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Background: Incorporation of molecular biomarkers and histological features could be applicated for more-precise tumour categorization and individualized treatment. IDH1/2 mutations, 1p/19q co-deletion and TERT-promoter mutations were requisite diagnostic biomarkers for gliomas classification. BRAF V600E mutation was common in a wide spectrum of brain tumors, including gliomas and glioneuronal tumors. Currently, FISH is the gold standard method for detecting 1p/19q co-deletion and qPCR was often used to detect hotspot mutations. However, all five genetic biomarkers couldn't be evaluated with same system. Here, we developed an all-in-one panel for brain cancer based on amplicon sequencing, which could detect all five biomarkers simultaneously. Methods: 155 specimens were collected from glioma patients in China. To evaluate the accuracy of five biomarkers panel (Genetron health), every specimen was analyzed by both five biomarkers panel and the combination method, which included qPCR analysis of hotspot mutations (IDH1 R132H/C, IDH2 R172K, TERT promoter C228T/C250T and BRAF V600E) and FISH analysis of 1p/19q co-deletion. For five biomarkers panel, DNA fragments containing hotspot mutations in four genes and 59 single-nucleotide polymorphisms (SNPs) in chromosome 1p/19q were amplified and the library was constructed. Sequencing was performed on Ion GeneStudio S5 system. The reads mapping, mutations calling and 1p/19q deletion calling were handled by in-house bioinformatic workflow. Results: Comparing performance of hotspot mutations detection, we found that five biomarkers panel was very consistent with qPCR but only in one specimen, in which BRAF V600E was detected by five biomarkers panel but not qPCR. For 1p/19q co-deletion analysis, 96% (149/155) of specimens were consistent. Only 1p deletion was identified by FISH in 2 specimens, in which 1p/19q co-deletion were identified by five biomarkers panel. In 2 specimens, 1p deletion or 19q deletion was detected by five biomarkers panel, but no deletion was detected by FISH. In the other 2 specimens, 1p deletion or 19q deletion could be detected by FISH, but not by 5 biomarkers panel. Furthermore, for multi-system combination analysis, each specimen was needed for DNA extraction and FISH slide preparation. Comparing to 10 ng DNA, which was enough for analysis by five biomarkers panel for brain cancer, much more FFPE orfresh frozen tumor tissues were required for multi-system combination method analysis. Conclusions: Glioma 5 biomarker panel, by which hotspot mutations of IDH1/IDH2, TERT promoter, BRAF and 1p/19q co-deletion can be detected with same high-throughput sequencing platform and analysis pipeline, required less biopsy and was highly consistent with current technologies such as qPCR and FISH. Citation Format: Min Shi, Sumin Geng, Yanling Niu, Jiao Feng, Xiao Shi, Le Li, Huiming Zhu, Min Chen, Yukun Zhang, Lin Teng, Qiaosong Zheng, Tonghui Ma. An amplicon sequencing based all-in-one genetic testing panel for molecular classification and guiding individualized treatment of brain cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 743.

Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12 and plays pivotal roles in transcriptional regulation through its histone H3K27 methyltransferase activity. Dysregulation of PRC2 is observed in multiple human cancers, for example, the catalytic subunit EZH2 is overexpressed in a wide range of human cancers and gain-of-function mutations of EZH2 within its catalytic site have been reported in human B-cell lymphoma, parathyroid carcinoma and melanoma. Small molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported and showed anti-lymphoma efficacy in pre-clinical studies. EED within the PRC2 complex allosterically activate the enzymatic activity by binding to tri-methylated H3K27 (H3K27me3). Here we report the discovery of EED226, a potent and selective PRC2 inhibitor directly binding to the H3K27me3 binding pocket of EED. EED226 induces conformational change upon binding EED leading to loss of PRC2 activity. EED226 shows similar activity as SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show EED226 inhibits PRC2 activity via an allosteric mechanism and offers opportunity for treatment of PRC2-dependent cancers. Citation Format: Wei Qi, Kehao Zhao, Justin Gu, Ying Huang, Youzhen Wang, Hailong Zhang, Man Zhang, Jeff Zhang, Zhengtian Yu, Ling Li, Lin Teng, Shannon Chuai, Chao Zhang, Mengxi Zhao, HoMan Chan, Zijun Chen, Douglas Fang, Fei Qi, Leying Feng, Lijian Feng, Yuan Gao, Hui Ge, Xinjian Ge, Andreas Lingel, Guobin Li, Ying Lin, Yueqin Liu, Fangjun Luo, Minlong Shi, Long Wang, Zhaofu Wang, Yanyan Yu, Jue Zeng, Chenhui Zeng, Lijun Zhang, Qiong Zhang, Shaolian Zhou, Counde Oyang, Peter Atadja, En Li. An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-288. doi:10.1158/1538-7445.AM2017-LB-288