Thursday, May 9, 2024
5/9/2024
PROJECT SUMMARY Leptomeningeal disease (LMD) is the metastasis of cancer cells into the pia arachnoid and cerebrospinal fluid (CSF), and is a devastating disease with survival rates of 4 months after diagnosis. Lumbar puncture (LP) with cytology is the gold standard for confirming the diagnosis of LMD; however, it has a low sensitivity with the first LP, requiring multiple additional samples to capture circulating tumor cells (CTCs). Flow cytometry and the CellSearch Assay are alternative methods for detection of CTCs in CSF, and they have demonstrated higher sensitivities with the first LP. These approaches detect CTCs and leukocytes as they both express epithelial cell adhesion molecule (EpCAM), and with additional labeled antibodies, such as those for CD45, they are able to distinguish between the two cell types. Although these approaches have significantly improved sensitivity and are quantitative, they unfortunately require liquid handling steps, costly equipment, and trained personnel. Samples therefore need to be sent to a central lab which is expensive and can take 1-2 weeks for results, which is unacceptable considering the low survival rates. Additionally, one LMD treatment corresponds to placing a CSF reservoir in the brain and injecting chemotherapeutics directly into the ventricle/CSF space, which can occur 2-3 times a week. Since LP with cytology is not quantitative, it currently takes months to know if such a treatment has failed, and by then, it is too late. Flow cytometry and the CellSearch Assay can quantify the concentration of CTCs, but they cannot be used for real-time monitoring of CTCs during treatment. An inexpensive, rapid, equipment-free, and user-friendly assay that can be used at the point of care (POC) is the lateral-flow immunoassay (LFA), which has been used to detect pregnancy and COVID-19. Although the LFA has many advantages, it unfortunately is limited to a yes/no answer and is not quantitative. We therefore propose to develop two types of next generation paper-based quantitative POC devices to allow the detection and quantification of CTCs during the LP and therefore eliminate delays in diagnosing LMD. Specifically, these devices will be able to determine the concentration of cells expressing EpCAM. Since these cells include both CTCs and leukocytes, our devices will also simultaneously determine the concentration of leukocytes through CD45. Similar to flow cytometry and the CellSearch Assay, the concentration of cells expressing CD45 (leukocytes) will be subtracted from the concentration of cells expressing EpCAM to estimate the concentration of CTCs. Such devices could also be easily integrated with treatment procedures, such as the one described above for injection of chemotherapeutics, to assess treatment efficacy. Our devices will be tested with synthetic CSF containing varying concentrations of human breast cancer cells and T-cells. Subsequently, a preliminary validation of the two types of devices will be conducted with clinical samples from approximately 25 LMD positive and 25 LMD negative patients at UCLA.
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