Professor Cornell University Ithaca, New York, United States
Introduction::Kaposi’s sarcoma (KS) is a cancer affecting both skin and internal organs, for which a virus (Kaposi’s sarcoma-associated herpesvirus; KSHV) is necessary but insufficient cause. In sub-Saharan Africa, KS is one of the most commonly occurring malignancies, and it confers high mortality. Early detection can mitigate the effects of KS, but the local infrastructure is insufficient for timely histopathological diagnosis. To address this, we have been pursuing KS diagnosis by quantifying KSHV DNA content in skin biopsies via a point-of-care device called TINY which performs loop-mediated isothermal amplification (LAMP). However, DNA extractions from skin biopsies require excessive time to digest the tissue chemical and a series of centrifugations to extract purified DNA; thus, the DNA extraction process is the rate-limiting step at the point of care. To streamline this process, we hypothesized that a small piece of tissue with a high surface area would facilitate a rapid, direct-to-LAMP (D2L) DNA extraction. To obtain optimal patient skin biopsy for D2L extraction, we developed SLICER, an inexpensive tool which cuts a biopsy into three pieces: two “halves” for histopathology and traditional DNA extraction plus a thin slice for D2L extraction. We implemented D2L extraction immersing the thin slice into an alkaline solution at room temperature and subsequently added a neutralizing buffer. At this point, the solution can be used directly in LAMP reactions in TINY. With the implementation of the proposed approach, we anticipate a total time to result of ~2 hours, which is about one-third of the current approach. At this point, its contents can be amplified and quantified using real-time LAMP. With this approach, we aim to bypass the lengthy methods and series of centrifugation steps for adequate DNA extraction, which would ultimately give a diagnosis in less than 2 hours.
Materials and Methods:: The rapid biopsy slicing mechanism SLICER was developed using off-the-shelf components. The device consists of two shaving razors positioned in parallel at 0.5 mm apart to cut the biopsy as well as three 3D printed parts – a piece for housing the blades, a piece for mounting the biopsy, and a piece in between the blades to aid in slice removal. The direct-to-LAMP solution is composed of a 20 mM NaOH solution and a Tris-EDTA solution (100 mM Tris-HCl and 0.5 mM EDTA, pH 8). The NaOH solution was diluted in water from a 0.1 M solution (obtained from VWR Cat. BJ71395-1L). The Tris-EDTA solution was prepared by adding Tris-HCl and 1X TE Buffer (VWR Cat. V6231). Porcine and human skin tissue were used for the optimization of the slicing mechanism and D2L (Obtained from the local butcher in Cortland, NY, and Weill Cornell Medicine). Optimization was assessed through DNA quantification through the GAPDH genes of both species. Once optimized, ~50 SLICERS were shipped to the Institute of Infectious Diseases (IDI) in Uganda, where clinical validation was performed against traditional DNA extraction.
In-house validation of the approach was done in Ithaca, NY, employing pig and human skin (obtained from the local butcher shop and Weill Cornell Medicine, respectively).
Results, Conclusions, and Discussions:: The SLICER mechanism was designed and optimized first using porcine skin, then tested on human belly and breast skin tissue. All the initial punch biopsies had a diameter of 5 mm (as it is currently done clinically). During bench testing on human tissue, SLICER consistently produced thin tissue slices averaging 7.1% (~0.27 mm thick) of the original biopsy mass. Further validation of the SLICER prototypes was done clinically in Uganda. On 25 patient skin biopsies, the prototype yielded tissue slices averaging 9 mg, or ~12% of the total biopsy mass. No damaged or miscut biopsy was recorded in the trials. In parallel, we optimized the D2L extraction using the same samples. All trials were done with a sample size of N=4, where we observed an increased DNA yield with incubation from 10 minutes to 1 hour, after which a plateau was observed. Currently, clinical validation of D2L using SLICER is undergoing in Uganda. Congruence between these results and gold-standard diagnostic methods would support the SLICER/D2L approach as a streamlined LAMP-based POC diagnostic for KS.
Acknowledgements (Optional): : We would like to thank our collaborators at the Institute for infectious diseases in Uganda, Weill Cornell Medicine, and University of California San Francisco
References (Optional): : Snodgrass, R., Gardner, A., Semeere, A., Kopparthy, V. L., Duru, J., Maurer, T., ... & Erickson, D. (2018). A portable device for nucleic acid quantification powered by sunlight, a flame or electricity. Nature biomedical engineering, 2(9), 657-665.
Gardner, A., Alvarez, J., Genovese, C., Snodgrass, R., Kopparthy, V., Erickson, D., & Cesarman, E. (2022). Simplified detection of Epstein-Barr virus for diagnosis of endemic Burkitt lymphoma. Blood Advances, 6(12), 3650-3654.
