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Deletion polymorphism of BCL-2-like protein 11 (BIM) is specifically found in East Asia. To explain some epidemiological discrepancies between Asian and Western countries, we analyzed a silent single nucleotide polymorphism (SNP) in exon 5 (c465C T) and a deletion site (2903 bp) in intron 2 in 77 patients with follicular lymphoma by the Q-invader method using PCR. In females, 5-year progression-free survivals (PFS) were 20.0% in the BIM deletion group, 66.7% in the SNP group and 81.5% in the wild-type (WT) group (p = .0012). In the WT group, 5-year PFS was 40.4% in males (p = .0448 vs. female PFS). This tendency was strengthened in patients receiving rituximab (26.9% vs. 84.2%, p = .006). Superior PFS in the WT females in Japan was comparable with the results of cohort studies in the United States and Sweden. Favorable prognosis in Japanese females may be masked by the BIM deletion polymorphism.

Hepatitis C virus (HCV) is a major worldwide public health problem, and mutations at amino acids 70 and 91 in the genotype 1b core region predict the effectiveness of combination therapy with peginterferon and ribavirin. An assay based on the Q-Invader technology was developed to determine the relative ratios of the mutant to wild-type virus with high sensitivity. The assay detected a minor type plasmid that constituted only 1% of a mixture of plasmids containing wild-type and mutant sequences. The calculated ratios agreed with those of the template DNA. A total of 123 serum samples of HCV in Japan were examined with the Q-Invader assay. The Q-Invader assay detected all of the mutations that were detected by direct sequencing and even some mutants that direct sequencing could not. PCR with mutant specific primers confirmed those mutations found by the Q-Invader assay and not by direct sequencing. The Q-Invader assay, thus, is a useful tool for detecting mutations at positions 70 and 91 in the HCV-1b core region.

BCR-ABL1 kinase domain mutations were evaluated in 60 imatinib-resistant patients with Philadelphia-positive (Ph + ) leukemia using PCR-Invader assay and direct sequencing. In chronic myelogenous leukemia (CML) – chronic phase (CP), 5 had P-loop mutations and 3 had T315I mutations. CML-CP patients with high Sokal score showed significantly higher incidence of mutations. P-loop mutations were associated with higher risk of disease progression. In CML-advanced phase, P-loop mutations and T315I mutation were associated with significantly shorter survival. In Ph + acute lymphoblastic leukemia, overall survival was poor irrespective of mutational status. The PCR-Invader assay is useful for screening of mutations and prediction of prognosis.

Early detection of resistant mutations of hepatitis B virus (HBV) is important for patients on nucleos(t)ide analog therapy. An assay based on the PCR-Invader technology was developed to detect resistant mutations with high sensitivity. The assay specifically detects mutations at codons 180, 181, 184, 202, 204, and 250 of the HBV polymerase reverse transcriptase domain. These mutations result in resistance to lamivudine and entecavir. In mixtures of plasmids containing wild-type and resistant mutants, fold-over-zero values for resistant mutations were detected in 2% of the total. Seventy-five serum samples from patients, whose treatment had been switched from lamivudine to entecavir, were examined by the PCR-Invader assay and direct sequencing. The PCR-Invader assay detected all resistant mutations that were detected by direct sequencing and even detected the presence of mutants that direct sequencing could not. Cloning sequencing confirmed those mutations found by the PCR-Invader assay and not by direct sequencing. The PCR-Invader assay is a useful tool for the early detection of drug-resistant mutations.

The Invader PLUS technology is a sensitive, rapid method for the detection and quantification of nucleic acid. While the original technology is based on the amplification by polymerase chain reaction (PCR) of the target sequence followed by its detection using the Invader technology, the current modification allows simultaneous PCR amplification and Invader reaction. The PCR primers and the Invader probes are designed to operate at the same temperature. This allows simpler design and faster results. This technology has been applied for the quantification of six periodontitis-related bacteria (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Toreponema denticola, Tannerella forsythensis and Fusobacterium nucleatum). Direct comparison of this modified Invader PLUS with real-time PCR demonstrated similar linear range. Furthermore, testing of 64 volunteers showed a good correlation between both technologies with correlation factors r2 spanning between 0.827 and 0.987. We demonstrated here that the proposed improvement of the Invader PLUS allows the detection and quantification of DNA sequences using a simple design and protocol that can be implemented in clinical testing.

With its broad effective range for fluorescence detection, real-time PCR is one of the most valuable techniques for quantitation in molecular biology. A modified real-time PCR assay is described for determining viral load. The assay uses fluorescence to measure the number of PCR amplicons by monitoring the Invader reaction in four steps in the thermal cycle. The Invader reaction with its cleavase was performed at moderate temperature after the amplicon was denatured at a high temperature. The method was as effective as real-time PCR with a TaqMan probe in determining the quantity of virus in samples of human papillomavirus type 16. Importantly, the assay allows the use of a common probe for multiple reactions. Thus, this method is a rapid inexpensive assay with a common fluorescence probe that does not depend on the conformation of the target DNAs.

Liver negative-strand hepatitis C virus (HCV) RNA is the most direct indicator of active viral replication but has only been examined in a few semiquantitative studies.Positive- and negative-strand HCV RNA in the right (R) and left (L) liver lobes was quantified by rTth-based strand-specific real-time polymerase chain reaction for 48 chronic hepatitis C patients.Close correlations between lobes were seen for positive- and negative-strand amounts (r = 0.950; P0.001 and r = 0.920; P0.001, respectively). The ratio of negative to positive strands (median, 0.14 for R and 0.13 for L) varied by 2 log directly in relation to HCV replication assessed by liver negative strands but had no relation to liver positive strands and circulating HCV. Only negative-strand quantitation was inversely correlated with age (r = -0.322; P = 0.026 for R and r = -0.340; P = 0.018 for L), while liver tissues with hepatitis B virus DNA contained larger amounts of each strand. In 27 patients treated with enhanced interferon monotherapy, the amounts of liver negative strands (4 log copies/100 ng RNA) were the only independent predictor of a sustained virologic response.Negative-strand quantitation is uniform in the liver and bears distinct relevance to the disease.

When quantifying periodontopathic bacteria, it is important to use a convenient method that does not produce false negative results. The Invader assay is a convenient method because it does not involve gene amplification. The purpose of this study was to evaluate the validity of the Invader assay to quantify periodontopathic bacteria. The Invader technology was applied in quantifying five periodontopathic bacteria (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola). The Invader assay produced a linear quantitative detection range over concentrations spanning seven exponential values, with a detection limit of 10(3.7) copies/tube and intra-day and inter-day variance of 0.1% to 4.7% and 0.1% to 3.4%, respectively, in quantifying five periodontopathic bacteria. We compared the results of the Invader assay with those of real-time polymerase chain reaction (PCR) performed for quantifying five periodontopathic bacteria in 22 patients with periodontitis. Among the Invader-detectable bacterial strains of each species, significant correlations were observed in the counts of concerned bacterial species between these two methods, with correlation coefficients ranging from 0.757 to 0.996. This study validated repeatability and reproducibility of the Invader assay in quantifying periodontopathic bacteria and demonstrated consistent agreement between the Invader assay and real-time PCR in quantifying periodontopathic bacteria.

Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts.

Human papillomavirus (HPV) is associated with several cervical diseases. A simple, rapid, cost-effective assay for identifying viral genotypes would greatly aid efforts for early detection and disease prevention. A real-time polymerase chain reaction monitoring Invader reaction assay (Q-Invader assay) was developed for genotyping and comparative quantitative analysis of 14 high-risk HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 67, and 68). A total of 131 cervical samples containing HPV in Japan were examined by Q-Invader assay, and the results were compared with those from sequencing with consensus and genotype-specific primers. Genotypes determined by Q-Invader agreed with those of sequencing in all samples. Coinfections with multiple high-risk genotypes were correctly identified by Q-Invader assay in 27 samples. In addition, the relative ratios of the genotypes were determined. Thus, Q-Invader assay is a useful tool for genotyping and comparative quantitative analysis of high-risk HPV types.

We recently developed an Invader assay combined with reverse transcriptase polymerase-chain-reaction in order to quantify T315I bcr-abl transcripts. Using this assay, we serially monitored T315I bcr-abl transcripts in chronic myeloid leukemia (CML) patients whose bcr-abl transcripts were still detectable at 6 months after starting imatinib therapy. Although, we continued to monitor bcr-abl transcripts in 14 CML patients (13 chronic phases and 1 accelerated phase) for up to 12 months, there were no patients who were apparently resistant to imatinib due to the T315I mutation. In contrast, in a case of Philadelphia chromosome-positive acute lymphoid leukemia being treated with chemotherapy including imatinib, we monitored both wild-type and T315I bcr-abl transcripts, and found increased levels of T315I transcripts during relapse (0% at the time of diagnosis and 54.8% at relapse). Thus, our new approach could be a useful tool to study the kinetics of mutant clones and the pharmacokinetics of drug resistance with regard to the T315I mutation.

Hepatitis B virus is a worldwide public health problem. A simple and effective test to identify viral genotypes would greatly aid efforts to understand and control the spread of this disease. A serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for distinguishing the known eight genotypes (A-H) and four subgenotypes (Aa, Ae, Ba, Bj) of hepatitis B virus (HBV). The preS/S and core regions were amplified by multiplex PCR and delivered to 12 wells containing genotype-specific Invader probes. By observing the fluorescence patterns in the wells, HBV sub/genotypes can be assigned. A total of 505 serum samples containing HBV/HBsAg in Japan was examined by PCR-Invader and compared the results with those from ELISA assays with monoclonal antibodies against epitopes on gene products of the preS2 region and with a genotype-specific probe assay (GSPA) based on the preS1 region. Genotypes determined by the PCR-Invader agreed with those of the ELISA method in 98.2% of cases and with the GSPA method in 97.1% of cases. Co-infection with two distinct genotypes was correctly identified by the PCR-Invader in four serum samples, as determined by GSPA. Thus, the PCR-Invader assay is a useful tool for detecting the 10 known HBV sub/genotypes.

Recently, it has been demonstrated that the proapoptotic protein BIM showed a deletion polymorphism at exon 3 in eastern Asian population, and some CML patients with the BIM deletion polymorphism are resistant to imatinib treatment (Ng et al. Nature Medicine, 2012). More recently, a BIM single nucleotide polymorphism (SNP) at exon 8 (c465C>T) has also been found in French CML patients and this SNP is associated with not only imatinib resistance but also the presence of BCR-ABL mutations (Mahon et al. ASH abstract, 2012). We aimed to investigate a possible association between such genetic variations of BIM and clinical manifestation in Japanese CML patients who experienced undetectable minimal residual disease (UMRD: so-called CMR4.5). In this study, we newly analyzed BIM SNP (c465C>T) in 47 CML-UMRD patients with known BIM deletion polymorphism status (Katagiri et al. Br J Haematol, 2013). Twenty normal subjects were used as controls. The frequency of either BIM SNP at exon 8 or BIM deletion polymorphism did not deviate from the normal subjects in the Japanese population (P = 0.7597 and P = 0.2880, respectively). None of the subjects showed both BIM SNP at exon 8 and BIM deletion polymorphism concomitantly. We then compared the clinical features among 3 CML-UMRD groups: patients with BIM SNP, patients with BIM deletion polymorphism, and patients who showed neither BIM SNP nor BIM deletion polymorphism (no genetic variations). The frequency of CML patients who maintained 400 mg imatinib dose until stopping was significantly higher in those without genetic variations than in those with BIM SNP or BIM deletion polymorphism (P = 0.0002). Moreover, the frequency of CML patients who switched to second tyrosine kinase inhibitors (2nd TKIs) was significantly higher in those with BIM SNP or BIM deletion polymorphism than in those without such polymorphisms (P = 0.0055).Number of CML patientsMaintained IM 400 mgChange of imatinib dose2nd TKIs switchingBIM SNP (c465C>T)11/474/115/112/11BIM deletion polymorphism6/471/63/62/6BIM SNP or deletion polymorphism17/475/178/174/17No BIM genetic variationss30/4725/305/300/30 This is apparently the first study to circumstantiate the BIM genetic variants in Japanese CML patients with UMPD. Although the number of patients is small, our results suggest that CML patients without BIM deletion polymorphism/SNP could be maintained under standard imatinib dose without switching to 2nd TKIs, and thereby, have a possibility to stop TKIs therapy. Disclosures: Ohyashiki: Norvartis KK: Research Funding, Speakers Bureau; Bristol Meyer Squibe KK: Research Funding, Speakers Bureau.

The carcinogenesis of colorectal cancer has been accepted by a model for a cascade of genetic alterations, named the adenoma-carcinoma sequence. In order to elucidate the carcinogenesis of the colorectal cancer more clearly, the genetic abnormalies of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated. It has been speculated that colonic Paneth cell metaplasia (PaM) is one of the pre-neoplastic mucosa of colonic cancer. Therefore, we studied the propria mucosa of the right colon with PaM from the standpoints of the frequency of the K-ras codon 12 mutations (K-ras), which is initial genetic abnormality in colorectal cancer, and the loss of heterozygosity of microsatellite markers (LOH-MS), which has a relationship to development of colorectal cancer.