1237P - Design and validation of a custom next-generation sequencing panel in melanoma, glioma and gastrointestinal stromal tumor

Background

Incidence of melanoma, glioma and gastrointestinal stromal tumor (GIST) is increasing worldwide, which results in public health problems. The increasing number of markers that are tested in a correct diagnostic procedure makes gene-targeted, next-generation sequencing (NGS) a powerful tool in routine pathology practice. However, special panels for melanoma, glioma and GIST (MGG) are scarce. In this study, we designed and validated a NGS panel which included 1064 amplicons in 57 genes covering targetable alterations associated with the diagnosis and potential treatment response of MGG.

Methods

Based on extensive literature review and integrating TCGA and COSMIC databases, we developed MGG panel that is associated with diagnosis and potential treatment response of MGG. We validated this NGS panel including accuracy, reproducibility/repeatability, limit of detection (Lod), a comparative analysis between conventional methodologies and NGS using reference mutated DNA, tumor and normal tissue samples that were previously characterized.

Results

The NGS panel achieved 100% accuracy for mutation detection. Intra-experiment repeatability and inter-experiment reproducibility showed 100% concordance for one reference mutated DNA and four tumor samples, and a high correlation was observed between inter- and intra-sequencing runs. The Lod was established at a 2% variant allele frequency (VAF) for single-nucleotide variants (SNVs) and insertion-deletions (indels), and the threshold for copy number amplification and deletion was 3 and 1.4, respectively. Subsequently, we performed a comparative analysis between conventional methodologies - such as Sanger sequencing or fluorescence in situ hybridization - and NGS on 119 tumor and normal tissue samples that had been previously tested in our laboratory. The comparison between conventional methodologies and NGS showed high concordance (116/119, 97.5%).

Conclusions

This NGS panel is an accurate and sensitive method. Moreover, it is a rapid and cost-effective approach that can simultaneously detect different genetic alterations useful for the diagnosis, prognosis and treatment of patients with melanoma, glioma or GIST.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Fudan university Shanghai Cancer Center.

Funding

Has not received any funding.

Disclosure

D. Zhao, Y. Jiao, X. Shi: Financial Interests, Personal, Full or part-time Employment: Zhengu Biotech Ltd. All other authors have declared no conflicts of interests.