci15080884. Nagano O, Okazaki S, Saya H. Redox regulation in stem-like cancer cells by CD44 variant isoforms. Oncogene. 2013; 32: 5191-8. https://doi.org/10.1038/onc.2012.638. Ishikawa K, Suzuki H, Kaneko MK, Kato Y. Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C(44)Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas. Curr Issues Mol Biol. 2023; 45: 5248-62. https://doi.org/10.3390/cimb45070333. Guo L, Ke H, Zhang H, Zou L, Yang Q, Lu X, et al. TDP43 promotes stemness of breast cancer stem cells through CD44 variant splicing isoforms. Cell Death Dis. 2022; 13: 428. https://doi.org/10.1038/s41419-022-04867-w. Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files DPHashimotoTTable1.xlsx DPHashimotoTTable2.xlsx DPHashimotoTTable3.xlsx DPHashimonoTTable4.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7744178","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":525355562,"identity":"a06a7b90-f768-480d-8167-676b9f97bc89","order_by":0,"name":"Taijiro Hashimoto","email":"","orcid":"","institution":"Kanazawa Medical University","correspondingAuthor":false,"prefix":"","firstName":"Taijiro","middleName":"","lastName":"Hashimoto","suffix":""},{"id":525355563,"identity":"85a6e505-e5ed-40fc-b59c-b2a0b4fdac8a","order_by":1,"name":"Takeru 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09:22:48","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126393,"visible":true,"origin":"","legend":"","description":"","filename":"00432500b6c940a08c1b87a8948b5c541structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/2e92cf44e36fbc975fda3e6a.xml"},{"id":93025176,"identity":"2f98caaf-1acb-45e8-9a07-900b19a1088d","added_by":"auto","created_at":"2025-10-08 09:22:48","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138214,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/8bcc05009a1d43ed9e702b85.html"},{"id":93025159,"identity":"058e0c10-4da5-45ab-8d51-1bf323bbb0f6","added_by":"auto","created_at":"2025-10-08 09:22:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":387873,"visible":true,"origin":"","legend":"\u003cp\u003eReceiver operating characteristic (ROC) curves for determining cutoff values of PRDX4 (A) and β‑catenin (B) expression levels by immunostaining in oral squamous cell carcinoma. The cutoff values of PRDX4 and β-catenin were both 10% each (area under the ROC curve: AUC, 58% and 53%; sensitivity, 33% and 58%; specificity, 93% and 60%).\u003c/p\u003e","description":"","filename":"DPHashimotoTFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/58753d5f38711649bd1ca441.png"},{"id":93027348,"identity":"df7f29fd-b407-4542-befe-d4dfd7a62356","added_by":"auto","created_at":"2025-10-08 09:38:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7168570,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative hematoxylin and eosin staining of tumors with high PRDX4-high expression and high β-catenin protein accumulation (A) and low PRDX4-low and low β-catenin protein accumulation (B). In the invasive front, high PRDX4 expression levels and high β-catenin protein accumulation formed smaller tumor cell nests in comparison to those with low PRDX4 expression levels and low β-catenin protein accumulation. Immunostaining of PRDX4 in the high (C) and low (E) expression groups. Immunostaining of β-catenin in the high (C) and low (F) expression groups. Higher PRDX4 expression levels and β-catenin protein accumulation were frequently observed in tumor cell nests in the invasive fronts. Scale bar = 100 mm\u003c/p\u003e","description":"","filename":"DPHashimotoTFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/fbe7965a2155a31a5c6c9fc4.png"},{"id":93026311,"identity":"0faa69f4-5a85-4f8d-9b05-8b0cf355937e","added_by":"auto","created_at":"2025-10-08 09:30:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":286775,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier curves for 2-year RFS in the PRDX4-high and PRDX4-low expression groups (A), β-catenin-high and β-catenin-low protein accumulation groups (B), and PRDX4 high-β-catenin high and other groups (C).\u003c/p\u003e","description":"","filename":"DPHashimotoTFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/f492346d253fd0d57f2f5511.png"},{"id":93026312,"identity":"e44fd8b7-5c90-424b-aec8-535ae477d57f","added_by":"auto","created_at":"2025-10-08 09:30:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5784981,"visible":true,"origin":"","legend":"\u003cp\u003ePerineural invasion in the microenvironment of a PRDX4-high tumor. Immunostaining of S-100 protein indicating peripheral nerves (red dashed circle) (A) and immunostaining of PRDX4 indicating tumor cell nests surrounding the peripheral nerves (red arrows) (B) Scale bar = 100 mm.\u003c/p\u003e","description":"","filename":"DPHashimotoTFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/72e99bc69bf7167eb2e7fea7.png"},{"id":98776984,"identity":"0d8fbf98-9772-4719-ac1c-1b3b87f1bbb5","added_by":"auto","created_at":"2025-12-22 12:24:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18543497,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/51ff2366-a69c-47a7-b823-5d0818132b61.pdf"},{"id":93025164,"identity":"f303d74a-4e24-474f-8b79-0db2565b5773","added_by":"auto","created_at":"2025-10-08 09:22:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15405,"visible":true,"origin":"","legend":"","description":"","filename":"DPHashimotoTTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/b6e1b84c734ba57b937a1100.xlsx"},{"id":93026309,"identity":"777b8311-e6da-4f57-bc4c-f027fd9aee5a","added_by":"auto","created_at":"2025-10-08 09:30:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11008,"visible":true,"origin":"","legend":"","description":"","filename":"DPHashimotoTTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/996fce83e638ea4bffc7ab81.xlsx"},{"id":93025163,"identity":"d68b1727-6a4c-4ff4-a4dd-022463fbaaa7","added_by":"auto","created_at":"2025-10-08 09:22:48","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13320,"visible":true,"origin":"","legend":"","description":"","filename":"DPHashimotoTTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/9960076ba78ef084ff214c86.xlsx"},{"id":93026314,"identity":"30c378c4-f6d1-4e89-bb71-b944628fe9f4","added_by":"auto","created_at":"2025-10-08 09:30:48","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12816,"visible":true,"origin":"","legend":"","description":"","filename":"DPHashimonoTTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7744178/v1/88a248b01a67dcb8f06a42b2.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"PRDX4 Can Potentially Serve as an Independent Prognostic Marker for Early Recurrence of Oral Squamous Cell Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOral squamous cell carcinoma (OSCC) is the main histological subtype of oral cavity cancer, accounting for approximately 90% of all cancer cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Globally, the incidence of OSCC in 2022 was 389,846, with 188,438 associated deaths (Global Cancer Observatory), and the rate is constantly increasing [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite easy access to clinical examinations, the majority of OSCCs are diagnosed at an advanced stage because the early stages of the tumor are frequently asymptomatic [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. OSCC is primarily treated with radical surgery with postoperative radiation and/or chemotherapy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Approximately one-third of patients experience postoperative recurrence, with the most common pattern being local recurrence [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which is a major risk factor for poor outcomes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recurrence usually occurs within the first 18 months after surgery, leading to poor overall survival (OS) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, patients with early recurrence have a significantly worse prognosis than those with late recurrence [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the identification of early recurrence in the postoperative state can be a clinical challenge until large and symptomatic tumors emerge [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Because of the detrimental properties of early recurrence, prognostic and predictive biomarkers are urgently needed.\u003c/p\u003e\u003cp\u003eWe focused on peroxiredoxins (PRDXs) as candidate biomarkers for the early recurrence of OSCC. PRDXs protect organisms against oxidative stress caused by the accumulation of reactive oxygen species (ROS) by catalyzing peroxide reduction to remove excessive hydroxyperoxide [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. PRDX4 is the only secreted subtype of PRDX localized in the endoplasmic reticulum (ER) and contributes to oxidative protein folding and the antioxidant function [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the ER, PRDX4, in collaboration with endoplasmic reticulum oxidoreductin 1 (ERO1) via protein disulfide isomerase (PDI), acts as a sulfhydryl oxidase to introduce disulfide bonds into nascent proteins that are transferred to the Golgi body for secretion [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Decreased capacity for oxidative protein folding by PRDX4 leads to a large amount of ER stress, resulting in a number of diseases, including wound healing [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], liver disease [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], atherosclerosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], diabetes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and metabolic syndrome [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMoreover, aberrations in the expression of PRDX4 have been detected in a large number of solid and hematopoietic neoplasms, and the majority of them were significantly upregulated, including lung cancer (lung adenocarcinoma) [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], liver cancer (hepatocellular carcinoma and hepatoblastoma) [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], pancreatic cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], gastric cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], renal cancer (renal papillary carcinoma) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], prostate cancer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], breast cancer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], ovarian cancer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], brain tumor (glioma) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], multiple myeloma [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and acute myeloid and promyelocytic leukemia [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Certain cancers with high PRDX4 expression levels showed a significantly close association with tumor initiation and progression, resulting in a poor prognosis. More recently, the overexpression of PRDX4 predicted shortened survival in hepatoblastoma [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and the strong immunohistochemical expression of PRDX4 predicted poorer survival in pancreatic ductal adenocarcinoma [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAberrations in the expression of PRDX4 have also been reported in squamous cell carcinomas including OSCC [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The overexpression of PRDX4 has been reported in human OSCC tissues and cell lines, based on an immunohistochemical analysis and a meta-analysis, and is associated with increased cell proliferation, migration, and invasion through the reduced expression of adhesion molecules \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, elevated expression levels of PRDX4 mRNA were detected using the Gene Expression Array based on quantitative reverse transcription PCR (qRT-PCR) in human OSCC tissue [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In the lung, the high expression of PRDX4 was observed in squamous cell carcinoma by immunostaining, which was significantly correlated with higher recurrence rates and shorter disease-free survival (DFS) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These studies revealed that PRDX4 is significantly overexpressed in squamous cell carcinomas from various organ types.\u003c/p\u003e\u003cp\u003eOne of the major mechanisms by which the overexpression of PRDX4 contributes to malignancy is activation of the Wnt/β-catenin signaling pathway [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In fact, knockdown of PRDX4 by short hairpin RNA (shRNA) inhibited the Wnt/β-catenin signaling pathway in two renal cell carcinoma cell lines [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The degradation of PRDX4 inactivated the Wnt/β-catenin signaling pathway in hepatocellular carcinoma cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These studies have shown that PRDX4 can promote cell growth, invasion, and migration through the Wnt/β-catenin signaling pathway, which plays a crucial role in the initiation and progression of malignant neoplasms. However, to our knowledge, no previous studies have disclosed the relationship between increased PRDX4 expression and recurrence, especially early recurrence, after surgical treatment of OSCC and its underlying mechanisms, including the Wnt/β-catenin signaling pathway. The detection of increased PRDX4 expression levels in postsurgical lesions might indicate the need for immediate therapeutic interventions for recurrent OSCC.\u003c/p\u003e\u003cp\u003eOur aim was to assess the potential application of the expression of PRDX4 and β-catenin as a biomarker, especially for the early recurrence after surgical treatment of OSCC. We investigated PRDX4/β-catenin expression levels and their association with the clinical characteristics and outcomes, including early local recurrence after surgical treatment of OSCC in 72 patients, based on an immunohistochemical analysis of PRDX4 and β-catenin.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSamples\u003c/h2\u003e\u003cp\u003eThis study was approved by the Medical Ethics Committee of Kanazawa Medical University [C133]. A total of 85 cases of formalin-fixed paraffin-embedded (FFPE) tissue blocks were retrieved from archives of the Pathological Department of Kanazawa Medical University Hospital. Corresponding clinicopathological information was also collected from hospital electronic records. Thirteen cases were excluded from the analysis because of a lack of either FFPE tissue blocks or clinicopathological information.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunohistochemical staining was performed using the antibody-linked dextran polymer method for antibody-bridge labelling with hematoxylin counterstaining (EnVision; Dako Cytomation Corporation, Glostrup, Denmark). Antibodies against PRDX4 (Life Technologies Corporation, Carlsbad, CA, USA), β-catenin (Life Technologies Corporation), and S-100 protein (Dako Cytomation Corporation, Glostrup, Denmark) were used at 1:200, 1:1000, and 1:100 dilutions, respectively. Human PRDX4 transgenic mouse pancreatic cells were used as positive controls for PRDX4 [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and well-differentiated tubular colorectal adenocarcinoma cells for β-catenin [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The 3-\u0026micro;m-thick unstained sections were deparaffinized with xylene, hydrated using a graded ethanol series (100, 90, and 80%) for 1 min each, and washed with PBS three times. The sections were unmasked in citrate buffer using Pascal (Agilent Technologies Japan, Ltd., Tokyo, Japan), incubated in 10% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol for 5 min to block endogenous peroxidase activity, and washed with PBS. The slides were then incubated with 1% serum for 1 h. First, the antibodies were applied and incubated overnight at 4\u0026deg;C. The second antibody-peroxidaselinked polymers were then applied and the sections were incubated with a solution consisting of 20 mg of 3.3\u0026rsquo;-diaminobenzidine tetrahydrochloride, 65 mg of sodium azide, and 20 mL of 30% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 100 mL of Tris-HCl (50 mM, pH 7.6). After counterstaining with Meyer\u0026rsquo;s hematoxylin, sections were observed under a light microscope. The sections were first scanned at low power for all fields (original magnification: \u0026times;40) with tumor and non-tumor tissues to account for the heterogeneity of distribution.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical analysis\u003c/h3\u003e\n\u003cp\u003eIn each case, PRDX4 and β-catenin immunoreactivity was assessed semi-quantitatively by evaluating the proportion of cells with clearly higher expression levels in the total neoplastic OSCC cells in comparison to the adjacent non-tumor squamous cells, which presented very weak or negative expression levels of both proteins. The cutoff values for immunohistochemical staining of PRDX4 (10%) and β-catenin (10%) were selected and validated based on a receiver operating characteristic (ROC) curve analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. All patients were divided into two groups based on the expression of PRDX4 as follows: high when PRDX4 staining was \u0026ge;\u0026thinsp;10% and low when staining was \u0026lt;\u0026thinsp;10%. All histological and immunohistochemical slides were evaluated by two independent observers (certified surgical pathologists in our department, A.S. and T.O.) using a blinded protocol design (observers were blinded to the clinicopathological data).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe interobserver agreement, as measured by the interclass correlation coefficient, was excellent (\u0026gt;\u0026thinsp;99%) for all antibodies investigated. For the few (\u0026lt;\u0026thinsp;1%) instances of disagreement, a consensus score was determined by a third board-certified pathologist (S.Y.) in our department.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe correlations between the PRDX4 and/or β-catenin expression status and clinicopathological characteristics were analyzed using the chi-squared test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Survival curves were generated using the Kaplan-Meier method. Log-rank and generalized Wilcoxon tests were used for the statistical comparison of RFS between a cohort with high PRDX4 expression levels and a cohort with low expression levels, using GraphPad Prism 10 (GraphPad software, Tokyo, Japan). Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eClinicopathological characteristics of the cases\u003c/h2\u003e\u003cp\u003eA total of 72 patients who underwent surgery at Kanazawa Medical University Hospital were included in the present study. \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e described the detailed clinicopathological characteristics of the patients. Subsequently, the cases were dichotomized into two groups according to clinicopathological characteristics (e.g., sex, age, tumor differentiation, maximum diameter, T-category of TNM staging system, and lymph node or distant metastasis) \u003cb\u003e(Table\u0026nbsp;2).\u003c/b\u003e The patients included 43 males and 25 females. The average age of the patients was 62.5 years (range, 26 to 93 years). Fourteen of 72 (19%) of the tumors were \u0026le;\u0026thinsp;30 mm in maximal diameter. Lymphatic, venous, and perineural invasion were observed in 19% (14/72), 22% (16/72), and 19% (14/72) of the tumors, respectively. The majority of tumors (96%; 69/72) were classified as well-to-moderately differentiated, while the remainder were poorly differentiated squamous cell carcinomas. Based on the TNM classification, 44% (32/72) of cases were classified as pathological T category\u0026thinsp;\u0026ge;\u0026thinsp;2 (pT2 or higher). Lymph node metastasis (pN1 or higher) was observed in 29% (21/72) of the tumors. Distant metastasis (M1) was observed in 7% (5/72) of the tumors; metastases were detected in the lung (3/5), liver (2/5), and spine (2/5). Local recurrence developed in 21% (15/72) of the patients. A smoking history was present in 71% (51/72) of the cases, and a drinking history was present in 64% (46/72).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAssociation of the overexpression of PRDX4 and β-catenin accumulation with clinicopathological characteristics\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows representative images of OSCC in the high-PRDX4 and high-β-catenin protein accumulation groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B,C\u003cb\u003e)\u003c/b\u003e, and the low-PRDX4 and low-β-catenin protein accumulation groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,E,F\u003cb\u003e)\u003c/b\u003e. The expression of PRDX4 and β-catenin protein accumulation were both higher, especially in the tumor cell nests of the invasive fronts \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,C\u003cb\u003e)\u003c/b\u003e, in comparison to the low expression group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE,F\u003cb\u003e)\u003c/b\u003e. Moreover, in the invasive front, tumors with the high PRDX4 expression levels and β-catenin protein accumulation formed smaller tumor cell nests, suggesting malignant properties, compared to those with low PRDX4 expression levels and β-catenin protein accumulation. \u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e shows that 51 (71%) samples showed high PRDX4 expression levels and 41 (57%) samples showed high β-catenin protein accumulation. The high expression of PRDX4 was significantly associated with a history of smoking (p\u0026thinsp;=\u0026thinsp;0.011) and drinking (p\u0026thinsp;=\u0026thinsp;0.009). All patients with distant metastasis were in the high β-catenin expression group (p\u0026thinsp;=\u0026thinsp;0.044), and the group showed a significant male predominance (p\u0026thinsp;=\u0026thinsp;0.016).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eSurvival analysis\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows 2-year-RFS in the PRDX4-high and PRDX4-low groups, β-catenin-high and β-catenin-low groups, and the combination of PRDX4-high and β-catenin-high vs. others. These analyses were performed using the Kaplan-Meier method. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows that the high expression of PRDX4 was significantly associated with poorer 2-year-RFS (P\u0026thinsp;=\u0026thinsp;0.043). However, Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC showed no significant differences in 2-year RFS according to the β-catenin expression status (p\u0026thinsp;=\u0026thinsp;0.931) or the combined high expression of PRDX4 and β-catenin (p\u0026thinsp;=\u0026thinsp;0.221). The median RFS of the PRDX4-high and PRDX4-low groups was 102 and 670 days, respectively, that of the β-catenin-high and β-catenin-low groups was 68 and 208 days, and that of the PRDX4-high-β-catenin-high-and other groups was 68 and 208 days, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCox regression analysis\u003c/h2\u003e\u003cp\u003eMultivariate Cox regression analyses were performed with sex, age, maximal tumor diameter, histological differentiation of SCC, pT category, lymph node and distant metastasis, perineural, lymphatic and venous invasion, and smoking and drinking history as covariates. \u003cb\u003eTable\u0026nbsp;4\u003c/b\u003e shows that the high expression of PRDX4 (P\u0026thinsp;=\u0026thinsp;0.047), maximal tumor size\u0026thinsp;\u0026gt;\u0026thinsp;30 mm (P\u0026thinsp;=\u0026thinsp;0.029), and perineural invasion (P\u0026thinsp;=\u0026thinsp;0.023) were independent prognostic factors for 2-year-RFS. Although perineural invasion was an independent prognostic factor for 2-year-RFS and was not correlated with the high expression of PRDX4, we additionally investigated the tumor microenvironment around the peripheral nerves by immunohistochemical staining of S-100 protein. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows that tiny PRDX4-positive tumor nests were frequently observed around S-100 protein-positive peripheral nerves.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study revealed that the high expression of PRDX4 in OSCC is a significant prognostic factor, particularly for early recurrence. Furthermore, univariate and multivariate analyses of the Cox proportional regression model revealed that the high expression of PRDX4 predicted significantly worse 2-year-RFS in comparison to the low expression of PRDX4 (P\u0026thinsp;=\u0026thinsp;0.043), indicating that an aberrantly increased PRDX4 expression level could be a candidate independent biomarker for early recurrence of OSCC. The early detection of PRDX4 in postoperative specimens might indicate the possibility of therapeutic intervention to improve patient survival. Moreover, PRDX4 could be a candidate serum biomarker to detect early recurrence of OSCC after surgical treatment, since the protein is known to be the only soluble PRDX subtype [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Indeed, the serum expression of PRDX4 can be detected in some tissues (e.g., pancreatic tissue), wherein protein secretion is critical for both exocrine and endocrine secretion [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present study had some limitations. This was a retrospective study conducted within a single institution with a relatively small study population. Furthermore, we only evaluated histopathological sections (but not serum) and could not comprehensively analyze the role of PRDX4 in the prognosis of OSCC. Additionally, identical studies using other antibodies may be necessary to validate the results of this study in the future. Finally, the pivotal effects of the overexpression of PRDX4 on OSCC were determined using human OSCC cell lines and/or OSCC animal models in human PRDX4 transgenic mice.\u003c/p\u003e\u003cp\u003eDespite these limitations, the univariate and multivariate Cox regression analyses in the present study revealed that perineural invasion was an independent prognostic factor for 2-year-RFS, although it was not associated with the increased expression of PRDX4 \u003cb\u003e(Table\u0026nbsp;4)\u003c/b\u003e. Perineural invasion has been recognized as one of the critical histopathological characteristics in various malignancies, including those of the breast, pancreas, colorectum, and prostate, and it correlates with higher tumor grade, increased risk of locoregional recurrence, and a potentially worse prognosis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, tiny OSCC nests surrounding the peripheral nerves in the tumor microenvironment showed high PRDX4 expression levels. Accordingly, the localized overexpression of PRDX4 surrounding the peripheral nerves, instead of the overall expression of the tumor, might be closely associated with poor outcomes, including early recurrence. Further studies are needed to investigate the association between microenvironmentally localized high PRDX4 expression levels (e.g., perineural invasion) and early recurrence.\u003c/p\u003e\u003cp\u003eIn this study, we revealed the association between high PRDX4 expression levels and early recurrence of OSCC; however, the underlying mechanisms could not be elucidated. Since PRDX4 in the ER lumen mainly catalyzes oxidative protein folding, its decreased expression results in the accumulation of unfolded proteins, which causes cell apoptosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, malignant cells such as OSCC cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, significantly upregulate PRDX4 to protect themselves from apoptosis caused by the accumulation of unfolded protein, which promotes tumor initiation and development, possibly through the Wnt/β-catenin signaling pathway [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Thus, inhibiting the overexpression of PRDX4, together with the Wnt/β-catenin signaling pathway, could be an attractive therapeutic target for OSCC. Furthermore, we speculate that high PRDX4 levels could affect malignant characteristics by maintaining stem cell-like properties mainly activated by the Wnt/β-catenin signaling pathway [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Such stem cell-like tumor cells have been reported to express splicing variant isoforms of CD44 (CD44v)[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], leading to increased production of the antioxidant glutathione, which removes excessive ROS in the tumor microenvironment and may enhance malignant features [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Similarly, the antioxidative PRDX4 expression can be upregulated, especially in stem cell-like tumor cell populations within malignant neoplasms, including OSCC. Therefore, PRDX4 may be a candidate biomarker for stem cell-like tumor cells, which aggressively enhance the occurrence and growth of malignant neoplasms. Further in-depth studies will be required in the near future.\u003c/p\u003e\u003cp\u003eIn conclusion, we demonstrated that high PRDX4 expression in OSCC is a significant and independent prognostic factor, particularly for early recurrence, and may serve as a useful biomarker for detecting early recurrence in postoperative histopathological specimens and potentially in serum samples. The underlying mechanisms of the effects of PRDX4 on early recurrence of OSCC remain to be elucidated; however, our future study will partly focus on the prevention of unfolded protein accumulation in the ER by PRDX4 and the undifferentiated state of stem-like tumor cells induced likely by the Wnt/β-catenin signaling pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll intended procedures in the present study, including the use of specimens obtained from human subjects, were approved by the Medical Ethics Committee of Kanazawa Medical University (permission number: C133). All patients provided informed written consent according to the guidelines of the Japanese Society of Pathology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided informed written consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no competing financial and/or non-financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by Japan Society for the Promotion of Science (20K07454 to S. Y. and 23K06474 to T. O.), by Hokkoku Cancer Foundation (to S. Y.) and by Grant for Promoted Research from Kanazawa Medical University (S2023-A4 to T.O.)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTaijiro Hashimoto, Yangxion Zhang, Takeru Oyama, Akihiro Shioya and Sohsuke Yamada conceptualized and designed the experiments. Mitsuaki Yoshida, Taijiro Hashimoto, Yangxion Zhang and Jia Han performed experiments. Taijiro Hashimoto, Jia Han, Yukinobu Ito and Takeru Oyama analyzed the data and drafted the manuscript. Masashi Okuro, Morimasa Kitamura and Sohsuke Yamada have reviewed and edited the manuscript. Sohsuke Yamada supervised the study. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Brian Quinn for editing the manuscript. We thank the members of the Department of Pathology, Kanazawa Medical University Hospital, for providing expert technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMuralidharan S, Nikalje M, Subramaniam T, Koshy JA, Koshy AV, Bangera D. A Narrative Review on Oral Squamous Cell Carcinoma. J Pharm Bioallied Sci. 2025; 17: S204-S6. https://doi.org/10.4103/jpbs.jpbs_593_25.\u003c/li\u003e\n\u003cli\u003eEsperouz F, Ciavarella D, Lorusso M, Santarelli A, Lo Muzio L, Campisi G, et al. Critical review of OCT in clinical practice for the assessment of oral lesions. Front Oncol. 2025; 15: 1569197. https://doi.org/10.3389/fonc.2025.1569197.\u003c/li\u003e\n\u003cli\u003eJagadeesan D, Sathasivam KV, Fuloria NK, Balakrishnan V, Khor GH, Ravichandran M, et al. Comprehensive insights into oral squamous cell carcinoma: Diagnosis, pathogenesis, and therapeutic advances. Pathol Res Pract. 2024; 261: 155489. https://doi.org/10.1016/j.prp.2024.155489.\u003c/li\u003e\n\u003cli\u003eBagan J, Sarrion G, Jimenez Y. Oral cancer: clinical features. Oral Oncol. 2010; 46: 414-7. https://doi.org/10.1016/j.oraloncology.2010.03.009.\u003c/li\u003e\n\u003cli\u003eHosni A, Huang SH, Chiu K, Xu W, Su J, Bayley A, et al. Predictors of Early Recurrence Prior to Planned Postoperative Radiation Therapy for Oral Cavity Squamous Cell Carcinoma and Outcomes Following Salvage Intensified Radiation Therapy. Int J Radiat Oncol Biol Phys. 2019; 103: 363-73. https://doi.org/10.1016/j.ijrobp.2018.09.013.\u003c/li\u003e\n\u003cli\u003eMontero PH, Patel SG. Cancer of the oral cavity. Surg Oncol Clin N Am. 2015; 24: 491-508. https://doi.org/10.1016/j.soc.2015.03.006.\u003c/li\u003e\n\u003cli\u003eLyu WN, Lin MC, Shen CY, Chen LH, Lee YH, Chen SK, et al. An Oral Microbial Biomarker for Early Detection of Recurrence of Oral Squamous Cell Carcinoma. ACS Infect Dis. 2023; 9: 1783-92. https://doi.org/10.1021/acsinfecdis.3c00269.\u003c/li\u003e\n\u003cli\u003eWang Y, Yang T, Gan C, Wang K, Sun B, Wang M, et al. Temporal and spatial patterns of recurrence in oral squamous cell carcinoma, a single-center retrospective cohort study in China. BMC Oral Health. 2023; 23: 679. https://doi.org/10.1186/s12903-023-03204-7.\u003c/li\u003e\n\u003cli\u003eLiao CT, Chang JT, Wang HM, Ng SH, Hsueh C, Lee LY, et al. Salvage therapy in relapsed squamous cell carcinoma of the oral cavity: how and when? Cancer. 2008; 112: 94-103. https://doi.org/10.1002/cncr.23142.\u003c/li\u003e\n\u003cli\u003eKernohan MD, Clark JR, Gao K, Ebrahimi A, Milross CG. Predicting the prognosis of oral squamous cell carcinoma after first recurrence. Arch Otolaryngol Head Neck Surg. 2010; 136: 1235-9. https://doi.org/10.1001/archoto.2010.214.\u003c/li\u003e\n\u003cli\u003eLiu SA, Wong YK, Lin JC, Poon CK, Tung KC, Tsai WC. Impact of recurrence interval on survival of oral cavity squamous cell carcinoma patients after local relapse. Otolaryngol Head Neck Surg. 2007; 136: 112-8. https://doi.org/10.1016/j.otohns.2006.07.002.\u003c/li\u003e\n\u003cli\u003eFujii J, Ochi H, Yamada S. A comprehensive review of peroxiredoxin 4, a redox protein evolved in oxidative protein folding coupled with hydrogen peroxide detoxification. Free Radic Biol Med. 2025; 227: 336-54. https://doi.org/10.1016/j.freeradbiomed.2024.12.015.\u003c/li\u003e\n\u003cli\u003eYamada S, Guo X. Peroxiredoxin 4 (PRDX4): Its critical in vivo roles in animal models of metabolic syndrome ranging from atherosclerosis to nonalcoholic fatty liver disease. Pathol Int. 2018; 68: 91-101. https://doi.org/10.1111/pin.12634.\u003c/li\u003e\n\u003cli\u003eHanaka T, Kido T, Noguchi S, Yamada S, Noguchi H, Guo X, et al. The overexpression of peroxiredoxin-4 affects the progression of idiopathic pulmonary fibrosis. BMC Pulm Med. 2019; 19: 265. https://doi.org/10.1186/s12890-019-1032-2.\u003c/li\u003e\n\u003cli\u003eYamaguchi R, Guo X, Zheng J, Zhang J, Han J, Shioya A, et al. PRDX4 Improved Aging-Related Delayed Wound Healing in Mice. J Invest Dermatol. 2021; 141: 2720-9. https://doi.org/10.1016/j.jid.2021.04.015.\u003c/li\u003e\n\u003cli\u003eZhang J, Guo X, Hamada T, Yokoyama S, Nakamura Y, Zheng J, et al. Protective Effects of Peroxiredoxin 4 (PRDX4) on Cholestatic Liver Injury. Int J Mol Sci. 2018; 19. https://doi.org/10.3390/ijms19092509.\u003c/li\u003e\n\u003cli\u003eHomma T, Kurahashi T, Lee J, Nabeshima A, Yamada S, Fujii J. Double Knockout of Peroxiredoxin 4 (Prdx4) and Superoxide Dismutase 1 (Sod1) in Mice Results in Severe Liver Failure. Oxid Med Cell Longev. 2018; 2018: 2812904. https://doi.org/10.1155/2018/2812904.\u003c/li\u003e\n\u003cli\u003eNawata A, Noguchi H, Mazaki Y, Kurahashi T, Izumi H, Wang KY, et al. Overexpression of Peroxiredoxin 4 Affects Intestinal Function in a Dietary Mouse Model of Nonalcoholic Fatty Liver Disease. PLoS One. 2016; 11: e0152549. https://doi.org/10.1371/journal.pone.0152549.\u003c/li\u003e\n\u003cli\u003eGuo X, Yamada S, Tanimoto A, Ding Y, Wang KY, Shimajiri S, et al. Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice. Antioxid Redox Signal. 2012; 17: 1362-75. https://doi.org/10.1089/ars.2012.4549.\u003c/li\u003e\n\u003cli\u003eDing Y, Yamada S, Wang KY, Shimajiri S, Guo X, Tanimoto A, et al. Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice. Antioxid Redox Signal. 2010; 13: 1477-90. https://doi.org/10.1089/ars.2010.3137.\u003c/li\u003e\n\u003cli\u003eNabeshima A, Yamada S, Guo X, Tanimoto A, Wang KY, Shimajiri S, et al. Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model. Antioxid Redox Signal. 2013; 19: 1983-98. https://doi.org/10.1089/ars.2012.4946.\u003c/li\u003e\n\u003cli\u003eShioya A, Guo X, Motono N, Mizuguchi S, Kurose N, Nakada S, et al. The Combination Of Weak Expression Of PRDX4 And Very High MIB-1 Labelling Index Independently Predicts Shorter Disease-free Survival In Stage I Lung Adenocarcinoma. Int J Med Sci. 2018; 15: 1025-34. https://doi.org/10.7150/ijms.25734.\u003c/li\u003e\n\u003cli\u003eZheng J, Guo X, Nakamura Y, Zhou X, Yamaguchi R, Zhang J, et al. Overexpression of PRDX4 Modulates Tumor Microenvironment and Promotes Urethane-Induced Lung Tumorigenesis. Oxid Med Cell Longev. 2020; 2020: 8262730. https://doi.org/10.1155/2020/8262730.\u003c/li\u003e\n\u003cli\u003eMizutani K, Guo X, Shioya A, Zhang J, Zheng J, Kurose N, et al. The impact of PRDX4 and the EGFR mutation status on cellular proliferation in lung adenocarcinoma. Int J Med Sci. 2019; 16: 1199-206. https://doi.org/10.7150/ijms.36071.\u003c/li\u003e\n\u003cli\u003eIwai S, Motono N, Oyama T, Shioya A, Yamada S, Uramoto H. The Clinical Relevance of the Expression of SGLT2 in Lung Adenocarcinoma. Oncology. 2024; 102: 710-9. https://doi.org/10.1159/000536060.\u003c/li\u003e\n\u003cli\u003eGuo X, Noguchi H, Ishii N, Homma T, Hamada T, Hiraki T, et al. The Association of Peroxiredoxin 4 with the Initiation and Progression of Hepatocellular Carcinoma. Antioxid Redox Signal. 2019; 30: 1271-84. https://doi.org/10.1089/ars.2017.7426.\u003c/li\u003e\n\u003cli\u003eLiu Y, Han J, Shioya A, Zhang YX, Dung VA, Oyama T, et al. The immunohistochemical combination of low SGLT2 expression and high PRDX4 expression independently predicts shortened survival in patients undergoing surgical resection for hepatoblastoma. Diagn Pathol. 2025; 20: 2. https://doi.org/10.1186/s13000-025-01596-4.\u003c/li\u003e\n\u003cli\u003eZheng J, Guo X, Shioya A, Yoshioka T, Matsumoto K, Hiraki T, et al. Peroxiredoxin 4 promotes embryonal hepatoblastoma cell migration but induces fetal cell differentiation. Am J Transl Res. 2020; 12: 2726-37.\u003c/li\u003e\n\u003cli\u003eHan J, Itoh T, Shioya A, Sakurai M, Oyama T, Kumagai M, et al. The combination of the low immunohistochemical expression of peroxiredoxin 4 and perilipin 2 predicts longer survival in pancreatic ductal adenocarcinoma with peroxiredoxin 4 possibly playing a main role. Histol Histopathol. 2023; 38: 1415-27. https://doi.org/10.14670/HH-18-666.\u003c/li\u003e\n\u003cli\u003ePark SY, Lee YJ, Park J, Kim TH, Hong SC, Jung EJ, et al. PRDX4 overexpression is associated with poor prognosis in gastric cancer. Oncol Lett. 2020; 19: 3522-30. https://doi.org/10.3892/ol.2020.11468.\u003c/li\u003e\n\u003cli\u003eJia W, Chen P, Cheng Y. PRDX4 and Its Roles in Various Cancers. Technol Cancer Res Treat. 2019; 18: 1533033819864313. https://doi.org/10.1177/1533033819864313.\u003c/li\u003e\n\u003cli\u003eZhou H, Li L, Chen J, Hou S, Zhou T, Xiong Y. Expression and prognostic value of PRDX family in colon adenocarcinoma by integrating comprehensive analysis and in vitro and in vivo validation. Front Oncol. 2023; 13: 1136738. https://doi.org/10.3389/fonc.2023.1136738.\u003c/li\u003e\n\u003cli\u003eKocaturk B. In silico analysis reveals PRDX4 as a prognostic and oncogenic marker in renal papillary cell carcinoma. Gene. 2023; 859: 147201. https://doi.org/10.1016/j.gene.2023.147201.\u003c/li\u003e\n\u003cli\u003eBasu A, Banerjee H, Rojas H, Martinez SR, Roy S, Jia Z, et al. Differential expression of peroxiredoxins in prostate cancer: consistent upregulation of PRDX3 and PRDX4. Prostate. 2011; 71: 755-65. https://doi.org/10.1002/pros.21292.\u003c/li\u003e\n\u003cli\u003eUmmanni R, Barreto F, Venz S, Scharf C, Barett C, Mannsperger HA, et al. Peroxiredoxins 3 and 4 are overexpressed in prostate cancer tissue and affect the proliferation of prostate cancer cells in vitro. J Proteome Res. 2012; 11: 2452-66. https://doi.org/10.1021/pr201172n.\u003c/li\u003e\n\u003cli\u003eJiang W, Wang M, Chen Q, Yu X, Liu G, He X, et al. Immune infiltration related PRDX4 facilitates the malignant features and drug resistance of breast cancer. Sci Rep. 2025; 15: 27507. https://doi.org/10.1038/s41598-025-13361-0.\u003c/li\u003e\n\u003cli\u003eSzeliga M. Comprehensive analysis of the expression levels and prognostic values of PRDX family genes in glioma. Neurochem Int. 2022; 153: 105256. https://doi.org/10.1016/j.neuint.2021.105256.\u003c/li\u003e\n\u003cli\u003eKim TH, Song J, Alcantara Llaguno SR, Murnan E, Liyanarachchi S, Palanichamy K, et al. Suppression of peroxiredoxin 4 in glioblastoma cells increases apoptosis and reduces tumor growth. PLoS One. 2012; 7: e42818. https://doi.org/10.1371/journal.pone.0042818.\u003c/li\u003e\n\u003cli\u003eDemasi AP, Martinez EF, Napimoga MH, Freitas LL, Vassallo J, Duarte AS, et al. Expression of peroxiredoxins I and IV in multiple myeloma: association with immunoglobulin accumulation. Virchows Arch. 2013; 463: 47-55. https://doi.org/10.1007/s00428-013-1433-1.\u003c/li\u003e\n\u003cli\u003ePalande KK, Beekman R, van der Meeren LE, Beverloo HB, Valk PJ, Touw IP. The antioxidant protein peroxiredoxin 4 is epigenetically down regulated in acute promyelocytic leukemia. PLoS One. 2011; 6: e16340. https://doi.org/10.1371/journal.pone.0016340.\u003c/li\u003e\n\u003cli\u003eCao R, Zhang W, Zhang H, Wang L, Chen X, Ren X, et al. Comprehensive Analysis of the PRDXs Family in Head and Neck Squamous Cell Carcinoma. Front Oncol. 2022; 12: 798483. https://doi.org/10.3389/fonc.2022.798483.\u003c/li\u003e\n\u003cli\u003eHwang JA, Song JS, Yu DY, Kim HR, Park HJ, Park YS, et al. Peroxiredoxin 4 as an independent prognostic marker for survival in patients with early-stage lung squamous cell carcinoma. Int J Clin Exp Pathol. 2015; 8: 6627-35.\u003c/li\u003e\n\u003cli\u003eChang KP, Yu JS, Chien KY, Lee CW, Liang Y, Liao CT, et al. Identification of PRDX4 and P4HA2 as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using iTRAQ technology. J Proteome Res. 2011; 10: 4935-47. https://doi.org/10.1021/pr200311p.\u003c/li\u003e\n\u003cli\u003ePedro NF, Biselli JM, Maniglia JV, Santi-Neto D, Pavarino EC, Goloni-Bertollo EM, et al. Candidate Biomarkers for Oral Squamous Cell Carcinoma: Differential Expression of Oxidative Stress-Related Genes. Asian Pac J Cancer Prev. 2018; 19: 1343-9. https://doi.org/10.22034/APJCP.2018.19.5.1343.\u003c/li\u003e\n\u003cli\u003eLi H, Wang Z, Chen X, Li S, Zhang F. Resveratrol Downregulated PRDX4 Expression to Inhibit the Progression of Renal Cell Carcinoma via Wnt/beta-Catenin Pathway. Food Sci Nutr. 2025; 13: e70352. https://doi.org/10.1002/fsn3.70352.\u003c/li\u003e\n\u003cli\u003eChen B, Lan J, Xiao Y, Liu P, Guo D, Gu Y, et al. Long noncoding RNA TP53TG1 suppresses the growth and metastasis of hepatocellular carcinoma by regulating the PRDX4/beta-catenin pathway. Cancer Lett. 2021; 513: 75-89. https://doi.org/10.1016/j.canlet.2021.04.022.\u003c/li\u003e\n\u003cli\u003eHugh TJ, Dillon SA, O\u0026apos;Dowd G, Getty B, Pignatelli M, Poston GJ, et al. beta-catenin expression in primary and metastatic colorectal carcinoma. Int J Cancer. 1999; 82: 504-11. https://doi.org/10.1002/(sici)1097-0215(19990812)82:4\u0026lt;504::aid-ijc6\u0026gt;3.0.co;2-6.\u003c/li\u003e\n\u003cli\u003eBahmad HF, Wegner C, Nuraj J, Avellan R, Gonzalez J, Mendez T, et al. Perineural Invasion in Breast Cancer: A Comprehensive Review. Cancers (Basel). 2025; 17. https://doi.org/10.3390/cancers17121900.\u003c/li\u003e\n\u003cli\u003eBhal S, Das B, Sinha S, Das C, Acharya SS, Maji J, et al. Resveratrol nanoparticles induce apoptosis in oral cancer stem cells by disrupting the interaction between beta-catenin and GLI-1 through p53-independent activation of p21. Med Oncol. 2024; 41: 167. https://doi.org/10.1007/s12032-024-02405-6.\u003c/li\u003e\n\u003cli\u003eEsteban-Roman NF, Taddei E, Castro-Velazquez E, Villafuentes-Vidal L, Velez-Herrera A, Rubio-Osornio M, et al. Redox-Regulated Pathways in Glioblastoma Stem-like Cells: Mechanistic Insights and Therapeutic Implications. Brain Sci. 2025; 15. https://doi.org/10.3390/brainsci15080884.\u003c/li\u003e\n\u003cli\u003eNagano O, Okazaki S, Saya H. Redox regulation in stem-like cancer cells by CD44 variant isoforms. Oncogene. 2013; 32: 5191-8. https://doi.org/10.1038/onc.2012.638.\u003c/li\u003e\n\u003cli\u003eIshikawa K, Suzuki H, Kaneko MK, Kato Y. Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C(44)Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas. Curr Issues Mol Biol. 2023; 45: 5248-62. https://doi.org/10.3390/cimb45070333.\u003c/li\u003e\n\u003cli\u003eGuo L, Ke H, Zhang H, Zou L, Yang Q, Lu X, et al. TDP43 promotes stemness of breast cancer stem cells through CD44 variant splicing isoforms. Cell Death Dis. 2022; 13: 428. https://doi.org/10.1038/s41419-022-04867-w.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7744178/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7744178/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOral squamous cell carcinoma (OSCC), a main histological subtype of oral cavity cancer, remains one of the most prevailing tumors worldwide with the increasing incidence and mortality. Since a high rate of early local recurrence is one of the major risk factors for poor outcome of OSCC, its prognostic biomarkers are urgently needed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePeroxiredoxin 4 (PRDX4), a member of PRDXs, which are involved in the antioxidant defense mechanism, is a unique secreted subtype of PRDXs. A certain number of previous studies have that the overexpression of the PRDX4 protein has a clear relationship with tumor initiation and progression in many cancers. Furthermore, recent studies have revealed that PRDX4 promotes tumor development through the Wnt/β-catenin signaling pathway. In the present study, to assess the status of the PRDX4/β-catenin expression and its association with clinical outcomes, including early local recurrence in OSCC, we immunohistochemically examined PRDX4 expression levels and cytoplasmic β-catenin protein accumulation levels in a total of 72 postoperative OSCC samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe high expression of PRDX4 was significantly correlated with smoking and alcohol intake and poorer early phase 2-year recurrence-free survival (RFS). In addition, a multivariate Cox regression analysis revealed that PRDX4 was an independent prognostic factor for 2-year-RFS. In addition, high β-catenin accumulation is significantly associated with distant metastasis. A comparison of the combination of a high expression of PRDX4 and high β-catenin protein accumulation groups with the other groups only showed a significant predominance among males. In conclusion, the increased expression of PRDX4 may be a useful independent prognostic biomarker for recurrence of OSCC, especially in the early postoperative phase.\u003c/p\u003e","manuscriptTitle":"PRDX4 Can Potentially Serve as an Independent Prognostic Marker for Early Recurrence of Oral Squamous Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 09:22:43","doi":"10.21203/rs.3.rs-7744178/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"88d240bf-7706-4588-b785-1e79bf7e0d5a","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-04T19:08:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 09:22:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7744178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7744178","identity":"rs-7744178","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}