Watanabe Jun, Natsumeda Manabu, Okada Masayasu, Kobayashi Daiki, Kanemaru Yu, Tsukamoto Yoshihiro, Oishi Makoto, Kakita Akiyoshi, Fujii Yukihiko
Niigata University, Niigata, Japan.
JCO Precis Oncol. 2019 Dec;3:1-13. doi: 10.1200/PO.18.00308.
Biopsy is the gold standard for the diagnosis of primary CNS lymphoma (PCNSL). However, surgical biopsy has problems of morbidity related to hemorrhagic complications and false-negative findings, so safer and more reliable diagnostic methods are required. The aim of this study is to detect the mutation, an important driver mutation, in the cerebrospinal fluid (CSF) of patients with CNS lymphoma.
Twenty-six patients with CNS lymphoma (20 primary CNS lymphoma and six CNS relapse from systemic lymphoma) were studied. We extracted cell-free DNA (cfDNA) from CSF by lumbar puncture. cfDNA was extracted from 1 mL of CSF, and Sanger sequencing and droplet digital polymerase chain reaction (ddPCR) were performed. Furthermore, we performed DNA sequencing of in 21 cases with available surgically obtained formalin-fixed paraffin-embedded (FFPE) tissue and compared the results.
The median cfDNA amount extracted from 1 mL CSF was 219 ng/mL (25th to 75th percentile, 129 to 333 ng/mL). mutations were detected from CSF in 76.9% (20 of 26 cases), and L265P in exon 5 was the most frequent mutation in 19 out of 20 (95.0%) cases. S219C in exon 3 was detected in one case. In four patients, mutation was confirmed by ddPCR but not by Sanger sequencing. In all 21 cases with sufficient FFPE tissue for DNA analysis, the detection of mutation from cfDNA was consistent with those of tumor-derived DNA from FFPE tissue.
This pilot study provided evidence that the somatic driver mutation can be reliably detected by combination of Sanger sequencing and ddPCR in the cfDNA taken from 1 mL of CSF in patients with CNS lymphomas.
活检是原发性中枢神经系统淋巴瘤（PCNSL）诊断的金标准。然而，手术活检存在与出血并发症相关的发病率问题以及假阴性结果，因此需要更安全、更可靠的诊断方法。本研究的目的是检测中枢神经系统淋巴瘤患者脑脊液（CSF）中的 突变，这是一种重要的驱动突变。
对26例中枢神经系统淋巴瘤患者（20例原发性中枢神经系统淋巴瘤和6例系统性淋巴瘤中枢神经系统复发）进行了研究。我们通过腰椎穿刺从脑脊液中提取游离DNA（cfDNA）。从1 mL脑脊液中提取cfDNA，并进行桑格测序和液滴数字聚合酶链反应（ddPCR）。此外，我们对21例有可用手术获取的福尔马林固定石蜡包埋（FFPE）组织的病例进行了 的DNA测序，并比较了结果。
从1 mL脑脊液中提取的cfDNA中位数为219 ng/mL（第25至75百分位数，129至333 ng/mL）。76.9%（26例中的20例）的脑脊液中检测到 突变，外显子5中的L265P是20例中的19例（95.0%）最常见的突变。外显子3中的S219C在1例中被检测到。在4例患者中，通过ddPCR证实了 突变，但桑格测序未证实。在所有21例有足够FFPE组织进行DNA分析的病例中，从cfDNA检测到的 突变与来自FFPE组织的肿瘤衍生DNA的检测结果一致。
这项初步研究提供了证据，表明通过桑格测序和ddPCR相结合，可以在中枢神经系统淋巴瘤患者1 mL脑脊液中获取的cfDNA中可靠地检测到体细胞驱动突变 。
