Single Allele Base Extension Reaction Research Articles

Comparative Analysis of Two Methods for the Detection of EGFR Mutations in Plasma Circulating Tumor DNA from Lung Adenocarcinoma Patients.

Mutations in the epidermal growth factor receptor (EGFR) are associated with various solid tumors. This study aimed to compare two methods for the detection of EGFR mutations in circulating tumor DNA (ctDNA) from lung adenocarcinoma (LUAD) patients and to evaluate the clinical significance of EGFR mutations in ctDNA. In this prospective cohort study, the EGFR mutation status of 77 patients with stage IIIB or IV LUAD was first determined using lung cancer tissue. The amplification refractory mutation system (ARMS) and single allele base extension reaction combined with mass spectroscopy (SABER/MassARRAY) methods were also used to detect EGFR mutations in plasma ctDNA from these patients and then compared using the EGFR mutation status in lung cancer tissue as a standard. Furthermore, the relationship between the presence of EGFR mutations in ctDNA after receiving first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy and survival was evaluated. The overall sensitivity and specificity for the detection of EGFR mutations in plasma ctDNA by ARMS and SABER/MassARRAY were 49.1% vs. 56% and 90% vs. 95%, respectively. The agreement level between these methods was very high, with a kappa-value of 0.88 (95% CI 0.77–0.99). Moreover, 43 of the patients who carried EGFR mutations also received first-line EGFR-TKI therapy. Notably, patients with EGFR mutations in plasma ctDNA had significantly shorter progression-free survival (9.0 months, 95% CI 7.0–11.8, vs. 15.0 months, 95% CI 11.7–28.2; p = 0.02) and overall survival (30.6 months, 95% CI 12.4–37.2, vs. 55.6 months, 95% CI 25.8–61.8; p = 0.03) compared to those without detectable EGFR mutations. The detection of EGFR mutations in plasma ctDNA is a promising, minimally invasive, and reliable alternative to tumor biopsy, and the presence of EGFR mutations in plasma ctDNA after first-line EGFR-TKI therapy is associated with poor prognosis.

A Simple and Highly Specific MassARRAY-Based Stool DNA Assay to Prioritize Follow-up Decisions in Fecal Immunochemical Test-Positive Individuals.

Background: Seventy-five percent of fecal immunochemical test (FIT)-positive individuals are false positives and undergo unnecessary colonoscopies. Here, we established a stool DNA (sDNA) test that uses the Single Allele Base Extension Reaction (SABER) MassARRAY platform to improve the accuracy of FIT-based CRC detection. Methods: Twenty-one variants in five CRC-associated genes were selected for the sDNA panel. Cell line DNA and matched mutation-confirmed tissue and stool samples from 34 patients were used for accuracy assessment (cohort 1). The clinical performance of the sDNA assay was further evaluated in 101 independent FIT-positive stool samples (cohort 2). Results: In cohort 1, we obtained a 62% mutation concordance rate in paired tissue and stool samples of the CRC group, regardless of the FIT status. In cohort 2, 100% specificity in normal controls with positive FIT results was observed. By weighting the FIT value and the presence of a given variant type in stool and then summing the two scores, we found that a one-increment increase in the score was associated with a 4.538-fold risk (95% CI = 2.121–9.309) for malignancy in the FIT-positive setting. Conclusions: Our highly specific sDNA assay can help prioritize the most at-risk FIT-positive persons to receive prompt colonoscopic confirmation of CRC.

Detection of epidermal growth factor receptor T790M mutation in plasma DNA from patients refractory to epidermal growth factor receptor tyrosine kinase inhibitor

A secondary epidermal growth factor receptor (EGFR) mutation, the substitution of threonine 790 with methionine (T790M), leads to acquired resistance to reversible EGFR-tyrosine kinase inhibitors (EGFR-TKIs). A non-invasive method for detecting T790M mutation would be desirable to direct patient treatment strategy. Plasma DNA samples were obtained after discontinuation of gefitinib or erlotinib in 75 patients with non-small cell lung cancer (NSCLC). T790M mutation was amplified using the SABER (single allele base extension reaction) technique and analyzed using the Sequenom MassARRAY platform. We examined the T790M mutation status in plasma samples obtained after treatment with an EGFR-TKI. The SABER assay sensitivity using mixed oligonucleotides was determined to be 0.3%. The T790M mutation was detected in 21 of the 75 plasma samples (28%). The presence of the T790M mutation was confirmed by subcloning into sequencing vectors and sequencing in 14 of the 21 samples (66.6%). In this cohort of 75 patients, the median progression-free survival (PFS) of the patients with the T790M mutation (n = 21) was not statistically different from that of the patients without the mutation (n = 54, P = 0.94). When patients under 65 years of age who had a partial response were grouped according to their plasma T790M mutation status, the PFS of the T790M-positive patients (n = 11) was significantly shorter than that of the T790M-negative patients (n = 29, P = 0.03). The SABER method is a feasible means of determining the plasma T790M mutation status and could potentially be used to monitor EGFR-TKI therapy.

Non-invasive prenatal diagnosis using cell-free fetal DNA in maternal plasma from PGD pregnancies

Preimplantation genetic diagnosis (PGD) is usually used to establish a non-affected pregnancy for those couples facing a genetic risk of having an affected child. However, an invasive test is still recommended to all PGD patients due to the risk of misdiagnosis. The discovery of cell-free fetal DNA in maternal plasma provides the possibility for noninvasive prenatal diagnosis. Studies have shown that fetal single-gene disorders can be detected in cell-free fetal DNA by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single-allele base extension reaction (SABER) approach or by the size-fractionation approach, whereby cell-free fetal DNA is enriched on the basis of its smaller size compared with maternal DNA fragments. Recent studies have indicated that a combination of the two approaches increases the accuracy of detection. This study combined the two methods and examined fetal paternally inherited gene mutations in maternal plasma obtained from four PGD-conducted pregnancies. The presence or absence of mutations was correctly detected in all cases. This combined method could be used for risk-free prenatal diagnosis of diseases caused by single-gene mutations, and in particular for couples who undergo PGD who opt not to perform invasive prenatal confirmation due to the risk of abortion.

Mass Spectrometry–Based Detection of Hemoglobin E Mutation by Allele-Specific Base Extension Reaction

The specific detection of a minor population of mutant DNA molecules requires methods of high specificity and sensitivity. While the single-allele base extension reaction (SABER) was shown to be useful for the detection of certain beta-thalassemia mutations, we encountered problems with false positivity during development of SABER for the noninvasive prenatal diagnosis of the hemoglobin E (HbE) disease. Systematic optimization resulted in an alternative protocol, the allele-specific base extension reaction (ASBER). An artificial model was established by mixing genomic DNA of HbE carriers and normal individuals. Effects of terminator concentration and annealing temperature on the nonspecificity of SABER were then studied. The use of a single relevant terminator and the other 3 types of dideoxynucleotide as competing terminators were also compared in the development of the ASBER protocol. Thirteen cases of HbE-susceptible pregnancies were tested to compare the SABER and the ASBER protocols. Decreasing the single relevant terminator concentration and increasing the annealing temperature in SABER were found to improve specificity. The use of the other 3 types of dideoxynucleotide as competing terminators was shown to offer better detection sensitivity than a single terminator in ASBER. Genotyping results were all correctly determined by ASBER, except one false-negative detection (sensitivity: 80%, specificity: 100%). An alternative mass spectrometry-based protocol for noninvasive prenatal diagnosis, ASBER, has been successfully developed to allow the detection of a minor DNA population with a point mutation.

Non‐invasive prenatal detection of achondroplasia in size‐fractionated cell‐free DNA by MALDI‐TOF MS assay

Achondroplasia is the most common form of short-limbed dwarfism in humans and is caused by mutations in the FGFR3 gene. Currently, prenatal diagnosis of this disorder relies on invasive procedures. Recent studies have shown that fetal single gene point mutations could be detected in cell-free DNA (cf-DNA) from maternal plasma by either the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single allele base extension reaction (SABER) approach or the size fractionation of cf-DNA in maternal plasma. Here, we combined the two approaches to non-invasively examine the fetal G1138A mutation in maternal plasma. cf-DNA was extracted from maternal plasma samples obtained from two pregnant women at risk for achondroplasia. The fetal G1138A mutation was determined by the analysis of size-fractionated cf-DNA in maternal plasma using MALDI-TOF MS with SABER approach and homogenous MassEXTEND (hME) assay, respectively. The fetal G1138A mutation was detectable in the two achondroplasia-affected pregnancies by the analysis of cf-DNA in maternal plasma using MALDI-TOF MS. However, the size-fractionation approach led to a more precise detection of the fetal mutation in both analyses. This analysis would be suitable for non-invasive prenatal diagnosis of diseases caused by fetal single gene point mutations.

Genotyping fetal paternally inherited SNPs by MALDI‐TOF MS using cell‐free fetal DNA in maternal plasma: Influence of size fractionation

The determination of fetal point mutations from fetal cell-free DNA (cf-DNA) in maternal plasma is technically challenging due to the preponderance of maternal sequences. It has recently been shown that fetal cf-DNA sequences are smaller than maternal ones and that the selection of small cf-DNA fragments by size fractionation by agarose gel electrophoresis leads to the enrichment of fetal cf-DNA sequences, thereby permitting the detection of otherwise masked fetal point mutations. In a separate development, the use of MALDI-TOF MS has also been shown to facilitate the detection of fetal point mutations from cf-DNA in maternal plasma. In this study, a combination of these approaches was examined. cf-DNA was extracted from 18 maternal plasma samples, 10 taken at term and 8 obtained early in the second trimester. A total of 41 SNP loci were examined in size-fractionated and total cf-DNA using either a conventional homogeneous MassEXTEND (hME) assay or a nucleotide-specific single allele base extension reaction (SABER) assay. The analysis of total cf-DNA indicated that size fractionation considerably enhanced the sensitivity of the standard hME assay, especially for samples taken early in pregnancy. Size fractionation also rendered the signals obtained by the SABER assay more precise.

Recent developments in fetal nucleic acids in maternal plasma: implications to noninvasive prenatal fetal blood group genotyping

The discovery of circulating cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Fetal DNA in maternal plasma has been used for the noninvasive prenatal determination of the RhD status of fetuses carried by RhD-negative pregnant women. In such analysis, the possible need of an internal control for the presence of detectable amounts of fetal DNA in a particular maternal plasma sample has been actively discussed. Recently, the development of a robust method for discriminating single nucleotide differences in plasma DNA using single allele base extension reaction (SABER) followed by matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has opened up the possibilities of using a panel of single nucleotide polymorphisms as such a positive control. A second approach is the recent successful development of fetal epigenetic markers which can be developed into universal fetal DNA markers. These developments hold promise to allow the eventual widespread utilization of maternal plasma DNA analysis for the noninvasive prenatal diagnosis of blood group mismatches between the mother and fetus.

MS analysis of single-nucleotide differences in circulating nucleic acids: Application to noninvasive prenatal diagnosis.

The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian beta-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the beta-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical beta-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation.