Identification of Clinically Insignificant or Significant Prostate Cancer With the miR Scientific Sentinel™ Platform
Identification of Clinically Insignificant or Significant Prostate Cancer With the miR Scientific Sentinel™ Platform
The purpose of the study is to validate the performance characteristics of the miR Scientific
 Sentinel™ PCC4 Assay, which consists of the Sentinel™ PCa, Sentinel™ CS and Sentinel™ HG
 Tests. This is a prospective, observational and non-interventional study. The informed
 participants will provide two or more urine samples over the course of the study, and consent
 to share relevant anonymized clinical data with the study team.

 Participants, between the age of 50 and 80 years with suspicion of prostate cancer for whom a
 core-needle biopsy is performed, and otherwise meeting the inclusion and exclusion criteria,
 will be enrolled and will provide urine samples for the miR Sentinel™ PCC4 Assay for the
 classification of no cancer, low-risk, intermediate-risk and high-risk prostate cancer.

 The miR Sentinel™ PCC4 Assay uses a Classification Algorithm to produce a Sentinel™ Score
 that is derived from the Sentinel™ PCa, Sentinel™ CS and Sentinel™ HG Tests. The test score
 is compared to a calculated cutoff that controls sensitivity for a future patient at a
 desired level (95%), to distinguish between cancer and no cancer, clinically significant
 (nominally Grade Group (GG) 2-5) and clinically insignificant cancer (low risk GG1), and high
 risk (nominally GG 3-5) and low to intermediate risk cancer (nominally GG1 and GG2). The
 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 known to not have prostate cancer based on the biopsy results; and (3) patients
 diagnosed with prostate cancer and whose core needle biopsy histopathology was reported as
 Grade Groups 1 through 5 prostate cancer.

 The "gold standard" assessment of cancer will be made from the results of core needle
 biopsies: participants with no positive cores will be designated "cancer-free"; participants
 with cancer in one or more cores will be designated as having "low-risk" prostate cancer
 provided all cores with cancer have no greater than Grade Group 1 histopathology;
 participants will be designated as having "intermediate-risk" prostate cancer if all positive
 cores have no greater than Grade Group 2 histopathology; and participants will be designated
 as having "high-risk" prostate cancer if any core is Grade Groups 3-5.
Primary Objective(s) To validate the performance of the miR Sentinel™ PCC4 (Prostate Cancer
 Classification 4) Assay for identifying patients with low-risk (nominally GG1),
 intermediate-risk (nominally GG2) and high-risk (nominally GG3-5) prostate cancer in men of
 age 50-80 years with suspicion of prostate cancer for whom a core-needle biopsy is performed.
 These classifications will be compared to the results of core needle biopsies, and of radical
 prostatectomy (where available). The sensitivity, specificity, positive and negative
 predictive values will be established.

 Time Perspective: Prospective This is an observational, non-interventional validation study.
 Patients between the age of 50 and 80 years who have a prostate core needle biopsy will be
 enrolled provided that they have provided a urine sample immediately prior to biopsy. Using
 the Pathological Grade Group system, the results of each biopsy will be grouped as either no
 cancer, Grade Group 1 (GG1) cancer, Grade Group 2 (GG2) cancer, and Grade Groups 3-5 (GG3-5)
 cancer. The performance of the miR Sentinel™ PCC4 Assay to classify the disease status of a
 future patient as either prostate cancer (which is further classified into low-risk,
 intermediate-risk, or high-risk) or no prostate cancer will be established; the sensitivity,
 specificity, positive and negative predictive values will be determined for each test in the
 context of classifying a future patient with unknown disease status.

 Statistical Analysis:

 To estimate the prevalence of no cancer, GG1 cancer and GG2-5 cancer in the target population
 of men, previously published data are referenced below. These published data use Prostate
 Specific Antigen (PSA) level as a group identifier, and it is used here for the sole purpose
 of the prevalence estimations below. Note that the proposed clinical study does not in any
 way use PSA testing or scores to define eligibility criteria.

 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; this means the probability (prevalence) of the event A among
 the subgroup of patients in a population all of whom have the event B.

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

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

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

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

 Published data from the European Randomized Study of Screening for Prostate Cancer (ERSPC)
 and the Prostate Cancer Prevention Trial (PCPT) are used below to estimate population
 prevalences. It is estimated that:

 1. The sensitivity of the PSA≥ 3.0 ng/mL assay for positive biopsy is 44%

 2. The specificity of the PSA≥ 3.0 ng/mL assay for positive biopsy is 68%

 3. The PPV of serum PSA≥ 3.0 ng/mL for positive biopsy is 24%

 4. The NPV of the serum PSA≥ 3.0 ng/mL assay for positive biopsy is 86%

 Using the numbers above, the population proportion estimate of core needle biopsy positive is
 0.14*(2/3) + 0.24*(1/3) = 0.18, and thus the population proportion with a negative core
 needle biopsy is 82%. Note that the ERSPC used sextant biopsies instead of 12-core and for
 this reason, it is considered that 18% to be a lower bound on the population prevalence of
 positive (12-core) biopsy.

 Also, using data that 66% of patients with core needle biopsy positive are in GG1, it is
 estimated the population prevalence of GG1 and GG2-5 core needle biopsies to be (0.18)*(2/3)
 = 0.12 and 0.18*(1/3) = 0.06.

 The following assumptions are used to estimate the misclassification rates of core-needle
 biopsy:

 i) P(true GG1 | Biopsy - ) = 0.12 ii) P(true GG2-5 | Biopsy -) = 0.02 iii) P(true GG2-5 |
 Biopsy GG1) = 0.25 iv) P(true GG1 | Biopsy GG2-5) = 0.08

 Therefore, at most 82% of participants that enter the study will have negative biopsy, at
 least 12% will have positive biopsy and GG1 cancer, and at least 6% will have positive biopsy
 and GG2-5 cancer. At most 10% of participants will actually be GG1 with a negative biopsy
 (false negative) and at most 2% will be GG2-5 with a negative biopsy (false negative).

 Of the participants labeled with GG1 cancer from biopsy, at least 3% to be truly GG2-5. A
 very small proportion of participants are expected to be labeled as GG2-5 on biopsy but truly
 GG1.

 Estimate of Sample Size. The proposed prospective study will serve to establish the
 performance of the miR Sentinel™ PCa Test on a representative sample of participants with any
 suspicion of prostate cancer for whom a core needle biopsy is ordered. The miR Sentinel™ PCa
 test is trained on retrospective data. The current proposed study is targeted to enroll 2000
 participants between the age of 50 and 80 years who receive a core-needle biopsy and who
 provided a urine sample immediately prior to biopsy being performed. At least 6 % of
 participants entering the study will be biopsy-positive and GG2 or above (120/2000), at least
 12% will be biopsy-positive and GG1 (240/2000) and at most 82% (1640/2000) will be biopsy
 negative.

 Regarding false negative misclassifications, at most 200/2000 will be biopsy negative but
 truly GG1 and at most 40/2000 will be biopsy negative but truly GG2-5. On the other hand, at
 least 60/2000 participants to be labeled as GG1 on biopsy but truly GG2-5 and at least
 10/2000 participants to be labeled GG2-5 on biopsy but truly GG1.

 For a cutoff of the miR Sentinel™ PCa Score that results in an observed sensitivity estimate
 of 0.97 among the 360 participants expected to be enrolled in this prospective study with
 prostate cancer, the one-sided upper 95% confidence interval (CI) for the population
 sensitivity of the miR Sentinel™ PCa test that uses this cutoff, denoted P.SE, would be P.SE
 > 0.95, that is, at least 95% sensitivity in the population after considering sample
 variation by only using 360 participants.

 For a cutoff of the miR Sentinel™ CS Test that results in an observed sensitivity estimate of
 0.97 among the 110+60+40 = 210 participants enrolled in this prospective study with GG2-5
 prostate cancer (taking into account the false-negative rate of biopsy), the one-sided upper
 95% confidence interval (CI) for the population sensitivity of the miR Sentinel™ CS Test that
 uses this cutoff, denoted P.CS.SE, would be P.CS.SE > 0.95, that is, at least 95% sensitivity
 in the population after considering sample variation by only using 210 participants in GG2-5.

 This prospective study will continue to enroll participants until a total of 2000
 participants are enrolled.

 Statistical Methodology. As outlined below, this clinical study will enroll participants
 between the ages of 50 and 80 years that are scheduled for a core needle biopsy based on any
 suspicion of prostate cancer. The study will evaluate the properties of the miR Sentinel™
 PCC4 Assay (comprises of Sentinel™ PCa, Sentinel™ CS, and Sentinel™ HG Tests) that is based
 on use of a proprietary Classification Algorithm to identify future patients with prostate
 cancer and to classify prostate cancer as low-risk, intermediate-risk and high-risk disease.

 The proprietary Classification Algorithm employed functions by controlling sensitivity 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 test, that is, the test is (incorrectly)
 negative for a patient who is truly positive.

 To describe how the cutoff for the miR Sentinel™ PCa Score is calculated to control
 sensitivity, for each participant in the training dataset, the miR Sentinel™ PCa Score will
 be calculated using the remaining members of the training dataset and only his small
 non-coding RNA (sncRNA) sequence; that is, the true disease status of each patient in the
 training dataset will be blinded, thereby mimicking the setting for classification of a
 future patient. The cutoff used in the miR Sentinel™ PCa Test is then calculated so that the
 empirical sensitivity over patients in the training dataset with prostate cancer corresponds
 to the value that provides an upper one-sided 95% confidence interval for population
 sensitivity for a future patient of at least 1-α.

 With this cutoff for the miR Sentinel™ PCa Score determined from the training dataset a
 priori, 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 participants 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 participants that are mis-labeled by
 biopsy in the prospective dataset. For example, a participant labeled as having no prostate
 cancer by biopsy and whose blinded miR Sentinel™ PCa Score indicates prostate cancer may be a
 participant that was potentially a false negative by core-needle biopsy (biopsy label
 negative) but a miR Sentinel™ PCa Test suggesting the presence of prostate cancer. The
 validation of this discordance will be addressed in the follow-up data analysis.

 The analysis for the miR Sentinel™ CS Test will employ a similar approach to that described
 above on the subset of the training dataset and the prospective data corresponding to
 participants labeled with GG1 and GG2-5 prostate cancer. Note that in the prospective data,
 all those participants determined to have cancer from the Sentinel™ PCa Test will be included
 in the analysis of the Sentinel™ CS Test. We will determine the performance of the
 proprietary Classification Algorithm for classifying a future patient as GG1 versus Grade
 Group 2 or above prostate cancer on the basis of the same 280 sncRNA sequences used in the
 miR Sentinel™ PCa Test. The corresponding sensitivity, specificity, positive and negative
 predictive values with 95% confidence intervals will be calculated over the prospective
 dataset using the cutoff for the miR Sentinel™ CS Score that was determined using the
 training dataset.

 The blinded Sentinel™ CS Score, calculated for each participant in the prospective dataset
 using only his sncRNA sequence and the training dataset, will enable estimation of the
 proportion who were labeled as GG1 by biopsy, but actually have a Sentinel™ CS Score
 indicating GG2-5, the grade groups that are typically considered high risk and in need of
 treatment. Similarly, when a participant is labeled as GG2-5 but who has a favorable
 Sentinel™ CS Score (GG1), may imply the presence of stable/indolent disease.

 The analysis of the miR Sentinel™ HG Test will use a similar approach to that described above
 while the overall performance of the Sentinel™ PCC4 Assay to classify subjects into low-risk,
 intermediate-risk and high-risk disease will be evaluated using the performance
 characteristics of all three tests.

 Each study participant enrolled will be followed for one year. Participants will provide
 urine samples during each visit, and all relevant clinical data, including re-biopsies, PSA
 results and pathology report from radical prostatectomy (if administered as part of clinical
 care) will be obtained. The follow-up results, if available will be used for outcome
 analysis. For each urine sample provided, the miR Sentinel™ PCC4 Assay result will be
 determined and compared with the available 1 year follow up outcome data to inform the
 sensitivity, specificity, positive and negative predictive values of the tests.
Inclusion Criteria:

 -  Males between the ages of 50 and 80 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 participants due to suspicion
 of prostate cancer.

 -  The participants provided a urine sample within 60 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.