Abstract 743: An amplicon sequencing based all-in-one genetic testing panel for molecular classification and guiding individualized treatment of brain cancer
Authors: Shi Min , Geng Sumin , Niu Yanling , Feng Jiao , Shi Xiao , Li Le , Zhu Huiming , Chen Min , Zhang Yukun , Teng Lin , Zheng Qiaosong , Ma Tonghui 
Published: September 2020

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.