Wolf Thibaut, Reita Damien, Deschuyter Marlène, Salmanli Chinar, Buffa Julie, Pencreach Erwan, Guérin Eric, Jeandidier Eric, Miguet Marguerite, Chenard Marie-Pierre, Geyer Lucas, Noel Georges, Todeschi Julien, Gauchotte Guillaume, Martin Sophie, Entz-Werlé Natacha, Lhermitte Benoît
Pathology Department, University Hospitals of Strasbourg, 67098, Strasbourg, France.
Laboratory of Biochemistry and Molecular Biology, Department of Cancer Molecular Genetics, University Hospitals of Strasbourg, 67200, Strasbourg, France.
Acta Neuropathol Commun. 2025 Aug 11;13(1):170. doi: 10.1186/s40478-025-02089-7.
Detecting homozygous deletion (HD) of CDKN2A is critical in BRAF-altered gliomas, as this molecular alteration has both diagnostic and prognostic significance. It is predominantly associated with BRAF-altered high-grade gliomas and has been associated with poorer prognosis in certain BRAF-altered low-grade glioma tumor types. The 2021 WHO classification of central nervous system tumors therefore recommends screening for this alteration in most BRAF-altered gliomas, but it does not recommend one specific technique over another. Here, we compare the performance of several detection methods, including p16 immunohistochemistry, fluorescence in situ hybridization (FISH), droplet digital PCR, next-generation sequencing and DNA methylation profiling-derived copy-number variation (CNV) analysis, in a retrospective cohort of 25 BRAF-altered gliomas. Ten cases showed diffuse p16 immunohistochemical expression (10/25) with no associated CDKN2A HD, whereas 15 cases had complete absence of p16 expression (15/25). In the latter group, a high level of discrepancy in CDKN2A HD detection when considering FISH versus other techniques was observed, suggesting a high false-negative rate with FISH. Using an original bioinformatic pipeline leveraging genome alignment of routinely available CNV raw data, we identified among most false-negative cases (4/5) a large and undeleted region encompassing MTAP, which is targeted by most commercial CDKN2A FISH probes. This is likely due to non-specific probe hybridization. Our finding suggests that FISH probes targeting the entire 9p21 locus may have lower sensitivity than anticipated among BRAF-altered gliomas and emphasizes the critical need for appropriate probe selection.
检测CDKN2A的纯合缺失（HD）在BRAF基因改变的胶质瘤中至关重要，因为这种分子改变具有诊断和预后意义。它主要与BRAF基因改变的高级别胶质瘤相关，并且在某些BRAF基因改变的低级别胶质瘤肿瘤类型中与较差的预后相关。因此，2021年世界卫生组织中枢神经系统肿瘤分类建议在大多数BRAF基因改变的胶质瘤中筛查这种改变，但并未推荐一种特定技术优于另一种技术。在此，我们在一个包含25例BRAF基因改变的胶质瘤的回顾性队列中比较了几种检测方法的性能，包括p16免疫组织化学、荧光原位杂交（FISH）、液滴数字PCR、下一代测序以及基于DNA甲基化谱的拷贝数变异（CNV）分析。10例病例显示p16免疫组织化学弥漫性表达（10/25），无相关的CDKN2A HD，而15例病例完全没有p16表达（15/25）。在后一组中，当考虑FISH与其他技术时，观察到CDKN2A HD检测存在高度差异，提示FISH存在较高的假阴性率。使用一种利用常规可用的CNV原始数据进行基因组比对的原始生物信息学流程，我们在大多数假阴性病例（4/5）中鉴定出一个包含MTAP的大的未缺失区域，大多数商业CDKN2A FISH探针靶向该区域。这可能是由于非特异性探针杂交所致。我们的发现表明，针对整个9p21位点的FISH探针在BRAF基因改变的胶质瘤中的敏感性可能低于预期，并强调了适当选择探针的迫切需求。
