Department of Neurosurgery, Division of Clinical Neuroscience, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
PLoS One. 2019 Jul 22;14(7):e0220146. doi: 10.1371/journal.pone.0220146. eCollection 2019.
Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVPs in PA contained tumor-derived cells and/or phenotypically distinct tumor cells expressing vascular markers. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was identified by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP contained tumor derived cell and/or phenotypically distinct tumor cells expressing vascular markers.
微血管增生（MVP）是一种异常的血管结构，包含多层有丝分裂活性的内皮和平滑肌细胞/周细胞，是胶质母细胞瘤多形性（GBM）的组织病理学标志。虽然 MVP 往往与高级别胶质瘤有关，但它也在 WHO 分级 I 毛细胞星形细胞瘤（PA）中被检测到。然而，关于其形成的机制知之甚少。使用 TP53 点突变作为肿瘤源性细胞的标志物，我们之前报道 MVP 部分通过间充质转化从肿瘤细胞中转化而来。在本研究中，我们使用 KIAA1549-BRAF 融合基因作为标志物，以评估 PA 中的 MVP 是否包含肿瘤源性细胞和/或表达血管标志物的表型不同的肿瘤细胞。对我院手术的 6 名 PA 患者的冷冻组织进行 cDNA 合成，通过逆转录聚合酶链反应（RT-PCR）检测 KIAA1549-BRAF 融合基因。通过长而准确的 PCR（LA-PCR）和基因组 DNA 的 Sanger 测序确定融合基因的断点。通过激光微切割获得 MVP 中的肿瘤细胞和细胞成分。进行基因组和数字 PCR 检测以定性和定量检测 KIAA1549-BRAF 融合基因。荧光原位杂交（FISH）用于评估 MVP 细胞成分中的基因融合。3 名 PA 患者的样本携带 KIAA1549 外显子 15、BRAF 外显子 9 融合基因。在两名 MVP 丰富的患者样本中，RT-PCR 检测到来自 KIAA1549-BRAF 融合基因的强条带在肿瘤细胞和 MVP 的细胞成分中均有出现。数字 PCR 显示，与肿瘤组织相比，其在 MVP 细胞成分中的相对表达在一个样本中为 42%，在另一个样本中为 76%。FISH 显示 MVP 中的肿瘤细胞和细胞成分均显示扩增信号，提示串联重复。我们的发现表明，在 PA 患者中，MVP 的一些细胞成分包含肿瘤源性细胞和/或表达血管标志物的表型不同的肿瘤细胞。
