Single-Circulating Tumor Cell Whole Genome Amplification to Unravel Cancer Heterogeneity and Actionable Biomarkers.

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called "liquid biopsies". Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection.

Circulating tumour cells in pancreatic cancer: A systematic review and meta-analysis of clinicopathological implications.

BACKGROUND: The detection and quantification of circulating tumour cells (CTCs) in pancreatic cancer (PC) has the potential to provide prognostic information. The aim of this review was to provide an overview of the literature surrounding CTCs in PC. METHODS: A systematic literature review on CTCs in PC between 2005-2020 was performed. Data based on peripheral vein samples were used to determine the positivity rate of CTCs, their prognostic significance and their relative numbers compared to portal vein (PV) samples. RESULTS: The overall CTC detection rate in forty-four articles was 65% (95%CI: 55-75%). Detection rate for CellSearch was 26% (95%CI: 14-38%), which was lower than for both filtration and microfluidic techniques. In nine studies with >50 patients, overall survival was worse with CTC positivity (HR 1.82; 95%CI: 1.61-2.05). Five of seven studies which described PV CTC collection provided patient-level data. PV CTC yield was 7.7-fold (95%CI 1.35-43.9) that of peripheral blood. CONCLUSIONS: CTCs were detected in the peripheral circulation of most patients with PC and may be related to prognosis and disease stage. PV blood contains more CTCs than peripheral blood sampling. This review points to the maturation of techniques of CTC enrichment, and its evidence base for eventual clinical deployment.

From Pediatric to Adult Brain Cancer: Exploring Histone H3 Mutations in Australian Brain Cancer Patients.

Genetic histone variants have been implicated in cancer development and progression. Mutations affecting the histone 3 (H3) family, H3.1 (encoded by HIST1H3B and HIST1H3C) and H3.3 (encoded by H3F3A), are mainly associated with pediatric brain cancers. While considered poor prognostic brain cancer biomarkers in children, more recent studies have reported H3 alterations in adult brain cancer as well. Here, we established reliable droplet digital PCR based assays to detect three histone mutations (H3.3-K27M, H3.3-G34R, and H3.1-K27M) primarily linked to childhood brain cancer. We demonstrate the utility of our assays for sensitively detecting these mutations in cell-free DNA released from cultured diffuse intrinsic pontine glioma (DIPG) cells and in the cerebral spinal fluid of a pediatric patient with DIPG. We further screened tumor tissue DNA from 89 adult patients with glioma and 1 with diffuse hemispheric glioma from Southwestern Sydney, Australia, an ethnically diverse region, for these three mutations. No histone mutations were detected in adult glioma tissue, while H3.3-G34R presence was confirmed in the diffuse hemispheric glioma patient.

PD-L1 Detection on Circulating Melanoma Cells.

The advent of personalized medicines targeting cell signaling pathways has radically improved melanoma patient outcomes. More recently, immune-modulating therapies disrupting the PD-1/PD-L1 axis have become a powerful tool in the treatment of a range of melanoma, showing a profound improvement in the overall survival outcomes. However, immune checkpoint inhibitors (ICIs) are associated with considerable toxicities and appear to only be efficacious in a subset of melanoma patients. Therefore, there is an urgent need to identify biomarkers that can determine if patients will or will not respond to ICI therapy. Here, we describe an optimized method for analyzing PD-L1 expression on circulating melanoma cells following immunomagnetic enrichment from patient blood samples.

Single-Cell Analysis of BRAFV600E and NRASQ61R Mutation Status in Melanoma Cell Lines as Method Generation for Circulating Melanoma Cells.

Molecular testing of tumor biopsies allows for the identification of the key mutations driving a patient's cancer. However, this is limited to singular nodes and may not accurately reflect cancer heterogeneity. Circulating tumor cell (CTC) analyses offer a noninvasive method of interrogating the genomic profile of patient-derived tumor material. To date, molecular analysis of CTCs has relied on the characterization of bulk or pooled CTC lysates, limiting the detection of minor tumorigenic CTC subclones. Here, we show a workflow enabling BRAFV600E/NRASQ61R mutation detection from single cultured melanoma cells by combining micromanipulation and genomic material amplification methods. This workflow can be directly integrated into circulating tumor cell analysis applications.

Immunomagnetic isolation of circulating melanoma cells and detection of PD-L1 status.

Personalised medicine targeted to specific biomarkers such as BRAF and c-Kit has radically improved the success of melanoma therapy. More recently, further advances have been made using therapies targeting the immune response. In particular, therapies targeting the PD-1/PD-L1 or CTLA-4 axes alone or in combination have shown more sustained responses in 30-60% of patients. However, these therapies are associated with considerable toxicities and useful biomarkers to predict responders and non-responders are slow to emerge. Here we developed a reliable melanoma circulating tumor cell (CTC) detection method with PD-L1 evaluation on CTCs. A set of melanoma cell surface markers was tested as candidates for targeted melanoma CTC isolation and a melanoma specific immunostaining-based CTC identification protocol combined with PD-L1 detection was established. In vitro testing of the effect of exposure to blood cells on melanoma cell PD-L1 expression was undertaken. Immunomagnetic targeting isolated melanoma CTCs in up to 87.5% of stage IV melanoma patient blood samples and 3 8.6% of these had some PD-L1 expressing CTCs. Our in vitro data demonstrate PD-L1 induction on melanoma cells in the blood.This study established a robust, reliable method to isolate melanoma CTCs and detect expression of PD-L1 on these cells.

Improved ovarian cancer EMT-CTC isolation by immunomagnetic targeting of epithelial EpCAM and mesenchymal N-cadherin.

Epithelial cell adhesion molecule (EpCAM)-targeted capture remains the most common isolation strategy for circulating tumor cells (CTCs). However, epithelial-to-mesenchymal transition (EMT) leads to decreased epithelial EpCAM expression affecting the optimal CTC capture. In this study, we tested a cohort of ovarian cancer cell lines using flow cytometry to identify N-cadherin as the additional immunomagnetic cell surface target for ovarian cancer cell isolation. Combined immunomagnetic targeting of mesenchymal N-cadherin and epithelial EpCAM enriched CTCs from advanced ovarian cancer patient blood approximately three times more efficiently than targeting of EpCAM alone. We also show that more EMT-phenotype CTCs are captured by including N-cadherin targeting into CTC isolation protocols. However, after N-cadherin-based CTC isolation, in some blood samples of healthy individuals, we also observed the presence of cells expressing markers common to CTCs. Our data show that these "false positives" can be largely distinguished from CTCs as circulating endothelial cells (CECs) by vascular endothelial-cadherin co-staining. CEC counts are highly variable in patients and healthy controls. Our data demonstrate that a combination of EpCAM with N-cadherin-targeted isolation can improve CTC detection and widen the EMT-phenotype spectrum of captured CTCs.