Urine-based nematode chemotaxis assay (N-NOSE) as a predictor of recurrence after curative surgery for resectable pancreatic cancer: preliminary data and single center experience

Abstract

Background

Pancreatic cancer (PC) remains one of the most lethal malignancies, with high recurrence rates even after curative-intent surgery. Current surveillance tools, including imaging and serum carbohydrate antigen 19–9, have limitations in specificity and sensitivity. N-NOSE is a urine-based assay using the chemotactic behavior of Caenorhabditis elegans to detect cancer-associated volatile organic compounds. This study prospectively evaluated the prognostic value of N-NOSE in patients undergoing curative surgery for resectable PC.

Methods

Twenty-four patients with resectable PC, all treated with preoperative gemcitabine plus S-1 chemotherapy followed by curative-intent pancreatectomy, were enrolled. The chemotaxis index was measured at three time points: before treatment, after preoperative chemotherapy, and postoperatively. Recurrence within two years was assessed. Univariate and multivariate logistic regression analyses were performed to identify predictors of recurrence, and recurrence-free survival was analyzed using Kaplan–Meier methods.

Results

During the two-year follow-up, 13 patients (54.2%) experienced recurrence. Univariate analysis identified pre-treatment platelet-to-lymphocyte ratio > 1.53 (p = 0.04), pathological lymph node metastasis (p = 0.04), and pre-treatment N-NOSE positivity (p = 0.007) as significant predictors. Multivariate analysis confirmed pre-treatment N-NOSE positivity as the only independent predictor (odds ratio: 3.10 × 10⁷; 95% CI: 11.38–; p = 0.03). In patients who recurred, the chemotaxis index increased significantly after surgery (p = 0.02), while remaining stable in non-recurrent cases.

Conclusions

Pre-treatment N-NOSE positivity and postoperative increases in chemotaxis index were associated with recurrence after curative-intent surgery for resectable PC. This simple, non-invasive urine assay shows promise as a novel prognostic biomarker to enable earlier detection of recurrence, refine postoperative surveillance, and support personalized patient management.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12893-025-03355-0.

Background

Pancreatic cancer (PC) is among the most lethal malignancies worldwide and is projected to become the second leading cause of cancer-related mortality in Western countries within the next decade [1]. In Japan, PC is currently the fourth leading cause of cancer-related deaths, with incidence expected to rise further due to the aging population [2]. Surgical resection remains the only potentially curative treatment for PC. However, even among patients classified as resectable according to National Comprehensive Cancer Network (NCCN) criteria [3], long-term survival is limited [4, 5]. Recurrence, either locoregional or distant, occurs in approximately 80% of cases, and more than half of patients relapse within 12 months after curative-intent surgery [6]. Several perioperative factors, such as tumor size, lymph node involvement, and elevated carbohydrate antigen 19–9 (CA19-9) levels, have been associated with an increased risk of recurrence and may assist in identifying high-risk patients [6–12].

Liquid biopsy has emerged as a key component of precision oncology, enabling minimally invasive detection of tumor-derived molecular information [13]. This approach includes the analysis of circulating tumor cells, extracellular vesicles, and cell-free nucleic acids, including circulating tumor DNA (ctDNA) and RNA, derived from body fluids such as blood, saliva, and urine [14]. Liquid biopsy facilitates serial sampling, allowing longitudinal monitoring of disease dynamics. Among these modalities, ctDNA is the most extensively studied in PC, with accumulating evidence linking cell-free DNA (cfDNA)–based biomarkers to tumor burden, histologic grade, and poor prognosis [15]. The presence of ctDNA may indicate micrometastatic disease and has been proposed as an early predictor of relapse before radiographic detection, making it a promising tool for recurrence monitoring [14, 16–18].

Beyond ctDNA-based methods, cancer-specific odor detection has recently gained attention as an alternative, non-invasive diagnostic approach [19–22]. Hirotsu et al. [23] developed a novel screening method using the olfactory behavior of the nematode Caenorhabditis elegans (C. elegans) to detect volatile organic compounds in human urine. This method demonstrated high diagnostic accuracy, with a reported sensitivity of 95.8% and specificity of 95%, even for early-stage malignancies. Kobayashi et al. [24] subsequently confirmed the clinical utility of this nematode-based assay, named “N-NOSE,” in pancreatic cancer diagnosis, particularly in early-stage disease, showing superior performance compared with conventional tumor markers.

Although N-NOSE has been evaluated for cancer detection in multiple settings, no studies have examined its prognostic value in pancreatic cancer. We therefore conducted a single-center prospective study to investigate whether N-NOSE results, both pre-treatment status and postoperative changes in chemotaxis index, are associated with recurrence risk following curative surgery for resectable PC.

Materials and methods

Preoperative chemotherapy

Patients without evidence of arterial involvement or distant metastases on contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) were classified as having initially resectable pancreatic cancer based on the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (version 1.2024) [25].

Preoperative chemotherapy consisted of intravenous gemcitabine (1,000 mg/m² on days 1 and 8) combined with oral S-1, dosed by body surface area (BSA) as follows:

• 40 mg for BSA < 1.25 m.²

• 50 mg for BSA 1.25–1.50 m.²

• 60 mg for BSA > 1.50 m.²

S-1 was administered twice daily on days 1–14 of a 21-day cycle. Two cycles of neoadjuvant chemotherapy were given as standard (Fig. 1).

Fig. 1.

Schematic diagram of the treatment protocol. Patients received two 21-day cycles of preoperative therapy prior to surgical resection. Each cycle comprised intravenous gemcitabine (1,000 mg/m²) on days 1 and 8, combined with oral S-1 (dose adjusted for body surface area) on days 1–14, followed by a 7-day rest period. Surgery, either pancreaticoduodenectomy (PD) or distal pancreatectomy (DP), was performed after completion of both cycles

Surgery and postoperative management

Following completion of chemotherapy, patients underwent restaging with preoperative imaging. In the absence of disease progression or metastasis, curative-intent pancreatectomy with regional lymph node dissection was performed, with surgical approach tailored to tumor location and extent.

Postoperatively, patients were advised to initiate adjuvant S-1 chemotherapy within 10 weeks of surgery and continue for 6 months.

Postoperative patient follow-up involved monthly tumour marker testing (carcinoembryonic antigen (CEA) and CA19-9) and CT scans every three months.

N-NOSE assay and chemotaxis index measurement

The N-NOSE assay was performed using previously reported methods [26, 27] (Fig. 2). Wild-type C. elegans (N2 strain) were cultured on nematode growth medium agar plates seeded with Escherichia coli. Sodium azide (0.5 μL, 1 M) was spotted at four positions on the plate to immobilize nematodes, minimizing adaptation effects.

Fig. 2.

A Clinical timeline illustrating the three urine sample collection points: baseline before treatment (1st sample), post-neoadjuvant chemotherapy (2nd sample), and post-surgery (3rd sample); (B) Schematic representation of the chemotaxis assay using C. elegans. Worms were cultured on nematode growth medium (NGM) plates, harvested at the young adult stage, and washed 2–3 times with chemotaxis buffer to remove bacteria. On the chemotaxis plate, 1 M sodium azide (0.5 μL) was placed at both the urine side and the non-urine side. Subsequently, 1 μL of diluted urine sample (tenfold) was applied to the urine side. Approximately 100 worms were placed at the center and allowed to migrate for 30 min. The chemotaxis index was calculated as CI = [n(a) − n(b)]/[n(a) + n(b)], where n(a) and n(b) represent the number of worms on the urine side and the non-urine side, respectively

Urine samples, diluted 1:10 with ultrapure water, were applied to two designated “urine-side” points. Approximately 100 adult nematodes were placed in the center of the plate and allowed to move freely for 30 min. The number of nematodes in the urine-side area (A) and the non-urine side area (B) was counted. The chemotaxis index was calculated as: "Chemotaxis Index" = (Number in A—Number in B)/total.

A positive index (0–1) indicated attraction, while a negative index (–1 to 0) indicated repulsion.

Study endpoints

The primary endpoint was the association between pre-treatment N-NOSE status and recurrence within two years after curative surgery.

Secondary endpoints also included:

• Changes in chemotaxis index after preoperative chemotherapy and after surgery.

• Associations between postoperative chemotaxis index changes and recurrence status.

Statistical analysis

Continuous variables are expressed as medians (range), and categorical variables as counts (percentages). Between-group comparisons were performed using Fisher’s exact test for categorical variables and the Mann–Whitney U test for continuous variables. Paired continuous variables were compared using the Wilcoxon signed-rank test. For the pre-treatment neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR), the cut-off value was set at the median value. For the tumor marker (CA19-9), the cut-off value was set at 100 U/ml. Variables with p < 0.05 in univariate analysis were included in multivariate logistic regression. Recurrence-free survival (RFS) was estimated using the Kaplan–Meier method, and group differences were evaluated with the log-rank test. Two-tailed p < 0.05 was considered statistically significant. Analyses were performed using JMP (SAS Institute Inc., Cary, NC, USA). The sample size of this study is based on the number of eligible cases during the study period.

Results

Patient cohort

A total of 31 patients were initially diagnosed with resectable pancreatic cancer and considered for study inclusion. Five patients developed disease progression after preoperative chemotherapy, and two were found to have unresectable disease at the time of surgery. The remaining 24 patients underwent curative-intent pancreatectomy and were included in the analysis (Fig. 3). All patients were followed for two years postoperatively to assess recurrence.

Fig. 3.

Patient selection and study cohort. Between October 2020 and May 2023, patients with initially resectable pancreatic cancer (n = 31) were prospectively enrolled at Gifu University Hospital and received neoadjuvant chemotherapy with gemcitabine plus S-1. Seven patients were excluded due to disease progression after chemotherapy (n = 5) or intraoperative findings of unresectable disease (n = 2). The remaining patients (n = 24) underwent curative-intent resection and were followed for two years postoperatively. During follow-up, 13 patients developed recurrence (Recurrence Group), and 11 patients remained disease-free (Non-Recurrence Group)

Baseline characteristics

Patient and tumor characteristics are summarized in Table 1. The median age was 76 years (range, 60–82), and 16 patients (66.7%) had pancreatic head cancer. The median tumor diameter in surgical specimens was 25 mm (range, 5–70 mm). Pathological lymph node metastasis was present in 12 patients (50.0%), and Stage II disease was the most frequent TNM classification (41.7%). According to the Evans classification of preoperative therapeutic effect, Grade Ⅱa (10–50% tumor cell destruction) was most common (66.7%).

Table 1.

Characteristics of the enrolled patients

	Number of patients (n = 24)
Age (years)	76 [60–82]
Sex	Male: 9 (37.5%) Female: 15 (62.5%)
Tumor location	Head: 16 (66.7%) Body or Tail: 8 (33.3%)
Postoperative severe complication (CD* grade≧3)	6 (25.0%)
Tumor size (mm)	25 [5–70]
Pathological T stage✝	T1: 6 (25.0%) T2: 15 (62.5%) T3: 3 (12.5%)
Pathological N stage✝	N0: 12 (50.0%) N1: 10 (41.7%) N2: 2 (8.3%)
Final Stage✝	1A: 4 (16.7%) 1B: 8 (33.3%) 2: 10 (41.7%) 3: 2 (8.3%)
v (+)§	19 (79.2%)
ly (+)§	16 (66.7%)
ne (+)§	17 (70.8%)
S (+)§	17 (70.8%)
RP (+)§	14 (58.3%)
PL (+)§	4 (19.0%)
Treatment effect of preoperative chemotherapy¶	Ⅰ: 3 (12.5%) Ⅱa: 16 (66.7%) Ⅱb: 3 (12.5%) Ⅲ: 2 (8.3%)
Postoperative adjuvant chemotherapy	22 (91.7%)
Recurrence within two-years after curative surgery	13 (54.2%)
Recurrent site	Lung: 5 (38.5%) Peritoneal dissemination: 4 (30.8%) Liver: 3 (23.1%) Local recurrence: 1 (7.7%) Bone: 1 (7.7%)
N-NOSE positive before treatment	20 (83.3%)
N-NOSE positive after preoperative chemotherapy	11 (50.0%)
N-NOSE positive after surgery	17 (73.9%)

Data are expressed as median (range) or number of patients (percentage)

^*the Clavien-Dindo classification

✝the UICC TMN classification (8th edition)

^§Japanese classification of pancreatic carcinoma by the Japan Pancreas Society: Eighth edition

^¶Evans Classification

v Pathological vessel invasion

ly Pathological lymphatic invasion

ne Pathological pancreatic nerve invasion

S Pathological serosal invasion

RP Pathological retroperitoneal invasion

PL Pathological nerve plexus invasion

Within two years after surgery, 13 patients (54.2%) experienced recurrence, most frequently in the lungs (5/13; 38.5%). N-NOSE positivity rates were 83.3% (20/24) before treatment, 45.8% (11/24) after neoadjuvant chemotherapy, and 73.9% (17/24) after surgery.

Recurrence vs. non-recurrence groups

Supplementary Table S1 compares baseline characteristics between recurrence and non-recurrence groups. There were no significant differences in patient-related variables. However, the recurrence group had significantly higher frequencies of pancreatic head cancer (84.6% vs. 45.5%, p = 0.04), pathological lymph node metastasis (69.2% vs. 27.3%, p = 0.008), and Stage II or higher disease (69.2% vs. 27.3%, p = 0.02). Histopathological findings did not differ significantly.

N-NOSE positivity before treatment was significantly higher in the recurrence group (100% vs. 63.6%, p = 0.02). Positivity rates at later time points were not significantly different between groups.

Predictors of recurrence

In univariate analysis, factors significantly associated with recurrence were:

• Pre-treatment PLR > 1.53 (p = 0.04)

• Pathological lymph node metastasis (p = 0.04)

• Pre-treatment N-NOSE positivity (p = 0.007)

Multivariate logistic regression identified pre-treatment N-NOSE positivity as the only independent predictor (odds ratio, 3.10 × 10⁷; 95% CI, 11.38–∞; p = 0.03) (Table 2). Kaplan–Meier analysis showed a trend toward shorter recurrence-free survival in patients positive for N-NOSE before treatment compared with those negative (log-rank p = 0.056) (Supplementary Figure S1).

Table 2.

Uni- and multivariate analysis of risk factors for recurrence within two years after curative surgery

	n	Univariate			Multivariate
		OR	95%CI	p-value	OR	95%CI	p-value
Age (years)
> 75	12	0.71	0.14–3.59	0.68
< 75	12	1
Sex
Male	9	2.29	0.43–14.31	0.34
Female	15	1
BMI (kg/m2)
> 22	9	0.25	0.04–1.36	0.11
< 22	15	1
PNI before NAC
> 45	16	0.60	0.10–3.33	0.56
< 45	8	1
NLR before treatment
> 2.11	12	1.40	0.28–7.31	0.68
< 2.11	12	1
PLR before treatment
> 1.53	12	6.0	1.11–41.10	0.04*	5.16	0.65–58.26	0.12
< 1.53	12	1			1
LMR before treatment
> 3.79	12	1.03	0.20–5.30	0.97
< 3.79	12	1
Diabetes mellitus
Yes	12	0.36	0.06–1.81	0.22
No	12	1
HbA1c before treatment
> 6.4	10	0.37	0.06–1.91	0.24
< 6.4	14	1
CA19-9 before treatment
> 100	13	0.97	0.19–4.96	0.97
< 100	11	1
CA19-9 after preoperative chemotherapy
> 100	6	0.60	0.08–4.18	0.60
< 100	16	1
CA19-9 after surgery
> 100	1	30,500,977	0.13-	0.28
< 100	22	1
Intraoperative blood loss (ml)
> 500	9	1.09	0.21–6.03	0.92
< 500	15	1
Postoperative severe complication
Yes	6	2	0.31–17.20	0.47
No	18	1
Tumor size (mm)
> 30	8	0.78	0.14–4.39	0.78
< 30	16	1
Pathological lymph node metastasis
Positive	12	6.00	1.11–41.10	0.04*	5.16	0.65–58.26	0.12
Negative	12	1			1
Pathological vessel invasion✝
Positive	19	6.86	0.81–149.30	0.08
Negative	5	1
Pathological lymphatic invasion✝
Positive	16	2.78	0.50–17.98	0.25
Negative	8	1
Pathological pancreatic nerve invasion✝
Positive	17	4.58	0.74–39.60	0.10
Negative	7	1
Pathological serosal invasion✝
Positive	17	0.84	0.13–5.02	0.85
Negative	7	1
Pathological retroperitoneal invasion✝
Positive	14	0.67	0.12–3.42	0.63
Negative	10	1
Pathological nerve plexus invasion✝
Positive	4	3.38	0.35–76.18	0.30
Negative	17	1
Treatment effect of preoperative chemotherapy¶
GradeⅡb/Ⅲa	5	0.15	0.007–1.23	0.08
GradeⅠ/Ⅱa	19	1
Postoperative adjuvant chemotherapy
Yes	22	7.15 × 1e-08	−5.41 × 1e−141	0.11
No	2	1
N-NOSE before treatment
Positive	20	232,127,930	2.56-	< 0.01 **	3.10 × 1e + 07	11.38-	0.03*
Negative	4	1			1
N-NOSE after preoperative chemotherapy
Positive	11	0.48	0.08–2.58	0.39
Negative	11	1
N-NOSE after surgery
Positive	17	1.43	0.21–9.88	0.71
Negative	6	1

Data are expressed as median (range) or number of patients (percentage)

OR Odds ratio, 95%CI 95% Confidence interval, BMI Body mass index, NAC Neo adjuvant chemotherapy, PNI Prognostic nutritional index = 10 × albumin(g/dl) + 0.005 × the absolute lymphocyte count, NLR Neutrophil to lymphocyte ratio, PLR Platelet to lymphocyte ratio, LMR Lymphocyte to monocyto ratio, CA19-9 Carbohydrate antigen 19–9

✝Japanese classification of pancreatic carcinoma by the Japan Pancreas Society: Eighth edition

^¶Evans classification

^*p < 0.05

^**p < 0.01

^***p < 0.001

Changes in chemotaxis index

We next investigated the dynamic changes in the chemotaxis index in response to treatment (Fig. 4). In the overall cohort, the chemotaxis index decreased significantly after preoperative chemotherapy (p = 0.02) and increased significantly after surgery (p = 0.01).

Fig. 4.

Changes in chemotaxis index (CI) across three time points in patients with resectable pancreatic cancer: (A) all patients (n = 24); (B) recurrence group (n = 13); (C) non-recurrence group (n = 11). Sample 1: baseline (before neoadjuvant chemotherapy); Sample 2: post-neoadjuvant chemotherapy; Sample 3: post-surgery. The CI was calculated as (number of nematodes on urine side − number on non-urine side)/(number on urine side + number on non-urine side). Lines connect individual patient values across time points. p-values were calculated using the Wilcoxon signed-rank test, with asterisks (*) indicating statistical significance at p < 0.05

When stratified by recurrence status, the non-recurrence group showed no significant changes in chemotaxis index either after chemotherapy (p = 0.51) or after surgery (p = 0.28). In contrast, the recurrence group exhibited a significant postoperative increase in chemotaxis index (p = 0.02), suggesting a possible association between this rise and disease relapse.

Next, comparing changes in chemotaxis index based on pre-treatment N-NOSE status, the positive group showed a significant decrease following preoperative chemotherapy (p < 0.001) and a significant increase postoperatively (p = 0.049). Conversely, the negative group consistently showed no significant changes. (Supplementary Figure S2).

Discussion

This prospective single-center study demonstrates that pre-treatment N-NOSE positivity is an independent predictor of recurrence within two years after curative-intent surgery for resectable PC. In addition, a postoperative increase in the chemotaxis index was significantly associated with recurrence, whereas patients without recurrence showed no significant postoperative change. These findings suggest that a urine-based, nematode chemotaxis assay may serve as a non-invasive prognostic tool that complements current postoperative surveillance strategies.

The prognosis for PC remains poor despite improvements in surgical techniques and perioperative therapy. Current follow-up protocols rely heavily on imaging modalities, such as CT and MRI, and serum CA19-9 measurements. While CA19-9 is widely used in clinical practice, its specificity is limited, and it is undetectable in Lewis antigen–negative patients [3, 28]. Blood-based liquid biopsy approaches, particularly ctDNA, have shown promise in detecting recurrence earlier than imaging [14, 16, 29, 30]. Still, these techniques remain costly, technically demanding, and are not yet integrated into routine clinical management of PC.

Urine-based assays offer distinct advantages: they are non-invasive, repeatable, and potentially more cost-effective than blood-based tests [31–33]. N-NOSE, which detects volatile organic compounds in urine via Caenorhabditis elegans chemotaxis, has previously demonstrated high diagnostic accuracy for various malignancies, including PC [23, 24]. However, previous investigations have primarily evaluated its diagnostic performance, and this study is the first to examine its prognostic utility in PC. The finding that all patients who experienced recurrence were N-NOSE–positive before treatment suggests that this assay may detect micrometastatic disease not visible on conventional imaging, similar to the predictive value of preoperative ctDNA detection in resectable PC [16].

Postoperative increases in the chemotaxis index occurred exclusively in patients who developed recurrence. This observation is consistent with prior studies in gastric, colorectal, and esophageal cancers, in which rising chemotaxis index values preceded radiologic or clinical evidence of relapse [34, 35]. Such changes may indicate minimal residual disease or early tumor regrowth, potentially allowing for earlier intervention than current surveillance modalities. On the other hand, the chemotaxis index may also be affected by some biostimulants including cytokines and chemokines for inflammation and repair of resected lesions at earlier phases of postoperation. Further investigation for frequency and timing of postoperative urine collection is warranted to improve the detection sensitivity of N-NOSE with minimizing any confounding effects by patients' physiological condition.

If validated in larger, multi-institutional cohorts, N-NOSE could be incorporated into postoperative surveillance to stratify recurrence risk and tailor follow-up intensity. Patients with baseline N-NOSE positivity and a postoperative increase in chemotaxis index might benefit from more frequent imaging, closer biochemical monitoring, or earlier initiation of systemic therapy. The simplicity of urine collection also allows for repeated testing during the high-risk period after surgery, potentially improving the timeliness of recurrence detection.

This study has several limitations. It was conducted at a single institution with a relatively small sample size, which may limit generalizability. The follow-up period was limited to two years, and longer observation is required to capture late recurrences. Although the N-NOSE assay follows a standardized protocol, variability in urine collection, storage conditions, or nematode responsiveness could affect results. Additionally, the biological mechanisms underlying nematode attraction to pancreatic cancer–related volatile compounds remain incompletely understood and warrant further investigation.

Future research should aim to confirm these findings in larger and more diverse patient populations, compare N-NOSE directly with established biomarkers such as CA19-9 and ctDNA, and assess whether N-NOSE–guided surveillance impacts survival outcomes. Metabolomic profiling of urine samples could help identify the specific volatile compounds responsible for nematode chemotaxis, potentially leading to assay refinement and discovery of novel molecular biomarkers.

Conclusions

In patients with resectable pancreatic cancer, pre-treatment N-NOSE positivity was independently associated with an increased risk of recurrence after curative-intent surgery. Furthermore, a postoperative increase in the chemotaxis index was observed exclusively in patients who relapsed, suggesting potential utility for early detection of recurrence. These findings indicate that this urine-based, nematode chemotaxis assay may serve as a simple, non-invasive prognostic tool to guide postoperative surveillance and personalized management strategies. Validation in larger, multi-center cohorts is warranted.

Authors’ contributions

Conceptualization: M.F. (Masahiro Fukada); validation: M.F. (Masahiro Fukada); formal analysis: M.F. (Masahiro Fukada); investigation: H.H.; resources: M.F. (Masahiro Fukada), N.M. (Noriki Mitsui), T.H. (Takeshi Horaguchi), I.Y., Y.S., J.Y.T., Y.T., M.F. (Manabu Futamura) and N.M. (Nobuhisa Matsuhashi); data curation: M.F. (Masahiro Fukada); writing—original draft preparation: M.F. (Masahiro Fukada); writing—review and editing: M.F. (Masahiro Fukada), N.M. (Noriki Mitsui), T.H. (Takeshi Horaguchi), I.Y., Y.S., J.Y.T., Y.T., H.H., A.H.A., M.M., T.H. (Takaaki Hirotsu), E.d.L., M.F. (Manabu Futamura) and N.M. (Nobuhisa Matsuhashi); supervision: M.F. (Masahiro Fukada) and N.M. (Nobuhisa Matsuhashi). All authors have read and agreed to the published version of the manuscript.

Funding

Not applicable.

Data availability

The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committees of Gifu University School of Medicine (ID: 2022–0107). Informed consent was obtained from all the patients before all study-related procedures.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.