Service de Cytologie et d'Anatomie Pathologique, Département de Biopathologie Moléculaire et de Département de Neuropathologie, Hospices Civils de Lyon, Groupement Hospitalier Est, Lyon, France.
University of Lyon, Université Claude Bernard Lyon 1, Lyon, France.
Mol Diagn Ther. 2019 Aug;23(4):537-545. doi: 10.1007/s40291-019-00403-3.
Genomic duplications and fusion involving BRAF and KIAA1549 that create fusion proteins with constitutive B-RAF kinase activity are a hallmark of pilocytic astrocytomas (PAs). The detection of KIAA1549-BRAF fusion transcripts is of paramount importance to classify these tumors and to identify patients who could benefit from BRAF inhibitors. In a clinical setting, the available material for molecular analysis from these pediatric tumors is often limited to formalin-fixed paraffin-embedded (FFPE) tissue. The aim of the present study was to develop a new method to detect the three most frequent KIAA1549-BRAF fusion transcripts, 15-9, 16-11, and 16-9, where numbers refer to the exons fused together, using a FFPE-compatible multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR).
We compared performance of the assay to a reference singleplex method on a collection of 46 FFPE PAs.
The results showed that both methods are comparable. The multiplex method had an overall 97% sensitivity and 100% specificity compared to the singleplex method, and agreement between the two techniques was almost perfect (Cohen's kappa: 0.97). There was no evidence of a significant difference between the qRT-PCR efficiencies of the multiplex technique and of the singleplex assay for all fusion transcripts and for GAPDH, the latter used as a reference gene. The multiplex method consumed four times less complementary DNA (cDNA), cost less, and required half the hands-on technical time.
The results show that it could be beneficial to implement the multiplex method in a clinical setting, where samples presenting low quantity of degraded RNA are not unusual.
涉及 BRAF 和 KIAA1549 的基因组重复和融合，可形成具有组成型 B-RAF 激酶活性的融合蛋白，是毛细胞星形细胞瘤（PA）的标志。检测 KIAA1549-BRAF 融合转录本对于对这些肿瘤进行分类以及确定可能受益于 BRAF 抑制剂的患者至关重要。在临床环境中，这些儿科肿瘤进行分子分析的可用材料通常仅限于福尔马林固定石蜡包埋（FFPE）组织。本研究的目的是开发一种新方法，使用 FFPE 兼容的多重定量逆转录聚合酶链反应（qRT-PCR）检测三种最常见的 KIAA1549-BRAF 融合转录本 15-9、16-11 和 16-9，其中数字指融合在一起的外显子。
我们将该检测方法与参考单重 qRT-PCR 方法在 46 例 FFPE-PA 中进行了比较。
结果表明两种方法具有可比性。与单重 qRT-PCR 方法相比，多重方法的总敏感性为 97%，特异性为 100%，两种技术之间的一致性几乎完美（Cohen's kappa：0.97）。对于所有融合转录本和用作参考基因的 GAPDH，多重技术的 qRT-PCR 效率与单重检测之间均无显著差异。多重方法消耗的 cDNA 减少了四倍，成本更低，实际操作技术时间减少了一半。
结果表明，在临床环境中实施多重方法可能是有益的，在这种环境中，常见的情况是样本中存在数量较少的降解 RNA。
