Evaluation of the Sentinel™ PCC4 Assay for Diagnosis, Prognosis and Monitoring of Prostate Cancer in Puerto Rico
Evaluation of the Sentinel™ PCC4 Assay for Diagnosis, Prognosis and Monitoring of Prostate Cancer in Puerto Rico
The miR Scientific Sentinel™ Prostate Cancer Classifier Platform (Sentinel™ PCC4 Assay) is a
 new molecular assay that interrogates 442 small non-coding RNAs extracted from urinary
 exosomes and provides urologists an early, very precise indication of disease status in men
 presenting with suspicion of prostate cancer. The assay classifies the disease status as
 having no molecular evidence of prostate cancer (NMEPC) or molecular evidence of low-,
 intermediate- or high-risk of aggressive, potentially lethal prostate cancer.

 The primary objective of this study is to assess the clarity of explanation of the biological
 background and clinical impact of the Sentinel™ PCC4 Assay, and the comfort level of
 urologists with the clinical data and report form, including the ease of understanding of
 results, and the potential use in clinical management of prostate cancer.

 Men being seen by urologists in the course of their normal practice, presenting with
 suspicion of prostate cancer (based on DRE and/or elevated Prostate Specific Antigen (PSA))
 and scheduled for core-needle biopsy will be asked to consent to the clinical study and
 provide one or more 50 mL sample of non-DRE urine along with relevant anonymized clinical
 data with the study team. The urine samples will be shipped to miR Scientific laboratories in
 Rensselaer NY, for exosomal sncRNA extraction and interrogation. The molecular status of the
 participant will be reported as a Sentinel™ PCC4 Risk Level.

 The second primary objective of this study is to establish the performance characteristics of
 the Sentinel™ PCC4 Assay in the Puerto Rican population for identifying men with NMEPC, and
 men with evidence of high-risk prostate cancer.

 Participants will receive the standard of care (SOC), including measurement of PSA, DRE and
 TRUS-guided systematic or MRI-guided targeted biopsy. Based on the histopathology report of
 the core-needle biopsy, read by pathologists associated with local institution, participants
 with no positive cores will be designated "cancer-free"; otherwise patients with cancer in
 one or more cores will have the clinical data associated with the SOC collected (including
 the Gleason grade group, tumor staging, PSA, race and CAPRA risk assessment score). The CAPRA
 score will be compared to the molecular classification provided by the Sentinel™ PCC4 Assay.
The Sentinel™ PCC4 Assay is a new molecular classification technology that provides an
 assessment of prostate cancer risk of advanced disease based on the expression of a cohort of
 442 small non-coding RNAs.

 At the start of the study, miR Scientific will schedule a teleconference call with all of the
 participating physicians in Puerto Rico (San Juan Bautista Hospital) to outline the study,
 the basis for the test and to explain the differences between this Sentinel™ PCC4 Assay and
 the calculators commonly used in risk assessment for prostate cancer (the CAPRA and/or
 D'Amico risk calculators and any other risk assessment methodology the uro-oncologists
 utilize on a routine basis).

 After the clinical data (see CRF form) and Sentinel™ result have been compiled for each of
 the first 50 patients who meet the inclusion/exclusion criteria, the CSO and/or Medical
 Director of miR Scientific will chair one or more teleconference call with the participating
 physicians to compare the results of the Sentinel™ Assay and the CAPRA and other risk
 assessment calculators, discuss the relative strengths and weaknesses of the tests and answer
 any questions regarding the performance of the Sentinel™ Assay and discordances (if any)
 between the test results. It is important to note that while the clinical data and the
 Sentinel Score are affiliated with specific barcodes, these data are fully anonymized and
 cannot be associated with specific patients. In addition to preserving the PHI
 confidentiality, this also ensures that the information related to the Sentinel™ Score will
 not influence the current standard of care for these patients.

 At the end of each session, the participating physicians will complete a questionnaire
 designed to evaluate the quality of the training and information related to the test result.
 This process will be repeated for every 50 new patients enrolled for the first 200 patients
 and then again after the data for 350 patients becomes available. The analysis of the
 complete data set for the 500 patient cohort will also be presented at the completion of the
 study, and a final questionnaire will be administered.

 To validate the performance of the miR Sentinel™ PCC4 Assay for identifying patients with
 prostate cancer in men of age 45-75 years with suspicion of prostate cancer for whom a
 core-needle biopsy is performed, the miR Sentinel™ PCC4 results with low-, intermediate- and
 high-risk prostate cancer (Grade Group 1, 2 or 3-5) will be compared to the results of the
 CAPRA score. The sensitivity, specificity, positive and negative predictive values (PPV, NPV)
 will be established.

 This clinical utility study is a prospective, observational, validation study. Patients
 between the age of 45-75-year who will have a prostate core needle biopsy and meet the
 inclusion/exclusion criteria listed below will be enrolled provided that they have provided a
 urine sample prior to biopsy. Using the Pathological Grade Group system, the results of each
 biopsy will be grouped as either no cancer, CAPRA low-, intermediate- or high-risk by the
 pathologists at the participating centers.

 The miR Sentinel™ PCC4 Assay uses a proprietary Classification Algorithm to produce a
 Sentinel™ PCC4 classification into one of four groups: NMEPC, low-, intermediate-, high-risk
 prostate cancer. The PCC4 Assay controls sensitivity of the diagnosis and prognosis of a
 future patient with unknown disease status at a desired level, usually at least 95%. The
 proprietary Algorithm has been trained on a dataset consisting of: (1) control subjects who
 presented in urology for conditions unrelated to prostate cancer; (2) subjects with suspicion
 of prostate cancer assumed to not have prostate cancer based on the negative biopsy results;
 and (3) patients diagnosed with prostate cancer and whose core needle biopsy histopathology
 was reported as Grade Groups 1 through 5.

 The performance of the miR Sentinel™ PCC4 Assay to classify the disease status of a future
 patient as either prostate cancer or no prostate cancer and for patients with prostate cancer
 as having low-risk (nominally Grade Group 1), intermediate-risk prostate cancer (nominally
 Grade Group 2) or high-risk prostate cancer (nominally Grade Group 3-5) will be established.
 The test reports will be provided to the consulting urologists who will also have the core
 needle biopsy reports. The discordances between core needle biopsies and the Sentinel™ PCC4
 Assay are well documented in Wang et al. (2020). At the start of the study the urologists
 will be briefed on the interpretation of the Sentinel™ PCC4 Assay, and will subsequently be
 asked to compare and contrast the information provided by the Sentinel™ PCC4 and the SOC (PSA
 +/- DRE and pathology report), using a validated questionnaire. To ensure that urologists
 associated with the project understand the Sentinel™ PCC4 platform, a review of the
 comparative results will be held when the first 50 patients have been accrued, and again for
 every additional 50 patients until 200 patients are accrued, and when 350 and 500 patients
 have been accrued, at which time the trial will have completed its enrollment. The survey
 instrument will be administered after each review to determine whether familiarity with the
 Sentinel™ PCC4 Assay imbues confidence in the risk assessment.

 Statistical Analysis:

 Definitions. The general notation P (A | B) = P(A , B)/P(B) is the probability of the event A
 given that the event B occurred; it is the probability (prevalence) of the event A among the
 subgroup of patients in a population encompassing all who have the event B.

 1. Sensitivity = P (screening test + | truly disease).

 2. Specificity = P (screening test - | truly no disease).

 3. PPV = P (truly disease | screening test +).

 4. NPV = P (truly no disease | screening test -).

 Estimate of Sample Size. The proposed prospective study will serve to establish the
 performance of the miR Sentinel™ PCC4 Assay on a representative sample of patients with any
 suspicion of prostate cancer for whom a core needle biopsy is ordered. The miR Sentinel™ PCC4
 Assay is trained on retrospective data as described above.

 The current proposed study is targeted to enroll at most 500 subjects between the age of
 45-75 years who receive a core-needle biopsy and who provided a urine sample prior to biopsy.

 For any subgroup with a minimum of 100 enrolled participants, if the observed (empirical)
 sensitivity of the PCC4 Assay for that subgroup is 95%, then the one-sided upper 95%
 confidence interval for the true population sensitivity will be 90% or greater.

 This prospective study will continue to enroll participants until the first of either 500
 total participants are enrolled, or at least 100 participants have enrolled in each of the
 four groups: NEPC; Grade Group 1; Grade Group 2; Grade Group 3 and above.

 Statistical Methodology. As outlined below, this clinical study will enroll patients between
 the ages of 45 and 75 years that are scheduled for a core needle biopsy based on any
 suspicion of prostate cancer. The study will evaluate the properties of the Sentinel™ PCC4
 Assay that is based on use of a proprietary Classification Algorithm to identify future
 patients with prostate cancer and to classify the risk of prostate cancer patients.

 The proprietary Classification Algorithm employed controls sensitivity for both identifying
 patients with prostate cancer and identifying high-risk prostate cancer at, or above, a
 pre-specified level, denoted 1-α; for example, the value that has been assumed in this design
 is α=0.05 so that sensitivity is at least 95% in the population. Note that the value of α
 represents the false-negative rate of the assay.

 To describe how the cut-offs for the Sentinel™ PCC4 Assay are calculated to control
 sensitivity, for each participant in the training dataset, the Sentinel™ PCC4 Assay result
 will be calculated using the remaining members of the training dataset and only the current
 participant's sncRNA sequence; that is, the true disease status of each participant in the
 training dataset will be blinded, thereby mimicking the setting for classification of a
 future patient. The tuning parameters used in the miR Sentinel™ PCC4 Assay are then
 calculated so that the empirical sensitivity over participants in the training dataset with
 prostate cancer and with high-grade prostate cancer correspond to the value that provides an
 upper one-sided 95% confidence interval for the corresponding population sensitivity for a
 future patient of at least 1-α.

 With these cut-offs for the Sentinel™ PCC4 Assay determined a priori from the training
 dataset, the corresponding values of sensitivity, specificity, positive and negative
 predictive values will be calculated, along with a corresponding upper 95% confidence
 intervals, on the prospective patient data accrued in this proposed study, with each biopsy
 result blinded, that is using only the participant's sncRNA sequence. Note that these error
 rates refer to the classification of a future patient with unknown disease status.

 The statistical analysis of the prospective data accrued that blinds the group label from
 biopsy will provide an estimate of the proportion of patients that are mis-labeled by biopsy
 in the prospective dataset. For example, patients labeled as having no prostate cancer by
 biopsy and whose blinded miR Sentinel™ PCC4 Assay result indicates prostate cancer may be a
 patient that was potentially a false negative by core-needle biopsy (biopsy label negative).
 The validation of this discordance and others are addressed below in the follow-up data
 analysis section.

 Follow-up Data Analysis. If a follow-up biopsy is scheduled for participants with negative
 biopsies as part of their standard clinical care, the follow-up Sentinel Assay and biopsy
 results will be deemed as the clinical truth for the outcome analysis.
Inclusion Criteria:

 -  Males between the ages of 45 and 75 years.

 -  Signed informed consent prior to initiation of any study-related procedures.

 -  Minorities are included in this protocol.

 -  A clinician has performed a core needle biopsy for the patient due to suspicion of
 prostate cancer.

 -  The patient provided a urine sample within 30 days prior to biopsy being performed.

 Exclusion Criteria:

 -  Persons incapable of providing informed consent.

 -  Persons presenting with clinical symptoms of urinary tract infection, including
 prostatitis at the time of enrollment.

 -  Persons with prior history of invasive treatment for benign prostatic hyperplasia
 within 3-6 months of study enrollment.

 -  Patients treated with finasteride or bicalutamide for BPH or male pattern baldness.