Validation of targeted next-generation sequencing for<i>RAS</i>mutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR

Gao, Jie; Wu, Huanwen; Wang, Li; Zhang, Hui; Duan, Hongfei; Lu, Junliang; Zeng, Liang

doi:10.1136/bmjopen-2015-009532

Cited by 43 publications

(39 citation statements)

References 22 publications

(30 reference statements)

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“…Thereafter, the mutation could be used as a noninvasive biomarker for disease progression monitoring in colorectal cancer. In the future, ddPCR can be developed in this field when considering the superior detection threshold of ddPCR for KRAS mutations in colorectal cancer (0.025%) [6] compared with Sanger sequencing (10−20%) [22], targeted NGS (1%) [23] and TagMeltPCR and High-resolution melt (HRM) (0.5%) [24].…”

Section: Cancer Progression and Treatment Response Monitoringmentioning

confidence: 99%

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Yu¹,

Shen²,

Jiang³

et al. 2017

Cell Physiol Biochem

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Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

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Section: Cancer Progression and Treatment Response Monitoringmentioning

confidence: 99%

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Yu¹,

Shen²,

Jiang³

et al. 2017

Cell Physiol Biochem

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“…Both Haley et al and Altimari et al demonstrated that NGS had better sensitivity and specificity than Sanger sequencing, pyrosequencing and ARMS-Scorpion PCR assays for the detection of known KRAS mutations (41,42). NGS has a sensitivity as low as 1% mutant allele frequency and had 100% concordance with a panel of KRAS mutant patients compared to 98% with Therascreen (43). Other major benefits of NGS will be discussed later when we address the roll of multiplex panels in mCRC.…”

Section: Next Generation Sequencing (Ngs)mentioning

confidence: 99%

Current companion diagnostics in advanced colorectal cancer; getting a bigger and better piece of the pie

Loree

Kopetz

Raghav

2017

J. Gastrointest. Oncol.

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While the treatment of colorectal cancer continues to rely heavily on conventional cytotoxic therapy, an increasing number of targeted agents are under development. Many of these treatments require companion diagnostic tests in order to define an appropriate population that will derive benefit. In addition, a growing number of biomarkers provide prognostic information about a patient's malignancy. As we learn more about these biomarkers and their assays, selecting the appropriate companion diagnostic becomes increasingly important. In the case of many biomarkers, there are numerous assays which could provide the same information to a treating physician, however each assay has strengths and weaknesses. Institutions must balance cost, assay sensitivity, turn-around time, and labor resources when selecting which assay to offer. In this review we will discuss the current state of companion diagnostics available in metastatic colorectal cancer and explore emerging biomarkers and their assays. We will focus on KRAS, BRAF, HER2, and PIK3CA testing, as well as microsatellite stability assessment and multigene panels.

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“…The Ion Torrent PGM sequencer has been recently validated as a reliable tool for the quantitative assessment of somatic mutations in KRAS [13]. The addition of a PNA that clamps WT KRAS to this NGS workflow increases the sensitivity for detecting KRAS G12D mutations, but also changes the nature of the assay from quantitative to qualitative as the true allelic frequency of this specific mutation cannot be determined in presence of PNA clamping.…”

Section: Limitationsmentioning

confidence: 99%

Peptide nucleic acid clamping to improve the sensitivity of Ion Torrent-based detection of an oncogenic mutation in <em>KRAS</em>

Rakhit

Ottolini

Jones

et al. 2017

Matters

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Detecting oncogenic changes in the genome of cancer patients is crucial for targeted therapy. Such changes include alterations to KRAS, a GTPase located upstream of several signalling transduction pathways implicated in cancer formation. While nextgeneration sequencing (NGS) allows for comprehensive analysis of a genome, the technology can struggle to detect low frequency variants. To improve the sensitivity of NGS for detecting mutations we created a peptide nucleic acid (PNA) clamp, validated by qPCR, designed to bind wild-type KRAS (WT KRAS) across codon 12 during the PCR amplification stage of a NGS library preparation. We tested the effect of clamping the wild-type KRAS sequence in a reference standard with a KRAS c.35G>A mutation (KRAS G12D ) at an allelic frequency (AF) of 1.3% and on circulating-free DNA from a patient harbouring a KRAS G12D mutation (at an AF of 3.2%). Runs were conducted using 10, 5, 2.5 and 1 ng of DNA input. The PNA increased the number of mutant reads and their frequency relative to wild-type calls, allowing for more sensitive detection at all tested concentrations of DNA input.

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Validation of targeted next-generation sequencing forRASmutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR

Cited by 43 publications

References 22 publications

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Current companion diagnostics in advanced colorectal cancer; getting a bigger and better piece of the pie

Peptide nucleic acid clamping to improve the sensitivity of Ion Torrent-based detection of an oncogenic mutation in <em>KRAS</em>

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