Team:Thrace/Human Practices

When we started looking for the idea that would become our project, we had many different thoughts. Something that was key to us all, was for our project to be impactful. In order for a project to have a real impact in the world, it would have to provide a solution to a crucial, contemporary problem and ultimately improve the quality and expectancy of human lives. After some contemplation, we decided that developing a diagnostic device for a disease that is hard to diagnose would meet this prerequisite that we had set for ourselves. Trying to decide on the disease that we would diagnose, many ideas were on the table. For example, we were considering creating a device for diagnosing IBD, since it is currently on the rise and it is difficult to diagnose it without invasive techniques. Soon after discussing with experts in the field such as Dr. Kouklakis and Dr. Skendros, we understood that physicians would never trust a device that is not as accurate as the golden standard method, colonoscopy.

Despite reaching a dead end with IBD, we did have one breakthrough. We realized that most diagnostic methods for diseases of the large intestine are either too invasive or involve unpleasant techniques. Hence, we came to the conclusion that our project could be the solution to the need for minimally invasive diagnostic methods when it comes to diseases of the large intestine.

How did we come up with our project?

Then we contacted Professor of Physiology at the Medical School of the University of Athens Dr. George Kollias, who introduced us to cutting edge innovations in biosciences, and helped us understand what it takes to create a truly innovative biotech project, that of course needs extensive research till its implementation. We asked, Assistant Professors of our department, Dr. Kolovos and Dr. Giannakakis about the kind of biomarkers that we were thinking to use in such a device and at the same time we trained and discussed with entrepreneurs that we met through Mindspace competition, Mrs. Markaki, Mr. Anapliotis and Mr. Kalligas about the market need and the tools to evaluate the impact that this device can have on our societies. We found that cancer stage at the time of diagnosis has a strong influence on the length of survival, as it is visible in the following diagram: Figure 1: SEER 18 2011–2017, All Races, Both Sexes by SEER Summary Stage 2000 The earlier it is detected, the better the chance of surviving five more years and the treatment is less extensive. Unfortunately, for colorectal cancer, only 38% are diagnosed at the local stage [2]. In our journey towards creating and determining the final version of our project, we further discussed with experts in colorectal cancer and manufacturing of diagnostic devices.

Understanding the Problem

First, we needed to understand the problem at its core. So we contacted gastroenterologists, who because of their specialty conduct screening examinations on an everyday basis. One of the people we talked to is Dr. Georgios Grammatikos. He highlighted that the problem is bigger than we first thought. Even though a percentage of the population does follow the recommended screening regimen, a big proportion unfortunately fails to do so. Having worked in both Germany and Greece, Dr. Grammatikos highlighted that the issue is especially apparent in Greece.

In order to further explore the extent of the issue and to look into it’s possible causes, we conducted a survey on Greek people’s attitude toward screening for colorectal cancer. We asked them if they follow the recommended regimen and if not, the reasons why they don’t.

The results showed that 51% of the people who took part in the survey don’t get a colonoscopy every 5 years, as it is recommended, and the main reason for their refraining is the discomfort of the examination.

Furthermore, 66% don't take a stool test, even though this examination causes less discomfort than colonoscopy. The main reason for not taking the test was that they were indifferent towards the exam, proving that more education is needed on the alternative methods of screening for colorectal cancer.

Almost all of the respondents noted that in order for them to get screened for colorectal cancer regularly, there should be a test that is not painful.

At this point, it became crystal clear that the way someone feels during an examination is of great importance for their adhesion to a screening regimen.

Designing the Solution

It is now obvious that most people avoid screening predominantly due to the uncomfortability of the exam. So in order to convince more people to get screened, a less invasive technique is needed. We decided that the solution would be a screening method that would be as easy and painless as possible.

To conduct screening for colorectal cancer, a biological sample is needed. We had meetings about the topic with Dr. Kyriaki Bakirtzi, the scientific director of the Experimental Educational and Research Center, with experience in research for colorectal cancer biomarkers. She suggested among many innovative screening ways, the use of saliva as one very accessible but still in early research stages biological sample. We all agreed that this biological fluid would be the perfect choice for our project, since it would eliminate the invasiveness and uncomfortability of screening. So, we opted for saliva as our chosen biological sample.

Before proceeding with our human practices and finding out the experts’ and people’s opinion and feedback on our choice, we first needed to check if screening for colorectal cancer with saliva was even possible. So we had to check if biomarkers for colorectal cancer in saliva exist. After extensive literature research, we concluded that while the research still has a long way to go, there is some evidence of changes in the composition of saliva correlated with colorectal cancer, enough to consider that it is worth supporting the actual development of such a device in the near future.

We also contacted professor Yiorgos Apidiannakis from the Biology department of the University of Cyprus, who has researched innovative ways of colorectal cancer diagnosis. He informed us about the advantages and disadvantages of diagnosing the disease in saliva and helped us take a closer look at the literature. This helped us look at our project from a new and more comprehensive point of view. Our choice of biological sample was also validated by the results of the survey that we conducted. The vast majority of respondents stated that they would use the device consistently.

Choosing Biomarkers

Since our choice to use saliva was official, we then started designing the device. It was clear that we had to detect biomarkers in saliva, but we had to choose which ones.

-Fusobacterium nucleatum: F.nucleatum is linked to colorectal cancer pathology and more than 40% of CRC patients exhibited identical strains of F.nucleatum in their CRC and saliva specimen [3], but it is also associated with other diseases of the oral cavity such as periodontal disease. To solve this firstly, we could assess the medical history of the tested individuals and rule out samples from individuals with periodontitis, as Fusobacterium nucleatum can be found in large quantities in the oral cavities of patients with periodontitis and this could produce a false positive result in our device. Secondly, testing just for one biomarker wouldn’t be enough to produce an accurate test result. Dr. Kyriaki Bakirtzi advised us to detect multiple biomarkers at the same time, since one biomarker could easily produce a false positive result.

-Αfter more bibliographical research for a second biomarker we opted for salivary microRNAs. There are a lot of different types of microRNAs so we had to find the one that matches our needs. We first found miR-21 which is, according to bibliography, a trustworthy biomarker for colorectal cancer [4]. On that point our instructor Dr. Georgios Tsekenis stated we also wanted our biomarker to be characterised with novelty and questioned the specificity of that particular microRNA. That’s why we decided to replace miR-21 with miR-766-5p [5].

-Knowing that our assay can be potentially used for quantification also of protein biomarkers, in order to make the device even more accurate we thought of implementing a third biomarker, IL-8 [6]. Then, we consulted Dr. Kolios who is a gastroenterologist and professor of pharmacology at the Democritus University of Thrace and has many years of experience in the field of biomarkers and inflammation related to the large intestine. We discussed with him our selection of biomarkers. He told us that IL-8 isn’t a good option and would give us many false positive results, since it is associated with a vast spectrum of inflammatory conditions. Taking his feedback into account, we decided to leave IL-8 out of our chosen pool of biomarkers.

Accuracy of results

Something that doctors like Dr. Mimidis and Dr. Grammatikos and scientists like Dr. Apidianakis emphasized to us since the early beginnings of our project is that the result of the device needs to be very accurate. Colorectal cancer is a serious disease, so a false negative could prove to be dangerous for the patient.

The lack of sufficient bibliography for biomarkers in saliva, the correlation of the chosen biomarkers with other diseases and the diversity in the composition of saliva during the day are all factors that can reduce the accuracy of the result.

For the above reasons and with a sense of responsibility towards patients, we struggled to design the device in a way that will almost eliminate the false negative results but there is still a long way to go. Our aim is not to develop a golden standard diagnostic tool, but as rapid tests for Covid-19 do, to promote the detection of colon cancer and thus allow the frequent screening of the population due to the accessibility of the device.

Implementation

Still there was one crucial piece missing from the puzzle that is our project. Since the technical aspects of the device were determined, we started thinking about the bigger picture: how can the device be implemented in the real world? And where exactly is there a need for our device? It was obvious to us that to encourage people to take the test, it needs to be as easily accessible as possible. For this reason, we thought that the most convenient way for someone to get tested would be if they had a device that they can use on their own, right from the comfort of their own home. We discussed our idea with manufacturers of medical devices such as Dr. Gizeli and Dr. Tsorbatzoglou and entrepreneurs such as Mrs. Markaki, Mr. Kalligas and Mr. Anapliotis.

Even though Dr. Gizeli and Dr. Tsorbatzoglou stated that we may face many challenges for a home-kit like the one we were thinking of, they did not discourage us and at the same time, the entrepreneurs saw greater value and a bigger market need for such a device that can be used as a kit for at-home use. In this way, the consumer can purchase the device from a pharmacy and use it from the comfort of their own home, without having to visit a medical facility, which can be more expensive and time-consuming.

Despite that fact, physicians such as Dr. Mimidis stated that the elderly wouldn’t be comfortable using the device on their own. Therefore, he suggested that the device should be designed for use at a primary care setting.

Almost 60% of the respondents exclaimed their preference for a home kit instead of having to visit a doctor’s office to take the test.

In the end, we decided to trust the public opinion and design the device so that it is more suitable for at-home use.

Changing our initial readout method

After deciding to create a kit for at-home use, we started designing each part of the device. Initially, we thought of using the SHERLOCK protocol, which has an electrochemical based readout. Following numerous discussions with aforementioned stakeholders and experts in assay and biosensor development, Dr.Tsekenis, Dr. Gizeli and Dr. Tsorbatzoglou, we had to reconsider our initial design. Taking into account their feedback, we decided to change the readout from electrochemical to fluorescent. There were many arguments to support this switch.

Firstly, the research technology of electrochemical detection using CRISPR Cas12a is still at its infancy, so it is difficult to implement. Secondly, multiplexing is easier and quicker with the fluorescent assay because it involves readily available hardware, like a smartphone camera instead of a custom potentiostat. Finally, the SHERLOCK protocol implements a pre-amplification step that results in a large increase of sensitivity that reaches the attomolar range, thus making it sufficient for the quantification of our biomarkers

References

Figure 1: Cancer of the Colon and Rectum - Cancer Stat Facts. SEER. (2021). Retrieved 21 October 2021, from https://seer.cancer.gov/statfacts/html/colorect.html

Colorectal cancer statistics | World Cancer Research Fund International. WCRF International. (2021). Retrieved 21 October 2021, from https://www.wcrf.org/dietandcancer/colorectal-cancer-statistics/
Colorectal Cancer - Statistics. Cancer.Net. (2021). Retrieved 21 October 2021, from https://www.cancer.net/cancer-types/colorectal-cancer/statistics
Komiya, Y., Shimomura, Y., Higurashi, T., Sugi, Y., Arimoto, J., & Umezawa, S. et al. (2018). Patients with colorectal cancer have identical strains of Fusobacterium nucleatum in their colorectal cancer and oral cavity. Gut, 68(7), 1335-1337. https://doi.org/10.1136/gutjnl-2018-316661
Sazanov, A., Kiselyova, E., Zakharenko, A., Romanov, M., & Zaraysky, M. (2016). Plasma and saliva miR-21 expression in colorectal cancer patients. Journal Of Applied Genetics, 58(2), 231-237. https://doi.org/10.1007/s13353-016-0379-9
Rapado-González, Majem, Álvarez-Castro, Díaz-Peña, Abalo, & Suárez-Cabrera et al. (2019). A Novel Saliva-Based miRNA Signature for Colorectal Cancer Diagnosis. Journal Of Clinical Medicine, 8(12), 2029. https://doi.org/10.3390/jcm8122029
Najdaghi, S., Razi, S., & Rezaei, N. (2020). An overview of the role of interleukin-8 in colorectal cancer. Cytokine, 135, 155205. https://doi.org/10.1016/j.cyto.2020.155205
Kellner, M., Koob, J., Gootenberg, J., Abudayyeh, O., & Zhang, F. (2019). SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature Protocols, 14(10), 2986-3012. https://doi.org/10.1038/s41596-019-0210