Pathways with an impact on effects of metronomic chemotherapy with a fluoropyrimidine prodrug, S-1

Pathways with an impact on effects of metronomic chemotherapy with a fluoropyrimidine prodrug, S-1 | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 21 April 2025 V1 Latest version Share on Pathways with an impact on effects of metronomic chemotherapy with a fluoropyrimidine prodrug, S-1 Authors : SHINICHIRO KINA [email protected] , Sho Miyamoto , Mika Kina-Tanada , Masaru Ogawa , Akira Arasaki , and Satoshi Yokoo Authors Info & Affiliations https://doi.org/10.22541/au.174521795.53239906/v1 196 views 106 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose Adjuvant chemotherapy with 5-fluorouracil (5-FU) prodrugs is now the standard of care for stage I–III oral squamous cell carcinoma (OSCC); however, it remains to be clarified for which patient neoadjuvant chemotherapy with 5-FU prodrugs may be an option. Experimental Approach In the Ryukyu cohort [neoadjuvant chemotherapy with 5-FU prodrugs (UFT or S-1) + bleomycin, neoadjuvant chemotherapy with UFT + bleomycin + cisplatin, and up-front surgery] and Gunma cohort [neoadjuvant chemotherapy with S-1 and up-front surgery], the incidence of distant metastasis was significantly higher in male patients or those with less well differentiated tumors. Key Results Based on these data, we compared the overall survival of male patients who received neoadjuvant chemotherapy and those who received up-front surgery in each cohort. Neoadjuvant chemotherapy with S-1 or S-1 + bleomycin significantly increased overall survival rates in male patients. The ratio of male patients correlated with less well differentiated tumors. Conclusion and Implications Data in The Cancer Genome Atlas revealed that the expression of DNA repair genes correlated with male sex and less well differentiated tumors, while higher Myd88 expression and type I interferon activity were associated with female sex and well-differentiated tumors. Pathways with an impact on effects of metronomic chemotherapy with a fluoropyrimidine prodrug, S-1 Shinichiro Kina, 1,2✉ Sho Miyamoto, 3 Mika Kina-Tanada, 4 Masaru Ogawa, 4 Akira Arasaki, 5 Satoshi Yokoo 4 1 Department of Medical Education and Development, Graduate School of Medicine, Gunma University, Maebashi, Japan. 2 Department of Pharmacology, Graduate School of Medicine, University of the Ryukyus, 1076 Kiyuna, Ginowan, Okinawa, 901-2720, Japan. 3 Department of Oral Surgery, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan. 4 Department of Oral and Maxillofacial Surgery, and Plastic Surgery, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan. 5 Department of Oral and Maxillofacial Functional Rehabilitation, Graduate School of Medicine, University of the Ryukyus, Japan ✉ E-mail: [email protected] Background and Purpose Adjuvant chemotherapy with 5-fluorouracil (5-FU) prodrugs is now the standard of care for stage I–III oral squamous cell carcinoma (OSCC); however, it remains to be clarified for which patient neoadjuvant chemotherapy with 5-FU prodrugs may be an option. Experimental Approach In the Ryukyu cohort [neoadjuvant chemotherapy with 5-FU prodrugs (UFT or S-1) + bleomycin, neoadjuvant chemotherapy with UFT + bleomycin + cisplatin, and up-front surgery] and Gunma cohort [neoadjuvant chemotherapy with S-1 and up-front surgery], the incidence of distant metastasis was significantly higher in male patients or those with less well differentiated tumors. Key Results Based on these data, we compared the overall survival of male patients who received neoadjuvant chemotherapy and those who received up-front surgery in each cohort. Neoadjuvant chemotherapy with S-1 or S-1 + bleomycin significantly increased overall survival rates in male patients. The ratio of male patients correlated with less well differentiated tumors. Conclusion and Implications Data in The Cancer Genome Atlas revealed that the expression of DNA repair genes correlated with male sex and less well differentiated tumors, while higher Myd88 expression and type I interferon activity were associated with female sex and well-differentiated tumors. Introduction In oral cancer, there were an estimated 389,485 new cancer cases and 188,230 deaths worldwide in 2022, with a higher incidence in male patients; moreover, the male sex is a predictor of mortality [1,2]. Up-front surgery is recommended for patients with localized oral squamous cell carcinoma (OSCC) [3]. Although the survival of oral cancer patients with recurrent distant metastasis has improved, the majority of patients die within 5 years after the diagnosis of recurrence [4], necessitating a multimodality treatment approach incorporating perioperative (neoadjuvant or adjuvant) chemotherapy. Multiple clinical trials have been conducted to evaluate the efficacy and safety of neoadjuvant chemotherapy for advanced HNSCC; however, none reported the prolongation of overall survival (OS) [5, 6]. All trials evaluating the efficacy of neoadjuvant chemotherapy in OSCC were based on the maximum tolerated dose (MTD) [7, 8]. MTD chemotherapy frequently suppresses anti-tumor immunity and may induce tumor recurrence [9]. Metronomic (low-dose and high-frequency mode of the continuous administration of chemotherapeutic drugs without prolonged drug-free intervals) [10] adjuvant chemotherapy was found to prolong the OS of patients by reducing distant metastasis [11], which represents a paradigm shift in the management of advanced OSCC. The efficacy of metronomic adjuvant chemotherapy for advanced HNSCC has stimulated interest in its potential in the neoadjuvant setting. Typical adjuvant metronomic chemotherapy for OSCC uses fluoropyrimidine prodrugs [12]. Treatment with 5-fluorouracil (5-FU) leads to replication fork collapse through the base excision repair (BER) system in tumor cells by increasing the nuclear localization of XRCC1, which activates XRCC1, thereby stalling the replication fork [13,14]. In addition to replication fork collapse, 5-FU-modified DNA specifically binds to mismatch repair protein 2 (MSH2) [15], which may lead to G2 arrest and cell death [16]. In 2021, a randomized study showed the prolongation of OS with adjuvant metronomic capecitabine [17]. Adjuvant metronomic chemotherapy with S-1 after curative resection is preferred for pancreatic ductal adenocarcinoma (PDAC) or gastric cancer [18, 19]. However, data on the metronomic neoadjuvant setting with S-1 are limited. Neoadjuvant chemotherapy with gemcitabine and S-1 have revolutionized treatment for early PDAC [19]. A randomized study on PDAC showed that OS was prolonged by 4 weeks of neoadjuvant chemotherapy with S-1 and gemcitabine [20]. The prevention of overtreatment is an increasingly important consideration due to the evolution of drug resistance and increasing toxicity with more intense and longer treatments. Therefore, there is an urgent clinical need to optimize treatment schedules and improve patient selection for 5-FU-based treatments. There are currently no biomarkers that predict which patients with OSCC will benefit from 5-FU prodrugs. The presence of deficient mismatch repair (dMMR) in tumors [21] and tumor differentiation [22, 23] were found to be predictive of responses to 5-FU/5-FU prodrugs in some cancer types. We recently showed that patients with poorly or moderately differentiated OSCC benefited from metronomic neoadjuvant chemotherapy with S-1 [24, 25]. Head and neck squamous cell carcinoma (HNSCC) is classified into three distinct histological types: well-, moderately, and poorly differentiated tumors [26]. These histological types are associated with progression-free survival and OS, with well-differentiated tumors having the best outcomes and poorly differentiated tumors having the worst. Between approximately 30 and 70% of OSCC are classified as the poorly and moderately differentiated types [6, 27]. A subset of well-differentiated HNSCC were found to include a high frequency of CASP8 mutations [28]. The knockdown of FAT1, CASP8, or both in OSCC cells promoted terminal differentiation in some cases [29]. We hypothesized that metronomic neoadjuvant chemotherapy with 5-FU prodrugs may prolong the OS of patients at a high risk of developing distant metastasis. Therefore, we herein investigated the characteristics of OSCC patients with distant metastasis and the efficacy of metronomic neoadjuvant chemotherapy with 5-FU prodrugs. The present study was based on data obtained from two independent institutions. Results Study design We herein report the results of two independent cohorts that investigated the characteristics of OSCC patients with distant metastasis (the Ryukyu large cohort and Gunma cohort, n = 539) and examined the efficacy of short-term neoadjuvant S-1 ± bleomycin (the Ryukyu main cohort and Gunma cohort, n = 358) in patients with stage I-III OSCC. Patients aged 18 years or older and 85 years or younger with clinical stage I-III OSCC were eligible for this study. Between August 1987 and July 2013, 303 patients were collected in Ryukyu University. Between 10 July 2008 and 28 March 2017, 236 patients were collected in Gunma University. In Ryukyu University, patients received bleomycin 15 mg twice a week for three weeks until surgery or unacceptable toxicity. During this period, (1) 450-mg UFT-E granules were administered orally three times a day or (2) 100-mg/day S-1 granules were administered orally. Some patients received neoadjuvant chemotherapy with cisplatin (Fig. 1). In Gunma University, patients received S-1 120 mg orally every day for 2 weeks until surgery or unacceptable toxicity. The treatment schedule with S-1 for the Gunma cohort was based on the well-established, effective, and tolerable adjuvant chemotherapy schedule for HNSCC [30]. Baseline clinical and pathological characteristics are summarized in Table 1. In the Ryukyu large cohort, the median age of patients was 68 years (range, 23–85). In total, 189 patients (62.4%) had well-differentiated OSCC, 100 (33.0%) had moderately differentiated OSCC, 5 (1.7%) had poorly to moderately differentiated OSCC, and 9 (3.0%) had poorly differentiated OSCC (Table 1A). In the Gunma cohort, the median age of patients was 71 years (range, 23–85). Overall, 151 patients (64.0%) had well-differentiated OSCC, 33 (14.0%) had well- to moderately differentiated OSCC, 41 (17.8%) had moderately differentiated OSCC, 9 (3.8%) had poorly to moderately differentiated OSCC, and 2 (0.8%) had poorly differentiated OSCC (Table 1B). Clinical characteristics of patients with distant metastasis To examine the characteristics of patients with distant metastasis, we compared the rate of patients with distant metastasis in each clinical characteristic. The rate of distant metastasis was significantly higher in male patients than in female patients in the Ryukyu large cohort ( P = 0.01) and Gunma cohort ( P = 0.007) (Table 2A, B). Furthermore, the rate of distant metastasis was significantly higher in patients with less well differentiated tumors than in those with well-differentiated tumors in the Ryukyu large cohort ( P = 0.01) and Gunma cohort ( P = 0.01) (Table 2A, B). The rate of distant metastasis was significantly lower in patients receiving neoadjuvant chemotherapy than in those who underwent surgery (the Ryukyu large cohort: P = 0.03, the Gunma cohort: P = 0.01). Furthermore, in the multivariate analysis of the incidence of distant metastasis in patients with stage I-III disease, male sex, well less differentiated tumors, and up-front surgery were identified as significant prognostic factors for an increased risk of distant metastasis in the Ryukyu large cohort (male: HR 7.091, 95% CI 1.481-33.937, P = 1.42×10 -2 , less well differentiated tumors: HR 3.946, 95% CI 1.253-12.418, P = 1.89×10 -2 , neoadjuvant chemotherapy: HR 0.22, 95% CI 0.068-0.709, P = 1.12×10 -2 )(Table 2C) and Gunma cohort (male: HR 11.125, 95% CI 1.374-90.057, P = 2.39×10 -2 , less well differentiated tumors: HR 3.845, 95% CI 1.079-13.702, P = 3.77×10 -2 , neoadjuvant chemotherapy: HR 0.167, 95% CI 0.041-0.677, P = 1.22×10 -2 )(Table 2D). These results indicate that sex and histological differentiation affected the primary and secondary endpoints of short-term neoadjuvant chemotherapy (S-1 ± bleomycin). We previously reported the prolongation of OS in patients with poorly or moderately differentiated tumors receiving metronomic neoadjuvant chemotherapy with bleomycin + 5-FU prodrugs [23] or S-1 alone [24] in each institution. Therefore, we herein investigated sex differences in the efficacy of metronomic chemotherapy with S-1 + bleomycin or S-1 alone. Efficacy of short-term S-1 + bleomycin or S-1 alone in early OSCC The primary endpoint of the present study was the 6-year OS rate. In the Ryukyu large cohort, 77 patients underwent up-front surgery, 22 received UFT and bleomycin as induction chemotherapy and then in combination with cisplatin-based chemotherapy, 159 received UFT and bleomycin, and 45 received S-1 and bleomycin due to the approval of S-1 in 2008. In Gunma, 100 patients underwent up-front surgery and 136 were treated with neoadjuvant chemotherapy with S-1 (Fig. 1). In the Ryukyu large cohort, 56 (18%) of 303 patients died (41 [18%] in the all neoadjuvant chemotherapy group and 15 [19%] in the up-front surgery group). The 6-year OS rate was 81.2% in the all neoadjuvant chemotherapy group and 79.5% in the surgery group ( P = 0.762; Fig. 2A). In the Ryukyu main cohort, 16 (13%) of 122 patients died (1 [2.2%] in the S-1 chemotherapy group and 15 [19%] in the up-front surgery group). Six-year OS rates were higher in patients receiving bleomycin plus S-1 (97.6% versus 79.5% for bleomycin plus S-1 and up-front surgery, respectively; Log-rank, P = 5.63×10 -3 ; Fig. 2B). In the Gunma cohort, 29 (29%) of 236 patients died (10 [7.4%] in the S-1 chemotherapy group and 19 [19%] in the up-front surgery group). Six-year OS rates were 92.4% in the S-1 group and 78.8% in the surgery group (p = 4.25×10 -3 ; Fig. 2C). Short-term neoadjuvant S-1 + bleomycin and S-1 may increase distant metastasis-free survival (DMFS) rates Sixteen (13%) of 122 patients in the Ryukyu main cohort had an event or died (15 [19%] in the up-front surgery group and 1 [2%] in the S-1 plus bleomycin chemotherapy group). In the Ryukyu main cohort, 6-year DMFS rates were 97.6% in the S-1 group and 79.5% in the surgery group (p = 5.57×10 -3 ; Fig. 2D). In the Gunma cohort, 32 (14%) of 236 patients had an event or died (10 [7.4%] in the S-1 group and 22 [22%] in the surgery group). In the Gunma cohort, 6-year DMFS rates were 91.7% in the S-1 group and 75.2% in the surgery group (p = 8.21×10 -4 ; Figure 2E). In the Ryukyu main and Gunma cohorts, baseline disease characteristics and demographics were generally balanced between groups (Table 3). Safety Although they were generally low grade, adverse events with pruritus (25.6%), white blood cell decreases (25.0%), and anemia (35.5%) were the most frequent in the S-1 plus bleomycin group in the Ryukyu main cohort (Table 4A). Grade 3 or worse adverse events of any cause occurred in 0 (0%) of 45 patients in the S-1 plus bleomycin group in the Ryukyu main cohort and in 1 (0.7%) of 136 patients in the S-1 group in the Gunma cohort; the most common events were thrombocytopenia (28.1%), anemia (28.6%), and white blood cell decreases (31.6%) (Table 4A, B). No deaths of any cause occurred in the neoadjuvant chemotherapy group in the Ryukyu main and Gunma cohorts. All patients proceeded with tumor resection. Subgroup analysis according to sex differences In the Ryukyu main male cohort, 11 (16%) of 68 patients died (0 [0%] in the S-1 plus bleomycin chemotherapy group and 11 [27%] in the up-front surgery group). Six-year OS rates were higher in male patients receiving neoadjuvant chemotherapy with S-1 plus bleomycin (100% versus 71.3% for neoadjuvant chemotherapy and up-front surgery, respectively; Log-rank, P = 2.89×10 -3 ; Fig. 3A). We then examined changes in OS rates in female patients between the two treatments, but did not observe significant differences (93.3% versus 86.6% for neoadjuvant chemotherapy and up-front surgery, respectively; Log-rank, P = 0.491). In the Gunma male cohort, 21 (16%) of 130 patients died (8 [10%] in the neoadjuvant chemotherapy with S-1 alone group and 13 [25%] in the up-front surgery group). Six-year OS rates were 89.4% in the S-1 alone group and 73.6% in the surgery group (p = 2.57×10 -2 ; Fig. 3B). A significant difference was also observed in the Gunma female cohort (96.4% versus 84.4% for neoadjuvant chemotherapy and up-front surgery, respectively; log-rank, P = 4.14×10 -2 ). The benefit of OS following S-1 versus surgery was generally consistent in the male subgroups (Fig. 3A, B). Eleven (16%) of 68 male patients in the Ryukyu main cohort had an event or died (11 [25%] in the up-front surgery group and 0 [0%] in the S-1 plus bleomycin chemotherapy group). In the Ryukyu main male cohort, 6-year DMFS rates were 100% in the S-1 group and 71.5% in the surgery group (p = 2.95×10 -3 ; Fig. 3C). In the Gunma cohort, 25 (19%) of 131 male patients had an event or died (8 patients [10%] in the S-1 group and 17 patients [32%] in the surgery group). In the Gunma male cohort, 6-year DMFS rates were 89.4% in the S-1 group and 66.9% in the surgery group (p = 1.93×10 -3 ; Fig. 3C). The DMFS benefit observed with S-1 versus surgery was generally consistent in the male subgroups in the Ryukyu main and Gunma cohorts (Fig. 3C). DNA repair genes show sex- and histological differentiation-specific expression The rate of distant metastasis was higher in male patients in the Ryukyu large and Gunma cohorts (Table 2) after surgery, suggesting the involvement of sex dimorphism in mediating the development of distant metastasis, which is consistent with previous findings [31]. We observed a relationship between the ratio of male patients and less well differentiated tumors in the Ryukyu large and Gunma cohorts and also in The Cancer Genome Atlas (TCGA) cohort (Fig. 4A). We investigated the underlying molecular mechanisms according to sex and histological differentiation as an exploratory objective. Reverse phase protein assay (RPPA) data from 211 patients and mRNA data, defined as RNA-Seq by Expectation-Maximization (RSEM), from 515 patients in TCGA were available for evaluation. We stratified patients based on sex and histological differentiation. We identified five sex- and histological differentiation-specific genes, two of which (MSH2 and MSH6) were involved in the mismatch repair (MMR) system [32] (Fig. 4B-E). Two other genes (XRCC1 and PCNA) were involved in the BER system [13,14] (Fig. 4B-F). A fifth specific gene (FOXM1) was a transcriptional regulator of XRCC1 and PCNA, exhibiting expression patterns similar to those of XRCC1 and PCNA (Fig. 4B-F). Therefore, the correlation between sex and histological differentiation may be explained by DNA repair systems. MSH2, MSH6, XRCC1, PCNA, and FOXM1 protein expression was significantly higher in tumors in male patients (P = 9.08×10 -3 , 2.93×10 -3 , 8.10×10 -3 , 2.17×10 -2 , and 1.30×10 -2 respectively; Fig. 4B), suggesting the involvement of DNA repair systems in mediating responses to 5-FU, which is consistent with previous findings [33]. These differences were driven by mRNA expression (Fig. 4C). Immunohistochemistry on OSCC samples revealed increases in the number of MSH2+ tumor cells in male patients (P = 0.049; Fig. 4D). Furthermore, tumors with distant metastasis showed the higher expression of MSH2 than those without distant metastasis (Fig. 4D), which may represent the correlation between the MMR status and synchronous metastases, as shown in colorectal cancer [34]. We also observed a relationship between the expression of DNA repair genes and less well differentiated tumors in the TCGA HNSCC cohort (Fig. 4E, F). The expression of PCNA was similar in tumors in male patients and less well differentiated HNSCC (Fig. 4B, C, E, F), which has been implicated in the development of distant metastasis [35]. Moreover, a linear regression analysis showed that increases in the mRNA expression of XRCC1 and PCNA correlated with the up-regulated expression of FOXM1 (Fig. 4G). These results suggest that the FOXM1-induced XRCC1 and PCNA pathways were more strongly activated in tumors in male patients and less well differentiated HNSCC. Accordingly, we hypothesized that differences in the expression of DNA repair genes between females and males may lead to different rates of somatic gene mutations between females and males. Whole-exome sequencing revealed higher rates of somatic gene mutations in females (P males. Specific somatic mutations were dependent on sex, including FAT1 , NOTCH1 , CASP8 , DMD , HUWE1 , PCDH11X , TENM1 , FBXW7 , ATRX , BRWD3 , and BIRC6 (Fig. 4H). Among these mutations, loss-of-function mutations in FAT1 and CASP8 were detected in the same tumor, and the knockdown of these genes induced terminal differentiation [28]. We showed that the ratio of well-differentiated tumors was significantly higher in patients with FAT1 mutations (18% in patients with mutations versus 11% in patients without mutations, P = 0.046) or CASP8 mutations (22% in patients with mutations versus 9.7% in patients without mutations, P = 0.0096) (Fig. 4I). We then investigated the relationship between the expression of MSH2, MSH6, XRCC1, and PCNA and the FAT1 and CASP8 DNA status. The mRNA expression of MSH2, MSH6, XRCC1, and PCNA was significantly lower in patients with FAT1 mutations (Fig. 4I) and also in patients with CASP8 mutations (Fig. 4I). Myd88 expression and type I interferon activity are higher in tumors in female patients or well-differentiated tumors Fusobacterium nucleatum has been reported to suppress MSH2 and MSH6 expression via the Myd88-dependent innate immune signaling pathway in HNSCC [36]; therefore, we hypothesized that the down-regulated expression of MSH2 or MSH6 in tumors in female patients or well-differentiated tumors may be induced by the Myd88 pathway. We performed an analysis of these genes in the TCGA cohort. RNA-seq in TCGA revealed higher Myd88 gene expression in tumors in female patients (P = 4.28×10 -10 ; Fig. 5A). We observed a negative relationship between Myd88 expression and less well differentiated tumors (Fig. 5B). Moreover, we found a negative relationship between the mRNA expression of MSH2 or MSH6 and that of Myd88 (Fig. 5C), suggesting the involvement of Myd88 in mediating the suppression of MSH2 and MSH6, which is consistent with previous findings [36]. We then examined the phenotypes of tumor cells with innate immunity activation in the HNSCC TCGA cohort. Tumors in female patients or well-differentiated HNSCC more highly expressed type I interferon signaling markers (IFIT and ISG15) (Fig. 5D) [37,38], which have roles in anti-angiogenesis and potentially contribute to the inhibition of distant metastasis [39]. Moreover, significant differences in the expression of Myd88, IFIT, and ISG15 were found between patients who harbored CASP8 mutations and those who did not (Fig. 5E). Collectively, these results suggest that innate immunity induced CASP8 mutations that retained resistance to cell death induced by type I interferon in tumors in some female patients or well-differentiated HNSCC [40, 41]. Pathways with an impact on effects of metronomic chemotherapy with S-1 Metronomic neoadjuvant chemotherapy with S-1 significantly increased OS rates in males (Fig. 3A-B) and patients with less well differentiated tumors [24, 25]. We confirmed the presence of sex- and histological differentiation-specific pathways that had an impact on the effects of metronomic chemotherapy, including the MMR and BER pathways [42]. Tumors in female patients or well-differentiated HNSCC highly expressed genes encoding for innate immunity signaling (Myd88, IFIT, and ISG15) and anti-apoptotic mutations ( CASP8 and FAT1 ) (Fig. 4H, I and Fig. 5A, D, G), which are related to type I interferon activity and terminal differentiation (Fig. 5H) [29, 37, 38]. In contrast, tumors in male patients or less well differentiated HNSCC highly expressed DNA repair genes (MSH2, MSH6, XRCC1, and PCNA) (Fig. 4C-E, 5I). PCNA has an inhibitory function in tumor immune microenvironment beyond its role in DNA repairment (35). Sex- and differentiation-specific genes included FOXM1 signaling in males or less well differentiated tumors (Fig. 4B-F, 5I), shown to be involved in VEGF upregulation (43). Wnt/β-catenin signaling was predicted in the female or well-differentiated HNSCCs, where tumors harbor higher frequency of FAT1 mutations which drive Wnt/β-catenin activation (Fig. 4H, I, 5G). Wnt/β-catenin signaling plays an important role in the formation of head and neck tissues. Conversely, Wnt/β-catenin dysregulation contributes to HNSCC (44). WNT7A mRNA expression shows enrichment in female (Fig. 5F) and well-differentiated HNSCCs (25), with higher expression in patients with FAT1 mutations (Fig. 5F). Given the known role of WNT7A in blood-brain barrier repair (45), we speculate that Wnt7A contributes to vascular normalization in the tumor microenvironment (Fig. 5G). Thus, vascular normalization via Wnt7A might contribute to the reduced distant metastasis. Type I interferons are signaling molecules that play an important role in inflammation. Type I interferon dysregulation contributes to cancer (46). Here we describe type I interferon activities with sexual and histological differences. By investigating the type I interferon marker expressions (IFIT and ISG15), we identified an enrichment of this signaling in female and well-differentiated HNSCCs (Fig. 5H). ISG15 has known roles in anti-angiogenesis and potentially contributing to inhibiting distant metastasis (47). Based on higher type I interferon activity and the higher frequency of CASP8 mutations, tumors in female patients or well-differentiated tumors were considered to be resistant to the 5-FU prodrug, S-1 (Fig. 4H, 4I, 5D, 5E, 5G). The synergistic inhibitory effects of type I interferon and 5-FU were previously demonstrated in in vitro studies [48]; however, the role and impact of type I interferon on the therapeutic effects of 5-FU for HNSCC have yet to be examined. Overall, correlations between sex difference and histological differentiations demonstrated the differences of DNA repair gene expressions and the frequency of somatic mutations, suggestive of effects on the tumor microenvironment. Discussion The present results showed a significant higher incidence of distant metastasis in male patients with OSCC or those with less well-differentiated tumors based on data collected from two independent institutions. In this retrospective study on two independent institutions, 6-year OS rates were significantly higher in patients at a high risk of distant metastasis, namely, male patients, treated with neoadjuvant chemotherapy with S-1 than in those who underwent up-front surgery in the Gunma cohort (89.4% versus 73.6%: P = 0.0257) and in the Ryukyu main cohort (100% versus 71.3%: P = 2.89×10 -3 ). Metronomic neoadjuvant chemotherapy with S-1 increased 6-year DMFS rates in male patients regardless of the institution. Male patients benefited from substantial disease control, with significant increases being observed in OS rates. Almost no safety signals were noted, although one patient developed adverse events toxicities, including a white blood cell decrease and thrombocytopenia. The present results showed that the expression of MSH2 proteins was higher in males than in females, which is consistent with previous findings on the role of MSH2 proteins in response to 5-FU [49]. DNA repair processes, such as BER and MMR, are crucial for responding to 5-FU [33]. XRCC1 and PCNA are involved in the BER process, while MSH2 and MSH6 play key roles in the MMR pathway [33]. The 5-FU treatment has been shown to down-regulate the expression of MSH2 and MSH6 in 5-FU-resistant colon cancer cell lines [50]. Collectively, these findings demonstrate that DNA repair systems may play a critical role in responses to 5-FU. Using immunohistochemistry, we showed that DNA repair gene, MSH2 expression, largely correlated with the frequency of developing distant metastasis, although the tendency of distant metastasis exists in line with specialized characteristics of tumor microenvironment (51), suggesting that DNA repair genes may affect the tumor microenvironment. A recent study on the abundance of F. nucleatum in HNSCC [36] enabled us to identify upstream regulators for MMR impairments in HNSCC. By using the suggested pathways reported by Hsueh et al., we demonstrated that Myd88 expression was dependent on sex and histological differentiation. Type I interferon activity, as measured by the expression of IFIT and ISG15 in HNSCC in TCGA, was higher in tumors in female patients or in well-differentiated HNSCC, in which the rate of distant metastasis was predicted to be low, corroborating previous findings highlighting the role of type I interferon in the inhibition of angiogenesis and vascular tumor growth [52]. An analysis of tumor characteristics in the HNSCC TCGA cohort revealed that the inflammatory phenotype and markers were also dependent on sex and histological differentiation. In the TCGA cohort, well-differentiated tumors and tumors in female patients had higher inflammatory gene expression profiles, including signatures for type I IFN responses and Myd88 up-regulation. This result suggests that profiling of the baseline inflammatory status facilitates the early identification of (non)responders to metronomic neoadjuvant chemotherapy and needs to be considered in addition to sex and histological differentiation. This study had some limitations that need to be addressed. This retrospective study did not include a balanced control group, which limits the ability to make proper comparisons with standard treatment options. Furthermore, the effects of neoadjuvant chemotherapy on females varied in the cohort under study, which revealed that the small sample size may have affected the study’s statistical power as well as the generalizability of the results obtained. Although this study did not show interactions between subgroups (male vs. female), the benefits observed in male patients were similar to those reported in other studies on adjuvant chemotherapy for HNSCC. Another limitation of this clinical study was the use of a previous staging system. While patients are now diagnosed with OSCC according to the AJCC 8th edition TNM staging system, patients in the present study were diagnosed according to the previous AJCC edition. Moreover, the pathological complete response rate was not investigated as an endpoint, but is an important measure of neoadjuvant therapy efficacy. In conclusion, several factors, including patient selection, need to be considered prior to the administration of neoadjuvant chemotherapy with S-1 in clinical practice. The results of this retrospective study across two independent institutions suggest the effectiveness of neoadjuvant chemotherapy including S-1 for male patients with stage I-III OSCC. The results obtained herein indicate that the guidance of neoadjuvant chemotherapy with S-1 may be personalized based on individual patient characteristics in clinical practice. The present study provides valuable insights for the selection of neoadjuvant chemotherapy, including S-1, to manage stage I-III OSCC. Methods Study design This was a retrospective study of 2 medical institutions in 2 prefectures in Japan. This study was approved by the institutional ethics committee at each study site (approval number 30 and HS2017-142). Patients in the Ryukyu large cohort were collected between August 4, 1987 and July 30, 2013, and those in the Gunma cohort between July 10, 2008 and March 28, 2017. Eligible patients were ≥18 years with previously untreated clinical stage I-III OSCC as defined by a T3 or smaller primary lesion, the presence of a single positive node, or the absence of positive nodes without evidence of distant metastatic disease per American Joint Committee on Cancer guidelines 7 version. Sex was self-reported by patients. Patients were excluded from the present study if they were not candidates for surgery. Written informed consent was provided by all study patients. Treatment The following four treatment cohorts were collected in the Ryukyu large cohort: bleomycin + S-1 (main cohort), bleomycin + UFT, bleomycin + UFT, and cisplatin-based chemotherapy, and up-front surgery (main cohort). The chemotherapy regimen was bleomycin, administered intravenously at a dose of 15 mg over a period of 1 h twice a week for 3 weeks. During this period, 450-mg UFT-E granules were administered orally three times a day or 100-mg/day S-1 granules were administered orally. After chemotherapy with bleomycin, some patients received cisplatin-based chemotherapy and underwent surgical dissection. Surgery was performed 1 week after the completion of neoadjuvant chemotherapy. The following two treatment cohorts were collected in the Gunma cohort: patients received 120 mg of S-1 orally twice daily for 2 weeks and surgery was performed 1 week after the completion of neoadjuvant chemotherapy, or patients underwent up-front surgery. Toxicity data were classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. Eligibility Patient inclusion criteria were as follows: 1. Histologically confirmed diagnosis of OSCC. 2. No distant metastasis. 3. 18 years or older and 85 years or younger. The patient exclusion criterion was as follows: 1. Previous history of OSCC. “‘latex Outcomes The primary endpoints assessed in the two cohorts were the 6-year rates of DMFS (time from the first visit to distant recurrence or death due to any cause) and OS (time from the first visit to death from any cause) RNA-seq data analysis in TCGA Gene expression data were obtained as RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) (log2) from TCGA [53]. Differentially overexpressed genes in well-differentiated tumors were identified in the HNSCC TCGA cohort. Immunohistochemistry Paraffin sections were cut at a thickness of 4 μm and deparaffinized. Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0) at 121℃ for 10 min. MSH2 was detected using the clone G219-1129 (1:100 dilution, at 4℃ overnight, Cell Marque, #286M-14). The bound antibody was detected using a horseradish peroxidase-conjugated secondary antibody (HISTOFINE #424134, Nichirei Bioscience Inc.) and DAB as the substrate. Slides were counterstained with hematoxylin. MSH2+ cells were defined based on the level of MSH2 protein expression in the nuclei of tumor cells. Slides were examined at ×400 magnification. Statistical analysis OS was defined as the time from the first visit to death due to any cause. DMFS was defined as the time from first visit to distant metastasis or death due to any cause. Patients were censored if no event occurred by the last follow-up. OS was estimated using the Kaplan–Meier method, and survival curves were compared using the Log-rank test. The distribution of continuous variables (for example, the RSEM values of MSH2) between the two groups was compared using the Student’s t -test. Comparisons of the frequency of mutations were performed using two-tailed Fisherʼs exact tests. Statistical analyses were conducted using EZR. Adverse events were tabulated. Figures were generated using EZR. Fig. 1. Study cohort. The main cohort includes patients who underwent up-front surgery or neoadjuvant chemotherapy with bleomycin plus S-1 in the Ryukyu large cohort. Table 1. Baseline and demographic characteristics in the Ryukyu large cohort (A) and Gunma cohort (B). a Median (range) and b number of patients (percentage). A B Table 2. Factors associated with the development of distant metastasis A. Clinical characteristics of patients with distant metastasis in the Ryukyu large cohort. B. Clinical characteristics of patients with distant metastasis in the Gunma cohort. C. Multivariable analysis of distant metastasis in the Ryukyu large cohort. D. Multivariable analysis of distant metastasis in the Gunma cohort. Multivariable logistic regression for distant metastasis. P values were calculated using multivariable logistic regression. Two-sided P values were calculated. a Less well differentiation includes well- to moderately, moderately, poorly to moderately, and poorly differentiated tumors. A B C D Fig. 2 Clinical responses of OSCC patients treated with neoadjuvant chemotherapy and up-front surgery. A, Kaplan–Meier curve showing OS in the Ryukyu large cohort. Six-year OS rates were 81.2% in the all neoadjuvant chemotherapy group and 79.5% in the surgery group ( P = 0.762). B, Six-year OS rates were 97.6% in the short-term neoadjuvant chemotherapy with S-1 plus bleomycin group and 79.5% in the surgery group ( P = 5.67×10 -3 ). C, Kaplan–Meier curve showing OS in the Gunma cohort. Six-year OS rates were 92.4% in the S-1 group and 78.8% in the surgery group (p = 4.25×10 -3 ). D, Kaplan–Meier curve showing DMFS in the Ryukyu main cohort. Six-year DMFS rates were 97.6% in the neoadjuvant chemotherapy with S-1 plus bleomycin group and 79.5% in the surgery group ( P = 5.57×10 -3 ). E, Kaplan–Meier curve showing DMFS in the Gunma cohort. Six-year DMFS rates were 91.7% in the neoadjuvant chemotherapy with S-1 alone group and 75.2% in the surgery group ( P = 8.21×10 -4 ). Table 3. Baseline and demographic characteristics in the Ryukyu main cohort; A, and Gunma cohort; B. Data are n (%) or medians (range). A B Table 4. Summary of adverse events in the neoadjuvant chemotherapy group in two cohorts. Data are n (%). Reported events occurred in at least 10% of patients in any group. A, Ryukyu main cohort B, Gunma cohort Fig. 3 Clinical responses of OSCC patients treated with neoadjuvant chemotherapy and up-front surgery according to sex. A, Kaplan–Meier curves showing OS in the Ryukyu main cohort of males and females. Six-year OS rates in male patients were 100% in the neoadjuvant chemotherapy with S-1 plus bleomycin group and 71.3% in the surgery group ( P = 2.89×10 -3 ). Six-year OS rates in female patients were 93.3% in the neoadjuvant chemotherapy with S-1 plus bleomycin group and 86.6% in the surgery group ( P > 0.05). B, Kaplan–Meier curves showing OS in the Gunma cohort of males and females. Six-year OS rates in male patients were 89.4% in the neoadjuvant chemotherapy with S-1 alone group and 73.6% in the surgery group ( P = 2.57×10 -2 ). Six-year OS rates in female patients were 96.4% in the neoadjuvant chemotherapy with S-1 alone group and 84.4% in the surgery group ( P = 4.14×10 -2 ). C, Kaplan–Meier curves showing DMFS in the Ryukyu main male cohort and that in the Gunma cohort. Six-year DMFS rates in male patients were 100% in the neoadjuvant chemotherapy with S-1 plus bleomycin group and 71.5% in the surgery group ( P = 2.95×10 -3 ) in the Ryukyu main cohort. Six-year DMFS rates in male patients were 89.4% in the neoadjuvant chemotherapy with S-1 alone group and 66.9% in the surgery group ( P = 1.93×10 -3 ) in the Gunma cohort. Fig. 4 Gene expression patterns associated with sex and histological differentiation. A, Ratios of male patients relative to all OSCC (Ryukyu large plus Gunma cohorts) and HNSCC (TCGA) patients in relation to histological differentiation. Variables were compared by the Cochran–Armitage trend test. n = 539 patients (Ryukyu large plus Gunma cohorts) and 494 patients (TCGA), respectively. B, DNA repair protein expression according to the RPPA in 62 females and 149 males from the HNSCC TCGA cohort. C, DNA repair gene mRNA expression according to the RSEM in the tissues of 135 female and 380 male HNSCC patients in the HNSCC TCGA cohort. D, Representative example of a MSH2-stained tissue section (hematoxylin = blue, MSH2 = brown) (Upper). MSH2 expression in the tumor cells of males and females patients. n = 41 patients (Lower, left). MSH2 expression in the tumor cells of patients with and without distant metastasis. n = 32 patients (Lower, right). E, DNA repair protein expression in the tissues of HNSCC patients in relation to histological differentiation in the HNSCC TCGA cohort. n = 16 (Well-differentiated), n = 134 (Moderately differentiated), n = 53 (Poorly differentiated). F, DNA repair gene mRNA expression according to the RSEM in the tissues of HNSCC patients with well-differentiated tumors (62), moderately differentiated tumors (301), and poorly differentiated tumors (123) in the HNSCC TCGA cohort. G, Correlation of XRCC1 and PCNA mRNA expression with FOXM1 mRNA expression. H, Relationship between sex and genetic mutations. The prevalence of the indicated genetic mutations in females and males, in addition to the log 10 P value for the difference in prevalence between females and males, are shown. I, FAT1 and CASP8 mutations correlated with well-differentiated tumors (P = 0.046 and 0.027, respectively). J, MSH2, MSH6, XRCC1, and PCNA mRNA expression in HNSCC patients with and without FAT1 mutations in the TCGA cohort (Upper). MSH2, MSH6, and XRCC1 mRNA expression in HNSCC patients with and without CASP8 mutations in the TCGA cohort (Lower). Error bars in D indicate the standard error. Boxplots display the median value, with the lower and upper hinges corresponding to the first and third quartiles, respectively. The upper whisker extends from the upper hinge to at most the 1.5× interquartile range and the lower whisker extends from the lower hinge to at most the 1.5× interquartile range. P values were derived using the two-sided Welch t -test. Correlations were estimated with Spearman’s rank correlation coefficient, two-sided. Fig. 5 Sex- and histological differentiation-specific signaling in HNSCC. A, Myd88 mRNA expression according to the RSEM in the tissues of 135 female and 380 male patients in the HNSCC TCGA cohort. B, Myd88 mRNA expression according to the RSEM in the tissues of patients with well-differentiated tumors (62), moderately differentiated tumors (301), and poorly differentiated tumors (123) in the HNSCC TCGA cohort. C, MSH2 and MSH6 mRNA expression using RSEM in relation to Myd88 mRNA expression in the tissues of HNSCC patients (TCGA). n = 515 patients. D, IFIT and ISG15 mRNA expression according to the RSEM in the tissues of 135 female and 380 male patients in the HNSCC TCGA cohort (Upper). IFIT and ISG15 mRNA expression according to the RSEM in the tissues of patients with well-differentiated tumors (62), moderately differentiated tumors (301), and poorly differentiated tumors (123) in the HNSCC TCGA cohort (Down). E, Myd88, IFIT, and ISG15 mRNA expression in patients with and without CASP8 mutations in the HNSCC TCGA cohort. F, Wnt7A mRNA expression according to the RSEM in the tissues of 135 female and 380 male patients in the HNSCC TCGA cohort (Left). Wnt7A mRNA expression in HNSCC patients with and without FAT1 mutations in the TCGA cohort (Right). Boxplots display the median value, with the lower and upper hinges corresponding to the first and third quartiles, respectively. The upper whisker extends from the upper hinge to at most the 1.5× interquartile range and the lower whisker extends from the lower hinge to at most the 1.5× interquartile range. P values were derived using a two-sided Welch t -test. 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Funding This work was supported by JSPS KAKENHI Grant Number 23K16141 and 24K13062 Author contributions Study design and assembly: S.K., M.K.T., M.O., A.A. and S.Y. Data generation— histology: S. K. and S.M. Data analysis: S.K. and S.M. Data interpretation: S.K. Manuscript writing: S.K. Manuscript review and editing: S.K., S.M., M.K.T., M.O., A.A., and S.Y. Competing interests The authors declare no competing interests. Supplementary Material File (image1.emf) Download 108.46 KB File (image10.emf) Download 51.02 KB File (image2.emf) Download 100.08 KB File (image3.emf) Download 79.93 KB File (image4.emf) Download 77.76 KB File (image5.emf) Download 217.83 KB File (image6.emf) Download 215.64 KB File (image7.emf) Download 77.30 KB File (image9.emf) Download 51.57 KB Information & Authors Information Version history V1 Version 1 21 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords biological sex cancer and carcinogenesis cancer pharmacology Authors Affiliations SHINICHIRO KINA [email protected] Graduate School of Medicine, University of the Ryukyus View all articles by this author Sho Miyamoto Sapporo Medical University School of Medicine View all articles by this author Mika Kina-Tanada Gunma University Graduate School of Medicine School of Medicine Faculty of Medicine View all articles by this author Masaru Ogawa Gunma University Graduate School of Medicine School of Medicine Faculty of Medicine View all articles by this author Akira Arasaki University of the Ryukyus Faculty of Medicine Graduate School of Medicine View all articles by this author Satoshi Yokoo Gunma University Graduate School of Medicine School of Medicine Faculty of Medicine View all articles by this author Metrics & Citations Metrics Article Usage 196 views 106 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation SHINICHIRO KINA, Sho Miyamoto, Mika Kina-Tanada, et al. 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