Dataset Information

Detection of the G17V RHOA mutation in angioimmunoblastic T-cell lymphoma and related lymphomas using quantitative allele-specific PCR.

ABSTRACT: Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) are subtypes of T-cell lymphoma. Due to low tumor cell content and substantial reactive cell infiltration, these lymphomas are sometimes mistaken for other types of lymphomas or even non-neoplastic diseases. In addition, a significant proportion of PTCL-NOS cases reportedly exhibit features of AITL (AITL-like PTCL-NOS). Thus disagreement is common in distinguishing between AITL and PTCL-NOS. Using whole-exome and subsequent targeted sequencing, we recently identified G17V RHOA mutations in 60-70% of AITL and AITL-like PTCL-NOS cases but not in other hematologic cancers, including other T-cell malignancies. Here, we establish a sensitive detection method for the G17V RHOA mutation using a quantitative allele-specific polymerase chain reaction (qAS-PCR) assay. Mutated allele frequencies deduced from this approach were highly correlated with those determined by deep sequencing. This method could serve as a novel diagnostic tool for 60-70% of AITL and AITL-like PTCL-NOS.

SUBMITTER: Nakamoto-Matsubara R

PROVIDER: S-EPMC4195681 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Detection of the G17V RHOA mutation in angioimmunoblastic T-cell lymphoma and related lymphomas using quantitative allele-specific PCR.

Nakamoto-Matsubara Rie R Sakata-Yanagimoto Mamiko M Enami Terukazu T Yoshida Kenichi K Yanagimoto Shintaro S Shiozawa Yusuke Y Nanmoku Tohru T Satomi Kaishi K Muto Hideharu H Obara Naoshi N Kato Takayasu T Kurita Naoki N Yokoyama Yasuhisa Y Izutsu Koji K Ota Yasunori Y Sanada Masashi M Shimizu Seiichi S Komeno Takuya T Sato Yuji Y Ito Takayoshi T Kitabayashi Issay I Takeuchi Kengo K Nakamura Naoya N Ogawa Seishi S Chiba Shigeru S

PloS one 20141013 10

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) are subtypes of T-cell lymphoma. Due to low tumor cell content and substantial reactive cell infiltration, these lymphomas are sometimes mistaken for other types of lymphomas or even non-neoplastic diseases. In addition, a significant proportion of PTCL-NOS cases reportedly exhibit features of AITL (AITL-like PTCL-NOS). Thus disagreement is common in distinguishing between AITL and PTCL-N ...[more]

PMID: 25310466

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Project description:BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

Dataset Information

Detection of the G17V RHOA mutation in angioimmunoblastic T-cell lymphoma and related lymphomas using quantitative allele-specific PCR.

Publications

Detection of the G17V RHOA mutation in angioimmunoblastic T-cell lymphoma and related lymphomas using quantitative allele-specific PCR.

Similar Datasets

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