Brief Summary:

This prospective, multicenter study aims to discover, develop, and validate a plasma exosomal RNA-based signature as a rule-out test for predicting bone metastasis in prostate cancer, using baseline treatment-naïve PSMA PET as the gold standard. The study is designed in four sequential phases:

Phase 1 (Discovery, n=250): High-throughput sequencing of plasma exosomal RNAs to identify differentially expressed candidate RNAs.

Phase 2 (Model Development, n=300): Digital droplet PCR (ddPCR) analysis of candidates in an independent cohort to construct and lock the final multi-RNA predictive signature using appropriate machine learning methods.

Phase 3 (Internal Validation, n=300): Independent validation of the locked signature in a consecutive cohort reflecting natural disease prevalence.

Phase 4 (External Validation, n=150): Final independent validation in a multi-center cohort enriched for bone metastasis.

Primary Outcome:

To evaluate the diagnostic performance of the signature as a rule-out test for PSMA PET-defined bone metastasis. The primary performance metrics are:

Sensitivity, with a prespecified target of ≥95% (to ensure minimal false negatives).

Specificity at the threshold that achieves the ≥95% sensitivity. A specificity of ≥30% will be considered supportive of clinical utility. A specificity of ≥30% (or a lower bound of the 95% confidence interval exceeding 20%) will be considered supportive of clinical utility.

Need:

Current biomarkers lack sensitivity and specificity for early detection of bone metastasis. More importantly, existing tools lack adequate negative predictive value to safely rule out bone metastasis in low-risk patients, leading to over-imaging or delayed detection. There is an urgent need for a non-invasive rule-out test to safely defer PSMA PET/CT in very-low-risk patients. Plasma exosomal RNAs offer a promising liquid biopsy approach, but prospective multicenter studies with rigorous validation are lacking.

Secondary Outcomes:

1. Secondary metrics include negative predictive value (NPV), positive predictive value (PPV), area under the ROC curve (AUC), calibration, and decision curve analysis.
2. Correlation between exosomal RNA levels and number of bone metastatic lesions (PSMA PET).
3. Association with PSA, PSMA PET SUVmax, and MRI findings.
4. Tissue-plasma correlation to confirm tumor origin (exploratory).
5. Mechanistic exploration of key candidates via in vitro/in vivo assays (exploratory).
6. Subgroup analyses by hormone sensitivity, metastatic pattern, Gleason grade (exploratory).

Inclusion Criteria:

1. Histologically confirmed prostate cancer scheduled for baseline PSMA PET.
2. PSMA PET performed prior to any prostate cancer-related treatment.
3. Blood samples collected prior to any treatment AND prior to prostate biopsy.
4. Willing to undergo prostate biopsy if clinically indicated (after blood collection).
5. Written informed consent.
6. Age ≥18 years.

Exclusion Criteria:

1. Any prior prostate cancer treatment before baseline PSMA PET.
2. Blood samples collected after prostate biopsy.
3. Other active malignancy within past two years (excluding non-melanoma skin cancer).
4. Inadequate blood sample quality or quantity.
5. Severe comorbidities interfering with study conduct.

Background Prostate cancer (PCa) is a leading cause of cancer-related morbidity worldwide, with bone metastasis being the most frequent and devastating complication. Early and accurate detection of bone metastasis is critical for timely intervention and improved patient outcomes. However, current biomarkers such as PSA lack sufficient sensitivity and specificity for early metastasis detection, and conventional imaging (e.g., bone scan, CT) often identifies metastases only after they become clinically apparent. PSMA PET has emerged as the most sensitive imaging modality for defining metastatic status at baseline, but its high cost, limited availability, and radiation exposure preclude its use as a universal screening tool.

Concurrently, liquid biopsy-particularly the analysis of plasma exosomal RNAs-offers a unique window into tumor biology. Exosomal RNAs are stable in circulation, reflect the molecular characteristics of the primary tumor, and can be detected using sensitive methods such as high-throughput sequencing and digital droplet PCR. Despite their promise, large-scale prospective multicenter studies with rigorous multi-phase validation are lacking.

Need There is a critical unmet need for a non-invasive, robust biomarker that can identify patients at very low risk of bone metastasis, thereby allowing safe deferral of PSMA PET/CT. Existing tools lack adequate negative predictive value to confidently rule out bone metastasis in low-risk populations. A successful rule-out test would reduce unnecessary imaging, lower healthcare costs, and minimize patient radiation exposure. This study implements a four-stage design with phase-specific sample size considerations aligned with contemporary standards for biomarker development.

Study Design and Technical Phases This is a prospective, multicenter, phase-sequential biomarker development and validation study. The gold standard for bone metastasis status is baseline, treatment-naïve PSMA PET/CT. All blood samples are collected prior to any prostate cancer-related treatment and prior to prostate biopsy (if performed) to avoid biopsy-induced contamination. Whole blood (approximately 10 mL in EDTA tubes) is processed within 2 hours to obtain plasma, which is stored at -80°C until analysis.

The four phases are defined as:

Phase 1 (Discovery, n=250): High-throughput RNA sequencing (e.g., small RNA-seq) of plasma exosomes. Differential expression analysis (e.g., DESeq2 or edgeR) identifies candidate RNAs distinguishing patients with versus without bone metastasis. Multiple testing correction (FDR <0.05) is applied.

Phase 2 (Model Development, n=300): An independent cohort enriched for bone metastasis (approximately 200 positive, 100 negative) is used for quantitative measurement of candidate RNAs using ddPCR. A continuous risk-scoring model is constructed using machine learning (e.g., regularized regression, random forests, or gradient boosting). The model is locked after internal cross-validation, before any validation data are examined.

Phase 3 (Internal Validation, n=300): A consecutive cohort reflecting natural disease prevalence (expected bone metastasis rate ~30%) is used to evaluate the locked model. In this phase, a single cut-off value is selected to achieve a sensitivity of ≥95%. The specificity at that cut-off is the primary endpoint. Secondary metrics (NPV, PPV, AUC, calibration, decision curve analysis) are also assessed.

Phase 4 (External Validation, n=150): A geographically distinct, multi-center cohort enriched for bone metastasis is used to assess generalizability, applying the same cut-off determined in Phase 3.

Statistical Considerations Sample Size Justification Sample sizes were chosen based on published recommendations for phased biomarker studies and on formal precision analysis for the primary endpoint (specificity at the ≥95% sensitivity threshold).

Phase 1 (Discovery, n=250): This sample size is typical for high-throughput discovery studies. With an expected bone metastasis prevalence of ~30%, approximately 75 positive and 175 negative cases will be available, providing sufficient power to detect differential expression with FDR <0.05.

Phase 2 (Model Development, n=300): Following the "events per variable" (EPV) rule (≥10 events per candidate predictor) and assuming a parsimonious final model (≤20 candidate RNAs after Phase 1), a minimum of 200 positive events is required. The cohort is enriched for bone metastasis (target ~67% positive), thus a total of 300 patients (≈200 positive, ≈100 negative) is planned.

Phase 3 (Internal Validation, n=300): The primary endpoint is specificity at the cut-off achieving ≥95% sensitivity. Assuming a true specificity of 35% at this threshold, a sample of approximately 210 negative patients (from total n=300 with ~30% bone metastasis prevalence) yields a 95% confidence interval half-width of approximately ±6-7%. This precision is more than sufficient to rule out a clinically useless specificity (e.g., <15%) and allows robust subgroup analyses.

Phase 4 (External Validation, n=150): This multi-center cohort is enriched for bone metastasis (target ~40-50% positive). The sample size of 150 (≈75 positive, ≈75 negative) allows precise estimation of specificity (95% CI half-width ±11% assuming 35% specificity) and sensitivity in an independent setting, confirming generalizability.

All sample sizes may be adjusted modestly based on actual recruitment; any adjustments will be documented.

Statistical Analysis Plan (High-Level) Analyses will be performed using R (version ≥4.2). The final analysis plan will be finalized before locking validation data.

Phase 1: Differential expression analysis (DESeq2/edgeR). Candidate selection based on fold change, adjusted p-value, and abundance.

Phase 2: Machine learning model building using cross-validation. The final continuous model is locked.

Phase 3: Apply the locked model to the internal validation cohort. Determine the cut-off value that achieves sensitivity ≥95%. Report specificity, NPV, PPV, AUC, calibration, and decision curve analysis at this cut-off.

Phase 4: Apply the same cut-off to the external validation cohort and repeat the performance evaluation.

Secondary/exploratory analyses: Correlation, subgroup analyses, mechanistic studies (as detailed in the secondary outcome measures).

Data Management Data will be stored centrally in a REDCap database with 3-step authentication. Data entry will occur approximately every 3-6 months. Patient confidentiality will be maintained.

Inclusion Criteria

1. Patients with histologically confirmed prostate cancer who are scheduled to undergo baseline PSMA PET imaging.

2. Patients who undergo PSMA PET imaging prior to any prostate cancer-related treatment (including androgen deprivation therapy, radiotherapy, or surgery).

3. Patients who provide blood samples for plasma exosomal RNA analysis collected prior to any treatment AND prior to prostate biopsy (if applicable).

   Whole blood samples (approximately 10 mL) will be collected in EDTA tubes at this specified time point. Samples will be processed within 2 hours to obtain plasma and stored at -80°C until analysis.This timing ensures circulating exosomal RNA profiles reflect tumor biology without biopsy-induced contamination.

4. Patients who are willing to undergo prostate biopsy if clinically indicated (biopsy performed after blood collection).

5. Patients who provide written informed consent to participate in the study.

6. Age ≥18 years.

Exclusion Criteria

1. Patients who have received any prior prostate cancer-related treatment before the baseline PSMA PET scan (including hormonal therapy, radiotherapy, chemotherapy, or surgery).
2. Patients whose blood samples were collected after prostate biopsy.
3. Patients with a history of other active malignancies within the past two years (excluding non-melanoma skin cancer).
4. Patients with inadequate blood sample quality or quantity for exosomal RNA extraction and analysis (e.g., hemolysis, insufficient volume <8 mL).
5. Patients with severe comorbidities or conditions that, in the judgment of the investigator, could interfere with study compliance or pose significant risk.

The study enrolls a total of approximately 1000 patients with histologically confirmed prostate cancer from multiple centers. All patients undergo baseline PSMA PET/CT and provide pre-treatment blood samples (collected before any therapy and before prostate biopsy). The single overall cohort is allocated into four sequential, independent phases: Phase 1 (Discovery, n=250), Phase 2 (Model Development, n≥300, bone-metastasis enriched), Phase 3 (Internal Validation, n≥300, natural prevalence), and Phase 4 (External Validation, n=150, multi-center enriched). Bone metastasis status is defined by PSMA PET/CT.