Anna Lyberopoulou

Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance.

MicroRNAs are a class of non‐coding molecules found to regulate a variety of cellular functions in health and disease. Dysregulation of microRNAs is involved in liver disease, especially hepatocarcinogenesis. Since primary hepatic malignancies are typically characterized by late diagnosis, frequent recurrence, and poor response to adjuvant therapy, there is a need for the discovery of novel biomarkers in order to achieve earlier diagnosis, predict tumor aggressiveness and response to adjuvant therapy. The purpose of this study is to evaluate the expression of certain microRNAs (miR‐21, ‐31, ‐122, ‐145, ‐146a, ‐ 200c, ‐221, ‐222 and ‐223) in patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), as well as to assess their prognostic significance. Micro‐RNA expression was assessed by reverse transcription and real‐time PCR (RT‐PCR). Clinicopathological data and survival rates were retrieved and analyzed. According to our results, miR‐21, miR‐31, miR‐122, miR‐221, miR‐222 were significantly up‐regulated in HCC tissues, whereas miR‐145, miR‐146a, miR‐200c, and miR‐223 were found to be down‐regulated. Concerning ICC samples, miR‐21, miR‐31, and miR‐223 were found to be over‐expressed, whereas miR‐122, miR‐145, miR‐200c, miR‐221, and miR‐222 were down‐regulated. Additionally, expression of miR‐21, miR‐31, miR‐122, and miR‐221 in HCC correlated with cirrhosis, while miR‐21 and miR‐221 associated with tumor stage and poor prognosis. In ICC tissues, miR‐21, miR‐31, and miR‐223 were found to be over‐expressed, but no correlation with clinicopathological features was found.

Expression of microRNAs in patients with pancreatic cancer and its prognostic significance.

Objectives Investigation of expression profile of well-established microRNAs in pancreatic adenocarcinoma, and its correlation with clinicopathological factors. Methods Eighty-eight samples of ductal pancreatic adenocarcinoma and 98 control samples were analyzed by real-time polymerase chain reaction for miR-21, miR-31, miR-122, miR-145, miR-146a, miR-155, miR-210, and miR-222 expressions. The results were normalized and then statistically analyzed using nonparametric statistical tests. Results According to our results, miR-21, miR-155, miR-210, miR-221, and miR-222, were overexpressed in diseased tissues than in the control samples, whereas miR-31, miR-122, miR-145, and miR-146a were underexpressed. Additionally, the expressions of miR-21 and miR-155 were associated with tumor stage and poor prognosis. Conclusions The tumorigenic role of miR-21 and miR-155 was confirmed, whereas down-regulation of miR-31, miR-145, and miR-146a, in dispute with current literature, renders necessary the revision of use of microRNAs as biological markers.

Quantum dots hold promise for early cancer imaging and detection

Despite all major breakthroughs in recent years of research concerning the complex events that lead to cancer expression and metastasis, we are not yet able to effectively treat cancer that has spread to vital organs. The various clinical phases originating from cancer diagnosis through treatment and prognosis require a comprehensive understanding of these events, to utilise pre‐symptomatic, minimally invasive and targeted cancer management techniques. Current imaging modalities such as ultrasound, computed tomography, magnetic resonance imaging and gamma scintigraphy facilitate the pre‐operative study of tumours, but they have been rendered unable to visualise cancer in early stages, due to their intrinsic limitations. The semiconductor nanocrystal quantum dots (QDs) have excellent photo‐physical properties, and the QDs‐based probes have achieved encouraging developments in cellular (in vitro) and in vivo molecular imaging. However, the same unique physical and chemical properties which renowned QDs attractive may be associated with their potentially catastrophic effects on living cells and tissues. There are critical issues that need to be further examined to properly assess the risks associated with the manufacturing and use of QDs in cancer management. In this review, we aim to describe the current utilisation of QDs as well as their future prospective to decipher and confront cancer.

Mutational Analysis of Circulating Tumor Cells from Colorectal Cancer Patients and Correlation with Primary Tumor Tissue

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.

Gallium-68 Labeled Iron Oxide Nanoparticles Coated with 2,3-Dicarboxypropane-1,1-diphosphonic Acid as a Potential PET/MR Imaging Agent: A Proof-of-Concept Study

The aim of this study was to develop a dual-modality PET/MR imaging probe by radiolabeling iron oxide magnetic nanoparticles (IONPs), surface functionalized with water soluble stabilizer 2,3-dicarboxypropane-1,1-diphosphonic acid (DPD), with the positron emitter Gallium-68. Magnetite nanoparticles (Fe3O4 MNPs) were synthesized via coprecipitation method and were stabilized with DPD. The Fe3O4-DPD MNPs were characterized based on their structure, morphology, size, surface charge, and magnetic properties. In vitro cytotoxicity studies showed reduced toxicity in normal cells, compared to cancer cells. Fe3O4-DPD MNPs were successfully labeled with Gallium-68 at high radiochemical purity (>91%) and their stability in human serum and in PBS was demonstrated, along with their further characterization on size and magnetic properties. The ex vivo biodistribution studies in normal Swiss mice showed high uptake in the liver followed by spleen. The acquired PET images were in accordance with the ex vivo biodistribution results. Our findings indicate that 68Ga-Fe3O4-DPD MNPs could serve as an important diagnostic tool for biomedical imaging.

Detection of colorectal circulating cancer cells with the use of a quantum dot labelled magnetic immunoassay method

Aim-BackgroundTo evaluate the incorporation of cadmium selenide quantum dots (QDs) in the detection of colorectal cancer (CRC) circulating tumour cell (CTC) surface antigens.Material and MethodsThe principle of this assay is based upon the separation of CTCs from body fluids using magnetic beads (MBs) coupled with specific antibody biomarkers, such as the Epithelial cell Adhesion Molecule Antibody (EpCAM) and Cytokeratin 19 (CK19) antibody. The detection signal was acquired from the fluorescence signal of QDs. To evaluate the performance, the method under study was used to isolate the human colon adenocarcinoma cell line (DLD-1) and CTCs from CRC patients’ peripheral blood samples. Peripheral blood samples were obtained from 9 CRC patients (Table 1) and 5 healthy donors who gave their informed consent to their inclusion in the study. Peripheral venous blood was sampled immediately after CRC patients had received general anaesthesia, moments before surgery. In all patients, an intravenous cannula was used to collect blood into 7 ml vacutainers containing sodium ethylenediaminetetraacetic acid (EDTA). The first 7 ml of blood was discarded in order to reduce the risk of contamination from skin epithelial cells. Subsequently, 30 ml samples were collected at one-minute intervals (five 6 ml aliquots per 30 ml sample).ResultsThe limit of detection (LOD) of the methodology described in the present study was determined at 10 DLD-1 cells/ml of sample as fluorescence measured with a spectrofluorometer. Fluorescence activated cell sorting (FACS) analysis and Real Time RT-PCR were also used to evaluate the performance of the method. Ultimately, in comparison to other methods currently in use, we developed a simple, sensitive, and more cost-effective method for the detection of CRC CTCs in human samples. Results were accomplished using magnetic bead isolation and subsequent QD fluorescence detection.ConclusionThe present method can be readily adjusted to target a variety of proteins of either the CTC or the host.

Nanotechnology‐Based Rapid Diagnostic Tests

Recently, various nanomaterials are used in order to develop nanotechnology‐based rapid diagnostic tests, such as metallic nanoparticles, quantum dots (QDs), silica nanospheres, magnetic nanoparticles, carbon nanotubes (CNTs), silicon nanowires (SiNWs), nanopores, graphene, nanostructured surfaces, and metal films. This novel nanodiagnostic approach will further develop point‐of‐care (POC) diagnostics and monitoring technologies. Nanobiosensors and microarrays of biosensors can create biochip systems and microfluidic platforms that are the most used nanofabrications for rapid diagnostic tests. These nanoplatforms are constructed for the rapid detection of various diseases or pathogen‐specific biomolecules/markers, such as DNA, proteins, whole cells (e.g., circulating tumor cells), and others. The fabrication of small‐scale portable devices with the incorporation of nanostructures will offer many advantages in the early detection of various diseases and health‐threatening infections by pathogens and in the treatment selection and treatment monitoring. The use of nanostructures in in vitro diagnostics gives the opportunity to augment the sensitivity and specificity required in clinical practice, lowers the cost and test time of the assays, and enables portable microfluidic platforms suitable for resource‐constrained settings. In this chapter, all the state‐of‐the‐art advantages in this field are discussed, starting with the nanostructures used for the fabrication of nanobiosensors, nanobiosensors arrays, and nanofluidic platforms and the nanodiagnostic use of rapid tests in the detection of pathogens, in cancer management, and glucose monitoring for the management of diabetes disease.