Cancer panel analysis of circulating tumor cells in patients with breast cancer.

Liquid biopsy using circulating tumor cells (CTCs) is a noninvasive and repeatable procedure, and is therefore useful for molecular assays. However, the rarity of CTCs remains a challenge. To overcome this issue, our group developed a novel technology for the isolation of CTCs on the basis of cell size difference. The present study isolated CTCs from patients with breast cancer using this method, and then used these cells for cancer gene panel analysis. Blood samples from eight patients with breast cancer were collected, and CTCs were enriched using size-based filtration. Enriched CTCs were counted using immunofluorescent staining with an epithelial cell adhesion molecule (EpCAM) and CD45 antibodies. CTC genomic DNA was extracted, amplified, and screened for mutations in 400 genes using the Ion AmpliSeq Comprehensive Cancer Panel. White blood cells (WBCs) from the same patient served as a negative control, and mutations in CTCs and WBCs were compared. EpCAM+ cells were detected in seven out of eight patients, and the average number of EpCAM+ cells was 8.6. The average amount of amplified DNA was 32.7 µg, and the percentage of reads mapped to any targeted region relative to all reads mapped to the reference was 98.6%. The detection rate of CTC-specific mutations was 62.5%. The CTC-specific mutations were enhancer of zeste polycomb repressive complex 2 subunit, notch 1, AT-rich interaction domain 1A, serine/threonine kinase 11, fms related tyrosine kinase 3, MYCN proto-oncogene, bHLH transcription factor, APC, WNT signaling pathway regulator, and phosphatase and tensin homolog. The technique used by the present study was demonstrated to be effective at isolating CTCs at a sufficiently high purity for genomic analysis, and supported the use of comprehensive cancer panel analysis as a potential application for precision medicine.

Evaluation of a novel approach to circulating tumor cell isolation for cancer gene panel analysis in patients with breast cancer.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

The mitochondrial hinge protein, UQCRH, is a novel prognostic factor for hepatocellular carcinoma.

Alterations in mitochondrial respiration contribute to the development and progression of cancer via abnormal biogenesis, including generation of reactive oxygen species. Ubiquinol-cytochrome c reductase hinge protein (UQCRH) consists of the cytochrome bc1 complex serving respiration in mitochondria. In the present study, we analyzed UQCRH abnormalities in hepatocellular carcinoma (HCC) and its association with clinical outcomes of patients. UQCRH expression in HCC was determined via semiquantitative and quantitative real-time reverse transcriptase polymerase chain reaction of 96 surgically resected HCC tissues positive for hepatitis B virus surface antigen. UQCRH was frequently overexpressed in HCC tissues (46.8%, based on 2.1-fold cutoff). UQCRH overexpression was observed in HCCs with larger tumor size, poorer differentiation, or vascular invasion. Kaplan-Meier analysis revealed significantly shorter overall (P = 0.005) and recurrence-free survival (P = 0.027) in patients with tumors overexpressing UQCRH. The prognostic impact of UQCRH was significant in subgroups of patients divided according to the α-fetoprotein (AFP) level. The patient subgroup with higher AFP levels (≥20 ng/mL) exhibited significant differences in 5-year overall (18.5% vs. 67.9%) and recurrence-free survival rates (11.1% vs. 46.4%) between groups with and without UQCRH overexpression. In contrast, no marked survival differences were observed between subgroups with lower AFP levels (<20 ng/mL). Multivariate analysis defined UQCRH as an independent poor prognostic factor. Conclusively, our results indicate that UQCRH overexpression is correlated with poor outcomes of HCC patients. Furthermore, in patients grouped as high risk based on elevated AFP, lack of UQCRH overexpression could be a useful indicator for clinical treatment.

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism.

BACKGROUND: Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment. METHODS: Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification. RESULTS: The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R²>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases. CONCLUSIONS: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Highly sensitive analysis of boron and lithium in aqueous solution using dual-pulse laser-induced breakdown spectroscopy.

We have applied a dual-pulse laser-induced breakdown spectroscopy (DP-LIBS) to sensitively detect concentrations of boron and lithium in aqueous solution. Sequential laser pulses from two separate Q-switched Nd:YAG lasers at 532 nm wavelength have been employed to generate laser-induced plasma on a water jet. For achieving sensitive elemental detection, the optimal timing between two laser pulses was investigated. The optimum time delay between two laser pulses for the B atomic emission lines was found to be less than 3 µs and approximately 10 µs for the Li atomic emission line. Under these optimized conditions, the detection limit was attained in the range of 0.8 ppm for boron and 0.8 ppb for lithium. In particular, the sensitivity for detecting boron by excitation of laminar liquid jet was found to be excellent by nearly 2 orders of magnitude compared with 80 ppm reported in the literature. These sensitivities of laser-induced breakdown spectroscopy are very practical for the online elemental analysis of boric acid and lithium hydroxide serving as neutron absorber and pH controller in the primary coolant water of pressurized water reactors, respectively.

Effect of Modulation of hnRNP L Levels on the Decay of bcl-2 mRNA in MCF-7 Cells.

It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.

hnRNP L binds to CA repeats in the 3'UTR of bcl-2 mRNA.

We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). A super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.

Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus.

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.