Enrichment of circulating tumor-derived extracellular vesicles from human plasma.

Extracellular vesicles (EVs) are gaining considerable traction within the liquid biopsy arena, as carriers of information from cells in distant sites that may not be accessible for biopsy. Therefore, there is a need to develop methods to enrich for specific EV subtypes, based on their cells of origin. Here we describe the development of an automated method to enrich tumor-derived EVs from plasma using the CellSearch technology compared to Total EVs isolated using differential ultracentrifugation (DUC). We use a modified CellSearch protocol to enrich EpCAM+ EVs from the plasma of patients with non-small cell lung carcinoma (NSCLC) and triple negative breast cancer (TNBC). As a test case, we examined PD-L1, an immune checkpoint ligand known to be expressed in some tumor tissues, to demonstrate enrichment for EpCAM+ EVs. For this purpose, we developed two custom immunoassays utilizing the Simoa HD-1 analyzer (Quanterix) to detect PD-L1 in EVs and interrogate specific EV populations from human plasma. PD-L1 was present in Total EVs from the plasma of healthy individuals and cancer patients, since it is also expressed on several immune cells. However, EpCAM+ EVs were only detectable from the plasma of cancer patients, suggesting these are tumor-derived EVs. As low as 250 µL of plasma could be used to reliably detect PD-L1 from patient-derived EpCAM+ EVs. In summary, this report demonstrates the development of a robust tumor-derived EV enrichment method from human blood. Furthermore, this proof-of-concept study is extendable to other known cancer-specific proteins expressed on EVs exuded from tumors.

Low-pass Whole-genome Sequencing of Circulating Cell-free DNA Demonstrates Dynamic Changes in Genomic Copy Number in a Squamous Lung Cancer Clinical Cohort.

PURPOSE: We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non-small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms. EXPERIMENTAL DESIGN: Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes. RESULTS: LP-WGS successfully detected CNVs in cfDNA with tumor fraction ≥10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib. CONCLUSIONS: LP-WGS offers an unbiased and high-throughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.

Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells.

The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of ∆Np63α, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the ∆Np63 promoter is sufficient for repression induced by H-RasV12. The suppression of ∆Np63α expression by these oncogenes concomitantly leads to an epithelial-to-mesenchymal transition (EMT). In addition, the depletion of ∆Np63α alone is sufficient to induce EMT. Both H-RasV12 expression and ∆Np63α depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ∆Np63α.

Lysines in the tetramerization domain of p53 selectively modulate G1 arrest.

Functional in a tetrameric state, the protein product of the p53 tumor suppressor gene confers its tumor-suppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including p21, but not others such as miR-34a and cyclin G1 to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest.

Pathway Regulation of p63, a Director of Epithelial Cell Fate.

The p53-related gene p63 is required for epithelial cell establishment and its expression is often altered in tumor cells. Great strides have been made in understanding the pathways and mechanisms that regulate p63 levels, such as the Wnt, Hedgehog, Notch, and EGFR pathways. We discuss here the multiple signaling pathways that control p63 expression as well as transcription factors and post-transcriptional mechanisms that regulate p63 levels. While a unified picture has not emerged, it is clear that the fine-tuning of p63 has evolved to carefully control epithelial cell differentiation and fate.