The prognostic impact of reduction of 5-hydroxymethyl-2'-deoxycytidine in non-tumoral tissue of upper tract urothelial carcinoma patients evaluated by mass spectrometry

The prognostic impact of reduction of 5-hydroxymethyl-2'-deoxycytidine in non-tumoral tissue of upper tract urothelial carcinoma patients evaluated by mass spectrometry | 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 Article The prognostic impact of reduction of 5-hydroxymethyl-2'-deoxycytidine in non-tumoral tissue of upper tract urothelial carcinoma patients evaluated by mass spectrometry Ayana Takemura, Yuto Matsushita, Yuji Iwashita, Hidetaka Yamada, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8812038/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Reduction of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) is related to poor prognosis in malignant tumor patients. However, reports on its role in urological cancers, particularly upper tract urothelial carcinoma (UTUC), are still limited. This study evaluated the relationship between 5hmdC levels and oncological outcomes including overall survival (OS) and metastasis-free survival (MFS) in patients with non-metastatic UTUC. Tumoral and non-tumoral tissues were obtained from patients who underwent nephroureterectomy between March 2012 and December 2024. 5hmdC accumulation was assessed by immunohistochemistry (IHC), and quantified using molar ratio by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Eighty-five and 82 paired samples were evaluated by IHC and LC-MS/MS, respectively. The significant reduction of 5hmdC accumulation in tumoral tissue compared with non-tumoral tissue was observed by both evaluations ( P < 0.001 and P < 0.001, respectively). In the investigation of prognostic impact, low molar ratio in non-tumoral tissue demonstrated significantly poor overall survival than the other (68.8 months vs. not reached, P = 0.035) and inferior trend of MFS (not reached vs not reached, respectively, P = 0.061). In conclusion, the reduction of 5hmdC in non-tumoral tissue may be associated with poor prognosis in patients with non-metastatic UTUC. Biological sciences/Cancer Health sciences/Oncology 5hmdC upper tract urothelial carcinoma liquid chromatography coupled with tandem mass spectrometry prognosis epigenetic biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In the development and progression of cancer, epigenetic modifications including DNA methylation and demethylation in CpG island have the critical role [ 1 ]. 5-hydroxymethyl-2'-deoxycytidine (5hmdC) is generated by the ten-eleven translocation (TET) enzyme family during DNA demethylation process [ 2 ]. Previously, the importance of hydroxymethylation of cytosine, detected as 5hmdC in cancer was reported. First, association with carcinogenesis were reported: 5hmdC level was reduced in tumoral tissue of various cancers [ 3 – 5 ] and its accumulation was reduced even in non-tumoral tissue of UTUC patient compared with non-UTUC patients [ 6 ]. Secondly, 5hmdC reduction was related to oncological prognosis in patients with melanoma, lung, hepatocellular head and neck, and prostate cancer [ 7 – 14 ]. Furthermore, 5hmdC modification in cell-free DNA (cfDNA) have emerged as an epigenetic marker in early cancer detection and monitoring cancer conditions [ 15 – 18 ]. However, there are limited research about the role of 5hmdC in urothelial carcinoma (UC), particularly upper tract urothelial carcinoma (UTUC). UTUC presents unique challenges due to its higher invasiveness at diagnosis compared to bladder urothelial carcinoma, resulting in poorer prognosis. Unlike bladder cancer, management of UTUC cannot be fully extrapolated from lower tract disease owing to anatomical, biological, and molecular differences. Epigenetic modifications, such as 5hmdC, have been implicated in urothelial carcinogenesis [ 19 ]. While global loss of 5hmdC has been observed in bladder cancer, its role in UTUC remains largely unexplored, highlighting the need for UTUC specific investigations to identify novel diagnostic and prognostic biomarkers. Understanding the relationships between 5hmdC levels and oncological outcomes in UTUC patients could provide insights into its potential as prognostic biomarker and guide the development of novel therapeutic strategies. We previously reported the 5hmdC reduction in non-tumoral urothelial tissue of UTUC patients [ 6 ], however, the relationships between 5hmdC reduction and oncological outcomes were not investigated. In this study, we investigated the relationship between oncological outcomes of non-metastatic UTUC patients and 5hmdC levels in tumoral tissue and non-tumoral tissues using immunohistochemical staining (IHC) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Results Patient characteristics and prognosis Patient characteristics are summarized in Table 1 . A total of 85 patients with UTUC were included, with a median age of 75 years. The most common pT category was pT3 (40.0%), and a small subset presented with concomitant carcinoma in situ (CIS) (9.4%) that including 5 cases were CIS only, 2 were T2 + CIS, and 1 was T1 + CIS. The predominant histological subtype was urothelial carcinoma without any histological subtype (83.5%). Neoadjuvant chemotherapy (NAC) was administered to 18 patients (21.1%): 8 received gemcitabine plus cisplatin and 10 received gemcitabine plus carboplatin. Adjuvant therapy (AC) was given to 17 patients (20.0%), including immune checkpoint inhibitor in 5 received nivolumab and platinum-based combinations therapy in 3 received gemcitabine plus cisplatin and 9 received gemcitabine plus carboplatin. Table 1 Patients characteristics in this study. Total 85 patients were investigated. *Only CIS patients were included. n = 85 Median age (years, range) 75 (49–90) Gender (%) Male 58 (68.2) Female 27 (31.8) Site of primary tumor (%) Renal pelvis 50 (58.8) Ureter 35 (41.2) Laterality (%) Left 50 (58.8) Right 35 (41.2) pT category (%) Ta 4 (4.7) Tis* 5 (5.9) T1 21 (24.7) T2 18 (21.2) T3 34 (40.0) T4 3 (4.7) Concomitant CIS (%) Yes 8 (9.4) No 77 (90.6) Histology (%) UC 71 (83.5) UC with squamous differentiation 10 (11.8) UC with glandular differentiation 1 (1.2) UC with sarcomatoid subtype 2 (2.4) UC with micropapillary subtype 1 (1.2) Neoadjuvant chemotherapy (%) Yes 18 (21.2) No 67 (78.8) Adjuvant therapy (%) Yes 17 (20.0) No 68 (80.0) In the observation period, metastatic recurrence and overall death was observed in 31 (28.3%), and 31 (28.3%) patients, respectively. The median OS and MFS were 48.8 months and 44.7 months, respectively. Difference of H-score of 5hmdC between tumoral and non-tumoral tissue Figure 1 illustrates the immunohistochemical staining of 5hmdC in tumoral tissue and non-tumoral urothelial tissues in UTUC patients. A total of 84 tissues were able to be evaluated, respectively (Supplementary Table S1 ). The median H-score in tumor tissue and non-tumoral tissue were 33 and 204, respectively and it was significantly lower in tumoral tissue than non-tumoral tissue ( P < 0.001) (Fig. 1 c). Relationship between H-score of 5hmdC and prognosis Patients were categorized based on the H-score into two groups. The cutoff values were determined by Youden’s index, and they were 30 for tumoral tissue and 150 for non-tumoral tissue. According to the H-score of tumoral tissue, OS nor MFS were not statistically different between the two groups ( P = 0.090 and 0.68, respectively). On the other hand, although the difference did not reach statistical significance, overall survival (OS) tended to be shorter in the low H-score group compared with the high H-score group in non-tumoral tissue (median OS was 65.4 vs not reached, respectively P = 0.075, HR = 1.92 (95%CI 0.92–3.99)) (Fig. 2 a). Metastasis free survival (MFS) was not statistically different between two groups (median MFS was not reached vs not reached, respectively, P = 0.13, HR = 0.88 (95%CI 0.38- 2.0)) (Fig. 2 b). Comparison of 5hmdC measurement by LC-MS/MS using FFPE and frozen tissue DNA In order to assess the feasibility of using FFPE instead of frozen tissues, we analyzed the 5hmdC molar ratio in FFPE and fresh-frozen tissue from same patient. In total, 15 paired non-tumoral tissue samples were analyzed. The median molar ratios in FFPE and fresh-frozen tissues were 1.4 × 10 − 3 and 1.4 × 10 − 3 , respectively (Fig. 3 a). Figure 3 b shows the significant correlation between FFPE and fresh-frozen tissues (correlation coefficient = 0.72, P = 0.0036). Reduction of 5hmdC molar ratio by LC-MS/MS in tumoral and non-tumoral tissues and their positive correlation with H-Score In 3 samples of tumor tissue and 12 samples of non-tumoral tissue, sufficient amount of DNA was not extracted because of the small amount of tissues. Then, we analyzed 82 tumor tissue samples and 73 non-tumoral tissue samples (Supplementary Table S1 ). The median molar ratios of 5hmdC were 7.7 × 10 − 4 , and 1.8 × 10 − 3 , respectively (Fig. 4 a), and, a significant reduction of 5hmdC molar ratio was shown in tumor tissue compared with non-tumoral tissue ( P < 0.001). A significant positive correlation was observed between H-score and 5hmdC molar ratio in UTUC patient tissues (correlation coefficient = 0.70, P < 0.001) (Fig. 4 b). Relationship between molar ratio of 5hmdC and prognosis Patients were divided into two groups according to the amount of 5hmdC molar ratio, based on cutoff values of 1.0 × 10 − 3 in tumoral tissue and 2.0 × 10 − 3 in non-tumoral tissue. In Supplementary Table S2, patients characteristics according to molar ratio in non-tumoral tissue were described. According to the molar ratio of tumoral tissue, OS nor MFS were not statistically different between the two groups ( P = 0.49 and 0.89, respectively). On the other hand, in the analysis using non-tumoral tissue, low molar ratio group also showed poor trend of MFS than high molar ratio group (both median MFS was not reached, P = 0.061, HR = 2.33 (95%CI 0.94–5.78)). In addition, the low molar ratio group had significantly shorter OS compared to the high molar ratio group (median OS was 68.8 months vs not reached, P = 0.035, HR = 2.57 (95%CI 1.04–6.36)) as shown in Fig. 5 . Discussion UTUC is relatively uncommon disease, in substance, it occupied only 5–10% of urothelial carcinoma. However, the incidence has risen because of the aging of population. For the patients with non-metastatic UTUC, radical nephroureterectomy is primary treatment option because kidney sparing surgery is adopted to limited low-risk patient [ 20 ]. The risk factors of poor prognosis are histological grade, multiplicity, presence of hydroureteronephrosis and tumor size and these are used for decision making of surgical treatment. According to the recurrent risk based on pT stage and pN stage, adjuvant systemic therapy is recommended after surgery based on the prospective randomized study, however, the oncological benefit was not described in UTUC subgroup [ 20 ]. Then, more precise risk stratification is expected because over treatment and treatment-related adverse event are inevitable for systemic therapy. In other words, investigation of prognostic factor in patients with non-metastatic UTUC is essential and major concern in clinical practice. 5hmdC is associated with both DNA methylation and demethylation: It is an oxidized derivative of 5mdC, generated during DNA demethylation by the TET family of enzymes [ 2 ]. It is known to contribute to the development of various cancers through epigenetic regulation [ 1 , 3 ] and it is highly enriched in promoter and enhancer regions upstream of transcriptional start sites and has been implicated in the regulation of gene expression [ 21 , 22 ]. 5hmdC reduction due to passive dilution during DNA replication [ 23 ] in tumor tissue is reported in a lot of kind of cancer [ 13 , 24 ], and association of 5hmdC reduction with cancer prognosis was also reported in melanoma, lung, hepatocellular, head and neck, parathyroid and prostate cancer [ 7 – 14 ]. Based on these studies, 5hmdC is expected to be a novel biomarker for cancer prognosis and response to immunochemotherapy [ 10 ]. Furthermore, 5hmdC has been proposed as a potential therapeutic target that could enhance the efficacy of conventional cancer treatments [ 21 ]. LC-MS/MS based analytical approaches have been proposed as reliable methods for the accurate detection and quantification of DNA adducts, including 5hmdC [ 25 ]. Not only 5hmdC, a wide range of DNA adducts can be comprehensively analyzed using this platform, and these adducts are increasingly recognized as potential biomarkers across various cancer types [ 26 – 29 ]. Accordingly, DNA adductome analysis represents a promising and rapidly developing research field with potential applications in cancer biomarker discovery [ 25 , 30 ]. Previously, we established the quantitation methods of 5hmdC using LC-MS/MS and reported 5hmdC reduction in non-tumoral tissue in UTUC patient [ 6 ]. In the present study, we investigated the prognostic impact of 5hmdC reduction on oncological outcomes in patents with non-metastatic UTUC. In the investigation of carcinogenesis, the evaluation of non-tumoral tissue serves as a critical methodology. In bladder cancer, which shares a common urothelial origin with UTUC, somatic mutations within non-tumoral urothelial tissue have been associated with cancer progression and clinical prognosis [ 31 – 35 ]. Furthermore, 5hmdC reduction in non-tumoral tissue of patients with gastric cancer or UTUC has been previously reported [ 5 , 6 , 30 ]. We hypothesized that 5hmdC depletion in non-tumoral tissue may be indicative of field cancerization and potentially correlates with oncological outcomes. Additionally, we posited that evaluating non-tumoral tissue might provide a more sensitive diagnostic indicator, given that 5hmdC levels in tumoral tissue are already substantially diminished. Consequently, we quantified 5hmdC levels in both tumoral and non-tumoral tissues. We demonstrated the reduction of 5hmdC in tumoral tissue compared to non-tumoral tissue in UTUC patients using IHC (Fig. 1 ), and it is similar to the other cancer [ 7 – 14 ]. So, genes with reduced 5hmdC levels are involved in proliferation, metabolism, and cell adhesion pathways, suggesting that 5hmdC loss may also contribute to epigenetic regulation of traits related to cell proliferation in UTUC. These findings indicate that, in UTUC, reduced 5hmdC may promote tumor progression through mechanism similar to those observed in other cancers [ 36 ]. Previous studies have reported that reduced 5hmdC levels in tumoral tissue which evaluated using H-score are associated with poor outcomes in several cancers as mentioned above [ 7 , 26 ]. In bladder cancer which are similar urothelial carcinoma to UTUC, Munari et al. reported the 5hmdC loss did not influence prognosis [ 24 ]. In contrast, Peng et al. showed that decreased 5hmdC levels predicted poorer prognosis, higher stage, and lymph node metastasis in bladder cancer [ 37 ]. In the present study, 5hmdC reduction evaluated by H-score in tumoral tissue showed no statistical significance on OS and MFS. Collectively, the association of 5hmdC reduction by IHC of urothelial tumoral tissue with prognosis is controversial, and it is not useful in patient with UTUC. In non-tumoral tissue, 5hmdC depletion evaluated by IHC also showed no statistically significant association with OS or MFS, however the OS tended to be shorter in the low H-score group (Fig. 2 ). Then, we evaluated 5hmdC using LC-MS/MS in FFPE tissues. Although fresh-frozen tissues are generally considered to better reflect physiological conditions [ 38 ], their limited availability restricts their use in large retrospective studies, particularly for rare malignancies such as UTUC. In contrast, FFPE tissues are more accessible in routine clinical practice but may be affected by fixation-related artifacts [ 39 ]. To address this concern, we examined the correlation of 5hmdC measurements between fresh-frozen and FFPE samples and observed a comparatively strong correlation in non-tumor urothelial tissues (Fig. 3 ). This finding supports the validity of FFPE based LC-MS/MS quantification of 5hmdC and highlights its utility for retrospective epigenetic analyses using archived tissues, thereby enabling robust investigation of prognostic biomarkers in rare diseases. Similar to 5hmdC level using IHC, 5hmdC level evaluated using LC-MS/MS was also reduced in tumor tissue compared to non-tumoral tissue. And the significant correlation of the result of these two methods was observed as shown in Fig. 4 . The impact of 5hmdC reduction measured by LC-MS/MS on oncological prognosis were analyzed. The molar ratio of 5hmdC in tumoral tissue showed no statistical significance on OS nor MFS. However, in non-tumoral tissue, low molar ratio group demonstrated significantly inferior OS and trend of shorter MFS. These results indicated 5hmdC molar ratio measured by LC-MS/MS in non-tumoral tissue may be useful for predicting poor prognosis. To our knowledge, this study provides the first evidence that describes the relationships between prognosis and amount of 5hmdC in non-tumoral tissue measured by LC-MS/MS in UTUC patients. The discrepancy between the prognostic performance of the molar ratio of 5hmdC and the H-score of 5hmdC may be explained by the fact that manual scoring in IHC is semi-quantitative and susceptible to observer bias [ 40 – 42 ], whereas LC-MS/MS is considered to provide more accurate quantification of 5hmdC levels in biological samples [ 43 – 45 ]. This study has several limitations that should be acknowledged. First, the comparatively small number of patients and retrospective design may introduce selection bias. Second, perioperative management was not uniform across patients, including variations in neoadjuvant and adjuvant treatments, which may have acted as potential confounding factors in the survival analyses. Third, we didn’t evaluate host-related factors such as smoking history, and chronic inflammation that may influence the accumulation of 5hmdC. Fourth, Multivariate Cox regression analysis was not performed due to the limited number of events relative to potential confounding variables. Fifth, FFPE sample is inevitable for variation in fixation and storage time which lead to DNA degradation. Finally, we analyzed only global levels of 5hmdC, therefore evaluating specific epigenetic alteration are expected in the future study. We showed 5hmdC reduction in tumoral tissue compared to non-tumoral tissue of UTUC patients. 5hmdC reduction evaluated by LC-MS/MS in non-tumoral tissue are associated with poor prognosis in patient with non-metastatic UTUC. Methods Patients and Tissue samples All procedures were performed in accordance with the Declaration of Helsinki and the use of residual pathological tissue in this research was approved by the Clinical Research Review Board of Hamamatsu University School of Medicine (No. 20–011). The requirement for written informed consent was waived by the same Clinical Research Review Board. An opt-out approach was used to obtain informed consent from all subjects. A total of 85 consecutive patients who underwent nephroureterectomy for non-metastatic upper tract urothelial carcinoma (UTUC) between January 2012 and December 2024 at Hamamatsu University Hospital were included in this study. Histologically confirmed tumor tissue and non-tumoral tissue samples were obtained from formalin-fixed paraffin-embedded (FFPE) specimens. Clinicopathological features and prognostic data were collected retrospectively by reviewing electronic medical records. The variables included age, sex, pathological diagnosis, peri surgical chemotherapy, MFS and OS. MFS and OS were defined as the time from nephroureterectomy to metastasis or death, and the time from surgery to overall death, respectively. Immunohistochemistry (IHC) Four-micron paraffin-embedded sections were deparaffinized using xylene and rehydrated in a graded series of distilled water and alcohol. Three hydrogen peroxide in distilled water was used to block endogenous peroxidase and slides were stained with anti-5-hydroxymethylcytosine (5hmC) antibody (1:2000 dilution; No.39069; Rabbit; Active Motif, Carlsbad, CA, USA) according to the below protocol. Antigen retrieval was performed using Tris/EDTA buffer, followed by incubation with 3.5N hydrochloric acid for 15 minutes at room temperature before primary antibody labeling. The anti-5hmC antibody as mentioned above was applied for 30 minutes at room temperature, followed by secondary antibody (MAX-PO (MULTI); Rat and rabbit; No.424154; Nichirei, Tokyo, Japan) labeling for 30 minutes. Immunocomplexes were detected with 3,3'-diaminobenzidine tetrahydrochloride (DAB) as a chromogenic system. Scoring systems of IHC We calculated H-score for scoring of presence of 5hmdC which is evaluated from 0 to 300 by the classification of the percentage of positive cells and the score of staining intensity (0 is negative, 1 is weekly positive, 2 is moderately positive, and 3 is strongly positive) [ 46 ] with QuPath software ver. 0.5.1 which is one of the open source digital pathology system [ 47 ]. We evaluated H-score of three spots for each cases, and calculated the average of these spots to quantify the 5hmC positive cells (Supplementary Figure S1 ). Verification of the feasibility of using FFPE instead of fresh-frozen tissue in non-tumoral tissue. We used FFPE in this retrospective study and verified the feasibility of data from FFPE by analyzing the correlation between FFPE and fresh-frozen tissue data obtained from our previous study [ 6 ]. Fresh-frozen samples obtained immediately after resection were frozen in liquid nitrogen and stored at − 80°C in a deep freezer until DNA extraction. DNA extraction from FFPE and enzymatic digestion DNA extraction was performed using the Quick-DNA™ FFPE kit (D3067; Zymo Research, Irvine, CA, USA). Two 20-um FFPE sections were deparaffinized by the protocol described above. Organizational processing was conducted by an experienced urologist, carefully removed excess tissue from tumoral and non-tumoral portions of each samples, following the kit protocol. The median specimen areas of tumoral tissue and non-tumoral tissue were 3.3 cm², and 1.8 cm², respectively, with median DNA yields of 11.4 µg and 0.99 µg, respectively. Nano Drop ONE spectrophotometer (Thermo Scientifis, Tokyo, Japan) was used to quantify the purified DNA amount by measuring absorbance at 260 nm. DNA solutions were adjusted to concentration of 250 ng/45 uL for liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using the 8-OHdG Assay Preparation Reagent Set (No.292-67801; Wako, Osaka, Japan). Adjusted DNA solution as mentioned above was incubated at 98°C for 2 minutes, cooled on ice for 5 minutes, and then mixed with 6.7 µL of Acetic Acid Buffer and 3.5 µL of Nuclease P1, followed by incubation at 37°C for 30 minutes. Subsequently, 7.0 µL of Tris buffer and 0.35 µL of Alkaline Phosphatase solution were added, and the mixture was incubated at 37°C for an additional 30 minutes. The solution was then transferred to a Nanosep 3K (Pall Life Science, Michigan, USA) and centrifuged at 15,000 × g at 20°C for 15 minute. DNA adduct identification and semi-quantification by LC/ MS DNA adduct were analyzed using a Triple Quad 5500 + QTRAP Ready mass spectrometer (SCIEX, Framingham, MA, USA) coupled with an ExionLC AD system (SCIEX, Framingham, MA, USA), as similar methods in our previous report [ 6 ]. Briefly, DNA samples (83 ng/20 uL) were injected and isolated through an Acquity UPLC HSS T3 column (2.1 × 100 mm, No.186003539; Waters, Tokyo, Japan). A gradient elution protocol (3% B from 0 to 5 min; 3–15% B from 5 to 10min; 15–80% B from 10 to 25min; 80% B from 25 to 30 min) was performed using 0.02% (v/v) acetic acid in water (mobile phase A) and methanol (mobile phase B), with a flow rate of 0.2 mL/min. Electrospray ionization (ESI) in positive-ion mode was used to detected the natural loss of 2' -deoxyribose from positively ionized 2’ -deoxy-nucleoside adducts, characterized by transition from [M + H] + → [M + H-116] + . The following MS parameters were applied: ion source temperature was 400°C, ion spray voltage was 5500 V, declustering potential was 36 V, entrance potential was 10 V, collision energy was 13 eV, and collision cell exit potential was 8 V. Standard compounds were used to validate each mass transition to evaluate chromatograms and detect DNA adducts [ 4 , 48 ]. For 5hmdC, the mass transition m/z 258.1→142.1 was monitored. Distilled water which was not enzymatically hydrolyzed was used as a background monitor in each experiment, and a blank sample, subjected to enzymatic hydrolysis like other DNA samples, was included. A standard compounds containing 24.5% 2'-deoxyadenosine (dA), 24.5% 2'-deoxythymidine (dT), 20.5% 2'-deoxyguanosine (dG), and 20.5% 2'-deoxycytidine (dC), along with a standard sample containing all standard compounds as above mentioned was used to monitor intra-day variability. Data processing LC-MS/MS data were analyzed using Analyst software v1.7.2 (SCIEX, Framingham, MA, USA). Peak boundaries for each adduct were defined using the minimum and maximum column retention times obtained from three repeated measurements of the standard sample performed on three discontinuous days. Chromatograms of all samples were visually evaluated, and confirmed that all peaks exhibited typical profiles comparable to those of the standard samples. The 5hmdC peak area in DNA samples from FFPE tissues was confirmed to be higher than the baseline, defined as the median value of repeated blank measurements (4.30×10 3 ), and no peaks were excluded from the analysis. Raw data is showed in Supplementary Table S1 . The molar ratios of 5hmdC was calculated as previously described [ 6 ]. Raw peak areas were normalized using the naturally occurring dC, dT, dA, and dG detected in each sample. To reduce intra- and inter-day variability, the median peak area of each nucleosides across all samples was calculated, and the peak area of each nucleoside in a given samples was divided by this median. The median of these four normalized values was defined as the correction coefficient for each sample. The 5hmdC peak area was divided by this correction coefficient to obtain the normalized area (Supplementary Table S1 ). The molar quantity of 5hmdC was obtained by multiplying the normalized area by the mol/area ratio, which was determined using the molecular weight of each DNA adduct and the mean peak area of a specified amount of the standard compound. The molar ratio was then calculated by dividing the molar quantity of 5hmdC by the mol amount of dC, calculated from the genomic DNA amount and its base composition containing 41% GC (Supplementary Table S1 ). We proceeded with the analysis assuming that 5hmC stained by H-score evaluation and 5hmdC measured by LC-MS/MS are identical molecules within genomic DNA. Statistical analysis All statistical examinations were performed by EZR software (Saitama Medical Center, Jichi Medical University, ver. 1.40). To analyzed the differences of H-sore and molar ratio between tumoral tissue and non-tumoral tissue, Mann-Whitney U test were performed. Spearman’s correlation tests were performed to evaluated the correlation between FFPE and frozen tissue or the results of H-score and LC/MS. The patients were divided into the two groups based on the H-score of 5hmC and molar ratio of 5hmdC, and the cut-off values were determined using ROC analysis (Youden index). MFS and OS were calculated by the Kaplan-Meier method, and these differences between groups were compared using the log-rank test. Hazard ratio was calculated by the Cox proportional hazard regression model. P value < 0.05 (two-sided) was considered statistically significant. Declarations Competing interests The authors declare no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution A.T., Y.M., and Y.I designed this study and developed the methodology. Y.M., Y.I., H.Y., R.M., and K.S provided administrative, technical, and material support. A.T., Y.M., H.T., S.W., K.W., H.W., K.T., and D.M. acquired the data. A.T., Y.M., Y.I., and H.Y. analyzed and interpreted the data. All authors contributed to the writing, review, and revision of this article. K.S. and T.I. supervised the study. Acknowledgements The authors are grateful to Ms. Miki Miyazaki and Ms. Keiko Ishino for their technical support. We are also grateful to Keitaro Matsuo, MD, PhD, for his insightful academic advice and constructive discussions. This project was supported by grants from Department of Urology, Hamamatsu University School of Medicine. Data Availability All data generated or analyzed during this study are included in this published article and its Supplementary Information files. References Jones, P. A. & Baylin, S. B. The epigenomics of cancer. Cell 128 , 683–692 (2007). Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324 , 930–935 (2009). Jin, S. G. et al. 5-Hydroxymethylcytosine is strongly depleted in human cancers but its levels do not correlate with IDH1 mutations. Cancer Res. 71 , 7360–7365 (2011). Du, C. et al. 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Global 5-Hydroxymethylcytosine Levels Are Profoundly Reduced in Multiple Genitourinary Malignancies. PloS One . 11 , e0146302 (2016). Kanaly, R. A. et al. Development of the Adductome Approach to Detect DNA Damage in Humans. Antioxid. Redox Signal. 8 , 993–1001 (2006). Storebjerg, T. M. et al. Dysregulation and prognostic potential of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) levels in prostate cancer. Clin. Epigenetics . 10 , 105 (2018). Tian, Y. et al. Global changes of 5-hydroxymethylcytosine and 5-methylcytosine from normal to tumor tissues are associated with carcinogenesis and prognosis in colorectal cancer. J. Zhejiang Univ. Sci. B . 18 , 747–756 (2017). da Santos, R. HPV infection and 5mC/5hmC epigenetic markers in penile squamous cell carcinoma: new insights into prognostics. Clin. Epigenetics . 14 , 133 (2022). Chen, M. L. et al. 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Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium. Cancers 16 , 961 (2024). Qi, J. et al. Regional gain and global loss of 5-hydroxymethylcytosine coexist in genitourinary cancers and regulate different oncogenic pathways. Clin. Epigenetics . 14 , 117 (2022). Peng, D. et al. Vitamin C increases 5-hydroxymethylcytosine level and inhibits the growth of bladder cancer. Clin. Epigenetics . 10 , 94 (2018). Dupont, M. E. et al. Fresh and frozen cardiac tissue are comparable in DNA methylation array β-values, but formalin-fixed, paraffin-embedded tissue may overestimate DNA methylation levels. Sci. Rep. 13 , 16381 (2023). Do, H. & Dobrovic, A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin. Chem. 61 , 64–71 (2015). Shi, S. R., Shi, Y. & Taylor, C. R. Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J. Histochem. Cytochem. Off J. Histochem. Soc. 59 , 13–32 (2011). O’Hurley, G. et al. Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers. Mol. Oncol. 8 , 783–798 (2014). Wen, Z. et al. Deep Learning-Based H-Score Quantification of Immunohistochemistry-Stained Images. Mod. Pathol. Off J. U S Can. Acad. Pathol. Inc . 37 , 100398 (2024). Liu, S. & Wang, Y. Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts. Chem. Soc. Rev. 44 , 7829–7854 (2015). Kisil, O., Sergeev, A., Bacheva, A. & Zvereva, M. Methods for Detection and Mapping of Methylated and Hydroxymethylated Cytosine in DNA. Biomolecules 14 , 1346 (2024). Dai, Y., Yuan, B. F. & Feng, Y. Q. Quantification and mapping of DNA modifications. RSC Chem. Biol. 2 , 1096–1114 (2021). Ram, S. et al. Pixelwise H-score: A novel digital image analysis-based metric to quantify membrane biomarker expression from immunohistochemistry images. PloS One . 16 , e0245638 (2021). Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Sci. Rep. 7 , 16878 (2017). Itahara, T. Oxidation of Cytosine and 5-Methylcytosine Nucleosides and 5-Methyl-2′-deoxycytidine 5′-Monophosphate with Peroxosulfate Ions. Chem. Lett. 20 , 1591–1594 (1991). Additional Declarations No competing interests reported. Supplementary Files supplementary.20260206xlsx.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 03 Apr, 2026 Reviewers invited by journal 04 Mar, 2026 Editor invited by journal 24 Feb, 2026 Editor assigned by journal 20 Feb, 2026 Submission checks completed at journal 20 Feb, 2026 First submitted to journal 20 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-8812038","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603045249,"identity":"6b11d05d-6ce2-4e04-9f8d-45de9dc08016","order_by":0,"name":"Ayana Takemura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYPACCR429uZjYCYbO4QipMVCjo/nWBoDQwJQLTNxWiqM5SRyzMBaGJgJqOWfkfzswQ8GicQ2iZxvDz7+2CbPx8zA+OEDA18eTk/cSDM37AFp4Xm73XBGwm3DNmYGZskZDGzFOK25kWAmwfsPqIU9d5s0T8JtRqAWNmYeBrbEBhw65G+kf5P8A7KFIecZSIs9QS0GN3LMpHkYJIzZOHLYQFoSCWoxPPOmTFqGQUKOjeeYmeSMtNvJbcyMzZIzDHD7Re54+jbJNwx1PPLtzc8kPtjctp3f3nzww4eKYzhDjEEgAUOIEegkg2OY4jDAfwC7eA1uLaNgFIyCUTDSAADnCkuQs2qcEgAAAABJRU5ErkJggg==","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ayana","middleName":"","lastName":"Takemura","suffix":""},{"id":603045253,"identity":"62cdcdc4-3917-49a4-80ad-de66a2e3fc7c","order_by":1,"name":"Yuto Matsushita","email":"","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuto","middleName":"","lastName":"Matsushita","suffix":""},{"id":603045254,"identity":"5520c282-9cce-4865-9567-7b54ebe125f6","order_by":2,"name":"Yuji Iwashita","email":"","orcid":"","institution":"Aichi Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Yuji","middleName":"","lastName":"Iwashita","suffix":""},{"id":603045255,"identity":"b3571a44-f261-40e9-bdb4-40700f537ed6","order_by":3,"name":"Hidetaka Yamada","email":"","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hidetaka","middleName":"","lastName":"Yamada","suffix":""},{"id":603045256,"identity":"6fc833de-96da-4aa6-b34d-6a12a1ca2646","order_by":4,"name":"Ryosuke Miyazaki","email":"","orcid":"","institution":"Hamamatsu University School of 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kyohei","middleName":"","lastName":"Watanabe","suffix":""},{"id":603045260,"identity":"528f53f5-a76f-4f71-887c-35d79f363e14","order_by":8,"name":"Hiromitsu Watanabe","email":"","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiromitsu","middleName":"","lastName":"Watanabe","suffix":""},{"id":603045261,"identity":"64b86ba7-8384-47ae-897c-863092cd54b9","order_by":9,"name":"Keita Tamura","email":"","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Keita","middleName":"","lastName":"Tamura","suffix":""},{"id":603045262,"identity":"779b3505-2342-430f-9a38-715d16b69bd6","order_by":10,"name":"Daisuke Motoyama","email":"","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Daisuke","middleName":"","lastName":"Motoyama","suffix":""},{"id":603045263,"identity":"69162764-a98f-420e-9fde-75e3a0d3bd19","order_by":11,"name":"Atsushi Otsuka","email":"","orcid":"","institution":"Iwata City Hospital","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Otsuka","suffix":""},{"id":603045264,"identity":"c1d57ac0-7691-4cc9-99b4-6bca9b6b380c","order_by":12,"name":"Kazuya Shinmura","email":"","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazuya","middleName":"","lastName":"Shinmura","suffix":""},{"id":603045265,"identity":"a3b6b8bc-a66b-4c51-95cf-92e98c6c73b5","order_by":13,"name":"Teruo Inamoto","email":"","orcid":"","institution":"Hamamatsu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Teruo","middleName":"","lastName":"Inamoto","suffix":""}],"badges":[],"createdAt":"2026-02-07 03:54:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8812038/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8812038/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104374035,"identity":"0017f745-3a64-4a9d-8ca8-5d4a7462d172","added_by":"auto","created_at":"2026-03-11 06:04:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5948702,"visible":true,"origin":"","legend":"\u003cp\u003eThe evaluation of 5hmC by immunohistochemistry. (a) Tumoral tissue demonstrating H-score was 53.4. (b) Non-tumoral tissue demonstrating H-score was 196.1. (c) Box plots of H-score. It was significantly reduced in tumoral tissue (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). *P \u0026lt; 0.001 by Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8812038/v1/e039edcea6f364c2fbf0e7c3.png"},{"id":104374038,"identity":"a736e292-cd5f-460c-bf1b-2d3d0035e797","added_by":"auto","created_at":"2026-03-11 06:04:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1074143,"visible":true,"origin":"","legend":"\u003cp\u003eThe Kaplan-Meier curves according to H-score of non-tumoral tissue and comparison using log-rank test. (a) Overall Survival (OS) tended to be shorter in the low H-score groups compared with the high H-score groups. (b) Metastasis Free Survival (MFS) did not differ between the high and low H-score groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8812038/v1/173ec1a4e6b12a55e359254d.png"},{"id":104374037,"identity":"f3275886-189c-4eec-939a-97ba9ce21fa7","added_by":"auto","created_at":"2026-03-11 06:04:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":577219,"visible":true,"origin":"","legend":"\u003cp\u003eMolar ratio of 5hmdC from FFPE and fresh-frozen non-tumoral tissue. (a) Box plots of molar ratio obtained according to the type of sample. (b) The scatter plot of molar ratio according to the tissue type. There was significant correlation between the two tissue types by Spearman’s correlation test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8812038/v1/33387abb3efec9dd5c76d5e2.png"},{"id":104374031,"identity":"f4591f93-1dd9-4b95-9b14-fc5e05299bbe","added_by":"auto","created_at":"2026-03-11 06:04:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":701823,"visible":true,"origin":"","legend":"\u003cp\u003e5hmdC molar ratio was reduced in non-tumoral tissue compared to tumoral tissue of the UTUC patients by LC-MS/MS. (a) The box plot of 5hmdC molar ratio. (b) 5hmdC molar ratio and H-score from FFPE of UTUC patients had significant correlation by Spearman's correlation test. *P \u0026lt; 0.001 by Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8812038/v1/f6346dab5cd4d3cff6c0656a.png"},{"id":104374032,"identity":"25c58324-d9ed-4c22-9a9a-31a9574d25ec","added_by":"auto","created_at":"2026-03-11 06:04:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":985297,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier curves according to molar ratio by the results of LC-MS/MS of non-tumoral tissue and comparison using log-rank test. (a) Overall survival (OS) was significantly shorter in low molar ratio group than high molar ratio group. (b) Metastasis free survival (MFS) tended to be shorter in low molar ratio group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8812038/v1/a4b58dfbab4799fd23994183.png"},{"id":104784141,"identity":"e36bccba-929e-40d7-8943-a3d2a3b5d4a5","added_by":"auto","created_at":"2026-03-17 08:05:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10132314,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8812038/v1/dcf03b60-68d1-4a0f-860c-fc6ae1224035.pdf"},{"id":104779848,"identity":"b728d545-87cd-4b28-ae4b-eaa4b17947dc","added_by":"auto","created_at":"2026-03-17 07:46:48","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5566770,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.20260206xlsx.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8812038/v1/a50f625252773e4f9a26ae7f.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The prognostic impact of reduction of 5-hydroxymethyl-2'-deoxycytidine in non-tumoral tissue of upper tract urothelial carcinoma patients evaluated by mass spectrometry","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the development and progression of cancer, epigenetic modifications including DNA methylation and demethylation in CpG island have the critical role [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. 5-hydroxymethyl-2'-deoxycytidine (5hmdC) is generated by the ten-eleven translocation (TET) enzyme family during DNA demethylation process [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previously, the importance of hydroxymethylation of cytosine, detected as 5hmdC in cancer was reported. First, association with carcinogenesis were reported: 5hmdC level was reduced in tumoral tissue of various cancers [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and its accumulation was reduced even in non-tumoral tissue of UTUC patient compared with non-UTUC patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Secondly, 5hmdC reduction was related to oncological prognosis in patients with melanoma, lung, hepatocellular head and neck, and prostate cancer [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, 5hmdC modification in cell-free DNA (cfDNA) have emerged as an epigenetic marker in early cancer detection and monitoring cancer conditions [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, there are limited research about the role of 5hmdC in urothelial carcinoma (UC), particularly upper tract urothelial carcinoma (UTUC). UTUC presents unique challenges due to its higher invasiveness at diagnosis compared to bladder urothelial carcinoma, resulting in poorer prognosis. Unlike bladder cancer, management of UTUC cannot be fully extrapolated from lower tract disease owing to anatomical, biological, and molecular differences. Epigenetic modifications, such as 5hmdC, have been implicated in urothelial carcinogenesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While global loss of 5hmdC has been observed in bladder cancer, its role in UTUC remains largely unexplored, highlighting the need for UTUC specific investigations to identify novel diagnostic and prognostic biomarkers. Understanding the relationships between 5hmdC levels and oncological outcomes in UTUC patients could provide insights into its potential as prognostic biomarker and guide the development of novel therapeutic strategies.\u003c/p\u003e \u003cp\u003eWe previously reported the 5hmdC reduction in non-tumoral urothelial tissue of UTUC patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], however, the relationships between 5hmdC reduction and oncological outcomes were not investigated. In this study, we investigated the relationship between oncological outcomes of non-metastatic UTUC patients and 5hmdC levels in tumoral tissue and non-tumoral tissues using immunohistochemical staining (IHC) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient characteristics and prognosis\u003c/h2\u003e \u003cp\u003ePatient characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A total of 85 patients with UTUC were included, with a median age of 75 years. The most common pT category was pT3 (40.0%), and a small subset presented with concomitant carcinoma in situ (CIS) (9.4%) that including 5 cases were CIS only, 2 were T2\u0026thinsp;+\u0026thinsp;CIS, and 1 was T1\u0026thinsp;+\u0026thinsp;CIS. The predominant histological subtype was urothelial carcinoma without any histological subtype (83.5%). Neoadjuvant chemotherapy (NAC) was administered to 18 patients (21.1%): 8 received gemcitabine plus cisplatin and 10 received gemcitabine plus carboplatin. Adjuvant therapy (AC) was given to 17 patients (20.0%), including immune checkpoint inhibitor in 5 received nivolumab and platinum-based combinations therapy in 3 received gemcitabine plus cisplatin and 9 received gemcitabine plus carboplatin.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatients characteristics in this study. Total 85 patients were investigated. *Only CIS patients were included.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;85\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian age (years, range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75 (49\u0026ndash;90)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58 (68.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 (31.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite of primary tumor (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal pelvis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 (58.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUreter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35 (41.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaterality (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 (58.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35 (41.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epT category (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (4.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTis*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (5.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (24.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18 (21.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34 (40.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (4.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcomitant CIS (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (9.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77 (90.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistology (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71 (83.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUC with squamous differentiation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (11.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUC with glandular differentiation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUC with sarcomatoid subtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (2.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUC with micropapillary subtype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeoadjuvant chemotherapy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18 (21.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67 (78.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdjuvant therapy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (20.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68 (80.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the observation period, metastatic recurrence and overall death was observed in 31 (28.3%), and 31 (28.3%) patients, respectively. The median OS and MFS were 48.8 months and 44.7 months, respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDifference of H-score of 5hmdC between tumoral and non-tumoral tissue\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the immunohistochemical staining of 5hmdC in tumoral tissue and non-tumoral urothelial tissues in UTUC patients. A total of 84 tissues were able to be evaluated, respectively (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The median H-score in tumor tissue and non-tumoral tissue were 33 and 204, respectively and it was significantly lower in tumoral tissue than non-tumoral tissue (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRelationship between H-score of 5hmdC and prognosis\u003c/h3\u003e\n\u003cp\u003ePatients were categorized based on the H-score into two groups. The cutoff values were determined by Youden\u0026rsquo;s index, and they were 30 for tumoral tissue and 150 for non-tumoral tissue. According to the H-score of tumoral tissue, OS nor MFS were not statistically different between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.090 and 0.68, respectively). On the other hand, although the difference did not reach statistical significance, overall survival (OS) tended to be shorter in the low H-score group compared with the high H-score group in non-tumoral tissue (median OS was 65.4 vs not reached, respectively \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.075, HR\u0026thinsp;=\u0026thinsp;1.92 (95%CI 0.92\u0026ndash;3.99)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Metastasis free survival (MFS) was not statistically different between two groups (median MFS was not reached vs not reached, respectively, P\u0026thinsp;=\u0026thinsp;0.13, HR\u0026thinsp;=\u0026thinsp;0.88 (95%CI 0.38- 2.0)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eComparison of 5hmdC measurement by LC-MS/MS using FFPE and frozen tissue DNA\u003c/h3\u003e\n\u003cp\u003eIn order to assess the feasibility of using FFPE instead of frozen tissues, we analyzed the 5hmdC molar ratio in FFPE and fresh-frozen tissue from same patient. In total, 15 paired non-tumoral tissue samples were analyzed. The median molar ratios in FFPE and fresh-frozen tissues were 1.4 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 1.4 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb shows the significant correlation between FFPE and fresh-frozen tissues (correlation coefficient\u0026thinsp;=\u0026thinsp;0.72, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0036).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eReduction of 5hmdC molar ratio by LC-MS/MS in tumoral and non-tumoral tissues and their positive correlation with H-Score\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn 3 samples of tumor tissue and 12 samples of non-tumoral tissue, sufficient amount of DNA was not extracted because of the small amount of tissues. Then, we analyzed 82 tumor tissue samples and 73 non-tumoral tissue samples (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The median molar ratios of 5hmdC were 7.7 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 1.8 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and, a significant reduction of 5hmdC molar ratio was shown in tumor tissue compared with non-tumoral tissue (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A significant positive correlation was observed between H-score and 5hmdC molar ratio in UTUC patient tissues (correlation coefficient\u0026thinsp;=\u0026thinsp;0.70, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRelationship between molar ratio of 5hmdC and prognosis\u003c/h3\u003e\n\u003cp\u003ePatients were divided into two groups according to the amount of 5hmdC molar ratio, based on cutoff values of 1.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e in tumoral tissue and 2.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e in non-tumoral tissue. In Supplementary Table S2, patients characteristics according to molar ratio in non-tumoral tissue were described. According to the molar ratio of tumoral tissue, OS nor MFS were not statistically different between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.49 and 0.89, respectively). On the other hand, in the analysis using non-tumoral tissue, low molar ratio group also showed poor trend of MFS than high molar ratio group (both median MFS was not reached, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.061, HR\u0026thinsp;=\u0026thinsp;2.33 (95%CI 0.94\u0026ndash;5.78)). In addition, the low molar ratio group had significantly shorter OS compared to the high molar ratio group (median OS was 68.8 months vs not reached, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035, HR\u0026thinsp;=\u0026thinsp;2.57 (95%CI 1.04\u0026ndash;6.36)) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUTUC is relatively uncommon disease, in substance, it occupied only 5\u0026ndash;10% of urothelial carcinoma. However, the incidence has risen because of the aging of population. For the patients with non-metastatic UTUC, radical nephroureterectomy is primary treatment option because kidney sparing surgery is adopted to limited low-risk patient [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The risk factors of poor prognosis are histological grade, multiplicity, presence of hydroureteronephrosis and tumor size and these are used for decision making of surgical treatment. According to the recurrent risk based on pT stage and pN stage, adjuvant systemic therapy is recommended after surgery based on the prospective randomized study, however, the oncological benefit was not described in UTUC subgroup [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Then, more precise risk stratification is expected because over treatment and treatment-related adverse event are inevitable for systemic therapy. In other words, investigation of prognostic factor in patients with non-metastatic UTUC is essential and major concern in clinical practice.\u003c/p\u003e \u003cp\u003e5hmdC is associated with both DNA methylation and demethylation: It is an oxidized derivative of 5mdC, generated during DNA demethylation by the TET family of enzymes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is known to contribute to the development of various cancers through epigenetic regulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and it is highly enriched in promoter and enhancer regions upstream of transcriptional start sites and has been implicated in the regulation of gene expression [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. 5hmdC reduction due to passive dilution during DNA replication [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] in tumor tissue is reported in a lot of kind of cancer [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and association of 5hmdC reduction with cancer prognosis was also reported in melanoma, lung, hepatocellular, head and neck, parathyroid and prostate cancer [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Based on these studies, 5hmdC is expected to be a novel biomarker for cancer prognosis and response to immunochemotherapy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, 5hmdC has been proposed as a potential therapeutic target that could enhance the efficacy of conventional cancer treatments [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLC-MS/MS based analytical approaches have been proposed as reliable methods for the accurate detection and quantification of DNA adducts, including 5hmdC [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Not only 5hmdC, a wide range of DNA adducts can be comprehensively analyzed using this platform, and these adducts are increasingly recognized as potential biomarkers across various cancer types [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Accordingly, DNA adductome analysis represents a promising and rapidly developing research field with potential applications in cancer biomarker discovery [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Previously, we established the quantitation methods of 5hmdC using LC-MS/MS and reported 5hmdC reduction in non-tumoral tissue in UTUC patient [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the present study, we investigated the prognostic impact of 5hmdC reduction on oncological outcomes in patents with non-metastatic UTUC.\u003c/p\u003e \u003cp\u003eIn the investigation of carcinogenesis, the evaluation of non-tumoral tissue serves as a critical methodology. In bladder cancer, which shares a common urothelial origin with UTUC, somatic mutations within non-tumoral urothelial tissue have been associated with cancer progression and clinical prognosis [\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, 5hmdC reduction in non-tumoral tissue of patients with gastric cancer or UTUC has been previously reported [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We hypothesized that 5hmdC depletion in non-tumoral tissue may be indicative of field cancerization and potentially correlates with oncological outcomes. Additionally, we posited that evaluating non-tumoral tissue might provide a more sensitive diagnostic indicator, given that 5hmdC levels in tumoral tissue are already substantially diminished. Consequently, we quantified 5hmdC levels in both tumoral and non-tumoral tissues.\u003c/p\u003e \u003cp\u003eWe demonstrated the reduction of 5hmdC in tumoral tissue compared to non-tumoral tissue in UTUC patients using IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and it is similar to the other cancer [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. So, genes with reduced 5hmdC levels are involved in proliferation, metabolism, and cell adhesion pathways, suggesting that 5hmdC loss may also contribute to epigenetic regulation of traits related to cell proliferation in UTUC. These findings indicate that, in UTUC, reduced 5hmdC may promote tumor progression through mechanism similar to those observed in other cancers [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have reported that reduced 5hmdC levels in tumoral tissue which evaluated using H-score are associated with poor outcomes in several cancers as mentioned above [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In bladder cancer which are similar urothelial carcinoma to UTUC, Munari et al. reported the 5hmdC loss did not influence prognosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In contrast, Peng et al. showed that decreased 5hmdC levels predicted poorer prognosis, higher stage, and lymph node metastasis in bladder cancer [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, 5hmdC reduction evaluated by H-score in tumoral tissue showed no statistical significance on OS and MFS. Collectively, the association of 5hmdC reduction by IHC of urothelial tumoral tissue with prognosis is controversial, and it is not useful in patient with UTUC. In non-tumoral tissue, 5hmdC depletion evaluated by IHC also showed no statistically significant association with OS or MFS, however the OS tended to be shorter in the low H-score group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThen, we evaluated 5hmdC using LC-MS/MS in FFPE tissues. Although fresh-frozen tissues are generally considered to better reflect physiological conditions [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], their limited availability restricts their use in large retrospective studies, particularly for rare malignancies such as UTUC. In contrast, FFPE tissues are more accessible in routine clinical practice but may be affected by fixation-related artifacts [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To address this concern, we examined the correlation of 5hmdC measurements between fresh-frozen and FFPE samples and observed a comparatively strong correlation in non-tumor urothelial tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This finding supports the validity of FFPE based LC-MS/MS quantification of 5hmdC and highlights its utility for retrospective epigenetic analyses using archived tissues, thereby enabling robust investigation of prognostic biomarkers in rare diseases.\u003c/p\u003e \u003cp\u003eSimilar to 5hmdC level using IHC, 5hmdC level evaluated using LC-MS/MS was also reduced in tumor tissue compared to non-tumoral tissue. And the significant correlation of the result of these two methods was observed as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The impact of 5hmdC reduction measured by LC-MS/MS on oncological prognosis were analyzed. The molar ratio of 5hmdC in tumoral tissue showed no statistical significance on OS nor MFS. However, in non-tumoral tissue, low molar ratio group demonstrated significantly inferior OS and trend of shorter MFS. These results indicated 5hmdC molar ratio measured by LC-MS/MS in non-tumoral tissue may be useful for predicting poor prognosis. To our knowledge, this study provides the first evidence that describes the relationships between prognosis and amount of 5hmdC in non-tumoral tissue measured by LC-MS/MS in UTUC patients. The discrepancy between the prognostic performance of the molar ratio of 5hmdC and the H-score of 5hmdC may be explained by the fact that manual scoring in IHC is semi-quantitative and susceptible to observer bias [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], whereas LC-MS/MS is considered to provide more accurate quantification of 5hmdC levels in biological samples [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study has several limitations that should be acknowledged. First, the comparatively small number of patients and retrospective design may introduce selection bias. Second, perioperative management was not uniform across patients, including variations in neoadjuvant and adjuvant treatments, which may have acted as potential confounding factors in the survival analyses. Third, we didn\u0026rsquo;t evaluate host-related factors such as smoking history, and chronic inflammation that may influence the accumulation of 5hmdC. Fourth, Multivariate Cox regression analysis was not performed due to the limited number of events relative to potential confounding variables. Fifth, FFPE sample is inevitable for variation in fixation and storage time which lead to DNA degradation. Finally, we analyzed only global levels of 5hmdC, therefore evaluating specific epigenetic alteration are expected in the future study.\u003c/p\u003e \u003cp\u003eWe showed 5hmdC reduction in tumoral tissue compared to non-tumoral tissue of UTUC patients. 5hmdC reduction evaluated by LC-MS/MS in non-tumoral tissue are associated with poor prognosis in patient with non-metastatic UTUC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Tissue samples\u003c/h2\u003e \u003cp\u003e All procedures were performed in accordance with the Declaration of Helsinki and the use of residual pathological tissue in this research was approved by the Clinical Research Review Board of Hamamatsu University School of Medicine (No. 20\u0026ndash;011). The requirement for written informed consent was waived by the same Clinical Research Review Board. An opt-out approach was used to obtain informed consent from all subjects.\u003c/p\u003e \u003cp\u003eA total of 85 consecutive patients who underwent nephroureterectomy for non-metastatic upper tract urothelial carcinoma (UTUC) between January 2012 and December 2024 at Hamamatsu University Hospital were included in this study. Histologically confirmed tumor tissue and non-tumoral tissue samples were obtained from formalin-fixed paraffin-embedded (FFPE) specimens.\u003c/p\u003e \u003cp\u003eClinicopathological features and prognostic data were collected retrospectively by reviewing electronic medical records. The variables included age, sex, pathological diagnosis, peri surgical chemotherapy, MFS and OS. MFS and OS were defined as the time from nephroureterectomy to metastasis or death, and the time from surgery to overall death, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eFour-micron paraffin-embedded sections were deparaffinized using xylene and rehydrated in a graded series of distilled water and alcohol. Three hydrogen peroxide in distilled water was used to block endogenous peroxidase and slides were stained with anti-5-hydroxymethylcytosine (5hmC) antibody (1:2000 dilution; No.39069; Rabbit; Active Motif, Carlsbad, CA, USA) according to the below protocol. Antigen retrieval was performed using Tris/EDTA buffer, followed by incubation with 3.5N hydrochloric acid for 15 minutes at room temperature before primary antibody labeling. The anti-5hmC antibody as mentioned above was applied for 30 minutes at room temperature, followed by secondary antibody (MAX-PO (MULTI); Rat and rabbit; No.424154; Nichirei, Tokyo, Japan) labeling for 30 minutes. Immunocomplexes were detected with 3,3'-diaminobenzidine tetrahydrochloride (DAB) as a chromogenic system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eScoring systems of IHC\u003c/h2\u003e \u003cp\u003eWe calculated H-score for scoring of presence of 5hmdC which is evaluated from 0 to 300 by the classification of the percentage of positive cells and the score of staining intensity (0 is negative, 1 is weekly positive, 2 is moderately positive, and 3 is strongly positive) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] with QuPath software ver. 0.5.1 which is one of the open source digital pathology system [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe evaluated H-score of three spots for each cases, and calculated the average of these spots to quantify the 5hmC positive cells (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eVerification of the feasibility of using FFPE instead of fresh-frozen tissue in non-tumoral tissue.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe used FFPE in this retrospective study and verified the feasibility of data from FFPE by analyzing the correlation between FFPE and fresh-frozen tissue data obtained from our previous study [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Fresh-frozen samples obtained immediately after resection were frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C in a deep freezer until DNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction from FFPE and enzymatic digestion\u003c/h2\u003e \u003cp\u003eDNA extraction was performed using the Quick-DNA\u0026trade; FFPE kit (D3067; Zymo Research, Irvine, CA, USA). Two 20-um FFPE sections were deparaffinized by the protocol described above. Organizational processing was conducted by an experienced urologist, carefully removed excess tissue from tumoral and non-tumoral portions of each samples, following the kit protocol. The median specimen areas of tumoral tissue and non-tumoral tissue were 3.3 cm\u0026sup2;, and 1.8 cm\u0026sup2;, respectively, with median DNA yields of 11.4 \u0026micro;g and 0.99 \u0026micro;g, respectively.\u003c/p\u003e \u003cp\u003eNano Drop ONE spectrophotometer (Thermo Scientifis, Tokyo, Japan) was used to quantify the purified DNA amount by measuring absorbance at 260 nm. DNA solutions were adjusted to concentration of 250 ng/45 uL for liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using the 8-OHdG Assay Preparation Reagent Set (No.292-67801; Wako, Osaka, Japan). Adjusted DNA solution as mentioned above was incubated at 98\u0026deg;C for 2 minutes, cooled on ice for 5 minutes, and then mixed with 6.7 \u0026micro;L of Acetic Acid Buffer and 3.5 \u0026micro;L of Nuclease P1, followed by incubation at 37\u0026deg;C for 30 minutes. Subsequently, 7.0 \u0026micro;L of Tris buffer and 0.35 \u0026micro;L of Alkaline Phosphatase solution were added, and the mixture was incubated at 37\u0026deg;C for an additional 30 minutes. The solution was then transferred to a Nanosep 3K (Pall Life Science, Michigan, USA) and centrifuged at 15,000 \u0026times; g at 20\u0026deg;C for 15 minute.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDNA adduct identification and semi-quantification by LC/ MS\u003c/h2\u003e \u003cp\u003eDNA adduct were analyzed using a Triple Quad 5500\u0026thinsp;+\u0026thinsp;QTRAP Ready mass spectrometer (SCIEX, Framingham, MA, USA) coupled with an ExionLC AD system (SCIEX, Framingham, MA, USA), as similar methods in our previous report [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Briefly, DNA samples (83 ng/20 uL) were injected and isolated through an Acquity UPLC HSS T3 column (2.1 \u0026times; 100 mm, No.186003539; Waters, Tokyo, Japan). A gradient elution protocol (3% B from 0 to 5 min; 3\u0026ndash;15% B from 5 to 10min; 15\u0026ndash;80% B from 10 to 25min; 80% B from 25 to 30 min) was performed using 0.02% (v/v) acetic acid in water (mobile phase A) and methanol (mobile phase B), with a flow rate of 0.2 mL/min. Electrospray ionization (ESI) in positive-ion mode was used to detected the natural loss of 2' -deoxyribose from positively ionized 2\u0026rsquo; -deoxy-nucleoside adducts, characterized by transition from [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e \u0026rarr; [M\u0026thinsp;+\u0026thinsp;H-116]\u003csup\u003e+\u003c/sup\u003e. The following MS parameters were applied: ion source temperature was 400\u0026deg;C, ion spray voltage was 5500 V, declustering potential was 36 V, entrance potential was 10 V, collision energy was 13 eV, and collision cell exit potential was 8 V. Standard compounds were used to validate each mass transition to evaluate chromatograms and detect DNA adducts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. For 5hmdC, the mass transition m/z 258.1\u0026rarr;142.1 was monitored.\u003c/p\u003e \u003cp\u003eDistilled water which was not enzymatically hydrolyzed was used as a background monitor in each experiment, and a blank sample, subjected to enzymatic hydrolysis like other DNA samples, was included. A standard compounds containing 24.5% 2'-deoxyadenosine (dA), 24.5% 2'-deoxythymidine (dT), 20.5% 2'-deoxyguanosine (dG), and 20.5% 2'-deoxycytidine (dC), along with a standard sample containing all standard compounds as above mentioned was used to monitor intra-day variability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData processing\u003c/h2\u003e \u003cp\u003eLC-MS/MS data were analyzed using Analyst software v1.7.2 (SCIEX, Framingham, MA, USA). Peak boundaries for each adduct were defined using the minimum and maximum column retention times obtained from three repeated measurements of the standard sample performed on three discontinuous days. Chromatograms of all samples were visually evaluated, and confirmed that all peaks exhibited typical profiles comparable to those of the standard samples. The 5hmdC peak area in DNA samples from FFPE tissues was confirmed to be higher than the baseline, defined as the median value of repeated blank measurements (4.30\u0026times;10\u003csup\u003e3\u003c/sup\u003e), and no peaks were excluded from the analysis. Raw data is showed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe molar ratios of 5hmdC was calculated as previously described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Raw peak areas were normalized using the naturally occurring dC, dT, dA, and dG detected in each sample. To reduce intra- and inter-day variability, the median peak area of each nucleosides across all samples was calculated, and the peak area of each nucleoside in a given samples was divided by this median. The median of these four normalized values was defined as the correction coefficient for each sample. The 5hmdC peak area was divided by this correction coefficient to obtain the normalized area (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The molar quantity of 5hmdC was obtained by multiplying the normalized area by the mol/area ratio, which was determined using the molecular weight of each DNA adduct and the mean peak area of a specified amount of the standard compound. The molar ratio was then calculated by dividing the molar quantity of 5hmdC by the mol amount of dC, calculated from the genomic DNA amount and its base composition containing 41% GC (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We proceeded with the analysis assuming that 5hmC stained by H-score evaluation and 5hmdC measured by LC-MS/MS are identical molecules within genomic DNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical examinations were performed by EZR software (Saitama Medical Center, Jichi Medical University, ver. 1.40). To analyzed the differences of H-sore and molar ratio between tumoral tissue and non-tumoral tissue, Mann-Whitney U test were performed. Spearman\u0026rsquo;s correlation tests were performed to evaluated the correlation between FFPE and frozen tissue or the results of H-score and LC/MS.\u003c/p\u003e \u003cp\u003eThe patients were divided into the two groups based on the H-score of 5hmC and molar ratio of 5hmdC, and the cut-off values were determined using ROC analysis (Youden index). MFS and OS were calculated by the Kaplan-Meier method, and these differences between groups were compared using the log-rank test. Hazard ratio was calculated by the Cox proportional hazard regression model. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (two-sided) was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eA.T., Y.M., and Y.I designed this study and developed the methodology. Y.M., Y.I., H.Y., R.M., and K.S provided administrative, technical, and material support. A.T., Y.M., H.T., S.W., K.W., H.W., K.T., and D.M. acquired the data. A.T., Y.M., Y.I., and H.Y. analyzed and interpreted the data. All authors contributed to the writing, review, and revision of this article. K.S. and T.I. supervised the study.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors are grateful to Ms. Miki Miyazaki and Ms. Keiko Ishino for their technical support. We are also grateful to Keitaro Matsuo, MD, PhD, for his insightful academic advice and constructive discussions. This project was supported by grants from Department of Urology, Hamamatsu University School of Medicine.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its Supplementary Information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJones, P. A. \u0026amp; Baylin, S. B. 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Lett.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 1591\u0026ndash;1594 (1991).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"5hmdC, upper tract urothelial carcinoma, liquid chromatography coupled with tandem mass spectrometry, prognosis, epigenetic biomarker","lastPublishedDoi":"10.21203/rs.3.rs-8812038/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8812038/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReduction of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) is related to poor prognosis in malignant tumor patients. However, reports on its role in urological cancers, particularly upper tract urothelial carcinoma (UTUC), are still limited. This study evaluated the relationship between 5hmdC levels and oncological outcomes including overall survival (OS) and metastasis-free survival (MFS) in patients with non-metastatic UTUC. Tumoral and non-tumoral tissues were obtained from patients who underwent nephroureterectomy between March 2012 and December 2024. 5hmdC accumulation was assessed by immunohistochemistry (IHC), and quantified using molar ratio by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Eighty-five and 82 paired samples were evaluated by IHC and LC-MS/MS, respectively. The significant reduction of 5hmdC accumulation in tumoral tissue compared with non-tumoral tissue was observed by both evaluations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). In the investigation of prognostic impact, low molar ratio in non-tumoral tissue demonstrated significantly poor overall survival than the other (68.8 months vs. not reached, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035) and inferior trend of MFS (not reached vs not reached, respectively, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.061). In conclusion, the reduction of 5hmdC in non-tumoral tissue may be associated with poor prognosis in patients with non-metastatic UTUC.\u003c/p\u003e","manuscriptTitle":"The prognostic impact of reduction of 5-hydroxymethyl-2'-deoxycytidine in non-tumoral tissue of upper tract urothelial carcinoma patients evaluated by mass spectrometry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 06:04:34","doi":"10.21203/rs.3.rs-8812038/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"263717048237584622116024404744079781635","date":"2026-04-03T20:28:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-05T03:39:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-24T14:38:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-20T14:49:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-20T09:44:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-20T09:40:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b45f037-c7cb-4afa-a1a7-a82d08f87a66","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64166378,"name":"Biological sciences/Cancer"},{"id":64166379,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-03-11T06:04:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 06:04:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8812038","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8812038","identity":"rs-8812038","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}