Tomokazu Ishikawa

Differential detection of KRAS mutations in codons 12 and 13 with a modified loop-hybrid (LH) mobility shift assay using an insert-type LH-generator.

BACKGROUND The loop-hybrid mobility shift assay (LH-MSA) was previously developed for the rapid detection of the EGFR mutation L858R for predicting clinical responses to gefitinib in lung cancer. Recently, clinical importance of determining KRAS mutations has been demonstrated in colorectal tumors as tumors harboring mutated KRAS genes were not responsive to therapy with EGFR-targeted antibodies such as cetuximab. METHODS We developed a new version of the LH-MSA using an insert-type LH generator that was capable of detecting all 12 KRAS mutations in codons 12 and 13. RESULTS Feasibility evaluation was performed with this new LH-MSA on 215 colorectal cancer specimens. KRAS codon 12 mutations were detected in 23% specimens and codon 13 mutations in 6.5% specimens by LH-MSA at a rate better than by direct sequencing. CONCLUSIONS Using the new method, the G13D mutation was readily distinguishable from other KRAS mutations in codon 12 and, therefore, would be advantageous for clinical applications.

An Automated Micro-Total Immunoassay System for Measuring Cancer-Associated α2,3-linked Sialyl N-Glycan-Carrying Prostate-Specific Antigen May Improve the Accuracy of Prostate Cancer Diagnosis

The low specificity of the prostate-specific antigen (PSA) for early detection of prostate cancer (PCa) is a major issue worldwide. The aim of this study to examine whether the serum PCa-associated α2,3-linked sialyl N-glycan-carrying PSA (S2,3PSA) ratio measured by automated micro-total immunoassay systems (μTAS system) can be applied as a diagnostic marker of PCa. The μTAS system can utilize affinity-based separation involving noncovalent interaction between the immunocomplex of S2,3PSA and Maackia amurensis lectin to simultaneously determine concentrations of free PSA and S2,3PSA. To validate quantitative performance, both recombinant S2,3PSA and benign-associated α2,6-linked sialyl N-glycan-carrying PSA (S2,6PSA) purified from culture supernatant of PSA cDNA transiently-transfected Chinese hamster ovary (CHO)-K1 cells were used as standard protein. Between 2007 and 2016, fifty patients with biopsy-proven PCa were pair-matched for age and PSA levels, with the same number of benign prostatic hyperplasia (BPH) patients used to validate the diagnostic performance of serum S2,3PSA ratio. A recombinant S2,3PSA- and S2,6PSA-spiked sample was clearly discriminated by μTAS system. Limit of detection of S2,3PSA was 0.05 ng/mL and coefficient variation was less than 3.1%. The area under the curve (AUC) for detection of PCa for the S2,3PSA ratio (%S2,3PSA) with cutoff value 43.85% (AUC; 0.8340) was much superior to total PSA (AUC; 0.5062) using validation sample set. Although the present results are preliminary, the newly developed μTAS platform for measuring %S2,3PSA can achieve the required assay performance specifications for use in the practical and clinical setting and may improve the accuracy of PCa diagnosis. Additional validation studies are warranted.

Simple and precise detection of UGT1A1 polymorphisms with a modified loop-hybrid mobility shift assay using Cy5-labeled loop probes

BACKGROUND Irinotecan, an inhibitor of topoisomerase I, has been widely used as an important anti-cancer therapeutic drug. Deleterious effects of the drug in hypersensitive patients are known to be associated with genetic polymorphisms of the UGT1A1 gene, namely the polymorphic variants, *28 and *6. METHODS A modified form of loop-hybrid mobility shift assay using a Cy5-tagged loop-hybrid probe was proposed as a precise and easy method of determining TA repeat polymorphisms at the *28 locus. RESULTS In this modified method, only loop-hybrid bands were detected by a Cy5-fluorescent signal, despite several irregular electrophoretic bands due to TA repeats in the PCR product. CONCLUSIONS When a loop-hybrid using a Cy5-tagged probe for the *28 locus and *6 locus were combined and used for mobility shift assay, simultaneous typing of the *28 and *6 variants was achieved in a single lane.

Use of transcriptional sequencing in difficult to read areas of the genome.

In genome and cDNA sequencing projects, current cycle sequencing often encounters difficult-to-sequence templates which have unique secondary structures due to GC-rich composition or repeated regions. Due to the formation of stable secondary structures, remarkable decreases in fluorescent signals are observed in cycle sequencing reactions. It is not easy to determine the nucleotide sequences of these regions. Although several modifications of sequencing reactions have been tried to overcome these problems, some unreadable regions remain as gaps in genome sequencing projects. Here, we further developed transcriptional sequencing technology and evaluated the sequencing accuracy in these regions. The method was successively applied to artificial GC cluster templates and putative secondary structure-forming templates from genomic and cDNA clones. Our results indicate that transcriptional sequencing is a powerful and accurate method for GC-rich regions, simple sequence repeats, hairpins (inverted repeats), tandem repeat DNA templates, and gap-closing in draft sequencing data.

Analysis of Human Androgen Receptor Polymorphism Using Fluorescent Loop-Hybrid Mobility Shift Technique

A fluorescent loop-hybrid mobility shift (LH-MS) technique was introduced for the analysis of polymorphic sites in exon 1 of the human androgen receptor gene. (CAG)17-31 or (CTG)17-31 loops were assumed to protrude from the sense- or antisense strands of the PCR products following hybridization with reverse or forward LH probes, respectively. When an array of male DNA was analyzed using Cy5-labeled LH probes, a unique linear correlation was established between CAG repeat lengths and the fluorescent LH band positions on polyacrylamide gels. This linearly shifting band patterns were used to assemble LH ladder size markers of CAG repeat lengths. Analysis of female DNA revealed that 87% of females are heterozygous for CAG repeat polymorphisms, which could be informative for clonality analysis of tumors in female cancer patients. As a proof of principle, heterozygous female colorectal tumor DNA was examined with fluorescent LH-MS technique and loss of one allele after treatment with methylation-sensitive restriction enzyme HpaII was clearly exhibited.