The portion of dialysis population in Taiwan is much higher than any other country for numerous years, and that leads to the fact that urothelial cancers here in Taiwan hold a higher prevalence than the world average. Urothelial cancers in Taiwan can be classified into mainly two groups, Bladder Cancer and Upper Tract Urothelial Cancer (UTUC). We perceived that solving problems of cancer detection would be a great way to help the society both globally and locally. Our aim is to improve current situations both scientifically and socially. After a deeper research, we found out that early detection of UTUC is more of a challenge than Bladder Cancer, the other common urothelial cancer in Taiwan, as it typically involves full anesthesia which patients are reluctant to opt for.
Stage 1：Project Purpose
Visiting Dr. Li at Chin-An Dialysis Center
In the early stages of our project, we met numerous problems and needed professional advice. Therefore, we turned to experts, especially the main stakeholders related to our project. Of which, the most directly linked to our project are nephrologists working at dialysis centers.
We first interviewed Dr. Li at Chin-An Dialysis Center regarding the incidence of urothelial cancers in Taiwan. We learned that UTUC stands a higher fraction in urothelial cancers locally at approximately 40% as compared to the world average, which is approximately 20%. We then further found out that the core reason behind this is the high incidence of kidney failure and undocumented usage of uncertified Chinese herbs. This has certainly inspired us to look deeper into the the situation of UTUC in Taiwan.
Fig.4 A group photo with Dr.Li
Fig.1-3 Our Team interviewing Dr. Li. on UTUC occurrence in Taiwan
Dr. Sung (Wen-Wei Sung)
As we chose to do research on urothelial carcinoma, many obstacles arose. Therefore, we decided to consult experts’ opinions.
Dr. Sung is a urologist in Chung Shan Medical University Hospital. At first, we wanted to detect microRNAs associated with UTUC in urine samples. However, we learned that it is difficult to detect microRNAs in urine samples as there are too many interfering substances in urine for it to develop into an accurate detection method. Therefore, he suggested blood serum instead as our body fluid sample of choice.
Fig.5 Photo taken with Dr. Sung before COVID-19 lockdown
Dr. Lin (Lin Po Hung)
During the session, Dr. Lin has verified the fact that delaying UTUC treatment will put patients at a signficantly higher risk of a reduced 5-year survival rate, which makes early detection of UTUC important.
Sadly, apart from Ureteroscopy, methods like CT and MRI have relatively low accuracy or are not suitable for patients with kidney failure. When we proposed our idea to him, regarding an in vitro UTUC detection kit, he shared some opinions on it. With regard to the specificity of microRNAs as biomarkers, Dr. Lin felt that using amplification can indeed overcome the problem of microRNAs being present in a very minute amount in blood serums, and has great potential in UTUC's early detection.
Fig.6 Dr.Lin pointing out our mistake after our presentation on the location where UTUC occurs
Fig.7 Our team members presenting the high risk groups of UTUC
Determining Detection Method with iGEM MingDao
We did some background research ourselves, and found two miRNAs hsa-miR-33b-3p and hsa-miR-664a-3p that exhibit 4 and 8 fold increases in early to mid stage UTUC patients as compared to healthy individuals. This was promising to us, as the significant levels of increase will better help to differentiate UTUC patients from others. Therefore, we went forth with choosing those two miRNAs mentioned above as the biomarkers of our detection kit.
Then, we discussed with iGEM Mingdaoabout our detection methods. Through our discussions, we determined and assured our method, which later on became RCA.
For more information, please see Partnership.
Stage 2：Project Design
During the process of designing our experiments, we consulted several professors to ensure that what we aimed for and the experiments we designed were practical and were able to be used clinically. We sincerely appreciate these experts for giving us useful advice and kind instructions.
Prof. Yao (Bo Yao)
After we decided to connect the extraction and amplification steps, we found a research paper by Professor Bo Yao on how to use magnetic beads to detect and capture miRNA. We then invited Professor Yao to have an online meeting with our team to provide us with some feedback. We really appreciate Professor Yao for sharing her thoughts on the topic and for giving us suggestions on how to test the efficiency of our kit and improve it’s specificity and sensitivity.
Fig.8 Our wet-lab members having a meeting with Professor Yao Bo virtually-1
Fig.9 Our wet-lab members having a meeting with Professor Yao Bo virtually-2
Our Senior: Kuan-Lin, Chen
Kuan-Lin Chen was the Dry-lab Leader of the iGEM team of CSMU Taiwan 2019, and is currently a graduate student studying at National Cheng Kung University.
Initially, we encountered numerous difficulties in this area, especially in ensuring no major loops in our circular DNA probe and our first model in circular DNA sequence design. Due to the fact that Kuan-Lin is part of the iGEM community as a past participant in a position where much professional expertise in this area is required, we decided to reach out to him for advice, and he gave us many feedbacks that were proven very useful throughout the course of our project. He first helped us alleviate obstacles we faced in the Dry-lab portion of our project. On top of these, he also gave us valuable advice on teamwork and the iGEM competition.
With his advice on these aspects, we have successfully used the python coding language, more efficiently and accurately, to improve our circular DNA design and generate the sequence we needed.
Prof. Chang (Chang Wen-Wei)
As the Professor and Head of Department of the Biomedical Sciences Department in Chung Shan Medical University specializing in Molecular Medicine, Tumor Biology, and Stem Cell Medicine, Professor Chang Wen-Wei has extensive knowledge on working with various types of cancers and micro-RNAs(the biomarkers of our choice). Therefore, our team turned to him for advice specifically regarding our wet-lab experiment procedures.
Through the consultation with Professor Chang, we had many takeaways in terms of the determination of the minimal concentration of micro-RNAs of choice in typical UTUC patients, the number of magnetic beads needed for optimal operation of GotCha, and the usage of micro-RNA mimics. With his professional advice, we then altered our protocol and provided a way to determine micro-RNA concentration in patients through working backward with the CT value provided by journals online using RT-PCR. This value derived will then, in turn, validate our product, GotCha, in the detection of UTUC.
Although we were unable to carry out on our RT-PCR experiments due to the constraints on lab accessibility put down by the COVID-19 pandemic, we have managed to provided a much more complete lab protocol to prove the validity of our product.
Fig.10 A photo of Professor Chang and some of our team members
Stage 3：Project Execution
Dr. Hsieh (Yi-Hsien, Hsieh)
Upon choosing miRNAs as biomarkers of our diagnostics tool on UTUC, we met several issues. As we were fairly inexperienced with verification experiments in the context of miRNA detection, we turned to Dr. Hsieh for assistance. As he has been doing research about miRNA for years, he gave us valuable advice on doing the miRNA experiments. Relating to the short lengths of the DNA sequences used in our experiments, he gave us advice on how to verify these small strands of DNA through gel-electrophoresis.
Prof. Yang (Yang Jyan Jou)
In order to revise bugs in our experiment design, we had a meeting with Professor Yang Jyan Jou. Professor Yang majors in Biomedical Sciences and Human Genetics. After discussing our project, Dr. Yang pointed out several problems:
- The design of probe sequence needs to prevent probe from binding with others.
- MiRNA concentrations used in our experiment should be similar to that of healthy individuals.
- It is important to make sure that the probe sequence used is stable to enable ligation.
We made great improvement after Professor Yang gave his advice.
Integrated Human Practices
Through multiple interviews and discussions with experts in diverse fields, we were able to re-exanimate our goal since the very beginning and improve our project. Our aim is to come up with a solution to the current problems of UTUC detection, and further help solve local problems.
Physical Interviews with Patients
UTUC potential patients are one of the main stakeholders and end-users of our project. As Taiwan experiences high incidence of kidney dialysis, this makes UTUC a heightened problem locally (see Description for more information). In this spirit, we paid a dialysis center a visit to know more on current day UTUC detection as well as their needs and requirements as patients in cancer detection methods.
Through presenting our idea and interviewing them, we gained many insights from the session. We gathered that potential patients like them, who are of high risks of getting UTUC, have requirements such as low-cost, non-invasive methods to be satisfied to overcome their inertia in getting checks on potential diseases they are vulnerable to, such as UTUC. Through this session, we have also realized the importance of tailoring our project to our end-users, as any diagnostic kit on the market is basically useless without the support of its end-users. With that principle in mind, we decided to cater to their needs as users of the diagnostic tool, in order to ensure the legitimate impact of our project in society.
Through mutual understanding of our project and their illness, we have managed to gain knowledge and their opinions on the management of UTUC. On top of their personal journeys of illness-battling, the patients that we managed to interview have also provided us with the emotional barriers they had to cross when it came to the detection of UTUC, which inspired us to continue to fully improve our project. Upon sharing GotCha with them, we have also received positive feedback and faith that we will introduce a product into the field that is indeed needed to elevate the chances of early detection of UTUC.
Fig.12 Our members documenting patients' answers to our questions
Fig.13 Photos are taken under patients' permission
Fig.14 Photos are taken under patients' permission
Fig.15 Photo-taking and recording are both done with consent of the patients
Experience sharing with Nantou Shiuhkuang Senior High School
This is one of the earliest events. We held a workshop, sharing our experiences through the process of iGEM, to high school students in Nantou Shiuhkuang Senior High School. Through the session, we shared extensively on our experience working with SynBio topics as well as our project on UTUC detection. We hope that with such activities we have managed to integrate our project back to the younger demographics of society.
See Communication and Education page for more information on how we took this workshop as a chance to publicize knowledge about iGEM and also SynBio.
Fig.16 A screenshot at the ending of our experience sharing
UTUC and Health Education Workshop in CSMU
Although the early detection of UTUC is an essential problem that must be solved, it is undeniable that UTUC remains to be one of the less known types of cancers in society. Not only is it an issue that the general public are not informed of it, the lack of awareness of UTUC can also put people at an increased risk of finding detection methods very redundant in the early stages when symptoms are not apparent. From day one, our team has strived to overcome the inertia in patients' willingness of getting checked for UTUC by coming up with a low-risk, low-cost and efficient test kit, this is merely one side of the work done. Beyond coming up with alternative options, it is important to propel the public towards awareness of the dangers late UTUC diagnosis poses to personal health through a bottom-up approach.
With this in mind, we decided to connect with the public to directly spread awareness through our sharing on the topic of UTUC, prevention measures, possible symptoms, and so on.
However, due to the COVID-19 pandemic, we were not able to carry out real life events and workshops with a large variety of populations. Though this was the case, we were still able to put together one UTUC workshop in our very own university (For more information, see Communication and Education).
Before and after the session, we requested attendees to fill out questionnaires regarding their understanding of UTUC. As seen below, it is evident that our workshop has indeed helped the public gain understanding and has brought awareness to the topic of UTUC.
Fig.17 Question 1 for our questionnaire: Did you gain a better understanding of UTUC after the workshop? (100% of the attendees answered "yes")
Fig.18 Question 2 for our questionnaire: Which of the following is true about iGEM?
(The color red is the correct answer: "teams choose their own topics, and do the design and modeling")
Fig.19 Question 3 for our questionnaire: Where is UTUC "not" located at?
(The color yellow is the correct answer: "renal pyramids")
Fig.20 Question 4 for our questionnaire: Which of the following is a potential factor that causes UTUC?
(The color green is the correct answer: "all of the above")
Fig.21 Question 5 for our questionnaire: Which of the following is "not" true about kidney health care?
(The color yellow is the correct answer: "going on a Mediterranean diet doesn't effectively prevent chronic kidney disease")
We hope that in the future when restrictions imposed by the pandemic have died down, we will be able to host bigger events to benefit the society in this direction.
Fig.22 Presenting the "Chronic Kidney Disease" page
Fig.23 Photo taken during the workshop
Dr.Lin (Lin Po Hung)
We also consulted Dr. Lin Po Hung, another experienced and esteemed urologist, on the need of our project on the medical field today.
After sharing our project idea with him, Dr. Lin was positive on using blood serum as a test sample for our detection kit due to the risk of inaccuracy caused by large amounts of interfering substances in urine samples. He did affirm that an early detection method for UTUC is in need in the medical field due to the lack of accuracy of the current methods for UTUC detection such as CT scans, as well as the inertia in patients that comes with invasive diagnostic tools such as Ureteroscopy.
After listening to our project sharing, Dr. Lin not only shared huge support for our idea but also pointed out that detection methods of similar nature probably cannot be incorporated into the mainstream health system locally just yet. Therefore, he suggested that we can adopt the Laboratory Developed Test (LDT) pathway, where tests are conducted in laboratories out of the main healthcare system.
Fig.24 A virtual group photo of Dr. Lin and some of our members
Prof. Hsieh (Ming-Chang, Hsieh)
After consulting Dr. Lin, we found out that our product has high potential on the LDT market. After more research on LDT methods, we decided that we needed professional advice in evaluating our diagnostic kit from an entrepreneur’s point of view. Therefore, we contacted Professor Hsieh, a clinical test instructor, and inquired him about the potential of our product.
Prof. Hsieh gave us very holistic advice and opinions on our product.
Firstly, accuracy of diagnostic tests is greatly prioritised on the LDT market.
Secondly, for LDT products to acquire human blood serum samples, products have get approval by the Taiwan FDA (TFDA) to collaborate with external laboratories.
Thirdly, it is adviced that we contact research foundations for Chronic Kidney Disease (CKD) patients to acquire test samples of UTUC patients as well as healthy individuals to do a holistic assessment of the accuracy of GotCha. Then, we can provide data for LDT standards assessment.
Through understanding the procedure and things to take note of in launching LDT kits, we hope that the development of GotCha will turn into a reality.
Fig.25 Our members giving a short presentation of our topic
Fig.26 Professor Hsieh and one of our members
Fig.27 A group photo of Professor Hsieh and some of our teammates
Feedback from LEADGENE BIOMEDICAL Inc.
Though we acquired detailed information on LDT products, we also wished to pave a long-term pathway to ensure the longevity of our product in the market. To achieve this, we contacted LEADGENE BIOMEDICAL Inc. and managed to book a session with their executive, Mr. Zhuang. Due to COVID-19, this session was held online on Microsoft Teams.
After listening to our sharing on our project, he thought that our idea definitely carried potential. However, he also thought that our project is more suitable for In Vitro Diagnostic Test. On the IVD market, our product has to develop into a qualitative test in order for more individuals to easily and regularly use GotCha. Therefore, though we will start with trying to enter the LDT market, through data collection and procedures, we strive to introduce GotCha as an IVD test in the long-term.
Fig.28 A screenshot of Mr. Zhuang and some of our members during our consultation