The use of microfluidic technology for cancer applications and liquid biopsy

© 2018 by the authors. There is growing awareness for the need of early diagnostic tools to aid in point-of-care testing in cancer. Tumor biopsy remains the conventional means in which to sample a tumor and often presents with challenges and associated risks. Therefore, alternative sources of tumor biomarkers is needed. Liquid biopsy has gained attention due to its non-invasive sampling of tumor tissue and ability to serially assess disease via a simple blood draw over the course of treatment. Among the leading technologies developing liquid biopsy solutions, microfluidics has recently come to the fore. Microfluidic platforms offer cellular separation and analysis platforms that allow for high throughout, high sensitivity and specificity, low sample volumes and reagent costs and precise liquid controlling capabilities. These characteristics make microfluidic technology a promising tool in separating and analyzing circulating tumor biomarkers for diagnosis, prognosis and monitoring. In this review, the characteristics of three kinds of circulating tumor markers will be described in the context of cancer, circulating tumor cells (CTCs), exosomes, and circulating tumor DNA (ctDNA). The review will focus on how the introduction of microfluidic technologies has improved the separation and analysis of these circulating tumor markers

Phenotypic Characterization of Circulating Lung Cancer Cells for Clinically Actionable Targets.

OBJECTIVES:In non-small cell lung cancers (NSCLC), tumour biopsy can often be an invasive procedure. The development of a non-invasive methodology to study genetic changes via circulating tumour cells (CTCs) is an appealing concept. Whilst CTCs typically remain as rare cells, improvements in epitope-independent CTC isolation techniques has given rise to a greater capture of CTCs. In this cross sectional study, we demonstrate the capture and characterization of NSCLC CTCs for the clinically actionable markers epidermal growth factor receptor (EGFR) alterations, anaplastic lymphoma kinase (ALK) rearrangements and programmed death ligand-1 (PD-L1) expression. The study identified CTCs/CTC clusters in 26/35 Stage IV NSCLC patients, and subsequently characterized the CTCs for EGFR mutation, ALK status and PD-L1 status. This pilot study demonstrates the potential of a non-invasive fluid biopsy to determine clinically relevant biomarkers in NSCLC

A Collective Route to Head and Neck Cancer Metastasis

© 2018 The Author(s). Distant metastasis (DM) from head and neck cancers (HNC) portends a poor patient prognosis. Despite its important biological role, little is known about the cells which seed these DM. Circulating tumour cells (CTCs) represent a transient cancer cell population, which circulate in HNC patients' peripheral blood and seed at distant sites. Capture and analysis of CTCs offers insights into tumour metastasis and can facilitate treatment strategies. Whilst the data on singular CTCs have shown clinical significance, the role of CTC clusters in metastasis remains limited. In this pilot study, we assessed 60 treatment naïve HNC patients for CTCs with disease ranging from early to advanced stages, for CTC clusters utilizing spiral CTC enrichment technology. Single CTCs were isolated in 18/60-30% (Ranging from Stage I-IV), CTC clusters in 15/60-25% (exclusively Stage IV) with 3/15-20% of CTC clusters also containing leukocytes. The presence of CTC clusters associated with the development of distant metastatic disease(P = 0.0313). This study demonstrates that CTC clusters are found in locally advanced patients, and this may be an important prognostic marker. In vivo and in vitro studies are warranted to determine the role of these CTC clusters, in particular, whether leukocyte involvement in CTC clusters has clinical relevance

The Use of Three-Dimensional DNA Fluorescent In Situ Hybridization (3D DNA FISH) for the Detection of Anaplastic Lymphoma Kinase (ALK) in Non-Small Cell Lung Cancer (NSCLC) Circulating Tumor Cells.

Tumor tissue biopsy is often limited for non-small cell lung cancer (NSCLC) patients and alternative sources of tumoral information are desirable to determine molecular alterations such as anaplastic lymphoma kinase (ALK) rearrangements. Circulating tumor cells (CTCs) are an appealing component of liquid biopsies, which can be sampled serially over the course of treatment. In this study, we enrolled a cohort of ALK-positive (n = 8) and ALK-negative (n = 12) NSCLC patients, enriched for CTCs using spiral microfluidic technology and performed DNA fluorescent in situ hybridization (FISH) for ALK. CTCs were identified in 12/20 NSCLC patients ranging from 1 to 26 CTCs/7.5 mL blood. Our study revealed that 3D imaging of CTCs for ALK translocations captured a well-defined separation of 3' and 5' signals indicative of ALK translocations and overlapping 3'/5' signal was easily resolved by imaging through the nuclear volume. This study provides proof-of-principle for the use of 3D DNA FISH in the determination of CTC ALK translocations in NSCLC

The prognostic role of circulating tumor cells (CTCs) in lung cancer

© 2018 Kapeleris, Kulasinghe, Warkiani, Vela, Kenny, O'Byrne and Punyadeera. Lung cancer affects over 1. 8 million people worldwide and is the leading cause of cancer related mortality globally. Currently, diagnosis of lung cancer involves a combination of imaging and invasive biopsies to confirm histopathology. Non-invasive diagnostic techniques under investigation include "liquid biopsies" through a simple blood draw to develop predictive and prognostic biomarkers. A better understanding of circulating tumor cell (CTC) dissemination mechanisms offers promising potential for the development of techniques to assist in the diagnosis of lung cancer. Enumeration and characterization of CTCs has the potential to act as a prognostic biomarker and to identify novel drug targets for a precision medicine approach to lung cancer care. This review will focus on the current status of CTCs and their potential diagnostic and prognostic utility in this setting

Generalised Apery numbers modulo 9

Whilst locoregional control of head and neck cancers (HNCs) has improved over the last four decades, long-term survival has remained largely unchanged. A possible reason for this is that the rate of distant metastasis has not changed. Such disseminated disease is reflected in measurable levels of cancer cells in the blood of HNC patients, referred to as circulating tumour cells (CTCs). Numerous marker-independent techniques have been developed for CTC isolation and detection. Recently, microfluidics-based platforms have come to the fore to avoid molecular bias. In this pilot, proof of concept study, we evaluated the use of the spiral microfluidic chip for CTC enrichment and subsequent detection in HNC patients. CTCs were detected in 13/24 (54%) HNC patients, representing both early to late stages of disease. Importantly, in 7/13 CTC-positive patients, CTC clusters were observed. This is the first study to use spiral microfluidics technology for CTC enrichment in HNC

Ethnic differences in lipid metabolism in two groups of obese South African women

There is a higher prevalence of ischemic heart disease (IHD) in South African white than black women. The objective of this study was to determine biochemical explanations for this prevalence. The study group contained 15 obese black women (OBW) and 14 obese white women (OWW), ah premenopausal, who were examined after an overnight fast. Anthropometric measurements and blood concentrations of glucose, non-esterified fatty acids (NEFAs), catecholamines, plasminogen activator inhibitor-1, C-peptide, proinsulin, lipograms, cortisol, growth hormone, and post-heparin Lipoprotein Lipase activity were measured during an oral glucose tolerance test (OGTT), Body composition was measured using bioelectrical impedance analysis, and subcutaneous and visceral fat mass were assessed with CT-scans. Visceral fat area was higher in OWW (139.7 +/- 10.7 cm(2)) than in OBW (72.3 +/- 3.9 cm(2)) (P < 0.01), as were fasting and 3 h triglyceride concentrations (P < 0.05 for all). OWW also had higher NEFA levels than OBW at 3 and 4 h compared, with OBW (P < 0.05 for both). Fasting cortisol (266 +/- 24 vs. 197 +/- 19 nmol/l; P < 0.05) was higher in OWW than in OBW. These data demonstrate that OWW have higher visceral fat mass than OBW, which may lead to a more atherogenic fasting and postprandial Lipid profile. The higher cortisol levels of the OWW may promote visceral fat deposition. - Punyadeera, C., M-T. van der Merwe, N.J. Crowther, M. Toman, C. P. Schlaphoff, and I. P. Gray. Ethnic differences in lipid metabolism in two groups of obese South African women

Short term ex-vivo expansion of circulating head and neck tumour cells.

Minimally invasive techniques are required for the identification of head and neck cancer (HNC) patients who are at an increased risk of metastasis, or are not responding to therapy. An approach utilised in other solid cancers is the identification and enumeration of circulating tumour cells (CTCs) in the peripheral blood of patients. Low numbers of CTCs has been a limiting factor in the HNC field to date. Here we present a methodology to expand HNC patient derived CTCs ex-vivo. As a proof of principle study, 25 advanced stage HNC patient bloods were enriched for circulating tumour cells through negative selection and cultured in 2D and 3D culture environments under hypoxic conditions (2% O2, 5% CO2). CTCs were detected in 14/25 (56%) of patients (ranging from 1-15 CTCs/5 mL blood). Short term CTC cultures were successfully generated in 7/25 advanced stage HNC patients (5/7 of these cultures were from HPV+ patients). Blood samples from which CTC culture was successful had higher CTC counts (p = 0.0002), and were predominantly from HPV+ patients (p = 0.007). This is, to our knowledge, the first pilot study to culture HNC CTCs ex-vivo. Further studies are warranted to determine the use of short term expansion in HNC and the role of HPV in promoting culture success

Isolation of Circulating Tumour Cells in Patients With Glioblastoma Using Spiral Microfluidic Technology – A Pilot Study

Glioblastoma (GBM) is the most common and aggressive type of tumour arising from the central nervous system. GBM remains an incurable disease despite advancement in therapies, with overall survival of approximately 15 months. Recent literature has highlighted that GBM releases tumoural content which crosses the blood-brain barrier (BBB) and is detected in patients' blood, such as circulating tumour cells (CTCs). CTCs carry tumour information and have shown promise as prognostic and predictive biomarkers in different cancer types. Currently, there is limited data for the clinical utility of CTCs in GBM. Here, we report the use of spiral microfluidic technology to isolate CTCs from whole blood of newly diagnosed GBM patients before and after surgery, followed by characterization for GFAP, cell-surface vimentin protein expression and EGFR amplification. CTCs were found in 13 out of 20 patients (9/20 before surgery and 11/19 after surgery). Patients with CTC counts equal to 0 after surgery had a significantly longer recurrence-free survival (p=0.0370). This is the first investigation using the spiral microfluidics technology for the enrichment of CTCs from GBM patients and these results support the use of this technology to better understand the clinical value of CTCs in the management of GBM in future studies

Progesterone regulation of implantation-related genes: new insights into the role of oestrogen

Genomic profiling was performed on explants of late proliferative phase human endometrium after 24-h treatment with progesterone (P) or oestradiol and progesterone (17β-E2+P) and on explants of menstrual phase endometrium treated with 17β-E2+P. Gene expression was validated with real-time PCR in the samples used for the arrays, in endometrium collected from early and mid-secretory phase endometrium, and in additional experiments performed on new samples collected in the menstrual and late proliferative phase. The results show that late proliferative phase human endometrium is more responsive to progestins than menstrual phase endometrium, that the expression of several genes associated with embryo implantation (i.e. thrombomodulin, monoamine oxidase A, SPARC-like 1) can be induced by P in vitro, and that genes that are fully dependent on the continuous presence of 17β-E2 during P exposure can be distinguished from those that are P-dependent to a lesser extent. Therefore, 17β-E2 selectively primes implantation-related genes for the effects of P