Quenching Probe Method Research Articles

Kras Gene Analysis Using Liquid-Based Cytology Specimens Predicts Therapeutic Responses and Prognosis in Patients with Pancreatic Cancer.

Simple SummaryNew therapeutic strategies are needed to improve the prognosis of pancreatic ductal adenocarcinoma (PDAC) and developing biomarkers that can guide individualized treatment decisions is an important part of these strategies. In this study, we found that unresectable PDAC patients harboring wild-type Kras had significantly longer progression-free survival (PFS) and overall survival (OS) than those harboring mutant Kras after undergoing first-line gemcitabine and nab-paclitaxel (GA) therapy and that wild-type Kras was a significant predictor of longer PFS and OS. This is the first report suggesting that Kras gene analysis has the potential to predict therapeutic responses to GA and the prognosis of unresectable PDAC.Background: Although several molecular analyses have shown that the Kras gene status is related to long-term survival of patients with pancreatic ductal adenocarcinoma (PDAC), the results remain controversial. Here, we examined the Kras gene status in a cohort of unresectable PDAC patients who underwent first-line therapy with gemcitabine and nab-paclitaxel (GA) and assessed differences in chemotherapy responses and survival. Methods: Patients with a histological diagnosis of PDAC (based on EUS-guided fine-needle aspiration) from 2017 to 2019 were enrolled. Tumor genomic DNA was extracted from residual liquid-based cytology specimens and Kras mutations were assessed using the quenching probe method. The relationships between the Kras status and progression-free survival (PFS) and overall survival (OS) were assessed. Results: Of the 110 patients analyzed, 15 had wild-type Kras. Those with the wild-type gene showed significantly longer PFS and OS than those with mutant Kras (6.9/5.3 months (p = 0.044) vs. 19.9/11.8 months (p = 0.037), respectively). Multivariate analyses identified wild-type Kras as a significant independent factor associated with longer PFS and OS (HR = 0.53 (p = 0.045) and HR = 0.35 (p = 0.007), respectively). Conclusions: The analysis of the Kras gene status could be used to predict therapeutic responses to GA and prognosis in unresectable PDAC patients.

Clinical Evaluation of a Novel Point-of-Care Assay To Detect Mycoplasma pneumoniae and Associated Macrolide-Resistant Mutations.

The recent increase in macrolide-resistant Mycoplasma pneumoniae in Asia has become a continuing problem. A point-of-care testing method that can quickly detect M. pneumoniae and macrolide-resistant mutations (MR mutations) is critical for proper antimicrobial use. Smart Gene (Mizuho Medy Co., Ltd., Tosu City, Saga, Japan) is a compact and inexpensive fully automatic gene analyzer that combines amplification with PCR and the quenching probe method to specify the gene and MR mutations simultaneously. We performed a clinical evaluation of this device and its reagents on pediatric patients with suspected M. pneumoniae respiratory infections and evaluated the impact of the assay on antimicrobial selection. Using real-time PCR as a comparison control, the sensitivity of Smart Gene was 97.8% (44/45), its specificity was 93.3% (98/105), and its overall concordance rate was 94.7% (142/150). The overall concordance rate of Smart Gene diagnosis of MR mutations in comparison with sequence analysis was 100% (48/48). The ratio of MR mutations was significantly higher at high-level medical institutions than at a primary medical clinic (P = 0.023), and changes in antibiotic therapy to drugs other than macrolides were significantly more common in patients with MR mutations (P = 0.00024). Smart Gene demonstrated excellent utility in the diagnosis of M. pneumoniae and the selection of appropriate antimicrobials for MR mutations at primary medical institutions, which play a central role in community-acquired pneumonia care. The use of this device may reduce referrals to high-level medical institutions for respiratory infections, thereby reducing the medical and economic burdens on patients.

Abstract 3782: Genetic analysis using a novel high-purity enrichment system for circulating tumor cells independent of epithelial cell antigen

Abstract Background Genetic analysis of circulating tumor cells (CTCs) is useful as a liquid biopsy. However, there are 3 challenging issues in processing of CTC samples for clinical use of the analysis: [1] numerous residual blood cells in processed samples, [2] loss of CTCs that do not express epithelial markers, and [3] very laborious process. Here, we developed a novel system capable of overcoming all of these problems. Methods Our CTC analysis system is composed of 3 steps: [1] filtering of whole blood followed by immunostaining and magnetic labeling of cells trapped on the filter, [2] depletion of white blood cells (WBCs) in the cells recovered from the filter by magnetic separation, and [3] trapping the resultant cells at an observation chamber in a microfluidic enrichment device using dielectrophoresis, followed by recovering them as a concentrated sample after fluorescence microscope observation. SNV and CNV analysis was conducted using the collected concentrated sample, and the system performance was substantiated. Genetic mutations of the cells in the collected sample were detected by the Quenching Probe method. On the other hand for CNV analysis, cells in the collected concentrated sample were immobilized on slide glass and analysis of gene amplification conducted by FISH. Results With SNV analysis, our system successfully detected EGFR, KRAS or PIK3CA mutations of cancer cell lines spiked in 8 mL of whole blood (Table). The detection sensitivity of our method was 1 cell/mL, and both the cancer cells and their genetic mutations were detected within 9 hours of starting the processing of whole blood. Additionally, with CNV analysis, HER2 gene amplification was confirmed with cell lines spiked in 8 mL of blood. This detection result was obtained within 48 hours. Conclusion Our system would be useful for the analysis of gene mutations in a wide range of CTC types independent of certain epithelial antigens, such as CK and EpCAM, and can be used for various genetic analyses. Table Results of spiking test for SNV analysisSpiked cell linesExpected count[cells/8ml]No. of detected spiked cells [cells]No. of detected remained WBCs [cells]Detected genotypesNCI-H197512979EGFR L858RNCI-H16508225EGFR exon19 deletionSW6208648KRAS codon12, 13SNU-1124154KRAS codon12, 13MCF-777341PIK3CA G1633A Citation Format: Hidenori Takagi, Masahiro Kozuka, Hiroshi Ito, Soo Hyeon Kim, Mitsuharu Hirai, Teruo Fujii. Genetic analysis using a novel high-purity enrichment system for circulating tumor cells independent of epithelial cell antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3782. doi:10.1158/1538-7445.AM2017-3782

Detection of the MYD88 mutation by the combination of the allele-specific PCR and quenching probe methods.

The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR. A lymphoma cell line heterozygous for the MYD88 mutation, two wild-type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS-PCR, PCR-RFLP, HRM, and QP, and their detection sensitivity was examined using the mixtures of the mutant and wild-type DNA. For mutation-carrying heterozygous samples, the QP method produced W-shaped melting profiles presenting curves derived from the wild-type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS-PCR and QP (AS-QP) improved the sensitivity to 0.62% of the mutant allele. The AS-QP analysis is rapid and minimally improves detection sensitivity compared to the AS-PCR.

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells.

Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61°C and 68°C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68°C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

Automated and rapid system for detection of ALK rearrangement genes in non-small cell lung cancer based on a Quenching Probe method

Automated and rapid system for detection of ALK rearrangement genes in non-small cell lung cancer based on a Quenching Probe method

An automated rapid detection system using the quenching probe method for detecting interleukin 28B and inosine triphosphatase single nucleotide polymorphisms in chronic hepatitis C

Single nucleotide polymorphisms (SNPs) in the interleukin 28B gene (IL28B) are good pretreatment predictors of anti-hepatitis C virus (HCV) therapy with interferon. SNPs of the inosine triphosphatase (ITPA) gene are associated with reduced haemoglobin levels during treatment with ribavirin. The i-densy™ (Arkray, Inc.), which is based on the quenching probe (QP) method, automatically detects target genes in blood samples by fluorescence quenching within 100 min. Using a QP and primer set, a gene amplification response is generated that can quickly and easily detect a specific gene's arrangement by fluorometry. The present study was conducted to compare the utility of i-densy (QP method) with that of conventional direct sequencing (DS) for detecting SNPs in the IL28B and ITPA genes in chronic hepatitis C patients. Between June 2011 and January 2012, 73 consecutive patients underwent genotyping of IL28B, and 54 patients underwent genotyping of ITPA. All of the patients were seropositive for HCV-RNA. The IL28B and ITPA genotypes were tested for bi-allelic polymorphisms in rs8099917 (T/T, T/G and G/G; minor allele, G) and rs1127354 (C/C, C/A and A/A; minor allele, A), respectively. The results obtained with the QP method were identical to those obtained with the conventional DS method. The frequency of the IL28B genotypes TT, GT and GG were 74%, 24.7% and 1.4%, respectively, and those of the ITPA genotypes CC, AC and AA were 68.5%, 29.6% and 1.9%, respectively. These results indicate that the i-densy using the QP method can automatically, quickly and easily identify genotypes of IL28B and ITPA.

ABO blood group genotyping by quenching probe method

We developed a multiplex ABO genotyping method with quenching probes (Q-probe). In this method, it is possible to discriminate the mutations, not only frequently used positions 261 and 796 but also position 703 in a single PCR. Each probe was designed to have cytosine residue at 5′ or 3′ end and labeled with three different fluorescence dyes, enabling the triplex detections of these polymorphisms. All polymorphisms were successfully detected by using fluorescence labeled Q-probe in a specifically amplified PCR product. Each Q-probe showed unique dissociation patterns depending on the polymorphism types. All of the results obtained with Q-probe were compared with standard serotyping and TaqMan PCR method and resulted in complete match with each other. Consequently, these results indicated that multiplex ABO genotyping method is quite accurate and convenient method for the determination of ABO genotype.

Advantages of the quenching probe method over other PCR-based methods for detection of the JAK2 V617F mutation

The detection of a V617F mutation (G to T exchange at nucleotide 1,849) in the JAK2 gene is crucial for the diagnosis of myeloproliferative neoplasms (MPN) such as polycythemia vera. Although sequence analysis is the standard method for detection, it is not suitable for clinical examinations due to the requirement of expensive equipment. In this study, we evaluated the efficiencies of four PCR-based methods to detect JAK2 V617F: allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRM) and the quenching probe method (QP). The HEL cell line, which harbors a homozygous JAK2 V617F mutation, as well as bone marrow samples from 16 MPN patients and normal control samples, were used in this assessment. The sensitivity of the detection limit of all four methods was also examined using samples of HEL cells mixed in a variety of ratios with cells containing wild-type JAK2. The results of all four methods were found to be concordant. AS-PCR was shown to be the most sensitive; however, it produced false positive results. Although PCR-RFLP demonstrated high specificity, it was time consuming. By contrast, results were obtained using HRM and QP in only 2 h. It was easier to recognize the curves derived from the mutant allele obtained using QP. QP is also suitable for the rough estimation of allele burden. JAK2 V617F assays are mainly used for diagnosis at presentation in clinical settings. We therefore conclude that in situations where high sensitivity is not required, QP is the preferable method for the detection of JAK2 V617F. To the best of our knowledge, this is the first report to demonstrate the efficiency of the QP method for the detection of JAK2 V617F using a standard thermal cycler.

A fully integrated, automated and rapid detection system for KRAS mutations

KRAS mutations are detected in tumors of various organs, and they are also markers of resistance for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and monoclonal antibodies against the EGFR. Thus, the accurate and rapid detection of KRAS mutations is crucial, not only for screening, but also for the prediction of the efficacy of molecular-targeted therapy. The aim of the present study was to establish a novel automated detection system for KRAS mutations. One hundred and thirty-six lung adenocarcinoma patients were genotyped for KRAS mutations with both the conventional direct sequence (DS) method and with the newly developed quenching probe (QP) method that obtains data automatically within 60 min. The detection limit of the QP method using a control plasmid containing the KRAS mutation was 50 copies, and 10% mutant plasmid was detected in the mixture of wild-type and mutants. The results obtained by the QP and DS methods were identical in all but two of the 136 cases. The two differentially identified samples, which consisted of substantially fewer lung cancer cells, were positive according to the QP method but negative as determined by DS for KRAS mutations. These findings characterize the QP method as an accurate and rapid detection system for KRAS mutations.

A Fully Integrated and Automated Detection System for Single Nucleotide Polymorphisms of UGT1A1 and CYP2C19

The need for examinations of single nucleotide polymorphisms (SNPs) on drug metabolizing enzymes is accelerating. Especially, SNPs of UTG1A1 and CYP2C19 are important for patients who are treated with irinotecan and proton pump inhibitors, respectively. Thus, a method for the rapid, fully automated, and accurate measurement of these SNPs is desired. We genotyped 109 Japanese volunteers for UGT1A1*6, UGT1A1*28, CYP2C19*2, and CYP2C19*3 with the quenching probe (QP) method. Only 90 min after whole blood was applied, QP method enabled to detect these SNPs automatically. The results obtained by QP method were absolutely identical to those examined by the conventional direct sequencing. These findings indicate that the QP method will enable point-of-care testing in clinical laboratories and patient-oriented therapy with its convenience and speed for patients who are treated with irinotecan or proton pump inhibitors.

Highly Sensitive Quenching Probe (QProbe) Method Is Useful to Detect c-Kit Mutation and to Predict Relapse of t(8;21)(q22;q22) Acute Myeloid Leukemia.

Abstract 2646Poster Board II-622▪▪This icon denotes an abstract that is clinically relevant. Background:t(8;21)(q22;q22) acute myeloid leukemia (t(8;21) AML) show a high rate of complete remission (CR) and prolonged CR duration, especially following consolidation chemotherapy with high-dose cytarabine (HD-ARAC), and are thought to have a better prognosis than other AML patients. Nevertheless, only approximately 50% of patients were alive at 5 years. This suggests that some patients have more aggressive leukemic phenotypes and indicates the need for predictive molecular marker of relapse and treatment optimization with novel and/or more aggressive therapies such as stem cell transplantation.Recently, several groups reported that c-kit mutation (MutKIT) defined an unfavorable subgroup in t(8;21) AML. However MutKIT has been reported approximately 20% at diagnostic samples of core binding factor leukemia. It is possible that MutKIT are more high frequency at relapse sample of t(8;21) AML. In the present study, we analyzed samples collected at diagnosis and relapse to investigate the role of MutKIT s and Flt3 internal tandem duplication (ITD) in t(8;21) AML. Methods:We analyzed MutKIT and Flt3 ITD among 32 t(8;21) AML patients diagnosed between 1991 to 2009 at the Nippon Medical School. All of them were succeeded achieving CR, but 18 patients (56.3%) relapsed, became refractory chemotherapy, and poor prognosis. Exon 8 and 17 of MutKIT were analyzed by direct sequence and QProbe-system (ARKRAY, Inc. Kyoto, Japan). QProbe-system was high sensitivity mutation screening method, detected approximately 1% MutKIT using mutation specific guanine quenching probe (Leukemia Res 32 (2008) 1462–1467). FLT3 ITD was analyzed by PCR amplification. Results:Using direct sequence, MutKITs were found in 5 (18.5%) of the 27 patients at diagnosis (D816V: 3 patients AN822K: 2 patients), and 7 (46.6%) of 15 patients at relapse. Interestingly 3 patients were detected MutKIT at only relapse (D816V: 2 patients AN822K: 1 patient). All mutations found in exon 17 clustered within the A-loop. Next we analyzed to detected very slight amount of mutation by QProbe-system. N822K were newly found of the 3 patients at diagnosis in spite of negative by direct sequence Finally MutKIT were found 8 (29.6%) at diagnosis. Flt3 ITD were found in 2 (9.5%) of the 21 patents at diagnosis, and none of 15 patients at relapse. 2 patients with Flt3 ITD were not accompanied MutKIT. All of N822K newly found by QProbe-system and Flt3 ITD positive patients were relapsed but turn to mutation negative. To evaluate the importance of MutKIT as a predicted relapse, we analyzed the cumulative incidence of relapse (CIR) and relapse free survival (RFS) of these patients with Kaplan-Meier method. The CIR was higher for patients with MutKIT (p=0.040, Wild type c-kit (WtKIT): 45.8% vs MutKIT: 87.5%). In direct sequencing method, The RFS tend to be shorter for patient with MutKIT, but no significant differences (p=0.260; WtKIT: 30 months vs MutKIT: 11 months).On the other hand, in QProbe-system, the median RFS was shorter for patients with MutKIT (p=0.033; WtKIT: 64 months vs MutKIT: 11 months). In addition of FLT3-ITD analysis, median RFS was shorter for patients with MutKIT or FLT3 ITD (p=0.005; Wild type of both genes: 64 months vs MutKIT or FLT3 ITD: 10 months). Additionally, we compared overall survival (OS) of wild type of both genes with MutKIT or FLT3 ITD. The OS tend to be shorter for patient with MutKIT or FLT3 ITD, but no significant differences. Discussion:In this study, we confirmed the prognostic significance of MutKIT in patients with t(8;21) AML when we use the highly sensitive quenching probe method (QProbe-system). We showed that QProbe-system is quite useful in predicting the prognosis of patients with t(8;21) AML and in determining its therapeutic strategy including tyrosine kinase inhibitors and/or up-front stem cell transplantation. We also revealed the important role of MutKIT (46.7%) at relapse, but several questions were still remained. It is unknown for some patients how to gain MutKIT at only relapse and why mutations detected at diagnosis were disappeared at relapse. We need further study to clarify the function and commitment of MutKIT in relapse of t(8;21) AML. Disclosures:No relevant conflicts of interest to declare.