Colocalization of cancer-associated biomarkers on single extracellular vesicles for early detection of cancer.

Detection of cancer early, when it is most treatable, remains a significant challenge due to the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. Detection of their presence is made more difficult by a lack of abundant tumor-specific indicators (i.e., protein biomarkers, circulating tumor DNA, etc.) that would enable detection using a non-invasive diagnostic assay. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. We demonstrate the technical feasibility of this approach in human cancer cell line-derived EVs where we show strong correlations between assay signal and cell line gene/protein expression for the ovarian cancer-associated biomarkers BST2, FOLR1, and MUC1. Furthermore, we demonstrate that detecting distinct colocalized biomarkers on the surface of EVs significantly improves discrimination performance relative to single biomarker measurements. Using this approach, we observe promising discrimination of high-grade serous ovarian cancer versus benign ovarian masses and healthy women in a proof-of-concept clinical study.

Improving specificity for ovarian cancer screening using a novel extracellular vesicle-based blood test - Performance in a training and verification cohort.

The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles (EVs) from blood. The novel OC Test employs immunoaffinity capture of tumor-associated EVs followed by proximity-ligation qPCR to detect combinations of up to three biomarkers to maximize specificity and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cut-off between cancer and non-cancer. Performance was verified and compared to CA125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI: 92.4%-99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI: 87.8%-99.2%), and an AUC of 0.97 (95% CI 0.93-0.99) and also detected 73.5% (61/83; 95% CI: 62.7%-82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, non-ovarian cancers, and inflammatory conditions when compared to CA125. The combined sensitivity and specificity of this new test suggests it may have potential in OC screening.

Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.

The transcription factor C/EBP alpha regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBP alpha gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBP alpha function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBP alpha is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBP alpha to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBP alpha via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBP alpha transactivation domain and does not change C/EBP alpha DNA binding. Ras acts on serine 248 of the C/EBP alpha transactivation domain, because it does not enhance the transactivation function of a C/EBP alpha serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBP alpha is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EB alpha by Ras. Ras signaling leads to phosphorylation of C/EBP alpha in vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBP alpha to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.

A B cell-based sensor for rapid identification of pathogens.

We report the use of genetically engineered cells in a pathogen identification sensor. This sensor uses B lymphocytes that have been engineered to emit light within seconds of exposure to specific bacteria and viruses. We demonstrated rapid screening of relevant samples and identification of a variety of pathogens at very low levels. Because of its speed, sensitivity, and specificity, this pathogen identification technology could prove useful for medical diagnostics, biowarfare defense, food- and water-quality monitoring, and other applications.