1.0 Project Summary:

Introduction: Prostate cancer (PCa) is the most commonly detected cancer in men and is the second leading cause of cancer death. Differences in race and ethnicity have been shown to have differences in PCa incidence, detection, and outcomes. Current prostate cancer screening involves PSA which is a nonspecific protein marker (aka kallikrein) that can often leads to unnecessary biopsies (up to 74% benign biopsies) and clinical overdiagnosis (with up to 22% clinically insignificant cancer). Recently more sophisticated tests have been developed for PCa screening in the United States such as the Prostate Health Index (PHI) and the 4k (kallikrein) score, as well as clinical models that use information from the patient clinical history. However, these tests utilize limited serum protein assays and none of the established screening protocols utilize genetic variables to help account for the likely inherited risks as seen in different ethnicities.

A recent Swedish, prospective, population-based study, published in the Lancet Oncology, developed a unique multivariable biopsy outcome prediction model within a Nordic population of nearly 60,000 men. This model, the Stockholm3, which incorporated plasma protein markers, germline DNA SNPs as well as clinical variables, was shown to be capable of reducing the number of biopsies by 44% compared to PSA while maintaining adequate sensitivity for detection of PCa.

It is unknown whether an approach developed in Sweden that incorporates protein markers, genetics, clinical variables, and genetic ancestry would be beneficial in a racially diverse cohort.

Hypothesis: We hypothesize that, a prospectively studied multiethnic cohort of men with the Stockholm3 test will identify unique and common risk factors that improve prostate cancer detection.

Aim: To assess the performance of the Stockholm3 test as compared to PSA and to identify unique features associated with PCa in Black (n=500), Asian (n=500), White Hispanic (n=500), and White Non-Hispanic (n=500) men.

Methods: We propose a prospectively identified cohort with participating institutions which have screened positive to undergo a prostate biopsy to have a retrospective analysis the Stockholm3 test and ancestry markers. Within this cohort we will examine several predetermined risk factors to investigate their relationship to prostate cancer.

This blood sample will be tested for quantitative levels of serum protein markers and DNA will be extracted and will be tested for germline mutations as defined by the Stockholm3 test and other ancestry informative markers. Results from the study will be presented in such a way that no individual information will be disclosed.

2.0 Background/Scientific Rationale

PCa is the most commonly detected cancer and the second leading cause of cancer death among US men overall; however, mortality rates among AA are nearly twice as high among black compared to white men. The Stockholm3 test is the first of its kind to incorporate protein level measurements and germline mutation assays that have been shown to improve PCa detection and reduce unnecessary biopsies in a European white cohort, however these objective measurements have not been assessed in a primarily multiethnic cohort.

3.0 Objective/Aims

To test blood samples from multiethnic populations with the Stockholm3 test to find associated risk factors in the detection of prostate cancer and show improved prediction over PSA alone.

3.1 Primary endpoint

• Detection of International Society of Urological Pathology Gleason Grade Group 2 Prostate Cancer on prostate biopsy

o Gleason Grade Group scale is used to grade prostate cancer if found on a scale from 1 to 5. A higher score means a worse outcome.

3.2 Secondary endpoint

• National Comprehensive Cancer Network Unfavorable intermediate prostate cancer or higher grade

o National Comprehensive Cancer Network risk stratification scores range from very low risk, low risk, intermediate risk, high risk and very high risk. Intermediate risk is stratified into favorable intermediate risk and unfavorable intermediate risk. The higher the risk the worse the outcome

4.0 Eligibility

4.1 Inclusion Criteria

• Men with age range (45.0 - 75.0 years), without prior evidence of prostate cancer
• Meeting standard of practice clinical criteria for a prostate biopsy. Able to obtain informed consent.

4.2 Exclusion Criteria

• There are no specific exclusion criteria. If there is a paucity of data with regards to the defined variables being analyzed, those patients might be excluded from sub analyses but not all.

5.0 Subject Enrollment

The subjects will be consented for prostate biopsy as part of standard of care. No additional advertising will be used for enrollment. Study enrollment goal will be 2,000 men who have a self-described race/ethnicity of: African/Black (n=500), Asian (n=500), White Hispanic (n=500), and White Non-Hispanic (n=500) men.

6.0 Study Design and Procedures

The research coordinator will explain the information contained within the consent. Additionally, patient's blood will be drawn prior to their biopsy.

Prior to the biopsy, blood will be collected in x2 EDTA 4 ml tubes after obtaining consent from the subjects. One tube will be immediately centrifuged (10 minutes at 2000G) and plasma decanted to a tube without additives (this typically produces 1.5 ml of plasma). The decanted tube (with plasma) and the remaining EDTA tube (with whole blood) is then frozen and stored at the designated participating institutional site. It will be stored at -20 Celsius until being shipped.

Patient data will be stored in a REDCap database, hosted on Sweden's secure server. Data will be stored for the duration of the study, and 5 years afterwards for data analysis purposes.

Consented patients will be tracked by patient logs by each participating institution. The medical record number will be collected to keep a consistent identifier for data collection by key site personnel. Once all the patient data is recorded the data will be exported from REDCap with the MRN removed. There will be no patient identifiers used at the Karolinska Institute or A23 lab. The following PHI and non-PHI information will be logged of the patient:

PHI:

Medical record number (MRN)

Non-PHI Demographic data

• Stockholm3 ID number
• Race
• Zip code

Clinical data

• Total PSA
• Age on sampling date [years]
• Family history of prostate cancer
• Use of 5-alpha reductase inhibitors
• Earlier biopsy conducted
• Prostate volume [Prostate volume as measure with US]
• DRE status [Benign/normal, Nodule/induration felt, Asymmetry, Not performed]

AND

Outcome data - Results from biopsy performed immediately after blood venipuncture, i.e.:

Results will be separated into targeted biopsy cores and systematic biopsy cores

• Gleason Score 1
• Gleason Score 2
• Gleason Sum
• Cancer length (mm) (total and highest grade)
• Number of cores
• Number of positive cores
• Time to perform biopsy after blood draw [days]
• Results from MRI, i.e. PIRADS (0, 1, 2, 3, 4, 5)

Permitted use:

To run the Stockholm3 test defined by Gronberg et al AND Ancestry informative genetic markers

Samples will be shipped to the Uppsala based laboratory (A23 Laboratory) in Sweden for analysis. Each patient will have two blood samples (plasma and whole blood) and will be frozen at -20 Celsius. The blood samples will then be tested for quantitative levels of serum protein levels and DNA will be extracted from white blood cells and will be tested for gene and small nucleotide polymorphic (SNPs) germline mutations and variants .

Genotyping will be performed using custom genotyping assays. Plasma will be used for protein analysis. Plasma protein analysis will be performed using a custom protein assays including total and free PSA, hK2, MSMB, and MIC-1. PSA will be tested with a commercial assay.

Based on the results from the plasma protein analysis, the genetic analysis and clinical data, the Stockholm3 Risk Score will be calculated. The participants' samples will be treated in accordance with the regulations of Sweden at the laboratory based in Uppsala, Sweden.

Results of the tests will not be shared with the patient, nor will the results change or impact medical decisions.

7.0 Expected Risks/Benefits

Anticipated Risks:

As this is retrospective analysis of deidentified patient information as well as deidentified biospecimens, there are few anticipated risks. A confidentiality breach as well as loss of privacy are possible, however every effort will be made to minimize this risk.

Anticipated Benefits:

Participants will advance scientific and clinical knowledge.

8.0 Data Collection and Management Procedures

This study will utilize REDCap (Research Electronic Data Capture), a software toolset and workflow methodology for electronic collection and management of clinical and research data, to collect and store data. The Karolinska Institute Information Technology Department will be used as a central location for data processing and management. REDCap is hosted by KI-IT in the Biomedicum (Solnavägen 9, Solna, Sweden 17165)

9.0 Data Analysis

Data analysis will be performed by the PI, co-investigators and/or key research personnel.

10.0 Quality Control and Quality Assurance

Key research personnel will be responsible for ensuring all data collected adheres to the protocol.

11.0 Data and Safety Monitoring

This study is minimal risk and all efforts will be made to ensure there are no confidentiality breaches as well as no loss of privacy.

12.0 Statistical Considerations

Power analysis This study is being conducted among several sites and thus pooled analysis will be performed. Based on the framework developed a two-sided alpha of 0.05, 250 men in each ethnicity gives 80% power to detect 10 percentage points differences in sensitivity and/or specificity of the Stockholm3 test across different ethnicities. Pooled data from several sites will allow for comparison between non-Hispanic White, Africa/Black, Asian, and Hispanic White men. Within each ethnicity group of 250 men, the same sample size gives a 90% power for detecting differences in AUC between Stockholm3 and PSA for detection of PC that are at least 10 percentage points (primary aim).

Goal accruement is 500 men within each race/ethnicity, interim analysis will be performed when 250 men in each race/ethnicity is enrolled.

Data Analysis Descriptive univariate statistics will be used to compare groups. Binary endpoints will be assessed with a logistic regression model. Statistical analysis will involve logistic regression modeling, AUC calculation, calibration analyses and calculation of basic performance characteristics (sensitivity, specificity and predictive values).

A genetic risk score will be calculated using allelic odds ratios based on GWAS studies. We will also test for differences in Stockholm3 score between these four groups using Wilcoxon tests, as well as construct ROC curves; AUCs and their 95% confidence intervals. Calibration analyses will be performed.

All statistical analyses will be performed using R (R Foundation for Statistical Computing, Vienna, Austria).

13.0 Regulatory Requirements

13.1 Informed Consent The participants indicate their consent to participate in the study by signing informed consents for accessing medical records, conducting genetic research and undergoing venipuncture for blood samples.

13.2 Subject Confidentiality Data used for this study will be stored in REDCaps and all data transferred between institutions will remain deidentified throughout the study.

13.3 Unanticipated Problems Any unanticipated problems will be immediately reported to the Site-specific ethical review board by designated research personnel.

Inclusion Criteria:

• Men, with age range (45.0 - 75.0 years), without prior evidence of prostate cancer
• Meeting standard of practice clinical criteria for a prostate biopsy. Able to obtain informed consent

Exclusion Criteria:

• There are no specific exclusion criteria. If there is a paucity of data with regards to the defined variables being analyzed, those patients might be excluded from sub analyses but not all.