Detection of PSMA expression on circulating tumor cells by blood-based liquid biopsy in prostate cancer.

Background: For patients with mCRPC, PSMA-targeted radioligand treatment has significantly improved the clinical outcome. A blood-based liquid biopsy assay for recognizing PSMA protein expression on circulating tumor cells may be beneficial for better informing therapeutic decision-making and identifying the patients most likely to benefit from PSMA-targeted radioligand therapy. Methods: Using high-throughput imaging and digital AI pathology algorithms, a four-color immunofluorescence assay has been developed to find PSMA protein expression on CTCs on a glass slide. Cell line cells (LNCaP/PC3s/22Rv1) spiked into healthy donor blood were used to study the precision, specificity, sensitivity, limit of detection, and overall accuracy of the assay. Clinical validation and low-pass whole-genome sequencing were performed in PSMA-PET-positive patients with high-risk mCRPC (N = 24) utilizing 3 mL of blood. Results: The PSMA CTC IF assay achieved analytical specificity, sensitivity, and overall accuracy above 99% with high precision. In the clinical validation, 76% (16/21) of the cases were PSMA positive with CTC heterogeneity, and 88% (21/24) of the patients contained at least one conventional CTC per milliliter of blood. Thirty-six low-pass-sequenced CTCs from 11 individuals with mCRPC frequently exhibited copy number increases in AR and MYC and losses in RB1, PTEN, TP53, and BRCA2 locus. Conclusions: The analytical validation utilizing Epic Sciences' liquid biopsy CTC platform demonstrated the potential to detect PSMA protein expression in CTCs from patients with mCRPC. This assay is positioned as an effective research tool to evaluate PSMA expression, heterogeneity, and therapeutic response in many ongoing clinical studies to target tumors that express PSMA.

A novel liquid biopsy assay for detection of ERBB2 (HER2) amplification in circulating tumor cells (CTCs).

Purpose: Circulating tumor cell (CTC)-based ERBB2 (HER2) assay is a laboratory test developed by Epic Sciences using single-cell genomics to detect ERBB2 (HER2) amplification in CTCs found in the peripheral blood of metastatic breast cancer (MBC) patients. Patients and methods: Peripheral blood was collected in Streck tubes and centrifugation was used to remove plasma and red blood cells. The remaining nucleated cells were deposited on glass slides, immunofluorescent-stained with proprietary antibodies, scanned by a high-definition digital scanner, and analyzed by a proprietary algorithm. In addition, single-cell genomics was performed on selected CTC. Analytical validation was performed using white blood cells from healthy donors and breast cancer cell lines with known levels of ERBB2 amplification. Clinical concordance was assessed on MBC patients whose blood was tested by the CTC ERBB2 (HER2) assay and those results are compared to results of matched metastatic tissue biopsy (immunohistochemistry [IHC] 3+ or IHC2+/in situ hybridization [ISH+]). Results: Epic's ERBB2 (HER2) assay detected 2-fold ERBB2 amplification with 85% sensitivity and 94% specificity. In the clinical concordance study, among the 50% of the cases that had ERBB2 status results from CTCs found to be chromosomally-unstable, the CTC ERBB2 (HER2) assay showed sensitivity of 69% and specificity of 78% when compared to HER2 status by metastatic tissue biopsy. Conclusions: The CTC ERBB2 (HER2) assay can consistently detect ERBB2 status in MBC cell lines and in the population of patients with MBC with detectable chromosomally unstable CTCs for whom tissue biopsy is not available or is infeasible.

Direct activation of human phospholipase C by its well known inhibitor u73122.

Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing membrane phospholipids. U73122 (1-(6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCß3 (hPLCß3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCß3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50=13.6±5 µm. Activation of hPLCß3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 µm) also activated hPLCγ1 (>10-fold) and hPLCß2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.

Quantification of isozyme-specific activation of phospholipase C-beta2 by Rac GTPases and phospholipase C-epsilon by Rho GTPases in an intact cell assay system.

Phospholipase C (PLC) catalyzes the hydrolysis of PtdIns(4,5)P2, which results in both formation of the second messengers Ins(1,4,5)P3 and diacylglycerol and alteration in the membrane association and/or activity of PtdIns(4,5)P2-binding proteins. The existence of 13 different PLC isozymes suggests multiple mechanisms of regulation of inositol lipid signaling, and the recent realization that Rho-family GTPases directly bind and activate certain PLC isozymes has added to this potential diversity of inositol lipid-related signal transduction. With the goal of delineating a less labor-intensive method for quantification of intracellular inositol phosphate production, we have applied a commercially available yttrium silicate RNA binding resin selective for inositol phosphates to develop a high-throughput inositol phosphate scintillation proximity assay (SPA). We highlight the utility of this assay using COS-7 cells robotically transfected in a 96-well format. This method is readily applied to quantify activation of PLC by receptors and G proteins, and we illustrate here the selective activation of PLC-beta2 by Rac but not by Rho GTPases and the selective activation of PLC-epsilon by Rho but not Rac GTPases.

PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling.

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.