Implementation

Overview

After consulting many experts in the field and affected persons, we determined the value and potential issues of the implementation of our novel approach to B-Cell Lymphoma detection. Current forms of B-Cell Lymphoma diagnosis typically involve invasive and expensive procedures.
A common form of traditional diagnosis, bone marrow biopsy, contains a wide range of drawbacks. The average cost of a bone marrow biopsy at Johns Hopkins University is $5,400 (The Johns Hopkins Hospital, 2023), a steep cost for a simple diagnosis of a very expensive disease to treat. Furthermore, bone marrow biopsies cause a significant amount of emotional and physical stress to the patient. Not only is the procedure very invasive, but according to a 2017 study, 70% of patients noted the operation to be painful, with 32% reporting the pain to be severe (Lidén et al., 2009).
Most current forms of B-Cell Lymphoma diagnosis, including bone marrow biopsy, require the disease to be later into development to identify. And although alternative methods like qtPCR are inexpensive notwithstanding the cost of equipment and training, according to Dr. Lu, a hematologist and medical oncologist at Stony Brook Hospital, these are not performed until after bone marrow biopsy and other diagnostics to confirm their results. Another effective way to determine B-Cell Lymphoma much earlier than traditional diagnosis would be to use biomarkers, more specifically small stranded miRNAs. The one with the earliest diagnosis ability, up to three years prior to traditional diagnosis, is by identifying miRNA-326 (Jørgensen et al., 2020).

Product Use

In order to produce a simple, easy-to-use and store system, we will be producing a cell-free system kit. This kit will contain the necessary protein components of a cell-free system along with a microfluidic assay plate and our genetic circuit. This system only requires refrigeration and micropipetting to be used.
The process begins with the mixture of the buffer, protein components, and genetic circuit, which simply requires micropipetting. This will allow for initial production of the Ago2 protein and GFP repressor mRNA strands. It is worth noting that a promising paper-based implementation of a similar system, utilizing a toehold switch to detect miRNA in an E. coli-based cell-free system, has already been achieved by the 2022 ICJFLS iGEM Team (Proposed Implementation, 2022), which would eliminate the need for this first step. A patient's blood sample must then immediately be added to the microfluidic assay plate in order to release the miRNA-326 from exosomes and filter out the rest of the patient's blood. The miRNA-326 will be bound to the Argonaute2 produced by the cell-free system. This bound Argonaute2 will be guided to the expressed mRNA and will cut at the binding site, preventing expression and thus allowing a certain amount of GFP expression relative to the miRNA-326 quantity. The resulting fluorescence from the GFP protein can be imaged using a plate reader or confocal microscope. This can then be compared with the concentration curve developed by our team to track whether or not a spike in miRNA-326 has occurred, which would indicate the development of B-Cell Lymphoma. This entire process would take less than a day, allowing for a rapid diagnosis when compared with other forms of identification.

Target Audience

Our target audience includes both healthcare providers and patients. This technology has the potential to help improve the healthcare experience of those from different countries and backgrounds.

Healthcare Providers

The cost of the components of a cell-free system for a single reaction is $23.70 (New England Biolabs, 2024), while the cost of the assay is much more difficult to determine. Assays for similar purposes and types of molecules can cost as low as ~$3 (Dou et al., 2019). In total, this system would cost the healthcare provider less than $30 and would require minimal additional equipment such as a micropipette and fluorescent imaging tool. Furthermore, the use of a cell-free kit does not require a hematologist or oncologist as a bone marrow biopsy would, but could instead be performed by a general practitioner or lab technician, further lowering the cost.

Patients

The requirements in order to conduct a bone marrow biopsy are much more physically and psychologically tolling on the patient when compared with the simple blood draw required for the cell-free system. The cell-free system also allows for early diagnosis shown in literature to be up to three years prior when detecting miRNA-326. This early detection would allow for earlier treatment and thus a less intensive treatment with better outcomes.

Rural/Low Income Communities

For rural/low-income communities, bone marrow biopsy may be unattainable or impossible due to the required specialists and equipment (oftentimes a CT scan will be used alongside the biopsy needle). Low-income groups tend to experience worse survival and diagnosis rates. In a cohort of 223,709 patients diagnosed with Large Diffuse B-cell Lymphoma from the National Cancer Database between 2004 and 2018, 87% of patients were White, 8% were Black, and 5% were other ethnicities. In patients 60 years or younger, overall survival outcomes were 76 months in White patients, but only 46 months in Black patients (Ermann, 2022). This is in part due to the large cost of alternative testing. As previously mentioned, bone marrow biopsies are both expensive and difficult for many communities to utilize due to the requirement for specialists. Current blood tests require immunophenotyping and flow cytometry, which requires expensive equipment. Most cytometers cost $50,000-$750,000 (Priya et al., 2023), an amount prohibitively expensive for many underrepresented groups, including lower economic classes and those in poor countries/rural communities.

Challenges

The largest challenge to implementation is the creation of a cheap and effective microfluidic assay. This assay would have to be inexpensive to make the cell-free method more expensive than a bone marrow biopsy. This may be difficult as the miRNA is kept within exosomes in the blood, which would have to be either lysed, centrifuged, or removed in some other method.
A challenge to implementation may be the increased cost if the cell-free system is used as a regular checkup test. The cell-free system may be used as a test at every doctor checkup; this would establish a baseline miRNA-326. A baseline miRNA-326 count would better help to identify spikes which would indicate the development of B-Cell Lymphoma. Despite the added accuracy, the use of this test as a regular checkup would increase the cost as a new test would have to be regularly purchased.

While miRNA-326 has been shown to be associated with B-Cell Lymphoma, it may not be as accurate as traditional diagnosis since cancer cells/growth cannot be directly observed. This may lower the accuracy of the cell-free test when compared with a bone marrow biopsy.

references

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Abhishek Maiti, Short, N. J., Srdan Verstovsek, Powers, C. A., Fullmer, C. A., Reyes, S. R., & Bueso-Ramos, C. E. (2017). Quality and cost comparison of powered versus manual bone marrow biopsy devices in patients with myelofibrosis. Leukemia & Lymphoma, 58(10), 2508–2510. https://doi.org/10.1080/10428194.2017.1295147

Cleveland Clinic. (2022). B-cell Lymphoma: Types, Symptoms & Prognosis. Cleveland Clinic. https://my.clevelandclinic.org/health/diseases/22030-b-cell-lymphoma

Dou, M., Sanchez, J., Tavakoli, H., Gonzalez, J. E., Sun, J., Dien Bard, J., & Li, X. (2019). A low-cost microfluidic platform for rapid and instrument-free detection of whooping cough. Analytica Chimica Acta, 1065, 71–78. https://doi.org/10.1016/j.aca.2019.03.001

Ermann, D. A. (2022, June 9). Racial disparities affecting Black patients with diffuse large B-cell lymphoma. Talk presented at: 2022 ASCO Annual Meeting; June 3-7, 2022; Chicago, IL. https://meetings.asco.org/abstracts-presentations/207250

Jørgensen, S., Paulsen, I. W., Hansen, J. W., Tholstrup, D., Hother, C., Sørensen, E., Petersen, M. S., Nielsen, K. R., Rostgaard, K., Larsen, M. A. H., Brown, P. de N., Ralfkiær, E., Homburg, K. M., Hjalgrim, H., Erikstrup, C., Ullum, H., Troelsen, J., Grønbæk, K., & Pedersen, O. B. (2020). The value of circulating microRNAs for early diagnosis of B-cell lymphoma: A case-control study on historical samples. Scientific Reports, 10(1), 9637. https://doi.org/10.1038/s41598-020-66062-1

Lidén, Y., Landgren, O., Arnér, S., Sjölund, K.-F., & Johansson, E. (2009). Procedure-related pain among adult patients with hematologic malignancies. Acta Anaesthesiologica Scandinavica, 53(3), 354–363. https://doi.org/10.1111/j.1399-6576.2008.01874.x

New England Biolabs. (2024). Neb.com. https://www.neb.com/en-us/products/e5360-nebexpress-cell-free-ecoli-protein-synthesis-system

Priya, S., Brundage, K., & Orlando, R. (2023). A Survey on Core Flow Cytometry Facilities: Instrument Maintenance, Usage, and Funding. Journal of Biomolecular Techniques, 34(4), 3fc1f5fe.557b2117–3fc1f5

Proposed Implementation | ICJFLS - iGEM 2022. (2022). Igem.wiki. https://2022.igem.wiki/icjfls/implementation