For targeted sequencing, hybridization-based and amplicon-based capture methods differ in their ability to uniformly capture and sequence targeted regions and identify mutations ( 11). A key issue for multigene sequencing is the choice of the design taking into account the relevant list of genes to be tested, the desire to detect in addition to mutations, gene copy number alterations, the heterogeneity of the tumour sample with a normal and tumour components and the type of tissue (DNA from frozen samples or formalin-fixed, paraffin-embedded samples or circulating tumour DNA ). Multigene sequencing is today used in precision medicine trials or screening programmes in comprehensive cancer centers worldwide aiming to orient patients towards appropriate early phase clinical trials. The prognostic value of multigene sequencing obviously depends on the molecular alterations that are being assessed. In summary, we cannot exclude that multigene sequencing has a prognostic value. The overall survival analysis of SHIVA suggests that patients with a druggable molecular alteration have indeed a better prognosis than those who have not (HR=0.85), although it did not reach statistical significance ( 10). Due to the lack of randomisation, nobody can tell whether the observed difference is due the predictive value of multigene sequencing or its prognostic value. Indeed, some non-randomised studies retrospectively comparing the outcome of patients treated with matched therapy based on the identification of a druggable molecular alteration with the outcome of patients who were not treated with matched therapy (one of the reasons being the lack of identification of a druggable molecular alteration) reported a statistically significant better outcome in the matched group ( 6, 7). The question of whether the molecular alterations detected when using multigene sequencing have a prognostic value is key. It is well established that some molecular alterations have a prognostic value, such as EGFR mutations in lung cancer. In summary, there is no level 1 evidence to date demonstrating the predictive value of multigene sequencing. However, the only randomised trial (SHIVA) did not confirm these results ( 9). Several non-randomised algorithm-testing trials have suggested that multigene sequencing might improve patient outcomes ( 5, 6, 7, 8). These trials are dichotomized into stratified trials that include basket trials (histologic stratification) and umbrella trials (molecular stratification), and algorithm-testing trials that mix molecular alterations, drugs and often tumour types ( 3). The predictive value of multigene sequencing is only assessed in trials using multigene sequencing to guide therapy. The key question behind the predictive value of multigene sequencing is whether the use of multigene sequencing improves patient outcomes (clinical utility). The predictive value of multigene sequencing cannot be interpreted the same way than for a single molecular alteration. WGS and WES also allow the evaluation of mutational and neoantigen loads that might be of interest to predict response to some immunotherapeutic agents, as well as genomic instability for DNA repair inhibitors ( 1, 2). WGS and WES require constitutional DNA in addition to tumour DNA for somatic variant calling. Different types of sequencing exist, including whole genome sequencing (WGS), whole exome sequencing (WES) and targeted sequencing. DNA gene sequencing allows seeking for mutations or gene copy number alterations, whilst RNA sequencing allows seeking for gene fusions, validate somatic mutations found in DNA as well as altered gene expression and splicing. From this breakthrough technology has emerged the term “precision medicine” in oncology, which is according to the National Cancer Institute “a form of medicine that uses information about a person’s genes, proteins, and environment to prevent, diagnose, and treat disease”. Multigene sequencing is the use of next-generation sequencing (NGS) with the ability to assess multiple gene molecular alterations in the same assay. Le Tourneau explains what we need in terms of multigene sequencing Definition
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