Circulating cell‐free microRNAs in cutaneous melanoma staging and recurrence or survival prognosis

Cutaneous melanoma is a skin cancer with increasing incidence. Identification of novel clinical biomarkers able to detect the stage of disease and suggest prognosis could improve treatment and outcome for melanoma patients. Cell‐free microRNAs (cf‐miRNAs) are the circulating copies of short non‐coding RNAs involved in gene expression regulation. They are released into the interstitial fluid, are detectable in blood and other body fluids and have interesting features of ideal biomarker candidates. They are stable outside the cell, tissue specific, vary along with cancer development and are sensitive to change in the disease course such as progression or therapeutic response. Moreover, they are accessible by non‐invasive methods or venipuncture. Some articles have reported different cf‐miRNAs with the potential of diagnostic tools for melanoma staging, recurrence and survival prediction. Although some concordance of results is already emerging, differences in analytical methods, normalization strategies and tumour staging still will require further research and standardization prior to clinical usage of cf‐miRNA analysis. This article reviews this literature with the aim of contributing to a shared focusing on these new promising tools for melanoma treatment and care.     

The evolution of molecular diagnosis using digital polymerase chain reaction to detect cancer via cell‐free DNA and circulating tumor cells

Cancer is one of the most important causes of death worldwide. The onset of cancer may be initiated due to a variety of factors such as environment, genetics or even due to personal lifestyle choices. To counteract this tremendous increase, the demand for a new technology has risen. By this means, the use of digital polymerase chain reaction (dPCR) has been shown to be a promising methodology in the early detection of many types of cancers. Furthermore, several researchers confirmed that the use of tumor cell‐free DNA (cfDNA) and circulating tumor cells (CTC) in peripheral blood is essential in revealing an early prognosis of such diseases. Besides this, it was established that dPCR might be used in a much more efficient, accurate, and reliable manner to amplify a variety of genetic material up to the identification of mutations in hematological diseases. Therefore, this article demonstrates the differences between conventional PCR and dPCR as a molecular technique to detect the early onset of cancer. Furthermore, CTC and cfDNA were officially approved by the Food and Drug Administration as new biological biomarkers in cancer development and monitoring.     

Mutational landscape and genetic signatures of cell‐free DNA in tumour‐induced osteomalacia

Tumour‐induced osteomalacia (TIO) is a very rare paraneoplastic syndrome with bone pain, fractures and muscle weakness, which is mostly caused by phosphaturic mesenchymal tumours (PMTs). Cell‐free DNA (cfDNA) has been regarded as a non‐invasive liquid biopsy for many malignant tumours. However, it has not been studied in benign tumours, which prompted us to adopt the targeted next‐generation sequencing approach to compare cfDNAs of 4 TIO patients, four patients with bone metastasis (BM) and 10 healthy controls. The mutational landscapes of cfDNA in TIO and BM groups were similar in the spectrum of allele frequencies and mutation types. Markedly, deleterious missense mutations in FGFR1 and loss‐of‐function mutations in MED12 were found in 3/4 TIO patients but none of BM patients. The gene ontology analysis strongly supported that these mutated genes found in TIOs would play a potential role in PMTs' process. The genetic signatures and corresponding change in expression of FGFR1 and FGF23 were further validated in PMT tissues from a test cohort of another three TIO patients. In summary, we reported the first study of the mutational landscape and genetic signatures of cfDNA in TIO/PMTs.     

Investigation of transrenal KRAS mutation in late stage NSCLC patients correlates to disease progression

Purpose: Using transrenal DNA to detect KRAS mutations in non-small cell lung cancer (NSCLC), the study addressed the clinical impact for longitudinal monitoring and prognostic value for disease outcome.

Methods: Digital droplet PCR was used to detect the mutant DNA. A total of 200 NSCLC patients were recruited with varying molecular profiles. To ascertain the specificity of transrenal DNA to accurately profile the disease, primary tissues were compared. Subsequently, serial samplings were performed at different treatment cycles to gauge the predictive value.

Results: Transrenal DNA was successfully detected in all 200 patients. Overall concordance rate for mutant KRAS DNA within urine specimens and primary tissue biopsies was 95% (k?=?0.87; 95% CI: 0.82–0.95). Patients with positive results at baseline had lower median overall survival (OS) than the wildtype group. More importantly, longitudinal monitoring of urine specimens showed an increase in the quantity of transrenal DNA, which were highly associated with disease progression and outcome.

Conclusions: Our study showed a highly associative link to the patient’s tumor KRAS profile. Monitoring its variations aided in stratifying patients with worse outcome. Urinary specimens that can be extracted non-invasively presents new opportunities to track patients with KRAS mutation undergoing therapy.     

Production of G protein‐coupled receptors in an insect‐based cell‐free system

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein‐coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell‐free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell‐free system, performed within a short time and in a cost‐effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect‐based cell‐free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell‐free synthesized MOR in comparison to MOR expressed in a human cell line by “one‐point” radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328–2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Analysis of monosomy‐3 in immunomagnetically isolated circulating melanoma cells in uveal melanoma patients

Monosomy‐3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno‐FISH assay to detect chromosome‐3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n = 44) with primary non‐metastatic UM, of which 58% were positive for monosomy‐3. The monosomy‐3 status of CMC corresponded to the monosomy‐3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy‐3 in the CMC was associated with an advanced tumor stage (P = 0.046) and was detected in all four patients who developed metastasis within the follow‐up period of 4 yr. This non‐invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.     

The diverse origins of circulating cell‐free DNA in the human body: a critical re‐evaluation of the literature

Since the detection of cell‐free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non‐invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle‐induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno‐ or gene therapy. This review presents an extensive compilation of the putative origins of cfDNA and then contrasts the contributions of cellular breakdown processes with active mechanisms for the release of cfDNA into the extracellular environment. The involvement of cfDNA derived from both cellular breakdown and active release in lateral information transfer is also discussed. We hope to encourage researchers to adopt a more holistic view of cfDNA research, taking into account all the biological pathways in which cfDNA is involved, and to give serious consideration to the integration of in vitro and in vivo research. We also wish to encourage researchers not to limit their focus to the apoptotic or necrotic fraction of cfDNA, but to investigate the intercellular messaging capabilities of the actively released fraction of cfDNA and to study the role of cfDNA in pathogenesis.     

A Combined test using both cell sediment and supernatant cell‐free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients

Introduction

The aim of this study was to examine whether a combined test using both cell sediment and supernatant cytology cell‐free DNA (ccfDNA) is more useful in detecting EGFR mutation than using cell sediment DNA or supernatant ccfDNA alone in pleural effusion of lung cancer patients.

Methods

A total of 74 lung adenocarcinoma patients with paired samples between primary tumour and corresponding metastatic tumour with both cell sediment and supernatant ccfDNA of pleural effusion cytology were enrolled in this study. Cell sediment and supernatant ccfDNA were analysed separately for EGFR mutations by polymerase chain reaction.

Results

Out of 45 patients with mutant EGFR in primary tumours, EGFR mutations were detected in 23 cell sediments of corresponding metastases (sensitivity; 51.1%) and 20 supernatant ccfDNA corresponding metastases (sensitivity; 44.4%). By contrast, the combined test detected EGFR mutations in 27 corresponding metastases (sensitivity; 60.0%), and had a higher sensitivity than the cell sediment or the supernatant ccfDNA alone (P < .05). Out of 45 patients with mutant EGFR, 24, three and 18 were cytologically diagnosed as positive, atypical or negative, respectively. The detection rate in the combined test was highest (95.8%) in the positive group, and mutant EGFR was also detected in four of 18 samples (22.2%) in the negative group.

Conclusions

A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.     

Therapeutic angiogenesis using tumor cell‐conditioned medium

Stem cell‐conditioned medium (CM), which contains angiogenic factors that are secreted by stem cells, represents a potential therapy for ischemic diseases. Along with stem cells, tumor cells also secrete various angiogenic factors. Here, tumor cells as a cell source of CM for therapeutic angiogenesis was evaluated and the therapeutic efficacy of tumor cell CM in mouse hindlimb ischemia models was demonstrated. CM obtained from a human fibrosarcoma HT1080 cell line culture was compared with CM obtained from a human bone marrow‐derived mesenchymal stem cell (MSC) culture. HT1080 CM contained higher concentrations of angiogenic factors compared with MSC CM, which was attributable to the higher cell density that resulted from a much faster growth rate of HT1080 cells compared with MSCs. For use in in vitro and in vivo angiogenesis studies, HT1080 CM was diluted such that HT1080 CM and MSC CM would have the same cell number basis. The two types of CMs induced the same extent of human umbilical vein endothelial cell (HUVEC) proliferation in vitro. The injection of HT1080 CM into mouse ischemic limbs significantly improved capillary density and blood perfusion compared with the injection of fresh medium. Although the therapeutic outcome of HT1080 CM was similar to that of MSC CM, the preparation of CM by tumor cell line culture would be much more efficient due to the faster growth and unlimited life‐time of the tumor cell line. These data suggest the potential application of tumor cell CM as a therapeutic modality for angiogenesis and ischemic diseases. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:456–464, 2016     

Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

The human bone morphogenetic protein‐2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell‐free synthesis in CHO cell lysates containing microsomal structures as sites of post‐translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153 pg/mL. Up to 280 ng/mL could be produced in the transient expression system. In both cases the rhBMP‐2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell‐free system, hBMP2 yields could be increased to almost 40 μg/mL, reached within three hours. The cell‐free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post‐translational processing.     

Label‐free indicator‐free nucleic acid biosensors using carbon nanotubes

Carbon nanotubes (CNTs) are promising components for electrical biosensors due to their high surface‐to‐volume ratio and improved electron transfer properties. This review surveys CNT‐based label‐free indicator‐free biosensing strategies that have been demonstrated for the sensitive detection of nucleic acids. After an introduction to CNTs, the fabrication of biosensors and techniques for the immobilization of probe nucleic acids are outlined. Subsequently, two major label‐free strategies namely electrochemical transduction and field‐effect detection are presented. The focus is on direct detection methods that avoid labels, indicators, intercalating agents, mediators, and even secondary receptors. The review concludes with a comparison between the various biosensors and presents ways of engineering them so that they can be deployed in realistic diagnostic applications.     

Toward cell‐free biofuel production: Stable immobilization of oligomeric enzymes

To overcome the main challenges facing alcohol‐based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell‐free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, β‐galactosidase, and an alcohol‐producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long‐term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH with keto‐acid decarboxylase free in solution. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:324–331, 2014     

Genome‐wide identification of urinary cell‐free microRNAs for non‐invasive detection of bladder cancer

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non‐invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT‐PCR. Whole‐genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR‐31‐5p, miR‐93‐5p and miR‐191‐5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2‐miRNA‐based urinary DxScore (miR‐93‐5p, miR‐31‐5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post‐operatively. In conclusion, we identified and independently validated cell‐free urinary miRNAs as promising biomarkers enabling non‐invasive detection of BCA.