Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes.

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called "the Eprobe leukemia assay," for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

PD-L1 mRNA expression in EGFR-mutant lung adenocarcinoma.

Molecular mechanisms of programmed death-ligand 1 (PD-L1) mRNA expression and roles of apoptosis and biomarkers are poorly understood in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma patients. Thirty-three patients with recurrent postoperative EGFR-mutant lung adenocarcinoma (exon 19 deletion in 16, L858R in 15, G719C in 2 patients) treated with gefitinib were studied. PD-L1 mRNA expression of formalin-fixed paraffin-embedded paratumoral and intratumoral tissues was quantified by PCR. Correlations of PD-L1 mRNA expression with BIM, p53 upregulated modular of apoptosis (PUMA), human epidermal growth factor receptor 2 (HER2), mesenchymal-epithelial transition (MET), EGFR, and vascular endothelial growth factor A (VEGFA) were determined. Eleven of the 33 patients (33.3%) and 14/33 patients (42.4%) expressed intratumoral and paratumoral PD-L1 mRNA, respectively. Patients with intratumoral PD-L1 mRNA expression had significantly higher BIM and lower VEGFA expression compared with paratumoral PD-L1 mRNA patients (P=0.049, P=0.009). PD-L1 mRNA expression was not associated with the expression of PUMA, HER2, EGFR and MET but was positively correlated with BIM expression (r=0.41, P=0.017) and inversely correlated with VEGFA expression (r=-0.33, P=0.043). Patients with intratumoral PD-L1 mRNA expression had significantly shorter median progression-free survival (PFS) after gefitinib therapy compared with no PD-L1 expression (255 vs. 732 days, respectively; P=0.032). Thus, PD-L1 mRNA expression in EGFR-mutant lung adenocarcinoma was associated with BIM and VEGFA mRNA expression and with shorter PFS after gefitinib therapy.

Control of cell adhesion and proliferation utilizing polysaccharide composite film scaffolds.

We have developed polysaccharide composite films made of anionic polysaccharides and chitosan (CHI) by utilizing hot press techniques. In order to demonstrate the versatility of these films as cell scaffolds, the present study investigated the adhesion and proliferation of fibroblasts on composite films prepared by using various kinds of anionic polysaccharides and that were modified with proteins. Cells were spread on heparin/CHI and alginic acid/CHI films and grew well, whereas those on chondroitin sulfate C (CS)/CHI and hyaluronic acid/CHI films were round in shape. The differences in adhesion and proliferation behaviors of cells could be explained by the differences in the biochemical function of the anionic polysaccharides and the physical properties of the films such as morphology, storage modulus, ζ-potentials, and swelling ratios. Among them, the number of cells on CS/CHI films remained almost unchanged. The mechanisms underlying growth suppression on CS/CHI films were investigated by using an integrin stimulator, the TNIIIA2 peptide, and platelet-derived growth factor-B. It was indicated that the growth suppression was due to the lack of fibronectin-integrin growth signaling. The surface modification of CS/CHI films with fibronectin promoted the adhesion and proliferation of cells. These results show that the chemical and physical properties of the polysaccharide composite films, which resulted from the chemical species of anionic polysaccharides or surface modifications of the films, can modulate cell adhesion and proliferation properties thereon.

Comparison of the Analytical Performance Between cobas EGFR Assay and PCR-Clamp Method in the Detection of EGFR Mutations in Japanese Non-Small Cell Lung Cancer Patients.

BACKGROUND: EGFR, a tyrosine-kinase, plays an important role in the progression of lung cancer. Since genetic abnormality of EGFR alters the effects of tyrosine-kinase inhibitors targeting EGFR, molecular analyses of EGFR have recently gained more attention in the treatment of lung cancer. However, several different techniques are available and which method is superior has not been determined. In this study, we compared two recently developed PCR-based techniques, PCR-clamp method and cobas EGFR assay. METHODS: Ninety-four surgical samples and 58 biopsy samples from patients suffering from non-small cell lung cancers (NSCLCs) were included in the study. Samples with positive and negative genetic abnormalities, 66 and 28 respectively, were chosen for PCR-Clamp methods. Those same samples were reanalyzed with cobas EGFR assay. RESULTS: The concordance between PCR-Clamp and cobas EGFR methods was 95.7%. PCR-Clamp failed to detect four mutations that were detected with cobas EGFR assay. These two methods were further tested by analyzing 58 random biopsy samples. The concordance for the biopsy samples was 93.1%, and PCR-Clamp, again, failed to detect three mutations that were detected with cobas EGFR assay. CONCLUSIONS: Our results showed both methods detected most of the known EGFR mutations and the concordance was similar to those previously reported in different ethnicities. However, in our study, PCR-Clamp method failed to detect a total of seven mutations that were detected with cobas EGFR assay. Thus, we concluded that cobas EGFR assay is an easier and more accurate screening assay than PCR-Clamp method in detecting EGFR genetic abnormalities.

Association of BIM Deletion Polymorphism and BIM-γ RNA Expression in NSCLC with EGFR Mutation.

AIM: This pilot study assessed the association of BIM deletion polymorphism and BIM RNA isoform in patients with EGFR-positive non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: The study included 33 patients with EGFR-positive NSCLC treated with gefitinib. BIM deletion polymorphism and BIM RNA isoform (EL/L/S/γ) were determined by polymerase chain reaction (PCR). RESULTS: BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism inside tumors (p=0.038) and around tumors (p=0.0024). Relative BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism (p=0.0017). Patients with BIM-γ had significantly shorter progression-free survival than those without BIM-γ (median: 304 vs. 732 days; p=0.023). CONCLUSION: Expression of BIM-γ mRNA and BIM deletion polymorphism were strongly associated. BIM-γ overexpression may have a role in apoptosis related to EGFR-tyrosine kinase inhibitor.

Emergence of two unrelated clones in acute myeloid leukemia with MLL-SEPT9 fusion transcript.

We report a case of acute myeloid leukemia (AML) with two unrelated clones, one of which was t(11;17)(q23;q25) carrying MLL-SEPT9 fusion transcripts. The patient was a 71-year-old man who was diagnosed with AML M0 and received multiple chemotherapy regimens, including DNA topoisomerase II inhibitors. Although the karyotype of bone marrow cells at the initial diagnosis was normal, two unrelated chromosomal aberrations concurrently appeared during the course of the disease, suggestive of t(11;17)(q23;q25) and add(1)(p36.1),del(6)(q?) by G-banding. Spectral karyotyping analysis identified a reciprocal translocation between chromosomes 11 and 17, and a translocation of the q arm of chromosome 6 to chromosome 1. Dual-color fluorescence in situ hybridization analysis that used probes specific for MLL in combination with tel 1p and tel 1q revealed a translocation of 1p-->pter to chromosome 6 and a translocation of 11q23-->qter to chromosome 17. Reverse transcriptase-polymerase chain reaction and sequencing analyses demonstrated MLL-SEPT9 fusion transcripts with the breakpoint of MLL exon 8/SEPT9 exon 2 and MLL exon 9/SEPT9 exon 2. Thus, the karyotype was defined as 46,XY,t(11;17)(q23;q25)/46,XY,t(1;6)(p36.3;q23). Our case represents an additional MLL-SEPT9-positive AML that was considered to be related to therapy.

[Gene testing of hereditary cancer on comprehensive gene medical examination support system].

It has been estimated that genetic factors or a combination of genetic and environmental factors play a role in the development of 10-15% of all cancers. A genetic cause of hereditary cancer has been identified in more than 40 diseases till now. For preventing this cancer, gene testing is essential because it has no definite clinical marker as in hereditary non-polyposis colorectal cancer: HNPCC. Much more experience must be accumulated in this testing at the clinical base in order to increase specificity and sensitivity while safeguarding ethical, legal and social issues (ELSI). Recently, the Personal Information Protection Law was enforced. Gene inspection involving hereditary cancer should be carried out under a comprehensive gene medical examination organization. It is important for the family doctor, medical specialist, and gene inspection person in charge to cooperate closely with one another, and this will be a subject of future study.

Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.