Molecular Diagnostic of Solid Tumor Using a Next Generation Sequencing Custom-Designed Multi-Gene Panel

Abstract

Next generation sequencing (NGS) allows parallel sequencing of multiple genes at a very high depth of coverage. The need to analyze a variety of targets for diagnostic/prognostic/predictive purposes requires multi-gene characterization. Multi-gene panels are becoming standard approaches for the molecular analysis of solid lesions. We report a custom-designed 128 multi-gene panel engineered to cover the relevant targets in 22 oncogene/oncosuppressor genes for the analysis of the solid tumors most frequently subjected to routine genotyping. A total of 1695 solid tumors were analyzed for panel validation. The analytical sensitivity is 5%. Analytical validation: (i) Accuracy: sequencing results obtained using the multi-gene panel are concordant using two different NGS platforms and single-gene approach sequencing (100% of 83 cases); (ii) Precision: consistent results are obtained in the samples analyzed twice with the same platform (100% of 20 cases). Clinical validation: the frequency of mutations identified in different tumor types is consistent with the published literature. This custom-designed multi-gene panel allows to analyze with high sensitivity and throughput 22 oncogenes/oncosuppressor genes involved in diagnostic/prognostic/predictive characterization of central nervous system tumors, non-small-cell lung carcinomas, colorectal carcinomas, thyroid nodules, pancreatic lesions, melanoma, oral squamous carcinomas and gastrointestinal stromal tumors.

miR-196B-5P and miR-200B-3P Are Differentially Expressed in Medulloblastomas of Adults and Children

“…Exon 3 of CTNNB1 of all cases, and exons 4, 5, 6, 7, 8, and 9 of TP53 of SHH subgroup samples, were sequenced using a TruSeq Custom Amplicon panel run on a MiSeq Illumina instrument (Illumina Inc., San Diego, CA, USA), according to established protocols [45].…”

Diagnostic and prognostic value of a 7-panel mutation testing in thyroid nodules with indeterminate cytology: the SWEETMAC study

“…This may have underestimated the number of positive cases although these mutations are uncommon [35]. Above all, a limitation was not having access to NGS multi-gene panel such as ThyroSeq v3 [36] or other recently reported technologies [37][38][39]. The use of multi-gene NGS panels makes it possible to analyse more than one hundred genes and to detect different classes of genetic alterations, including mutations, insertions and deletions, gene fusions, gene expression alterations and copy number variations to improve diagnostic accuracy and potentially prognostic value of genetic testing on cytologically indeterminate nodules.…”

ARID1A and CTNNB1/β-Catenin Molecular Status Affects the Clinicopathologic Features and Prognosis of Endometrial Carcinoma: Implications for an Improved Surrogate Molecular Classification

“…DNA was extracted using the Quick Extract Kit (Epicentre, Madison, WI, USA) and quantified using the “Qubit” fluorometer (ThermoFisher Scientific, Waltham, MA, USA). Samples were analyzed using a customized panel of genomic regions and sequenced using the Gene Studio S5 sequencer (ThermoFisher Scientific), according to the manufacturer’s instruction (ThermoFisher Scientific) as previously published [ 35 ]. Template preparation was performed using the Chef Machine instrument (ThermoFisher Scientific) and then sequenced using an Ion 530 chip.…”