Department of Advanced Diagnostics, Quest Diagnostics, San Juan Capistrano, CA, United States of America.
Department of Molecular Oncology, Med Fusion, Lewisville, TX, United States of America.
PLoS One. 2021 Apr 28;16(4):e0243683. doi: 10.1371/journal.pone.0243683. eCollection 2021.
Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9-99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes.
通过分子检测鉴定基因组突变在髓系肿瘤的诊断、预后和治疗中起着重要作用。下一代测序（NGS）是一种高效的方法，可同时以高灵敏度检测具有临床意义的基因组突变。各种基于 NGS 的内部开发和商业髓系肿瘤面板已整合到常规临床实践中。然而，髓系恶性肿瘤中经常发生突变的一些基因特别难以用 NGS 面板进行测序（例如，CEBPA、CARL 和 FLT3）。我们报告了一种 48 基因 NGS 面板的开发和验证，该面板包括在髓系肿瘤（包括急性髓系白血病（AML）、骨髓增生异常综合征（MDS）和骨髓增生性肿瘤（MPN）的分子分析中具有挑战性的基因。通过与互补生物素化 DNA 诱饵杂交捕获靶区，然后在 Illumina NextSeq500 仪器上进行 NGS。使用内部开发的生物信息学管道检测单核苷酸变异（SNVs）、插入/缺失（indels）和 FLT3 内部串联重复（FLT3-ITD）。对 184 个独特样本进行了等位频率≥5%的变体的分析验证研究。使用另外 137 个独特样本，将 48 基因面板鉴定的变体与 35 基因血液肿瘤面板鉴定的变体进行了比较。该检测方法应用于一个大型疑似髓系肿瘤患者队列（n=2053）。分析验证的灵敏度为 99.6%（95%CI：98.9-99.9%），特异性为 100%（95%CI：100%）。两种测试面板检测到的变体的一致性为 100%。在疑似髓系肿瘤患者（n=2053）中，54.5%的患者至少携带一种具有临床意义的突变：AML 患者中为 77%，MDS 患者中为 48%，MPN 患者中为 45%。总之，这些发现表明，该检测方法即使在技术上具有挑战性的基因中，也能识别与髓系肿瘤的诊断、预后和治疗选择相关的突变。
