Introduction:
Our study
protocol is designed to assess the ability of five biomarkers, previously
identified as having variable sensitivity and specificity in detecting cancer,
when combined in analysis of urine specimens to demonstrate improved results in
detecting the presence of prostate cancer.
The overall goal is to rule in or out prostate cancer with a simple
urine screening test with improved accuracy thereby reducing the need for
prostate biopsy, currently a huge drain on resources, facility use and health
care costs.
Originally intended to use panels of biomarkers for their ability to
detect prostate cancer in urine specimens, the expanded scope now addresses the
clinical need for better patient screening for both diagnosis and treatment, and
to generate sufficient information to possibly create a new, cost effective and
accurate clinical test. To fulfill this
role, new and more rigorous demands must be addressed, including:
1. Comparison to available screening methods
for prostate cancer in order to demonstrate improved sensitivity and cost
effectiveness, just justifying clinical use and widespread adoption.
2. 1. Demonstrating consistency and reproducibility
3. 2. Demonstrating transportability and
preservation of samples over 3-5 days, since sophisticated biomarker testing
will be performed at central specialized facilities.
Further development
of this testing is anticipated to create a reliable, cost effective, rapid and
point of care test. However, each step
in developing such a test will require changes in protocol and corresponding
investigation 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 and
specificity with overall cost savings to the healthcare system, resulting, in
part from declining need for prostate biopsy.
Objectives and Purpose:
This
Research Protocol is intended to assess the role of five biomarkers. We will
attempt to detect these in the urine in screening for prostate cancer. The immediate goal is to develop a physician
ordered, urine based screening test for prostate cancer, as well as for active
monitoring cancer patients, both those undergoing surveillance and those under
therapy. Finally, this could be
widespread means of distinguishing Benign Prostatic Hypertrophy from
Cancer. Our hypothesis and long-term
goal 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 of
men over age 45 who are scheduled for a prostate biopsy. To be included, they must be suspected of
having prostate cancer, and have had ultrasound or MRI determination of the
prostate size. They will have had a physical examination, including a DRE. In addition, they will have recorded the 4K
score, including Total PSA, Free PSA, Intact
PSA, and HK2 (Human kallikrein 2). PSAD
will be determined. This allows
comparison of our biomarker test with PSA, PSAD and the 4K score, all in common
clinical use to assess likelihood of prostate cancer.
No clinical decisions will be based on tests performed in this study and
patient’s care will not be affected in any manner by his participation in this
study. It is reasonable then to assert
there are no identifiable risks from participation. All clinical data required for the study,
including the above biomarkers, the prostate volume and the results of the
prostate biopsy will be anonymized. The
prostate pathology slides will be divided into those used for the patients
care, and the study slides to be read by a single pathologist for all research
subjects. These slides will have been
labelled according to the randomization code described below and reported using
only the code for identification.
Upon signing the consent form, the subject will contribute a urine
specimen no more than 3 days before their scheduled biopsy. This urine specimen will be divided into four
aliquots. If separate urinations are
required that would be allowed. One
urine specimen will be used for routine clinical purposes; the second will be
used 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 active
participation in the study is completed.
If that urine is needed for the patient care, a portion will be sent to
the clinical lab for analysis. All
aspects of the patient management from this time on are dictated by the care
being administered by his physicians and is uninfluenced by the study.
Urine Collection, Handling and Testing
1. 1. First
morning, urine collection immediately upon rising in the morning, recommended
for panel of biomarkers requiring concentration for detection in our sandwich
Elisa assay.
2. 2. Urine
collections should be maintained on ice or refrigerated for the duration of the
collection.
3 3. Approximately
45 ml urine will be voided into a 50-ml urine collection tube in the Assay
Assure (Thermo Fisher Scientific, India).
4. 4. The urine
will be centrifuged at 1,000 × g for 10 min, then washed with
phosphate-buffered saline followed by a second centrifugation at 1,000 × g for
10 min. The urine will be further processed for Sandwich Elisa test or
immediately frozen on dry ice and stored at −80◦C.
The sandwich ELISA requires two antibodies that bind to epitopes that do
not overlap. This can be accomplished by using the affinity-purified monoclonal
and polyclonal antibodies that will be used for panels of biomarker proteins.
One antibody (the "capture" antibody) will be purified and bound to a
solid phase. Antigen is then added and allowed to complex with this bound
antibody. Unbound products will be removed by washing, and a labelled second
antibody (the "detection" antibody) is allowed to bind to the antigen,
thus completing the "sandwich". The assay is then quantified by
measuring the amount of labelled secondary antibody bound to the matrix by
usage of a colorimetric substrate. After successful establishment of the
sandwich ELISA, we will then be ready to detect panels of biomarker protein in
patient urine specimen, which would be a major asset towards the usage of
randomized double blinded study as a rapid diagnostic marker in the field of
prostate 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 coding
table.
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