EU Instand-NGS4P: Pre-analytical multimodal workflows for NGS research and future precision cancer care

INTRODUCTION: The EU-funded Pre-Commercial Research project Instand-NGS4P (GA no. 874719) aims to improve cancer research by developing complete workflows for NGS, from specimen collection through data analysis and reporting. Multimodal testing involves analyzing multiple specimen types from one individual and/or multiple analytes (e.g., RNA, circulating cell-free DNA [ccfDNA], gDNA) from a single specimen. This can improve diagnostics by combining complementary results and increase sensitivity for earlier cancer detection. We developed 23 novel integrated NGS workflows for research purposes. Here, we present six exemplary workflows that may inform future innovation in clinical applications. METHODS: Different specimen types were obtained from lung cancer patients at the Medical University of Graz (Austria) under local ethics committee approval. Whole blood specimens were collected and stabilized in PAXgene ® Blood ccfDNA Tubes. Urine specimens were collected and stabilized using the PAXgene Urine Liquid Biopsy Set. Tumor tissue was preserved by PAXgene Tissue or formalin fixation followed by paraffin embedding (PFPE and FFPE) as well as cryopreservation. Stabilized specimens were shipped to QIAGEN, Hilden, Germany. Blood ccfDNA and gDNA were extracted using the QIAamp ® DSP Circulating Nucleic Acid Kit and QIAsymphony ® DSP DNA Mini Kit. Urine cell-free DNA (cfDNA) was isolated upon arrival at QIAGEN and after additional storage using the QIAsymphony DSP Circulating DNA Kit. Tissue gDNA was extracted using the PAXgene Tissue DNA Kit for PFPE samples, QIAamp DNA FFPE Advanced UNG Kit for FFPE samples and QIAamp DNA Mini Kit for cryopreserved samples. Mutation analysis of cfDNA was performed with QIAseq ® Targeted cfDNA Ultra Human Lung Cancer Panel, while gDNA was analyzed with QIAseq Targeted DNA Pro Human Lung Cancer Focus Panel. Sequencing runs were performed using the Illumina ® NextSeq ® 500/550 Mid Output Kit v2.5 (300 cycles). RESULTS: The developed workflows encompass specimen collection, stabilization, storage, analyte isolation, quality control (QC), library preparation, and sequencing. A novel QC concept enables traceability and process standardization through systematic metadata documentation. Specimens yielded high-quality libraries and successful sequencing performance across all analyte runs. Comparative mutational profiling of blood ccfDNA, urine cfDNA, and tissue DNA revealed both overlapping as well as complementary variants in key tumor genes. This gain in molecular information may lead to further development of multimodal liquid biopsy-based approaches with urine as a valuable complementing specimen type. CONCLUSIONS: Our NGS workflows support multi- and single-mode testing for cancer research. They are developed in alignment with recognized international regulatory principles to enable comprehensive validation from sample collection to assay output.