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Introduction

Prostate cancer (PCa) is the most commonly diagnosed cancer type among men in the United States, with an estimated 288,300 new cases and 34,700 deaths expected in 2023 (1). Early detection of PCa is crucial, as it improves survival rates significantly (2). Unfortunately, in the early stages of PCa, patients may not present with any symptoms.

Serum prostate-specific antigen (PSA) is the most widely used biomarker for PCa screening. A study that followed >20,000 middle-aged and older men for 10 years found that the sensitivity of the PSA test for PCa detection was 46% at a cut-off of 4.0 ng/ml (3). The issue is that elevated PSA levels are not exclusively indicative of PCa; indeed, they can be caused by benign prostatic hyperplasia (BPH), urinary tract infections, urinary retention and ejaculation (3–6). However, despite its limitations, PSA remains a key biomarker for PCa diagnosis and monitoring.

The low specificity of the PSA test results in a high rate of negative biopsies, particularly in patients with serum PSA levels between 3.0–10.0 ng/ml, a range known as the ‘PSA gray zone’. For patients within this PSA gray zone, the positive predictive value (PPV) of PSA for PCa detection by biopsy is between 30–42% (7). This implies that ~60–70% of these patients undergo an unnecessary prostate biopsy.

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules of ~18-22 nucleotides in length that regulate gene expression at the post-transcriptional level by degrading or repressing target mRNAs. Genetic alterations in cancer play significant roles in the specific activity of miRNAs. In PCa, 30% of epigenetic silencing regions contain miRNA loci. DNA methylation of promoter regions and p53 mutations lead to miRNA downregulation. As a result, tissue-derived and circulating miRNAs have been proposed as biomarkers for the diagnosis and prognosis of PCa (8). Herpes simplex virus (Hsv)2-miR-H9 is an miRNA derived from the herpes virus, while hsa-miR-3659 is a human miRNA. The biological functions of hsv2-miR-H9 and hsa-miR-3659 have remained largely elusive. However, a small number of reports have demonstrated the tumor-promoting roles of these miRNAs. Hsv2-miR-H9 may be associated with metastasis in lung cancer (9). miR-3659 was upregulated in drug-resistant squamous cell carcinomas of the esophagus (10). A previous study by our group demonstrated that increased levels of hsv1-miR-H18 and hsv2-miR-H9 may be associated with PCa, and suggested that these two viral miRNAs may be relevant diagnostic biomarkers for PCa, thereby reducing the biopsy burden (11–13). Indeed, miR-3659 shows similar and stable expression levels in patients with PCa and in BPH controls (14). Recent studies by our group suggest that hsv2-miR-H9 and the human miRNA hsa-miR-3659 may serve as potential diagnostic markers for PCa (11–14). In the present study, a single-center, retrospective, evaluator-blinded, pilot and pivotal clinical trial was conducted to assess the clinical performance of the mirCaP kit (Urotech, Inc.), which detects hsv2-miR-H9/hsa-miR-3659, to assist physicians in making decisions regarding further tests for patients within the PSA gray zone.

Patients and methods

Patients and samples

In total, 388 urine samples were collected from the National Biobank of Korea. The biospecimens and data used in this study were provided by the Biobank of Chungbuk National University Hospital (Cheongju, Korea), a member of the KoreaBiobank Network (project no. 2024ER051000). All patients had undergone prostate biopsy to determine whether they had PCa or not. A positive diagnosis of prostate cancer was confirmed by prostate biopsy, whereas the diagnosis was deemed negative when no tumor was identified after a transrectal 12-core prostate biopsy. The initial clinical trial enrolled 70 PCa-positive and 70 biopsy-negative samples, along with 5 cases each of bladder cancer (BC) and renal cell carcinoma (RCC). The checklist with the inclusion and exclusion criteria for the initial clinical trial is provided in Table SI. The pivotal clinical trial enrolled 109 PCa-positive and 139 biopsy-negative samples. The checklist with the inclusion and exclusion criteria used in the pivotal clinical trial is provided in Table SII. For the initial trial, the collection periods were as follows: PCa from February 2012 to December 2020; biopsy-negative from December 2018 to December 2021; bladder cancer from November 2019 to December 2021; and renal cell carcinoma from October 2019 to September 2022. For the pivotal trial, the collection periods were as follows: PCa from September 2010 to October 2023; and biopsy-negative from August 2020 to June 2023. Serum PSA concentrations between 3–10 ng/ml were classified as the ‘PSA gray zone’. The pivotal clinical trial enrolled only patients with PSA levels within this gray zone, categorizing them as positive or negative by biopsy. Urine samples selected from positive or negative patients were collected during the period from the date of the PSA test to the date of prostate biopsy or surgery. In general, voided urine samples were collected prior to surgery; however, in cases that underwent biopsy, urine samples were collected immediately before the procedure. In addition, positive urine samples were obtained from patients who underwent radical prostatectomy, palliative transurethral resection of the prostate (TURP) or prostate biopsy, and all had histologically-confirmed adenocarcinoma. Negative urine samples were obtained from patients with BPH who underwent TURP or had no tumors reported after prostate biopsy. Urine samples were obtained from patients with BC who underwent transurethral resection of bladder tumor with histologically confirmed urothelial carcinoma, and from patients with RCC who underwent radical or partial nephrectomy and had histologically-confirmed clear-cell RCC.

All tissue specimens were examined by an experienced senior pathologist. The pathologist who was responsible for diagnosing PCa is highly experienced; they have been diagnosing urologic tumors for >20 years and have completed a 2-year urologic oncology fellowship. Gleason grading and TNM 2002 staging were performed (15). Patients with urinary tract infections or other diagnosed cancers were also excluded. The study design is presented in Fig. 1.

Figure 1. Study design of the initial and pivotal clinical trials. PCa, prostate cancer; BC, bladder cancer; RCC, renal cell carcinoma; UTI, urinary tract infection; PSA, prostate specific antigen; RT-PCR, real-time polymerase chain reaction; hsv2-miR, herpes simplex virus 2 microRNA; hsa-miR, Homo sapiens microRNA; AUC, area under curve; ROC, receiver operating characteristic.

Basis for determining sample size in the initial clinical trial

The sample size for this clinical performance test was determined based on a literature review, which indicated that studies using comparable devices typically collected a certain number of specimens. For instance, the study by Byun et al (14) collected ~40 positive and 40 negative specimens. Likewise, Kang et al (13) gathered ~60 specimens of both positive and negative samples. Based on these studies, the present study collected 140 specimens for the initial clinical trial (70 positive and 70 negative specimens), thereby exceeding the sample sizes reported in the referenced literature.

Basis for determining the sample size for the pivotal clinical trial

The number of positive specimens to be used for the pivotal clinical trial was calculated based on a one-sided significance level of 0.025 and a power of 0.95. The study aimed to achieve a minimum sensitivity of 65% and specificity of 60%. The performance of this medical device for clinical performance testing is expected to have a sensitivity of 81% and a specificity of 75%. Therefore, the study required 98 positive specimens. To account for a potential dropout rate of 10%, this number was adjusted to 109 positive specimens. Similarly, the number of required negative samples was 125, increasing to 139 after adjusting for a dropout rate of 10%. The formula used to calculate sample numbers was as follows:

where Zα is the Z-value for a significance level of 0.025 (≈1.96), Zβ is the Z-value for a power of 0.95 (≈1.645), p0 is the minimum sensitivity (=0.65), q0 is calculated as 1-p0 (1–0.65=0.35), p1 is the expected sensitivity (=0.81), q1 is calculated as 1-p1 (1–0.81=0.19) and δ is the effect size. When calculating the difference in sensitivity in this clinical trial, the difference between the two sensitivities is δ=p1-p0=0.81–0.65=0.16.

Isolation of miRNA from urine

Urine samples were processed for miRNA purification using the Genolution Nucleic Acid Extraction Kit (Genolution Pharmaceuticals, Inc.) For each urine sample, 500 µl of supernatant was transferred to a tube containing the proprietary miRNA separation solution from Genolution and then vortexed for 20 sec. Subsequently, 200 µl of chloroform was added to the mixture, followed by vortexing for another 10 sec. The samples were then centrifuged at 15,928 × g for 10 min at 4°C. From the resulting mixture, 650 µl of the upper aqueous phase was carefully removed without disturbing the underlying white precipitate and transferred to a new 1.5-ml tube. To this, 0.8 ml of isopropanol was added and the mixture was centrifuged again for 20 min at 21,206 × g at 4°C. The supernatant was carefully decanted so as not to disturb the RNA pellet. The pellet was washed with 1 ml of 70% ethanol and centrifuged for an additional 20 min at 21,206 × g at 4°C. After discarding the residual ethanol, the RNA pellet was resuspended in 40 µl of RNase-free water and stored at −80°C until further analysis.

Synthesis of cDNA from urinary cell-free RNA

The concentration and purity of the isolated RNA were assessed using a spectrophotometer. A cell-free RNA concentration ≥5 ng/µl was deemed suitable for further processing. A ratio of the absorbance at 260 nm (A260)/A280 of ≤2.0 for cell-free RNA was considered to be indicative of acceptable purity. Following these assessments, cDNA synthesis was performed using the mirCaP Kit™ cDNA Synthesis Kit (Urotech, Inc.) according to the manufacturer's protocol. cDNA synthesis is performed using the maximum allowed template volume of 3.75 µl due to the minimal amount of miRNA present in the extracted cell-free RNA, 5 µl of Urotech buffer (Urotech, Inc.) and 1.25 µl of Urotech enzyme (reverse transcriptase; Urotech, Inc.), resulting in a final volume of 10 µl. The reverse transcription reaction was carried out at 37°C for 60 min, followed by inactivation of the reverse transcriptase at 85°C for 5 min. The synthesized cDNA was then stabilized at 4°C for 20 min. Subsequently, 90 µl of 1X Tris-EDTA buffer was added to the cDNA solution and the mixture was stored at 2–8°C for a minimum of 2 h prior to use in the experiments.

Real-time (RT) PCR

To measure the levels of urinary hsv2-miR-H9 and hsa-miR-3659, RT-qPCR was conducted using a Rotor-Gene Q instrument (Qiagen, Inc.) and UROTECH TB premix (Urotech, Inc.). The reactions were conducted by mixing 10 µl of Urotech TB premix, 0.4 µl of Urotech RT primer (Urotech, Inc.), 7.2 µl of distilled water (buffer), and 0.4 µl of the 20 ng/µl solution of the mirCaP (A or B) primer to create a master mix, followed by the addition of 2 µl of cDNA template for a final reaction volume of 20 µl in micro tubes (Corbett Research). The PCR conditions were as follows: A single cycle of denaturation at 95°C for 180 sec, followed by the amplification process consisting of 35 cycles of 95°C for 5 sec and 60°C for 20 sec. Finally, a single cycle of dissociation was conducted at 95°C for 60 sec, 55°C for 30 sec and 95°C for 30 sec. The melting program was performed by heating from 65°C to 95°C, increasing by 1°C increments. A standard curve was established using the Vector Standard containing the target miRNA, ranging from 2.25×103 to 2.25×106 copies (2,250 to 2,250,000). The mirCaP A and B primers, which are specific for hsv2-miR-H9 (A gene) and hsa-miR-3659 (B gene), respectively (Urotech, Inc.), were used for amplification. The following forward primers were used to amplify the miRNAs: hsv-miR-H9, 5′-CTCGGAGGTGGAGTCGCGGT-3′; and hsa-miR-3659, 5′-TGAGTGTTGTCTACGAGGGCA-3′. The reverse primer used was the UROTECH RT primer (Urotech Inc.). All samples were analyzed in triplicate. RT-PCR was performed according to the manufacturer's instructions. Rotor-Gene Q software 2.3.4.3 (Qiagen, Inc.) was used to acquire and analyze the spectral data.

Statistical analysis

The expression ratio of the two urinary miRNAs was calculated by using the upregulated miRNA (hsv2-miR-H9-5p) as the numerator and the downregulated miRNA (hsa-miR-3659) as the denominator. Currently, there are no reliable housekeeping genes for miRNAs in urine samples. Expression ratio analysis does not require normalization based on housekeeping genes in urine (11–14,16–18).

An independent two-samples t-test was used to compare the ages of the two groups. The Wilcoxon rank-sum test was used to compare PSA levels, expression of hsv2-miR-H9 and hsa-miR-3659, and the expression ratio of urinary hsv2-miR-H9 to hsa-miR-3659 between the groups. To determine the cut-off values for the initial clinical trial, the area under the curve (AUC) of the receiver operating characteristic curve was calculated using the Euclidean method, based on the expression ratio of hsv2-miR-H9 and hsa-miR-3659. All statistical analyses were performed using SAS software (SAS Institute). P<0.05 was considered to indicate statistical significance.

Results

Baseline characteristics

The clinical characteristics of patients in the initial and pivotal clinical trials are presented in Table SIII. In the initial clinical trial, the mean age of the positive group was 67.50±6.19 years, with a median baseline PSA level of 8.72 [interquartile range (IQR), 5.08–13.57] ng/ml. The mean age of the negative group was 63.97±9.30 years, with a median baseline PSA level of 5.21 (IQR, 3.34–9.13) ng/ml. In the pivotal clinical trial, the mean age of the positive group was 69.19±6.79 years, with a median baseline PSA of 5.92 (IQR, 4.63–7.63) ng/ml. The mean age of the negative group was 64.56±7.92 years, with a median baseline PSA level of 5.80 (IQR, 4.50–7.23) ng/ml. However, when dividing the ages into <60 years, 60–69 years and ≥70 years, no statistically significant differences in the expression ratio determied with the mirCaP kit were observed in neither the positive group nor the biopsy-negative group (P=0.293 and P=0.110, respectively).

Diagnostic value of the mirCaP kit in the initial clinical trial

The ratio of hsv2-miR-H9 to hsa-miR-3659 was significantly higher in the positive group than in the negative group (P<0.0001; Table SIV). The AUC for the expression ratio of urinary hsv2-miR-H9 to hsa-miR-3659 was 0.8237 (95% CI, 0.7539–0.8934; Fig. 2A), which was better than that for PSA (AUC, 0.6622; 95% CI, 0.5723–0.7522; Fig. 2B). The optimal cut-off value for the miRCaP kit, as determined by the Euclidean method, was 80.64 (Table SV). The sensitivity, specificity, accuracy, PPV and NPV were 77.14, 72.86, 75.00, 73.97 and 76.12%, respectively (Table I). Applying this cut-off value increased the diagnostic value in patients within the PSA gray zone; in these patients, the sensitivity, specificity, accuracy, PPV and NPV were 94.29, 77.50, 85.33, 78.57 and 93.94%, respectively (Table II).

Figure 2. ROC curves for discriminating the positive group from the negative group in the initial clinical trial. (A) ROC curve for the miRCaP kit. (B) ROC curve for PSA. ROC, receiver operating characteristic; PSA, prostate-specific antigen.

Table I. Diagnostic value of the miRCaP kit in the initial clinical trial.

Table I.

Diagnostic value of the miRCaP kit in the initial clinical trial.

	Reference standard (n=140)
Item	Positive (n=70)		Negative (n=70)
Results with the miRCaP kit (cut-off=80.64)
Positive (n=73)	54		19
Negative (n=67)	16		51
Sensitivity, % (95% CI)		77.14 (65.55, 86.33)
Specificity, % (95% CI)		72.86 (60.90, 82.80)
Accuracy, % (95% CI)		75.00 (66.98, 81.93)
Positive predictive value, % (95% CI)		73.97 (62.83, 83.55)
Negative predictive value, % (95% CI)		76.12 (64.14, 85.69)

Table II. Diagnostic value of the miRCaP kit for samples in the PSA Gray zone (3–10 ng/ml) in the initial clinical trial.

Table II.

Diagnostic value of the miRCaP kit for samples in the PSA Gray zone (3–10 ng/ml) in the initial clinical trial.

	Reference standard (n=75)
Item	Positive (n=35)		Negative (n=40)
Results with the miRCaP kit (cut-off=80.64)
Positive (n=42)	33		9
Negative (n=33)	2		31
Sensitivity, % (95% CI)		94.29 (80.84, 99.30)
Specificity, % (95% CI)		77.50 (61.55, 89.16)
Accuracy, % (95% CI)		85.33 (75.27, 92.44)
Positive predictive value, % (95% CI)		78.57 (63.19, 89.70)
Negative predictive value, % (95% CI)		93.94 (79.77, 99.26)

Diagnostic value of the mirCaP kit for patients within the PSA gray zone in the pivotal clinical trial

The ratio of hsv2-miR-H9 to hsa-miR-3659 was significantly higher in the positive group than in the negative group (P<0.0001; Table SVI). The sensitivity, specificity, accuracy, PPV and NPV were 94.50, 82.73, 87.90, 81.10 and 95.04%, respectively (Table III). There was no observed difference in sensitivity according to the Gleason grade (P=0.736; Table SVII).

Table III. Diagnostic value of the miRCaP kit for samples in the PSA Gray zone (3–10 ng/ml) in the pivotal clinical trial.

Table III.

Diagnostic value of the miRCaP kit for samples in the PSA Gray zone (3–10 ng/ml) in the pivotal clinical trial.

	Reference standard (n=248)
Item	Positive (n=109)		Negative (n=139)
Results with miRCaP kit (cut-off=80.64)
Positive (n=127)	103		24
Negative (n=121)	6		115
Sensitivity, % (95% CI)		94.50 (88.40, 97.95)
Specificity, % (95% CI)		82.73 (75.41, 88.61)
Accuracy, % (95% CI)		87.90 (83.18, 91.69)
Positive predictive value, % (95% CI)		81.10 (73.20, 87.50)
Negative predictive value, % (95% CI)		95.04 (89.52, 98.16)

Discussion

In a previous study, our group showed that the urinary hsv2-miR-H9 to hsa-miR-3659 expression ratio is a reliable non-invasive diagnostic marker for PCa (13). In the present study, the diagnostic utility and cut-off values of the ‘miRCaP’ kit, which measures the hsv2-miR-H9/hsa-miR-3659 ratio, was evaluated in an initial clinical trial, and its performance as a non-invasive diagnostic marker was then validated in a pivotal clinical trial enrolling only patients within the PSA gray zone.

The PSA gray zone is a well-established concept in urology. In general, the PSA gray zone is considered to be 4–10 ng/ml. However, Cho (19) reported that Korean men tend to have slightly lower PSA levels and smaller prostate volumes compared to Caucasians. The age-specific reference ranges for serum PSA levels in Korean men were found to be lower. The predictive factors and characteristics of prostate cancer in patients with serum PSA levels of 4.0 ng/ml or less are very similar in Korean men. Therefore, Cho (19) suggested lowering the PSA cutoff for biopsy recommendations in prostate cancer screening for Korean men. Following this report, our institution adopted a policy of performing prostate biopsies in patients with PSA levels >3 ng/ml.

Given the low specificity of PSA, numerous studies have attempted to overcome these limitations. Adjustments such as age-adjusted PSA, free PSA, PSA density, PSA velocity and PSA doubling time have been explored to optimize the accuracy of PSA or PSA derivatives (20–23). In addition, the prostate health index (PHI), the 4K score (four-kallikrein panel), PCA gene 3 (PCA3) levels, ExoDX Prostate IntelliScore and SelectMDX have been used; however, they show a weak clinical performance in real-world clinical practice (24–28). In a biopsy-naïve population, the PHI detects aggressive PCa with greater specificity than total and % free PSA. Although the sensitivity of PHI is 95%, its specificity was only 36.0% (that for total and % free PSA is 17.2 and 19.4%, respectively). These data show that the PHI, PSA and % free PSA have high sensitivity but low specificity (29). In the initial biopsy group, the diagnostic sensitivity and specificity of PCA3 were 42 and 91%, respectively, indicating that PCA3 has high specificity but low sensitivity (26). The performance of the ExoDx Prostate IntelliScore showed a sensitivity of 91.89% and specificity of 33.96%, again suggesting high sensitivity but low specificity (27). SelectMDX, a tool for diagnosing PCa by measuring the expression of homeobox C6 and distal-less homeobox 1 from mRNA, demonstrated a sensitivity of 91% and a specificity of 36% (28). The mirCaP kit showed both high sensitivity (94.5%) and high specificity (82.73%), making it a highly effective diagnostic marker for PCa. Therefore, in comparison to other tests where high sensitivity often coincides with low specificity, or high specificity comes at the cost of low sensitivity, the mirCaP kit, having both high sensitivity and specificity, is expected to be superior. This hypothesis should be confirmed through future head-to-head studies.

Recently, liquid biopsy (LB), which detects tumor-derived material circulating in blood or urine, has been developed as a tool for detecting PCa; an obstacle is that, during early-stage PCa, circulating tumor cells (CTCs) and ctDNA are either undetectable or detected at rates similar to biopsy-negative controls (30,31). However, CTC PSA mRNA is a potential biomarker that could replace serum PSA, and also provide information about the metastatic stage of PCa (32). The ExoDx™ Prostate IntelliScore, an established LB diagnostic marker for early PCa, was shown to prevent 26% of unnecessary biopsies, with a negative predictive value of 89%, in men aged >50 years with borderline PSA levels (2–10 ng/ml) (27,33). A panel that includes miR-141, miR-1290, miR-100 and miR-335 could serve as urine and/or tissue biomarkers for diagnosing BPH and PCa (34). The present study demonstrates the diagnostic performance of urinary miRNAs as a potential LB. Although RNA is highly unstable, miRNAs are stable in both serum and urine, and are resistant to RNase activity, extreme pH, extreme temperatures and multiple freeze-thaw cycles (16,35). A previous study showed that the urinary miR-H9 to miR-3659 ratio could be a relevant non-invasive biomarker for PCa diagnosis (13). In the present study, an accurate cut-off value was calculated for the mirCaP kit (miR-H9 to miR-3659) and its clinical performance was validated in a cohort of patients within the PSA gray zone. The results show that the mirCaP kit has higher sensitivity and specificity than PSA, suggesting its potential use as a urinary miRNA diagnostic marker for PCa. Of note, the NPVs in the two cohorts within the PSA gray zone were 93.94 and 95.04%, respectively, indicating that if patients received a negative result from the mirCaP Kit, the likelihood of them being tumor-free was 95%. This suggests that the mirCaP kit can reduce the number of unnecessary prostate biopsies by >90%.

This present study has both limitations and strengths. One limitation is that these two clinical trials were not multicenter studies. Another limitation is that this is a retrospective study, which may have introduced bias due to different enrollment periods between the groups. In order to achieve the initially planned sample size, it was required to enroll patients in different groups with samples provided during different periods. However, a strength of the study is that sequential evaluator-blinded, pilot and pivotal studies were conducted. An evaluator-blinded study has several advantages. First, it minimizes expectation bias, as the researchers do not know whether a participant has cancer or not. Second, evaluator-blinded studies increase the credibility of the study results. Blinding of researchers helps ensure that the findings are based on the actual effects of the markers. Third, blinding of researchers helps ensure that the results of the study can be more easily generalized to a broader population. Finally, the sample size required for each trial was calculated by statistical experts. The importance of sample size calculations cannot be overemphasized. Regardless of the aim of a research study, one can draw a precise and accurate conclusion only with an appropriate sample size. The two trails were managed by the Expert Contract Research Organization (Synex Consulting Ltd.), although prior approval by the Korean National Institute of Food and Drug Safety Evaluation was not required.

In conclusion, the present study shows that the expression ratio of urinary hsv-miR-H9 to hsa-miR-3659 (mirCaP Kit) could serve as a highly effective non-invasive diagnostic marker for PCa in patients within the PSA gray zone. Thus, the mirCaP kit shows promise as a tool for determining the need for prostate biopsy in these patients.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

This research was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant no. 2020R1I1A3062508); the NRF Regional Innovation Strategy funded by the Ministry of Education (grant no. 2021RIS-001); and the NRF BK21 FOUR program funded by the Ministry of Education (grant no. 5199990614277).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

Conception and design: WTK; data analysis and interpretation: WTK, YSH, JK and IYK; data acquisition: KK, HWK, YJB, XMP, YJK, SCL and SJY; drafting the manuscript and statistical analysis: WTK; checking and confirming the authenticity of the raw data: WTK, KK and HWK. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

This study adhered to the applicable laws and regulations, good clinical practice and the ethical principles outlined in the Declaration of Helsinki. The study protocol was approved by the Ethics Committee of Chungbuk National University Hospital (Cheongju, Korea). The Institutional Review Board (IRB) approval no. for the initial clinical trial is 2022-09-038, and for the pivotal clinical trial, it is 2023-06-034. The requirement of consent was waived prior to enrollment, as the samples were obtained from the National Biobank of Korea, where patients had already provided consent at the time of donation. The IRB of Chungbuk National University Hospital (Cheongju, Korea) approved all procedures related to sample collection and analysis. This clinical trial is retrospective and non-invasive, thus it does not fall under the category requiring medical device clinical trial plan approval. In such cases, the Ministry of Food and Drug Safety (MFDS) conducts a pre-review of the protocol. This study was not subject to prior approval by the Korean MFDS; however, it underwent a preliminary review. The clinical trial has been submitted to the Korean MFDS. The registration number for approval is 20240050719, and it was submitted on March 15, 2024. The data have been submitted as a ‘Results report’. The review is still in progress and it will take more time for the results to be finalized.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Use of artificial intelligence tools

AI tools were used to improve the readability and language of the manuscript.

Glossary

Abbreviations

Abbreviations:

References