Research protocol for the evaluation of TriNetra™ prostate: a circulating tumor cell-based diagnostic tool for prostate cancer

Abstract

Prostate cancer is one of the most prevalent malignancies among men, with early and accurate diagnosis is essential for optimizing clinical outcomes. The Trinetra-Prostate^TM study is a prospective multicenter observational trial designed to evaluate the diagnostic performance of a novel blood-based test for detection of clinically significant prostate cancer. The test, developed by Datar Cancer Genetics, identifies circulating tumor cells (CTCs) in peripheral blood and is assessed against standard diagnostic modalities, including multiparametric magnetic resonance imaging and prostate biopsy. The primary objective of this study is to determine the sensitivity and specificity of the CTC assay for detecting cancer in men with elevated prostate-specific antigen levels and/or abnormal digital rectal examinations. Secondary objectives include assessing concordance with histopathological findings, potential to reduce unnecessary biopsies, and integration with existing diagnostic pathways. The study aims to enroll 250 men aged 45–75 years across multiple centers, applying the defined inclusion and exclusion criteria to ensure methodological rigor. The protocol includes detailed procedures for blood sampling, imaging, biopsy, and data analysis. Ethical approval was obtained and data protection measures are in place in accordance with international research standards. If validated, the CTC-based assay may offer a noninvasive and accurate alternative to current diagnostic approaches, improving patient stratification and reducing the burden of invasive procedures in prostate cancer diagnostics.

Introduction

Prostate cancer is currently the second-most diagnosed malignancy among men worldwide, following lung cancer, with an estimated 1.5 million new cases and 396 792 deaths reported in 2022^[1,2]. It accounts for 14.1% of newly diagnosed cancers in men globally^[2]. It is the fifth leading cause of cancer-related deaths among men in 2020^[2]. The risk of developing prostate cancer increases significantly with age, with over 85% of new diagnoses occurring in individuals over 60 years of age^[1,2]. The median age at diagnosis is 67 years^[3]. Although the exact etiology of prostate cancer remains uncertain, more than 50% of prostate cancer risk is attributed to genetic factors^[1]. Established risk factors include advancing age, family history of this malignancy, Black race, and certain genetic mutations such as BRCA1, BRCA2, and conditions like Lynch syndrome^[1,2].

Between 1990 and 2019, the number of incident cases, deaths, and disability-adjusted life years increased by 116.11%, 108.94%, and 98.25%, respectively^[4]. Thus, the impact of prostate cancer has risen substantially globally, although a sharp reduction has been noted for a few years because of growing concerns about overdiagnosis and overtreatment^[3,4]. Recent projections estimate that the annual number of new cases will nearly double, rising from 1.4 million in 2020 to 2.9 million by 2040^[5].

Despite advances in numerous therapies that have led to improved survival and symptom control in prostate cancer, reliable methods for predicting and monitoring treatment response remain underdeveloped^[6]. While the clinical landscape has seen the emergence of advanced imaging modalities, enhanced biopsy techniques, and the development of novel systemic therapies, prostate-specific antigen (PSA) continues to serve as the primary biomarker for disease monitoring and is the initial clinical suspicion tool for prostate cancer^[6]. PSA testing, while widely used, does not consistently correlate with tumor burden or biological activity, and is associated with a significant rate of false positives and overdiagnosis, particularly in detecting indolent tumors that may never become clinically significant^[7]. Particularly, its utility in population-based screening remains controversial owing to inconsistent guidelines, clinical gaps, and variations in recommendations across geographic regions and professional organizations^[3,6,7]. Studies report that PSA screening leads to false positive rates ranging from 12% to 63%, depending on the PSA threshold and patient age, which often results in unnecessary biopsies and patient anxiety^[8] (US Preventive Services Task Force, 2018). Overdiagnosis rates have been estimated at 20%–50%, which underscores the potential harm of PSA-driven diagnostic pathways^[9].

Additionally, although multiparametric MRI (mpMRI) has significantly improved prostate cancer detection, it still presents diagnostic limitations. The reported negative predictive value of mpMRI ranges from 76% to 95%, indicating that clinically significant cancers may still be missed, particularly when mpMRI findings are classified as PI-RADS 1–2^[10,11]. False negative rates for mpMRI can be as high as 20% in some studies, particularly for smaller or anteriorly located tumors. These diagnostic gaps justify the exploration of novel, complementary, and minimally invasive tools such as circulating tumor cell (CTC)-based assays to improve accuracy and reduce unnecessary biopsies.

Histological confirmation via solid tissue biopsy remains the diagnostic gold standard because of its high analytical accuracy, sample stability, and reproducibility due to a high level of laboratory standardization^[12]. Nevertheless, tissue biopsies present several limitations and drawbacks, as they are invasive, time-consuming, and often unsuitable for serial sampling, limiting their utility in dynamic disease monitoring^[13,14].

In this context, the term liquid biopsy, which can be summarized as a minimally invasive technique involving the analysis of tumor-derived material such as CTCs and circulating tumor DNA in peripheral blood, has gained significant traction as a potential alternative^[13]. CTCs are cancer cells that are found in the blood, originally detached from a primary tumor, or an already established metastasis, which subsequently extravasate from the bloodstream to distant sites^[13,15]. Technological advancements over the past decade have enabled the ultrasensitive detection of these biomarkers, offering new avenues for noninvasive cancer profiling^[14,16]. CTCs, in particular, provide valuable molecular insights into tumor biology and metastatic progression at the molecular level^[13]. Specifically, CTCs have been demonstrated to be present and provide relevant diagnostic accuracy and utility across multiple cancers, with accuracy comparable to that of histopathologic examination^[17]. Several studies have demonstrated that the presence of CTCs is associated with poor prognosis with respect to progression-free survival and worse overall survival in different solid tumor entities, including breast, esophageal, pancreatic, and colorectal cancers^[13].

The clinical potential of liquid biopsy spans a wide range of applications, including early detection, refined cancer staging, early relapse detection, therapeutic efficacy monitoring, therapeutic targets detection, resistance mechanisms, and prognostication^[13,14]. CTC detection has been shown to be a reliable predictive medium for stratifying asymptomatic individuals with a high risk of developing cancer within the following year, thus minimizing radiological or invasive screening^[18]. Furthermore, CTCs have shown significant prognostic value in the context of metastatic prostate cancer19.

However, despite its promise and growing use in clinical research, CTC enumeration and molecular profiling have not yet been incorporated into routine clinical practice as a standalone diagnostic tool, as the results regarding their clinical use are still early^[14,20].

TriNetra™-Prostate is a noninvasive, blood-based diagnostic specifically designed for the detection of prostate adenocarcinoma using CTCs in peripheral blood^[15,21]. Unlike traditional CTC assays that predominantly rely on epithelial markers such as EpCAM, which may fail to detect CTCs that have undergone epithelial-to-mesenchymal transition, TriNetra™-Prostate employs a proprietary functional enrichment technology that isolates CTCs independent of epithelial marker expression. This approach enhances the likelihood of capturing a broader and more clinically relevant population of CTCs. Following enrichment, the test uses a highly specific multiplexed immunocytochemistry (ICC) protocol that targets prostate-specific biomarkers, including prostate-specific membrane antigen (PSMA) and alpha-methylacyl-CoA racemase (AMACR), in addition to EpCAM, to achieve superior diagnostic specificity^[15,21,22]. These innovations differentiate TriNetra™-Prostate from prior CTC platforms and position it as a potentially more accurate and robust tool for prostate cancer detection, by improving diagnostic accuracy, reducing false positives, and facilitating early detection of clinically significant prostate cancer^[15,23]. Recent validation studies have further supported the unique performance characteristics of this assay.

The aim of this article is to outline a protocol for evaluating the diagnostic performance of the TriNetra™ Prostate compared with histopathological confirmation.

Materials and methods

This is an experimental study to evaluate the performance characteristics of the TriNetra^TM Prostate test for prostate cancer detection in men aged 45–75 years with >3 ng/ml total PSA and/or abnormal digital rectal exam (DRE) who are scheduled for prostate biopsy as part of the standard diagnostic workup for prostate cancer. The total enrollment at the center will be 250 subjects. This test has CE certification (CE/IND/2020/07/16).

All subjects enrolled in the study will undergo blood extraction for TriNetra^TM Prostate after PSA or DRE evaluation. Blood samples must be collected within 60 days of the detection of elevated PSA or abnormal DRE before prostate biopsy. Prostate biopsy must be performed within 60 days of blood sample collection for TriNetra^TM Prostate.

The results of the TriNetra^TM Prostate will be compared with the PSA results of triaging patients for prostate biopsy. Histopathological results from prostate biopsies obtained according to standard procedures will be used to assess the performance of the TriNetra^TM Prostate. Blood samples collected from the participants and sent to the sponsor’s laboratory facilities for analysis. The sponsor and all individuals performing the TriNetra^TM Prostate test will be blinded to the subject’s clinical information, including PSA results, DRE, mpMRI, ultrasound, and other imaging tests.

Researchers, subjects, and radiologists interpreting clinical evaluations will be blinded to the TriNetra^TM Prostate test results, and these results will not be used for the clinical management of study subjects, nor will they be accessible to patients or their healthcare providers. Interim analyses will be used to evaluate the sample size or determine utility or effectiveness as per the statistical analysis plan.

Objectives

The primary objective of this prospective study is to determine the performance characteristics of the TriNetra^TM Prostate in an intended use population. Secondary outcomes include: detection rates by TNM stage, performance based on Gleason score, performance based on Gleason grade, performance of the TriNetra^TM Prostate for prostate biopsy triage, and performance of the TriNetra^TM Prostate compared with Multiparametric Magnetic Resonance Imaging (mpMRI).

Enrollment

The target enrollment will be 250 evaluable subjects.

Inclusion criteria

• Men aged 45–75 years with PSA >3 ng/ml and/or abnormal DRE for whom a urologist has recommended a biopsy based on current clinical standards.

• All patients will be recommended to perform mpMRI before biopsy. The final decision whether to perform it or not will be evaluated on the availability of the test.

• No prior diagnosis of any cancer.

• No current suspicion of any other cancer.

• Any previous prostate biopsy must have been performed at least 6 months prior.

• Signed informed consent.

• Capable of providing a sufficient medical history.

• Willing and able to participate in the study and providing a blood sample as per the protocol.

• No comorbidities that would interfere with participation in the study or sample collection.

• Able and willing to undergo prostate biopsy as part of the standard diagnostic workup within 60 days after TriNetraTM Prostate blood sample collection.

• Provision of anonymized clinical information, including TNM staging, Gleason score, and Gleason grade group, if prostate cancer is diagnosed in the biopsy.

• Blood collection for TriNetra^TM Prostate within 60 days of elevated PSA or abnormal DRE findings.

• Willing to accept follow-up contacts every 6 months for up to 2 years.

• Consent to share future biopsy or imaging data for up to 2 years after blood sample collection for the study.

While PSA-based screening has known limitations, including false positives, overdiagnosis, and limited correlation with tumor aggressiveness, it continues to be the primary tool for initiating diagnostic workup in current clinical practice. The decision to use PSA as an enrollment criterion in this study reflects its current widespread utilization and is intended to allow the TriNetra™ Prostate test to be evaluated within an authentic clinical workflow.

Exclusion criteria

• No recommendation for prostate biopsy.

• Inability or unwillingness to provide written informed consent.

• Previous diagnosis or clinical suspicion of any cancer.

• Inability or unwillingness to undergo prostate biopsy.

• Inability or unwillingness to provide a blood sample.

• Currently receiving any investigational agent.

• Blood transfusion within 30 days prior to blood sample collection for the study.

• Non-compliance with inclusion criteria.

In cases where the TriNetra™ Prostate test yields an indeterminate result or the test fails (e.g. sample clotting, hemolysis, test performance control failure), repeat blood sampling will be requested if it can be performed within the protocol-defined timeframe (within 60 days of PSA or DRE assessment and prior to biopsy). If a repeat sample cannot be obtained, these cases will be excluded from the primary endpoint analysis but will be included in a secondary sensitivity analysis to assess the impact of test failures on the assay’s clinical applicability.

Subjects may be withdrawn from the study for any of the following reasons: voluntary withdrawal of consent at any time during the study, failure to provide a blood sample within 60 days of the PSA test, and/or failure to perform prostate biopsy within 60 days of TriNetra^TM Prostate blood sample collection.

All available data from enrollment to subject withdrawal will be collected, unless the subject withdraws consent before a blood sample is collected. Data from all study visits of subjects who discontinue but do not withdraw consent will also be collected. The withdrawal of consent must be documented by the site personnel in the subject’s source records (see Supplemental Digital Content Appendix 1.0, available at: http://links.lww.com/ISJP/A17).

Timeframe

The duration of each patient’s participation in the study procedure will span from inclusion, defined as an elevated PSA result and/or abnormal DRE, until biopsy results and other relevant clinical data are obtained, including TNM staging, Gleason score, and Gleason grade. Follow-up will extend for 2 years from the date of blood collection, with brief contact by telephone or email every 6 months and review of medical records via the hospital database.

All enrolled subjects will undergo a blood draw for the TriNetra™ test within 60 days of PSA and/or DRE assessment prior to prostate biopsy. Biopsies will be performed only when clinically indicated and in accordance with the standard protocols of the participating centers.

Study procedure

Men who meet the inclusion and exclusion criteria are invited to participate in the study. Approximately 250 patients will be enrolled in this study (see Supplemental Digital Content Appendix 2.0, available at: http://links.lww.com/ISJP/A18, and Supplemental Digital Content Appendix 3.0, available at: http://links.lww.com/ISJP/A19). Subjects are considered enrolled once written informed consent is obtained and their eligibility has been confirmed.

All subjects enrolled in the study will undergo blood extraction for TriNetra^TM Prostate within 60 days following serum PSA evaluation and no more than 60 days before prostate biopsy. The sample collection adheres to the sample collection protocol (see Supplemental Digital Content Appendix 4.0, available at: http://links.lww.com/ISJP/A20, Supplemental Digital Content Appendix 5.0, available at: http://links.lww.com/ISJP/A21, and Supplemental Digital Content Appendix 6.0, available at: http://links.lww.com/ISJP/A22). Shipments will be tracked and blood samples will be delivered to the designated laboratory (see Supplemental Digital Content Appendix 7.0, available at: http://links.lww.com/ISJP/A23). Once the sample arrives at the laboratory, it will be inspected to check its acceptability according to the specifications described in the TriNetra^TM Prostate prostate sample collection criteria. If the blood sample was not collected according to the usage instructions or is damaged in a way that compromises the sample, a new sample may be requested if it can be obtained within 60 days following enrollment and before prostate biopsy is performed. The center will be asked to contact the subject to request another blood sample if the patient has not yet undergone prostate biopsy.

All blood samples received from Datar Cancer Genetics will be used exclusively for the execution of the designated study protocol. Blood samples will not be retained after completion of the study. Consequently, any remaining samples will be properly discarded once the associated analysis and data generation procedures are concluded.

Immunohistochemistry protocol

The TriNetra™ Prostate test utilizes a proprietary functional enrichment process that selectively isolates CTCs from peripheral blood without reliance on density gradient centrifugation or epithelial marker-based capture alone. The iICC panel includes monoclonal antibodies against PSMA (clone 3E6), AMACR (clone 13H4), and epithelial cell adhesion molecule (EpCAM, clone Ber-EP4). Antibody staining is performed according to standardized protocols using fluorophore-conjugated secondary antibodies, with validation conducted across multiple healthy donor and prostate cancer-positive samples.

Assay specificity was confirmed through extensive preclinical testing, ensuring no cross-reactivity with non-prostate tissues. Further technical details, including the analytical sensitivity, specificity, and reproducibility of the TriNetra™ Prostate platform, have been previously validated and published in independent studies by Limaye et al^[22].

Tests performed

The test results will be recorded as positive, negative, indeterminate, sample failure (e.g. clots in the blood sample/hemolysis/microbial contamination), and test failure (e.g. failure of the test performance control).

The results of the TriNetra^TM Prostate test will not be provided to investigators for the clinical management of the study subjects. The TriNetra^TM Prostate test results will remain blinded to the study subjects.

Multiparametric MRI

It is preferable to enroll patients who have undergone mpMRIs. The clinical decision to perform prostate biopsy may be based on an abnormal PI-RADS score or, in the case of a normal PI-RADS score, the decision may be made due to other indications, such as elevated PSA or abnormal DRE.

Biopsy

A minimum of 12 samples are expected to be obtained via transrectal or transperineal biopsy. If mpMRI is performed and the report shows a PI-RADS score of 3–5, at least three biopsy samples must be taken from the target lesion.

Histopathology

Tissue samples must be interpreted according to standard histopathological practices, including Gleason score and grading of cancer samples. Subjects without a confirmed histopathological diagnosis will not be included in the performance analysis because they cannot be classified conclusively. In case of discrepancies between the TriNetra^TM Prostate results and the histopathological results, slides will be made available to an external pathologist to review and resolve discrepancies if requested by the sponsor (see Supplemental Digital Content Appendix 8.0, available at: http://links.lww.com/ISJP/A24).

Data collection

Subject data will be collected in case report forms (CRFs) and provided to the sponsor once the tests of the enrolled subjects’ specimens have been completed. The results will be shared with the principal investigator (PI). After the subjects provided informed consent, demographic data, clinical history, social history, and family history will be recorded on the case information form. The clinical center will send a copy of the de-identified reports available, such as ultrasound and mpMRI (if available), and de-identified histopathology reports and/or other diagnostic information to the PI.

Mandatory information

Demographic data and medical history

In addition to the list of requirements for participation in the study, the CRF also includes requests for demographic information and medical history. Guidelines for completing the CRF have been drafted to assist in its proper completion. The guidelines will be distributed and discussed during the initial visit to training sessions at the center. Demographic information included the following: year of birth and race/ethnicity.

The required medical history includes, among others, the following data: date and results of the most recent serum PSA evaluation (including details of the platform and assay used for the PSA test), DRE results (if available), ultrasound results (if available), family history of cancer, dates and results of the most recent mpMRI and PI-RADS score (if available), FDG PET/CT (if available), PSMA PET-CT (if available), CT scan (if available), and bone scan (if available).

Study blood sample data

Data relating to the study blood sample, such as the date and time of collection, must be documented in the CRF, as well as in the Sample Submission Form (SSF) (see Supplemental Digital Content Appendix 9.0, available at: http://links.lww.com/ISJP/A25). Once collection is completed, all information related to the study blood sample is recorded exclusively in the SSF, which follows the blood sample throughout its journey to the requesting analysis center. The information collected includes the date of collection, time of collection, date of submission to the requester, and signatures and/or initials of the participants.

A SSF will be provided to the centers and must be completed for each sample shipment. The manifest lists the study sample ID of the samples being sent, including tracking numbers, shipment dates, etc., for shipment tracking.

Study completion

A subject’s participation will be considered complete when they have provided the completed and monitored study documents, the required blood sample, the MRI report (if available), the histopathology report, or when the subject withdraws. Participants with discordant results between histopathological tissue and TriNetra (negative device results and positive biopsy results), as well as equivocal TriNetra results, will be followed up for 2 years after the study.

The centers will ensure that all data are evaluated, entered into the CRF and related study documents, and will report any adverse events (AEs) and/or protocol deviations, if necessary.

Sample retention

The TriNetra™ prostate protocol requires 10 ml of blood. The additional 10–12 ml of blood will be used for research purposes, analytical validation, or device improvement.

AE reporting

An AE is any unfavorable or unintended sign, symptom, or disease temporally associated with the use of an investigational product or intervention imposed by the protocol, regardless of attribution. No serious adverse events (SAEs) are expected during the study. An AE may be investigated as a unanticipated device effect (UDE), if applicable. Commonly occurring AEs associated with mpMRI or other standard care procedures, such as tissue biopsy that does not involve the device, will not be collected.

Only AEs that occur during blood collection for the study or are related to it will be recorded. AEs will be collected from the time of enrollment until a blood sample is collected. All AEs will be reported on the AE page(s) of CRF.

No serious AEs are expected in this study, but they will be reported to the sponsor if they occur. A SAE is an AE that results in one or more of the following outcomes: death, potentially life-threatening illness or injury, permanent impairment of body structure or function, hospitalization or prolongation of an existing hospitalization, and/or need for medical or surgical intervention to prevent permanent impairment of body structure or function.

An unexpected AE of a medical device is any serious AE related to health, safety, or any potentially life-threatening problem or death caused by a medical device or associated with it. If such an effect, problem, or death was not previously identified in terms of its nature, severity, or incidence rate in the study plan or research request (or in any supplementary study plans or requests), or any other serious unexpected problem associated with a medical device that affects the rights, safety, or well-being of the subjects.

Information requirements

SAE reporting requirements

Blood extraction is the only additional intervention assigned to the study, and it is not expected to increase the risk of AEs. Although SAEs are not expected, they must be reported to the CDR within 10 days of being notified by the sponsor. If an SAE occurs, the PI must immediately notify Datar Cancer Genetics Inc. regardless of the extent of the information available about the AE. This timeframe also applies to new additional information (follow-up) regarding previously reported AEs.

For all SAEs, the PI is required to seek and provide information to Datar Cancer Genetics Inc. or their designs, according to the specified reporting timelines. Additionally, PI may be requested by Datar Cancer Genetics Inc. or by their design to obtain specific follow-up information on an expedited basis. This information may be more detailed than that collected from the AE notification forms. Generally, this will include a description of the AEs with sufficient detail to allow a full medical evaluation of the case and an independent determination of possible causality. Information regarding other potential causes of the event, such as medications and concomitant diseases, must also be provided. SAEs may also be reported to the local institutional review board (IRB) according to the institutional requirements.

Non-serious AE reporting requirements

All AEs will be reported on the AE page of the CRF.

Reporting requirements for adverse and unexpected effects of medical devices

All UADEs must be reported by the investigator to the sponsor within 3 days of identifying or becoming aware of the event. Investigators are responsible for preparing and submitting complete, accurate, and timely reports to the reviewing IRB regarding any UADE that occurs during the investigation as soon as possible, but in no case later than ten business days after becoming aware of the effect.

Requirements for sponsor reporting to regulatory authorities

Datar Cancer Genetics Inc. will immediately assess UADE. If Datar Cancer Genetics Inc. determines that a UADE poses a reasonable risk to the subjects, it will terminate all investigations or parts of the investigations that present that risk as soon as possible. The termination will occur no later than 5 business days after the sponsor makes this determination and no later than 15 business days after the sponsor receives the first notification of the effect.

Efficacy endpoints criteria

Endpoints

The primary definitions are presented in Table 1. The primary endpoint of this study is to assess the overall performance characteristics of the TriNetra^TM Prostate test, where the diagnosis is confirmed by histopathology as the reference method. Sensitivity and specificity analyses will be conducted (Tables 2 and 3).

Table 1.

Definitions

Sensitivity (SE)	Sensitivity (SE) or the true positive fraction of the test is defined as: SE = TP/(TP + FN)
Specificity (SP)	Specificity (SP) or the true negative fraction of the test is defined as: SP = TN/(TN + FP)
False positive fraction (FFP)	The false positive fraction (FFP) is defined as: FFP = FP/(TN + FP)
Negative predictive value (NPV)	The negative predictive value (NPV) is defined as: NPV = TN/(FN + TN) and/or NPV = (SP * (1 - Prevalence))/((1 - SE) * (Prevalence) + SP * (1 - Prevalence))
Positive predictive value (PPV)	The positive predictive value (PPV) is defined as: PPV = TP/(TP + FP) and/or PPV = (SE * Prevalence)/(SE * Prevalence + (1—SP) * (1—Prevalence))

Table 2.

Diagnostic performance of TriNetra™ Prostate using histopathology including PIN+

TriNetraTM – prostate	Reference test (histopathological diagnosis)
	PIN+/prostate cancer+/prostate cancer with PIN+	PIN-/prostate cancer-
TriNetraTM+	A (TP)	B (FP)
TriNetraTM-	C (FN)	D (TN)

Table 3.

Diagnostic performance of TriNetra™ Prostate using histopathology excluding PIN+

TriNetraTM – prostate	Reference test (histopathological diagnosis)
	Prostate cancer+/prostate cancer with PIN+	Prostate cancer
TriNetraTM+	A (TP)	B(FP)
TriNetraTM-	C (FN)	D(TN)

Subjects who do not complete the recommended tissue diagnosis will be excluded from all performance analyses, and subjects with rejected samples for TriNetra^TM Prostate analysis due to suboptimal blood samples will be excluded from the primary endpoint analysis. Subjects with histopathological findings indicative of prostate adenocarcinoma will be considered to have a positive result and will be included in sensitivity calculations.

Sample size and lower threshold justification

For the lower bound of the 95% confidence interval (CI) above the threshold, where the threshold is 85% for specificity and 80% for power with a 5% type 1 error, 283 negative prostate cancer diagnosis samples (nonmalignant) will be required. (expected test specificity, 90%). For the lower bound of the 95% CI above the threshold, where the threshold is 65% for sensitivity and 80% for power with a 5% type 1 error, 133 positive prostate cancer diagnosis samples are required. (expected test sensitivity, 75%). Prostate biopsy has resulted in negative malignancy findings in approximately 50%–70% of patients (non-prostate cancer group) and positive cancer findings in approximately 25%–50% of patients (prostate cancer group). Loss to follow-up or the unavailability of a conclusive tissue diagnosis may occur in approximately 20% of patients. Unmasking will be conducted for the first 250 samples to determine the percentage of benign vs. malignant representation in the recruited patients to determine the final sample size required to reach evaluable non-malignant and evaluable prostate cancer cases.

Analysis approach, primary efficacy analysis

Primary efficacy analysis

The specificity and sensitivity will be evaluated, with a lower confidence limit exceeding 65%. Rejection of the null hypothesis will demonstrate that specificity is no worse than the prespecified lower limit of 65%. Specificity and sensitivity will be calculated by considering equivocal results as positive and will also be recalculated by considering equivocal results as negative.

Additional analyses

The analysis will be repeated to include ethnicity, race, and age, with modelling based on regression, including sensitivity distribution analysis by TNM stage, sensitivity distribution analysis by Gleason grade, sensitivity distribution analysis by Gleason score, performance distribution analysis by PSA level, performance distribution analysis by race/ethnicity, and performance distribution analysis by age.

The negative and positive predictive values will be estimated using the observed results of the study.

Handling of missing data

Primary analyses will be conducted using complete cases without imputing the missing values. Secondary analyses will use multiple imputations to handle missing data. Missing data regarding TriNetra^TM Prostate results and prostate cancer occurrence will be imputed using other available study data based on exclusion patterns and imputation of demographic data, such as age, race, and ethnicity will be considered.

General statistical methods

Test results from all centers will be compiled and data analysis will be performed based on the compiled results, as described in this protocol. Summaries will also include the baseline characteristics of the subjects enrolled in the study by center and then grouped.

These data will be presented in the form of 2 × 2 tables (Table 4). The CIs for sensitivity and specificity will be reported as described above.

Table 4.

Outcomes in patients diagnosed with prostate cancer

TriNetraTM – prostate	Reference test (histopathological diagnosis)
	PrAD+	PrAD-
TriNetraTM+	A(TP)	B(FP)
TriNetraTM-	C(FN)	D(TN)

Control and quality assurance

During the course of the study, Datar Cancer Genetics Inc. or its agent will conduct periodic remote monitoring visits to ensure that the protocol and Good Clinical Practice are being followed. Center supervisors may review source documents to confirm that the data recorded in the CRFs are accurate. The investigator and institution will allow the center supervisors and relevant regulatory authorities direct access to source documents for verification purposes.

The study center may be subject to review by the IRB/Ethics Committee (EC) and/or quality assurance audits by Datar Cancer Genetics Inc., companies working with or on behalf of Datar Cancer Genetics Inc., and/or inspection by relevant regulatory authorities.

Data handling and record maintenance

All references to the sponsor in this section include all designees, such as Contract Research Organizations or consultants acting on behalf of the sponsor.

Protocol deviations

The center should make every effort to adhere to the procedures or requirements described in this protocol, except when necessary, to eliminate immediate risks to the subjects. Any deviation from this protocol must be reported to the sponsor and appropriately documented. Protocol deviations must also be communicated to the IRBs in accordance with their policies.

Direct access to source data/documents

Investigator institutions must allow for monitoring, audits, IRB reviews, and regulatory inspections related to the trial by providing direct access to data and source documents. These may include electronic medical record systems and/or original paper records.

Case report forms

As used in this protocol, the term CRF refers to either a paper form, electronic data record, or both, depending on the data collection method used in this study. The CRF must be completed for each enrolled subject.

Document archiving

The consolidated good clinical practice (GCP) guidelines define source documents as “original documents, data, and records (e.g. hospital records, clinical and office charts, laboratory notes, memorandums, subject diaries or checklist evaluations, pharmacy dispensing records, data recorded from automated instruments, certified copies or transcripts after verification as accurate copies, microfiche, photographic negatives, microfilms, magnetic media, X-rays, subject files, and records kept in the pharmacy, laboratories, and medical-technical departments involved in the clinical trial).” To allow evaluations and/or audits by regulatory authorities or Datar Cancer Genetics Inc., the investigator agrees to maintain records, including the identity of all subjects (sufficient information to link records, e.g. CRFs and hospital records), all signed informed consent forms, copies of all CRFs, serious AE forms, source documents, and detailed records of treatment disposition, as well as proper documentation of relevant correspondence (e.g. letters, meeting minutes, telephone call reports). The investigator or sponsor will retain study records for a period of 2 years from the latest of the following three dates: the date the investigation ends or concludes, or the date the records are no longer necessary to support a pre-market approval submission. If the investigator cannot, for any reason, continue retaining study records for the required period (e.g. retirement or relocation), they must notify Datar Cancer Genetics Inc. in advance. The study records must be transferred to a qualified design acceptable to Datar Cancer Genetics Inc., such as another investigator, institution, or an independent third party agreed upon. The investigator must obtain written permission from Datar Cancer Genetics Inc. before disposing any records, even if the retention requirements have been met.

Ethics

Risk analysis

The blood extraction process is not expected to present any significant risk to patients. However, it may involve minor risks, such as local bleeding or hematoma. The results of the TriNetra^TM Prostate will not be provided to the investigator or the subject. Consequently, there is no direct benefit to the participants enrolled in this study. The results of the TriNetra^TM Prostate will not be used in the subject’s clinical treatment, and both the subject and investigator will remain blinded to the results.

Institutional review board/Ethics Committee)

The investigator’s responsibility to obtain prospective approval for the study protocol, protocol amendments, informed consent forms, and other relevant documents, such as recruitment advertisements, if applicable, from the IRB. All correspondence with the IRB should be retained in the investigator’s file. Copies of IRB/EC approval must be sent to Datar Cancer Genetics Inc. or a designated person.

Ethical conduct of the study

The study will be conducted in accordance with legal and regulatory requirements as well as the general principles set forth in the International Ethical Guidelines for Biomedical Research Involving Human Subjects (Council for International Organizations of Medical Sciences 2002), the Declaration of Helsinki (World Medical Association), and applicable local regulatory requirements and laws.

Subject information and consent

All parties will ensure the protection of personal health information (PHI). Forms, reports, and publications by the sponsor, as well as any other type of disclosure, will not include PHI unless required by law. The informed consent form must comply with GCP, local regulatory requirements, and legal requirements. The informed consent form used in this study and any changes made during the study must be prospectively approved by both the IRB and Datar Cancer Genetics Inc. before use. The PI must ensure that each participant is fully informed about the nature and objectives of the study and the potential risks associated with participation. The investigator or designated person will obtain written informed consent from each subject or their legal representative before performing any study-specific activity. The investigator will retain the original signed informed consent form for each participant.

Sponsor’s criteria for termination

Premature termination of this study may occur because of a decision by the regulatory authority, change of opinion by the IRB, or Datar Cancer Genetics Inc.’s discretion. Additionally, Datar Cancer Genetics Inc. reserves the right to discontinue the development of TriNetra^TM Prostate for the intended population at any time.

If the study is prematurely terminated or discontinued, Datar Cancer Genetics Inc. will promptly notify the investigator. Upon notification, the investigator must contact all enrolled subjects who have not completed or withdrawn within 30 days, gather all study materials, and complete all CRFs to the extent possible following Datar Cancer Genetics Inc.’s instructions.

Limitations

One limitation of this study is the exclusion of patients with prior cancers or significant comorbidities, which may restrict the generalizability of our findings to broader, real-world populations where prostate cancer often coexists with other health conditions. This exclusion was intentional to minimize potential confounders and to allow a focused evaluation of the TriNetra™ Prostate diagnostic performance in this initial phase.

Additionally we must note that the reliance on PSA levels for study inclusion, while clinically appropriate for this validation phase, may reinforce preexisting diagnostic biases. However, this design choice was made to mirror current clinical practices and facilitate integration of the TriNetra™ Prostate assay into the established diagnostic pathway.

Regarding further evaluation, the future integration of artificial intelligence (AI) with CTC-based diagnostics, such as TriNetra™ Prostate, may offer additional opportunities to improve diagnostic precision. AI-driven models could potentially assist in pattern recognition, risk stratification, and clinical decision support when combining CTC results with imaging and clinical data. Although this study does not involve AI-based analysis, future research may explore this interdisciplinary approach to further optimize prostate cancer diagnostic pathways.

Ethical approval

Ethical approval for this study (internal code°23/469-EC_P) was provided by the Ethical Committee NAC of Hospital Clínico San Carlos, Madrid, Spain on September 13, 2023.

Consent

Written informed consent is prospectively obtained from each patient involved in the study. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.

Sources of funding

This study is supported by Datar Cancer Genetics, the developer of the TriNetra™ Prostate test.

Author contributions

Conceptualization: J.G.R., M.R.S., F.G.S.; formal analysis: J.G.R., V.C.R., P.M.D., M.R.S.; writing – original draft preparation: P.M.D., A.P.; supervision: J.G.R., J.M.S., L.F.M., F.E.; project administration: V.C.R., I.G., F.G.S. All authors have read and agreed to the published version of the manuscript.

Conflicts of interest disclosure

The authors have no conflicts of interest to declare.

Research registration unique identifying number (UIN)

Not applicable.

Guarantor

Dr. Juan Gómez Rivas.

Provenance and peer review

This paper was not invited.

Data availability statement

Not applicable.