Team:SDU-Denmark/Description
Project Description
Introduction to PROSTATUS
In the last 20 years, the amount of men diagnosed with prostate cancer (PCa) has been steadily increasing. This correlates with the implementation of screening for the concentration of Prostate Specific Antigen (PSA) in the blood. The PSA-test is highly unreliable with a specificity of 69% and sensitivity of 58%, leaving a 31% risk of false positives and a 42% risk of false negatives [1].
Moreover, an increase of PSA levels in the blood can be caused by other factors apart from the prostate. These factors include having a urinary infection, ejaculating, and taking certain medications. These are all problematic characteristics, where ethical considerations should be taken into account. After initial testing for PSA-levels in the bloodstream, the patient is examined through digital rectal examination. This is performed by inserting a finger into the rectum and touching the prostate.
If there is further grounds for examination, a tissue-sample is taken with a spring-loaded needle. Though local anesthesia is applied, the procedure can be painful as the needle breaks the intestinal barrier. As the barrier is broken, it becomes possible for bacteria from the rectum to enter the blood stream, causing severe infection and even death [2]. In short, the established procedure can be majorly improved.
All this information made a powerful impression on our team during our ideation process, where we were first introduced to the difficulties accompanying PCa. When our team got to know one another more, we learned that our team had a personal stake and this inspired us even more to investigate the possibilities of incorporating synthetic biology and the PCa. After further research and discussions, meetings with a project ideation expert, and exertion of democracy, we came up with PROSTATUS.
PROSTATUS offers a non-invasive way of testing for PCa biomarkers, in order to ensure that more men get tested as they progress in age. Detecting the presence of PCa biomarkers without invasive procedures allows the user to avoid unpleasant and potentially dangerous invasive tests, which can lead to side effects. PROSTATUS works through utilization of specifically designed guide RNAs that target three PCa biomarkers found in urine: TMPRSS2:ERG [2, 3], AMACR [4], and PCA3 [5], and two single-nucleotide polymorphisms (SNPs), rs6983267 and rs16901979, in saliva [6]. RNA biomarkers were chosen based on their high specificity and sensitivity, to ensure a higher accuracy of our test than presently found in the PSA-test. The specificity and sensitivity of TMPRSS2:ERG (71% and 75%), AMACR (81% and 79%), and PCA3 (79% and 69%) all outperform the current PSA-test.
The two SNPs were chosen, as they result in an increased risk of developing PCa when having one or two of the risk alleles. For rs6983267, being homozygous for the G;G risk allele results in a 1.6x increased risk of developing PCa, while being homozygous for the A;A risk allele of rs16901979 increases the risk with 1.5x.
Prostate Malignancy Test (PMT)
PROSTATUS has developed two tests for detection of PCa biomarkers. Prostate Malignancy Test (PMT) utilizes a CRISPR-Cas13a mediated system to detect RNA biomarkers present in urine and provides results through a lateral flow strip.
If the test result is negative, no target sequence will bind and activate the Cas-protein, leaving the reporter sequence intact. Therefore, the biotin molecule will bind to its receptor and the Gold-particle will bind to the FAM end, producing a line at the control line. If, on the other hand, the test result is positive, Cas13a will cleave the reporter sequence. This leads to only biotin binding to the control line, while the gold-antibody and FAM end will bind to the test line making a visible line [7].
Of course, problems with false positives and false negatives are still to be taken into account. Therefore, we also considered how the test and test results would be handled.
Congenital Risk Assessment Test (CRAT)
Apart from the Cas13-mediated system, our team focused on generating another test based off SNPs. SNPs describe genetic variations and have recently been found to influence the progression of PCa [5]. A similar method that utilizes Cas12 was adapted to saliva samples in order to evaluate the genetic risk of getting PCa.
We have chosen to focus on rs6983267 from chromosome 8q24. Through in-vitro transcription of the SNP, we aim to end up with purified RNA that can both be used as a control and as a proof of concept that shows that our Cas13a system has worked. While the urine-based test is targeted at men in the risk group, age 50+, the saliva-based test is targeted towards younger men, in the hopes of spreading awareness and breaking taboos in relation to PCa.
PROSTATUS User Manual
This is the manual that we made for the CRAT and PMT. We carefully considered the user-friendliness, the user-segment (the elderly) and the practical problems of our test.
Manual for PMT
Manual for CRAT
Gamification
Alongside the development of the test, we also worked on methods to encourage talking about PCa and normalizing such conversations for men. This was done through the concept of gamification, where we designed playing cards and created a card game that focuses on early detection. Accompanying the cards are words of encouragement and trivia about PCa, which open for opportunity of conversing on the matter. This is an integral part of the project, as it also aids the normalizing of early testing and knowledge of PCa, allowing for earlier detection.
References
[1] Benecchi, L. PSA velocity and PSA slope. Prostate Cancer Prostatic Dis 9, 169–172 (2006). https://doi.org/10.1038/sj.pcan.4500866[2] Loeb, Stacy et al. “Systematic review of complications of prostate biopsy.” European urology vol. 64,6 (2013): 876-92. doi:10.1016/j.eururo.2013.05.049
[3] Nicholas TR, Strittmatter BG, Hollenhorst PC. Oncogenic ETS Factors in Prostate Cancer. Adv Exp Med Biol. 2019;1210:409-36.
[4] Sreekumar A, Laxman B, Rhodes DR, Bhagavathula S, Harwood J, Giacherio D, et al. Humoral immune response to alpha-methylacyl-CoA racemase and prostate cancer. J Natl Cancer Inst. 2004;96(11):834-43.
[5] Yang Z, Yu L, Wang Z. PCA3 and TMPRSS2-ERG gene fusions as diagnostic biomarkers for prostate cancer. Chin J Cancer Res. 2016;28(1):65-71.
[6] Zheng SL, Sun J, Wiklund F, Smith S, Stattin P, Li G, et al. Cumulative Association of Five Genetic Variants with Prostate Cancer. New England Journal of Medicine. 2008;358(9):910-9.
[7] Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019;14(10):2986-3012.
