Team:Thessaloniki/Implementation

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Implementation





Overview




METIS is a diagnostic kit for the screening for Pancreatic Ductal Adenocarcinoma at the first two stages of the disease. Its function is based on the detection of specific miRNAs in urine samples with a safe, cheap, and user-friendly procedure. We suggest that this tool can be used in a variety of different settings, such as hospitals and diagnostic centers without any expensive equipment. Here, we will explain how we determined the characteristics of an ideal diagnostic, as well as the factors we took into consideration to incorporate these features in our project design.



Our project design


From our first steps, after the conception of our basic idea, we brainstormed in order to lay the cornerstones of our project. Through literature research, we found that one of the most important challenges, related to the late diagnosis, is the inconsistency of the symptoms [1]. Most of the patients are either asymptomatic at the onset of the disease or present non-specific symptoms such as weight loss, jaundice, anorexia, or diarrhea, leading to a delay of the diagnosis of the disease [2], [3]. For this reason, we decided to design a diagnostic tool for the screening of both symptomatic and healthy populations.
Consequently, we realized that since we intended to design a diagnostic tool appropriate for the preventative screening, before the onset of symptoms, the method should be easily accepted by the patient. That means it should be safe, non-invasive, cost-effective, and provided in an easily reached by the patient place. Through the research we conducted for the safety of our project, we had the chance to confirm these ideas and discover more essential attributes of an ideal diagnostic.



The desired characteristics


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Safety







Our solution


Before we move on to the implementation of our project, it is essential to provide a short description of our basic idea. For more information, you can read our Project Description. METIS is a kit for the early diagnosis of Pancreatic Ductal Adenocarcinoma (PDAC), which is based on the detection of specific miRNA molecules that are upregulated in the urine of patients at stages I and II. These miRNAs are hsa-miR-143-3p, hsa-miR-30e-5p [6] and hsa-miR-1246 [7].
For the detection of these molecules, we are using Toehold Switches, RNA sequences that lead to the expression of a reporter gene, which produces an easily measured signal in the presence of the miRNAs.Toehold Switches are RNA molecules that contain the RBS and “AUG” codon within a hairpin structure. As a result, the binding of the ribosomes to the RBS -and subsequently the translation is inhibited. However, the selected miRNAs are able to bind to the toehold and make the hairpin unfold. In this way, the ribosomes are free to bind to the RBS and begin the translation [8], [9], [10]. Here, this leads to the production of eGFP, a fluorescent protein.
In conclusion, the workflow of our method consists of three main parts: First of all, the sample should be collected and the whole RNA should be isolated by ultracentrifugation. The next step is the amplification of the selected miRNAs specifically and finally, the reaction of the amplification product with the toehold switches and the measurement of fluorescence.
The fluorescence is easy to measure and interrelate with the quantity of the biomarkers. For the measurement of the fluorescence, we are designing a user-friendly device, to minimize the errors that could happen due to the different equipment of labs and handling of the personnel.
To further increase the accuracy of our method, it is important to use the necessary calibrators, to correctly quantify the selected microRNAs. For this reason, we need a negative control sample, to determine the fluorescence that corresponds to the cell-free system or other components of the reaction. This sample should contain the reagents of the in vitro protein synthesis protocol and the toehold switch for each miRNA and the reagents of EXPAR reaction without the miRNA. Furthermore, we need three positive control samples for each miRNA. This sample should consist of all the necessary reagents for the reaction, plus the miRNA. Each positive control sample should contain the miRNA in a different known quantity. We need a sample with a very low quantity of the miRNA, one with an average quantity and one with the highest quantity of miRNA that produces a detectable change in the fluorescent signal.

The exact quantities of miRNAs for these control samples should be determined through clinical trials, based on the measurements of fluorescence and the concentration of miRNAs in the urine of patients with PDAC. Although we understand that this is a critical step for the function of our method, we have decided not to run these experiments, due to safety and bioethical issues. However, we hope that in the future we will be able to test our toeholds with real urine samples.


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Project Description



After the Diagnosis


According to the World Health Organization, the screening for cancer, either in an early or late state, ideally should be accompanied by an efficient method for the management of the disease [11]. Although many statistics suggest that the early diagnosis would contribute to a more effective treatment, we learned that the resection of a tumor at such an early stage is still difficult with the current non-specific imaging techniques.
For this reason, we decided to use a combination of three miRNAs, to narrow down the possible results to one type of cancer. According to Dr. Emmanouilides Christos, Medical Oncologist in Interbalkan European Medical Center, this will allow the doctor, the validation of the results with an imaging technique such as a pancreas-specific CT and the resection of the tumor, to suggest a specific, short, yet efficient cycle of chemotherapy and immunotherapy for the treatment of the disease. This will reduce the risk of false-positive results.
Taking this idea one step further, we suggest that in the future more combinations of biomarkers can be used for the diagnosis of either precursors of PDAC, such as PanIN, MCN and IPMN, to further improve the prognosis of the disease, or different cancer types.


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Integrated Human Practices



Who will use it?


Many factors, both genetic and modifiable, have been identified to be related to the development of PDAC. In 10% of PDAC cases a genetic background is identified, related with mutations in BRCA1 and BRCA2, STK11/LKB1 and other genes commonly involved in other types of cancer as well. On the other hand, modifiable factors include smoking, excessive alcohol use, chronic pancreatitis, obesity or diabetes [11].

Another important factor affecting the incidence of PDAC is age: It has been shown that the risk of developing PDAC increases with age [11], [12]. More specifically, according to the SEER program, the incidence of PDAC starts rising from approximately the age of 50 [13]. This is also, in agreement with the statistics from Cancer Research UK [14]. In the next diagram, we can see the PDAC appearance by age compared by sex. Although the incidence is slightly higher for males, the trend is the same for females, leading to the conclusion that this examination would be beneficial for both men and women above the age of 60.







As mentioned before, the most efficient treatment of PDAC is the surgical removal of the tumor. However, only a small proportion of patients are diagnosed at an early stage before the metastasis of cancer cells [15]. The survival rate even for these patients is low, however, this fact highlights the importance of preventative screening, especially for the patients with an increased risk of developing PDAC. Based on this fact, we suggest that our diagnostic test can be included in a regular check-up for high-risk populations after the age of 60. For more information you can read our Entrepreneurship page.


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Entrepreneurship








Improving accessibility & efficiency: Why toehold switches?





Improving safety: Choosing the appropriate sample and biomarker


As described above we decided to design an in vitro diagnostic, to increase the safety of the patient: This way the patient does not come in contact with hazardous reagents. To further improve the safety of our method we decided to use a sample that can be collected with a non-invasive method. The most suitable sample types for this purpose are urine and saliva.
After conducting an extensive literature research, we determined that the most appropriate miRNAs to use as biomarkers are hsa-miR-143-3p, hsa-miR-30e-5p, has-miR-1246, because of the high specificity and sensitivity that the combination of these three molecules provide. In addition, according to literature these molecules are excreted and upregulated in urine during PDAC stages I and II. This way, not only we utilize a non-invasive type of sample but also, contribute to the earlier diagnosis of the disease.



Improving affordability: Choosing the appropriate amplification method


As described above, the second step in the workflow of our method is the amplification of the specific miRNAs. This step is needed because although the miRNAs in the sample are upregulated, their quantity is still so low that the signal produced from our system is not detectable. The gold-standard amplification technique is Polymerase Chain Reaction (PCR). PCR is widely used for diagnostic purposes and it is known to be accurate and sensitive. However, the thermocyclers needed for the PCR reaction are expensive and this increases the cost of these diagnostic methods [19], [20].
To make the technique more affordable both for the patients and the medical service providers, we decided to use an isothermal amplification method. For isothermal methods there is no need for expensive equipment, lowering the overall cost of our method. Exponential Amplification Reaction (EXPAR) is an isothermal amplification method, designed to amplify small nucleic acid molecules, such as miRNAs [21]. EXPAR seemed the most appropriate method for our diagnostic kit, although there were some problems that we had to solve.



Moving towards a POC diagnostic






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Hardware



Safety & Future Challenges





Summary





References