15 research outputs found
Detection of the G17V RHOA Mutation in Angioimmunoblastic T-Cell Lymphoma and Related Lymphomas Using Quantitative Allele-Specific PCR
<div><p>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 <i>RHOA</i> 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 <i>RHOA</i> 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.</p></div
Analysis of genomic DNA samples.
<p>*<sup>1</sup>amp, amplified; *<sup>2</sup>not-amp, not-amplified.</p><p>Analysis of genomic DNA samples.</p
Correlation between qAS-PCR and MiSeq.
<p>*<sup>1</sup>N, number; *<sup>2</sup>RCC, rank correlation coefficient; *<sup>3</sup>PPV, positive predictive value; *<sup>4</sup>NPV, negative predictive value, *<sup>5</sup>WGA, whole-genome amplification. *<sup>6</sup>FFPE, formalin-fixed/paraffin-embedded.</p><p>Correlation between qAS-PCR and MiSeq.</p
Design of primers used in the study.
<p>A WT allele-specific primer forward primer (Upper), a mutant allele-specific forward primer (Lower), and a common primer were designed. The 3′ end of the forward mutant primer was specific to the mutant site (G to T) and an internal mismatch at the second nucleotide from 3′ end (G to A) was introduced to improve specificity.</p
Primer sequences for making PCR amplicons of FFPE samples.
<p>Primer sequences for making PCR amplicons of FFPE samples.</p
Sequence of allele-specific primers used for this study.
<p>*<sup>1</sup> WT, wild-type; *<sup>2</sup> MUT, mutant.</p><p>Sequence of allele-specific primers used for this study.</p
qAS-PCR for 275 tumor and control samples.
<p>qAS-PCR was performed for tumor samples, including 43 AITL (a), 52 PTCL-NOS (b), 5 T-cell lymphoma other than AITL and PTCL-NOS (c), 19 B-cell lymphomas (d), 129 myeloid malignancies (e) and 27 control samples (f).</p