Establishment and Validation of Urine Biomarkers before Prostate Biopsy: A Sequential-Enrollment, Blinded Study

Introduction:

Our studyprotocol is designed to assess the ability of five biomarkers, previouslyidentified as having variable sensitivity and specificity in detecting cancer,when combined in analysis of urine specimens to demonstrate improved results indetecting the presence of prostate cancer. The overall goal is to rule in or out prostate cancer with a simpleurine screening test with improved accuracy thereby reducing the need forprostate biopsy, currently a huge drain on resources, facility use and healthcare costs.

 Originally intended to use panels of biomarkers for their ability todetect prostate cancer in urine specimens, the expanded scope now addresses theclinical need for better patient screening for both diagnosis and treatment, andto generate sufficient information to possibly create a new, cost effective andaccurate clinical test.  To fulfill thisrole, new and more rigorous demands must be addressed, including:

1.     Comparison to available screening methodsfor prostate cancer in order to demonstrate improved sensitivity and costeffectiveness, just justifying clinical use and widespread adoption.

2.    1. Demonstrating consistency and reproducibility

3.   2. Demonstrating transportability andpreservation of samples over 3-5 days, since sophisticated biomarker testingwill be performed at central specialized facilities.

 Further developmentof this testing is anticipated to create a reliable, cost effective, rapid andpoint of care test.  However, each stepin developing such a test will require changes in protocol and correspondinginvestigation as the methods evolve. Therefore, this pilot study is intended to satisfy proof of concept,early adoption, and clinical benefit as measured by improved sensitivity andspecificity with overall cost savings to the healthcare system, resulting, inpart from declining need for prostate biopsy.

Objectives and Purpose**:**

ThisResearch Protocol is intended to assess the role of five biomarkers. We willattempt to detect these in the urine in screening for prostate cancer.  The immediate goal is to develop a physicianordered, urine based screening test for prostate cancer, as well as for activemonitoring cancer patients, both those undergoing surveillance and those undertherapy.  Finally, this could bewidespread means of distinguishing Benign Prostatic Hypertrophy fromCancer.  Our hypothesis and long-termgoal is that the use of urine for a combined biomarker test will allow a simple,point of care and cost-effective screening test for prostate cancer.

            Methods:

This will be a sequential, non-randomized, single arm, blinded study ofmen over age 45 who are scheduled for a prostate biopsy.  To be included, they must be suspected ofhaving prostate cancer, and have had ultrasound or MRI determination of theprostate size. They will have had a physical examination, including a DRE.  In addition, they will have recorded the 4Kscore, including Total PSA, Free PSA, IntactPSA, and HK2 (Human kallikrein 2).  PSADwill be determined.  This allowscomparison of our biomarker test with PSA, PSAD and the 4K score, all in commonclinical use to assess likelihood of prostate cancer.

No clinical decisions will be based on tests performed in this study andpatient’s care will not be affected in any manner by his participation in thisstudy.  It is reasonable then to assertthere are no identifiable risks from participation.  All clinical data required for the study,including the above biomarkers, the prostate volume and the results of theprostate biopsy will be anonymized.   Theprostate pathology slides will be divided into those used for the patientscare, and the study slides to be read by a single pathologist for all researchsubjects.  These slides will have beenlabelled according to the randomization code described below and reported usingonly the code for identification.

Upon signing the consent form, the subject will contribute a urinespecimen no more than 3 days before their scheduled biopsy.  This urine specimen will be divided into fouraliquots.  If separate urinations arerequired that would be allowed.   Oneurine specimen will be used for routine clinical purposes; the second will beused for immediate biomarker determinations; a third specimen will be frozen to-70*C and kept indefinitely for possible future determinations.

As soon as the patient has produced his urine sample, his activeparticipation in the study is completed. If that urine is needed for the patient care, a portion will be sent tothe clinical lab for analysis.  Allaspects of the patient management from this time on are dictated by the carebeing administered by his physicians and is uninfluenced by the study.

            Urine Collection, Handling and Testing

1. 1. Firstmorning, urine collection immediately upon rising in the morning, recommendedfor panel of biomarkers requiring concentration for detection in our sandwichElisa assay.

2. 2. Urinecollections should be maintained on ice or refrigerated for the duration of thecollection.

3  3. Approximately45 ml urine will be voided into a 50-ml urine collection tube in the AssayAssure (Thermo Fisher Scientific, India).

4.  4. The urinewill be centrifuged at 1,000 × g for 10 min, then washed withphosphate-buffered saline followed by a second centrifugation at 1,000 × g for10 min. The urine will be further processed for Sandwich Elisa test orimmediately frozen on dry ice and stored at −80â—¦C.

  The sandwich ELISA requires two antibodies that bind to epitopes that donot overlap. This can be accomplished by using the affinity-purified monoclonaland polyclonal antibodies that will be used for panels of biomarker proteins.One antibody (the "capture" antibody) will be purified and bound to asolid phase. Antigen is then added and allowed to complex with this boundantibody. Unbound products will be removed by washing, and a labelled secondantibody (the "detection" antibody) is allowed to bind to the antigen,thus completing the "sandwich". The assay is then quantified bymeasuring the amount of labelled secondary antibody bound to the matrix byusage of a colorimetric substrate. After successful establishment of thesandwich ELISA, we will then be ready to detect panels of biomarker protein inpatient urine specimen, which would be a major asset towards the usage ofrandomized double blinded study as a rapid diagnostic marker in the field ofprostate cancer before biopsy.

 Patient Coding

Immediately after signing the informed consent,every patient will be de-identified for the study.  Each site will have a subject codingtable.