Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors.
The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.
Department of Clinical Biology, Jessa Hospital, Hasselt, Belgium.
Guy
G
Froyen
Department of Clinical Biology, Jessa Hospital, Hasselt, Belgium.
An
A
Broekmans
Department of Clinical Biology, Jessa Hospital, Hasselt, Belgium.
Femke
F
Hillen
Department of Pneumology, Jessa Hospital, Hasselt, Belgium.
Karin
K
Pat
Department of Pathology, Jessa Hospital, Hasselt, Belgium.
Ruth
R
Achten
Department of Medical Oncology, Jessa Hospital, Hasselt, Belgium.
Jeroen
J
Mebis
Department of Clinical Biology, Jessa Hospital, Hasselt, Belgium.
Jean-Luc
JL
Rummens
Department of Clinical Biology, Jessa Hospital, Hasselt, Belgium.|Department of Clinical Biology, AZ Turnhout, Turnhout, Belgium.
Johan
J
Willemse
Department of Clinical Biology, Jessa Hospital, Hasselt, Belgium.
Brigitte
B
Maes
D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057230:Limit of Detection; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity