Hakho Lee

Empowering the on-site detection of nucleic acids by integrating CRISPR and digital signal processing

AbstractAddressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing) for on-site NA detection. The CreDiT platform integrates i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals and ii) a robust fluorescent detection based on digital communication (encoding/decoding) technology. These features enable a rapid assay (<35 minutes) in a single streamlined workflow. We demonstrate the sensitive detection of cell-derived HPV DNA targets down to single copies and accurate identification of HPV types in clinical cervical brushing specimens (n = 121).

Inaugurating High‐Throughput Profiling of Extracellular Vesicles for Earlier Ovarian Cancer Detection

AbstractDetecting early cancer through liquid biopsy is challenging due to the lack of specific biomarkers for early lesions and potentially low levels of these markers. The current study systematically develops an extracellular‐vesicle (EV)‐based test for early detection, specifically focusing on high‐grade serous ovarian carcinoma (HGSOC). The marker selection is based on emerging insights into HGSOC pathogenesis, notably that it arises from precursor lesions within the fallopian tube. This work thus establishes murine fallopian tube (mFT) cells with oncogenic mutations and performs proteomic analyses on mFT‐derived EVs. The identified markers are then evaluated with an orthotopic HGSOC animal model. In serially‐drawn blood of tumor‐bearing mice, mFT‐EV markers increase with tumor initiation, supporting their potential use in early cancer detection. A pilot clinical study (n = 51) further narrows EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. The combined expression of these markers distinguishes HGSOC from non‐cancer with 89% sensitivity and 93% specificity. The same markers are also effective in classifying three groups (non‐cancer, early‐stage HGSOC, and late‐stage HGSOC). The developed approach, for the first time inaugurated in fallopian tube‐derived EVs, could be a minimally invasive tool to monitor women at high risk of ovarian cancer for timely intervention.

Diagnostic Accuracy of Serum P16ink4A and FOX‐P3 Concentrations for Detection of Cervical Lesions Among Women Attending a Cervical Cancer Clinic in Western Uganda: A Case‐Control Study

Introduction: Expression of P16ink4A and FOXP3 is correlated with the grades of cervical lesions. In this study, we determined the diagnostic accuracy of serum P16ink4A and FOXP3 concentrations for detection of cervical intraepithelial neoplasia (CIN) and cervical cancer (CC) in a rural setting in Southwestern Uganda. Material and Methods: CIN and CC cases (93 each before treatment), and 93 controls were identified. Clinical and demographic data were documented before quantifying serum P16ink4A and FOXP3 concentrations using quantitative ELISA kits. Cases were confirmed by cytology and/or histology. We employed descriptive statistics, cross‐tabulation, and receiver operating curves (ROC) using statistical software for data science (STATA) 17.  p ‐values <0.05 were considered statistically significant. Results: Serum FOXP3 concentration of 0.0545 ng/mL < showed moderate sensitivity (32.22% and 57.78%) for detection of CIN and CC from healthy controls, respectively. It also showed a moderately high specificity of 68.89% for detection of both CIN and CC from healthy controls (AUC‐0.6014 and 0.7679, respectively). Serum P16ink4A concentration of 0.946 ng/mL < showed moderate sensitivities (50.00% and 60.00%) and specificities (56.67% and 55.56%) for the detection of CIN and CC from healthy controls, respectively (AUC‐0.6085 and 0.7592, respectively). A combination of elevated serum FOXP3 and P16ink4A showed very low sensitivities of 18.89% in detecting CIN from healthy controls and 33.33% for detecting CC from healthy controls. This combination showed high specificity of 83.33% in detecting both CIN and CC from healthy controls (AUC‐0.5992 and 0.7642, respectively). Conclusion: Although serum P16ink4A and FOXP3 concentrations showed moderate accuracy, their combination was more specific than sensitive. This combination has a high potential to be applied for diagnosis rather than screening for cervical lesions, at least in the Ugandan population. Combinations of P16ink4A and FOXP3 with other biomarkers could improve diagnostic accuracies. Additionally, studies could be conducted to assess the performance of these biomarkers in the detection of cervical lesions in specific populations, say Human Immunodeficiency Virus (HIV)‐positive and HIV‐negative populations.