Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Multivariate analysis approach to the plasma protein profile of patients with advanced colorectal cancer

The aim of the present study was to identify the pattern of plasma protein species of interest as markers of colorectal cancer (CRC). Using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), the plasma protein profile was determined in nine stage IV CRC patients (study group) and nine clean-colon healthy subjects (control group). Multivariate analysis methods were employed to identify distinctive disease patterns at protein spectrum. In the study and control groups, cluster analysis (CA) on the complete MALDI-MS spectra plasma protein profile showed a distinction between CRC patients and healthy subjects, thus allowing the identification of the most discriminating ionic species. Principal component analysis (PCA) and linear discriminant analysis (LDA) yielded similar grouping results. LDA with leave-one-out cross validation achieved a correct classification rate of 89% in both the patients and the healthy subjects.     

On the design of deterministic dielectrophoresis for continuous separation of circulating tumor cells from peripheral blood cells

Detection and analysis of circulating tumor cells (CTCs) have emerged as a promising way to diagnose cancer, study its cellular mechanism, and test or develop potential treatments. However, the rarity of CTCs among peripheral blood cells is a big challenge toward CTC detection. In addition, in cases where there is similar size range between certain types of CTCs (e.g. breast cancer cells) and white blood cells (WBCs), high‐resolution techniques are needed. In the present work, we propose a deterministic dielectrophoresis (DEP) method that combines the concept of deterministic lateral displacement (DLD) and insulator‐based dielectrophoresis (iDEP) techniques that rely on physical markers such as size and dielectric properties to differentiate different type of cells. The proposed deterministic DEP technology takes advantage of frequency‐controlled AC electric field for continuous separation of CTCs from peripheral blood cells. Utilizing numerical modeling, different aspects of coupled DLD‐DEP design such as the required applied voltages, velocities, and geometrical parameters of DLD arrays of microposts are investigated. Regarding the inevitable difference and uncertainty ranges for the reported crossover frequencies of cells, a comprehensive analysis is conducted on applied electric field frequency as design's determinant factor. Deterministic DEP design provides continuous sorting of CTCs from WBCs even with similar size and has the future potential for high throughput and efficiency.     

Increased levels of multiple forms of dihydrofolate reductase in peripheral blood leucocytes of cancer patients receiving haematopoietic colony-stimulating factors: interim analysis

The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean +/- SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 +/- 0.4 ng/mg protein and 0.99 +/- 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 +/- 0.32 ng/mg protein and 1.18 +/- 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P < 0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 +/- 97 ng/mg protein and 605 +/- 475 ng/mg protein before and after stimulation with GM-CSF, and 115 +/- 165 ng/mg protein and 1,054 +/- 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.     

Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString

Quantitative real-time PCR (qPCR) is commonly used for gene expression analyses with defined documentation guidelines to compare published results. To minimize the impact of variances from qPCR performance, sample handling and processing reference genes are used. Their selection process cannot be completely aligned due to variations in experimental conditions. Furthermore, the named sources of error are also present when determining the stability of the reference genes themselves. Even software applications that are used to identify the best reference genes rarely coincide on their rankings and can be misleading under certain conditions. In previous experiments, peripheral blood mononuclear cells (PBMC) were analyzed to identify the most stable reference gene(s). Twelve of the 13 investigated genes showed sample type specific differences in the expression. Direct mRNA measurement was performed in the form of a NanoString analysis, a multiplexed absolute quantification method. The external validation showed a high concordance of the reference gene expression levels. However, it identified the same sample type specific expression pattern for only some of the tested reference genes. By comparing various combinations of reference genes with both methods we are able to suggest a set of well-performing reference genes. Figure

We here compare the expression of reference genes for qPCR and NanoString data and determine the value of the latter method as a bias-free mRNA quantification method     

Molecular features of ascitic ovarian cancer cells detected by immunofluorescence assay using flow cytometry

Peritoneal dissemination and growth of tumor cells in the ascitic fluid in stages III and IV of recurrent ovarian cancer is difficult to treat; resistance to many anticancer drugs used in the treatment of solid ovarian cancer is observed at this stage. The author’s hypothesis that the differences in the molecular phenotype of tumor cells for various types of the disease could be an explanation for this. A comparative evaluation of the expression and coexpression of a number of molecular markers (immunofluorescence assay with flow cytometry) was performed using solid and ascitic ovarian cancer cells. In contrast to solid ovarian cancer, ascitic ovarian cancer cells present leukocyte common antigen CD45 and mesenchymal marker vimentin in addition to epithelial marker cytokeratin. In addition to the inhibition of anoikis (specific mechanism of epithelial cell death in a liquid medium in the absence of contact with the substrate), ascitic ovarian cancer cells are characterized by (1) emperipolesis (intracellular migration of leukocytes without damage to the tumor cell), and (2) phenotype of epithelial-mesenchymal transition. Thus, the data on clinically significant molecular differences between solid and recurrent ascitic ovarian cancer was first obtained, opening up opportunities for anticancer therapy, which previously was not used in the treatment of ovarian cancer.     

Identification of differential metabolic characteristics between tumor and normal tissue from colorectal cancer patients by gas chromatography–mass spectrometry

Colorectal cancer (CRC) is one of the most common human malignancies and encompasses cancers of the colon and rectum. Although the gold‐standard colonoscopy screening method is effective in detecting CRC, this method is invasive and can result in severe complications for patients. The purpose of this study was to determine differences in metabolites between CRC and matched adjacent nontumor tissues from CRC patients, to identify potential biomarkers that may be informative and developed screening methods. Metabolomic analysis was performed on clinically localized CRC tissue and matched adjacent nontumor tissue from 20 CRC patients. Unsupervised analysis, supervised analysis, univariate analysis and pathway analysis were used to identify potential metabolic biomarkers of CRC. The levels of 25 metabolites in CRC tissues were significantly altered compared with the matched adjacent nontumor tissues. Four metabolites (lactic acid, alanine, phosphate and aspartic acid) demonstrated good area under the curve of receiver‐operator characteristic with acceptable sensitivities and specificities, indicating their potential as important biomarkers for CRC. Alterations of amino acid metabolism and enhanced glycolysis may be major factors in the development and progression of CRC. Lactic acid, alanine, phosphate, and aspartic acid could be effective diagnostic indicators for CRC.     

Isotopic analysis of Fe in human red blood cells by multiple collector-ICP-mass spectrometry.

Precise 56Fe/54Fe and 57Fe/54Fe isotopic ratios on human red blood cell (RBC) samples have been measured using multiple collector-ICP-mass spectrometry (MC-ICPMS). The mass spectrometric interferences on Fe isotopes (e.g., 56ArO+ and 57ArOH+) were successfully minimized by a dry plasma condition achieved by a desolvating nebulizer sample-introduction technique. In order to eliminate possible variations in the measured isotopic ratios due to non-mass spectrometric interferences, Fe was separated from remaining organic compounds and major co-existing elements using an ion chromatographic technique. The resulting precisions of the 56Fe/54Fe and 57Fe/54Fe ratio measurements were 0.12 per thousand and 0.20 per thousand, respectively, which were high enough to detect the isotopic variation of Fe in nature. For an interlaboratory comparison, all of the Fe isotopic ratio data were normalized by the ratios for the IRMM-014 international isotopic standard. A series of 12 RBC samples were collected from one person through monthly-based sampling over a period of one year. These were analyzed to test possible seasonal changes in the 56Fe/54Fe and 57Fe/54Fe ratios. Moreover, in order to test possible variations in the 56Fe/54Fe and 57Fe/54Fe ratios among different people, RBC samples were collected from five volunteers (four males and one female). The 56Fe/54Fe and 57Fe/54Fe ratios for a series of 12 RBC samples collected over a one-year period show 3.06 per thousand and 4.51 per thousand lower than the values of IRMM-014, and no significant seasonal change could be found in the ratios. The lack in seasonal changes in the Fe isotopic ratios could be explained by a small contribution of the daily net-intake of Fe (1 - 2 mg/day) onto the total amount of Fe in the human body (2 - 4 g). The 56Fe/54Fe and 57Fe/54Fe ratios for RBC samples collected from four male samples did not vary measurably, whereas the Fe isotopic ratios for a female RBC were 0.3 per thousand/amu heavier than the mean value of four male samples. This difference in Fe isotopes among the individuals can be the result of a difference in uptake efficiency of the Fe through a dietary process from the digestive tract. The data obtained here demonstrate that the isotopic ratios of trace metals can provide new information about metabolic efficiencies of the metallic elements.     

Pharmacodynamic assay of thymidylate synthase activity in peripheral blood mononuclear cells

A simple, selective, and sensitive method utilizing tritium (³H) release from ³H-deoxyuridine 5′-monophosphate (dUMP) substrate for accurate and precise determination of the low basal thymidylate synthase activity (TSA) in normal healthy peripheral blood mononuclear cells (PBMCs) was developed and validated. The method is based on the removal of the remaining substrate after the TSA reaction by absorption onto activated carbon and measurement of the supernatant fluid by liquid scintillation counting. The method background was substantially decreased by using lyophilized substrate and optimized binding conditions of remaining substrate onto carbon after TSA reaction. The concentration of cofactor N ₅,N ₁₀ methylene-(6R,S)-tetrahydrofolate was increased to obtain maximal TSA. Method sensitivity was further increased by omission of ethylenediaminetetraacetic acid from the reaction mix and by using longer reaction times. The validation parameters included specificity, linearity, sensitivity, precision, and stability. The lower limit of quantification was 25 μg PBMC cytosolic lysate, which released 1.4 pmol?³H/h. TSA was stable in PBMC pellets stored for 6 months at ?80 °C. The applicability of the method was demonstrated by the successful determination of TSA in PBMC cytosolic lysates from ten healthy volunteers with and without the specific TSA inhibitor FdUMP.

Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC‐MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC‐MS/MS) method has been developed for the determination of 5,10‐methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5‐methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC‐MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut‐off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC‐MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right‐ and left‐sided tumors follows different pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Ex vivo identification of circulating tumor cells in peripheral blood by fluorometric “turn on” aptamer nanoparticles

The detection of the circulating tumor cells (CTCs) detached from solid tumors has emerged as a burgeoning topic for cancer diagnosis and treatment. The conventional CTC enrichment and identification mainly rely on the specific binding of the antibodies on the capture interface of the magnetic nanoparticles with the corresponding biomarkers on the cell membranes. However, these methods could easily generate false-negative results due to the extremely low concentration of CTCs and the internal heterogeneity of the tumor cells. Herein, with the aim of selectively identifying CTCs and improving the detection accuracy in peripheral blood, we designed the fluorometric “turn on” Au nanoparticles (DHANs) with the modification of a tumor-targeted moiety, dehydroascorbic acid (DHA) and a fluorometric aptamer, which could be “switched-on” by an over-expressed intracellular protein, namely hypoxia-inducible factor-1α (HIF 1α). This novel nanoformulated detection platform demonstrated the great capacity for visualizing various CTCs in peripheral blood with significantly improved detection efficiency and sensitivity. As a result, the nanoplatform has a great potential to be further applied for CTC detection in vitro or in vivo, which holds promise for extensive CTC studies.

The detection of the circulating tumor cells (CTCs) detached from solid tumors has emerged as a burgeoning topic for cancer diagnosis and treatment.     

Cyclophosphamide-induced changes in plasma and red blood cells detected by differential scanning calorimetry (DSC) in guinea pigs

Cyclophosphamide is one of the most widely used drugs, but it has severe side effects. Considering the variability of kinetics and metabolism of cyclophosphamide in the daily use, it is an important question how could we predict these undesirable consequences. The purpose of this study was to introduce calorimetry for the first time in the detection of cyclophosphamide-induced changes in plasma and red blood cells in an experimental animal model. Adult guinea pigs (n = 55, in 11 different groups) were injected intraperitoneally with the dose of cyclophosphamide that is comparable to the human dosage. Animals were euthanized; plasma and formed elements of blood were analyzed by a SETARAM Micro DSC-II calorimeter. The denaturation temperatures were measured, and the calorimetric enthalpies were calculated based on the areas under thermal absorption curves. The results show a definite dose dependence in both denaturation temperature and calorimetric enthalpy, that is, the DSC seems to be a suitable technique to detect the damage caused by chemotherapy.     

Study of urinary nucleosides as biological marker in cancer patients analyzed by micellar electrokinetic capillary chromatography

Thirteen normal and modified nucleosides, primarily degradation products of transfer ribonucleic acid (tRNA), were evaluated as potential tumor markers for cancer patients. Their urinary concentrations were determined by means of micellar electrokinetic capillary chromatography (MEKC) in the urine from 54 healthy adults and 70 cancer patients, then quantitatively expressed as a function of creatinine excretion. It was found that urinary nucleosides for cancer patients were on the average significantly higher than those for healthy controls, however, no significant differences were found between male and female or between different ages. Based on 13 urinary nucleoside concentrations, principal component analysis (PCA) could be used to classify 72% of cancer patients from the healthy controls. The present study shows that the precise measurement of urinary nucleosides by MEKC in combining with PCA technique may provide a clinically useful approach for diagnosis of cancer.     

Inhibition of phospholipase D2 induces autophagy in colorectal cancer cells

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.     

Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.     

Detection of circulating tumor cells in patients with gastrointestinal tract cancer using RT-PCR and its clinical implications

To investigate the relationship between the presence of circulating tumor cells in different stages of gastrointestinal tract cancer and the subsequent relapse or distant metastasis, circulating levels of CEA mRNA was serially examined at an interval of 10.6+/-4.5 or 13.7+/-3.0 months in gastric or colorectal cancer patients, respectively. CEA mRNA was measured by means of RT-PCR amplification as an indicator for micrometastatic malignant cells. Seven of twenty-nine respectable gastric cancer patients (24.1%) [EGC: 2/9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 4/15 (26.6%)] were positive for CEA mRNA on the initial test and 10 of 29 patients (34.4%) [EGC: 2/ 9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 7/15 (46.7%)] were positive on a follow-up test. Only in AGC IIIb, the positive rate for CEA mRNA increased about twice and 6 of 7 positive cases (85.7%) relapsed within 2.6+/-2.4 months after the follow-up test. In colorectal cancer, 4 of 19 patients (21.1%) [B2: 1/6 (16.7%), C2: 3/13 (23.0%)] were positive on the initial test and 10 of 19 patients (52.6%) [B2: 4/6 (66.7%), C2: 6/13 (46.2%)] were positive on a follow-up test showing an increase in positive rates during a follow-up, however, no significant correlation between CEA mRNA positivity and subsequent relapse was demonstrated. These results suggest that an early tumor cell dissemination may occur in gastrointestinal tract cancer without subsequent relapse, however, the serial regular examination of CEA mRNA level may contribute to predicting a subsequent relapse in AGC IIIb in gastric cancer.     

Aging-related changes in the calorimetric profile of red blood cells from women with miscarriages

The backward-facing step or the sudden expansion in internal flows is an important problem in different areas. In this study, a porous baffle is mounted on the opposite wall of a sudden expansion to enhance heat transfer near the step. Unlike the solid baffle, which is extensively studied in the literature, the porous baffle has a lower pressure drop, and its properties can be tuned to reach the optimal prospected performance. Effects of different porous baffle geometrical parameters including its normalized height (H_b?=?0.5, 1.0, 1.5, 1.75), width (W_b?=?0.5, 1, 1.5, 2.0, 2.5), porous baffle-step relative distance (D?=?1, 2, 3, 4), Darcy number (10^?2, 10^?3, 10^?4, 10^?6), and Reynolds number (100, 200, 300, 400, 500) on the heat transfer and pressure drop are investigated. The simulation indicates that higher Reynolds numbers enhance more the heat transfer (35% improvement at Re?=?500 with respect to 10% at Re?=?100). Also, longer baffles can lead to higher heat transfer rates (5% improvement in H_b?=?0.5 with respect to 32% at H_b?=?1.5).

     

Synthesis of three fluorescent boronic acid sensors for tumor marker Sialyl Lewis X in cancer diagnosis

Sialyl Lewis X (sLex) is a carbohydrate that is considered not only a marker for cancer, but also an antigen associated with the malignant behavior of cancerous cells. We have synthesized three fluorescent boronic acid sensors as potential sensors for sLex. Photoinduced electron transfer by a fluorescence analyzer was used to assess sensor-sLex antigen binding. The reaction was carried out in mixed aqueous solution, and Sensor 3 was identified as showing the strongest fluorescence enhancement upon binding to sLex at 10 nM. In cell-line culture experiments, Sensor 1 was shown to label sLex expression positively for HepG2, Colo 205, and COS-7 cells, and negatively for MDA-MB-231 cells; Sensor 2 did so positively for HepG2, PLC/PRF/5, and Colo 205 cells, and negatively for MDA-MB-231 and COS7 cells; and Sensor 3 did so positively for PLC/PRF/5 and HepG2 cells, and negatively for MDA-MD-231 and COS7 cells. MTT cytotoxicity experiment results showed that the three sensors are nontoxic, and Hoechst 33258 experiments showed that no apoptosis occurred.     

Determination of trace elements in blood samples of patients affected by multiple sclerosis from Iran by neutron activation analysis

Multiple sclerosis (MS) is a neurological autoimmune disease in which the immune system attacks the central nervous system for unknown reasons and causes several damages to human body by demyelinating the nerve cells. One of the possible cause of this disease is the abnormality levels of some trace elements such as Br, Fe, Rb, Sb, As, and Zn in human body. This study attempts to measure the levels of four trace elements of Br, Fe, Rb, and Zn in the patients?? blood samples and compare them with control samples from healthy individuals. It should be noted that the objectives set out partly met. According to the obtained results, the differences between the levels of Br, Fe, and Rb in patients?? blood samples and control was not significant (P?>?0.05). However, the average level of Zn between samples and controls showed a significant difference (P?<?0.05). Therefore the lower level of Zn in blood is likely to be a major cause of MS emergence. Furthermore, by using the concentration level of Zn as indicator, it was revealed that the risk of MS infection rises as the number of pregnancies increase.