Clinical evidence demonstrates that treatment with immune checkpoint inhibitor immunotherapy agents can have considerable benefit across multiple tumours. However, there is a need for the development of predictive biomarkers that identify patients who are most likely to respond to immunotherapy. Comprehensive characterisation of tumours using genomic, transcriptomic, and proteomic approaches continues to lead the way in advancing precision medicine. Genetic correlates of response to therapy have been known for some time, but recent clinical evidence has strengthened the significance of high tumour mutational burden (TMB) as a biomarker of response and hence a rational target for immunotherapy. Concordantly, immune checkpoint inhibitors have changed clinical practice for lung cancer and melanoma, which are tumour types with some of the highest mutational burdens. TMB is an implementable approach for molecular biology and/or pathology laboratories that provides a quantitative measure of the total number of mutations in tumour tissue of patients and can be assessed by whole genome, whole exome, or large targeted gene panel sequencing of biopsied material. Currently, TMB assessment is not standardised across research and clinical studies. As a biomarker that affects treatment decisions, it is essential to unify TMB assessment approaches to allow for reliable, comparable results across studies. When implementing TMB measurement assays, it is important to consider factors that may impact the method workflow, the results of the assay, and the interpretation of the data. Such factors include biopsy sample type, sample quality and quantity, genome coverage, sequencing platform, bioinformatic pipeline, and the definitions of the final threshold that determines high TMB. This review outlines the factors for adoption of TMB measurement into clinical practice, providing an understanding of TMB assay considerations throughout the sample journey, and suggests principles to effectively implement TMB assays in a clinical setting to aid and optimise treatment decisions.

The clinical demand for mutation detection within multiple genes from a single tumour sample requires molecular diagnostic laboratories to develop rapid, high-throughput, highly sensitive, accurate and parallel testing within tight budget constraints. To meet this demand, many laboratories employ next-generation sequencing (NGS) based on small amplicons. Building on existing publications and general guidance for the clinical use of NGS and learnings from germline testing, the following guidelines establish consensus standards for somatic diagnostic testing, specifically for identifying and reporting mutations in solid tumours. These guidelines cover the testing strategy, implementation of testing within clinical service, sample requirements, data analysis and reporting of results. In conjunction with appropriate staff training and international standards for laboratory testing, these consensus standards for the use of NGS in molecular pathology of solid tumours will assist laboratories in implementing NGS in clinical services.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-016-2025-7) contains supplementary material, which is available to authorized users.

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

PURPOSE Liquid biopsy specimen genomic profiling is integrated in non–small-cell lung cancer (NSCLC) guidelines; however, data on the clinical relevance for ALK /ROS1 alterations are scarce. We evaluated the clinical utility of a targeted amplicon-based assay in a large prospective cohort of patients with ALK/ROS1-positive NSCLC and its impact on outcomes. PATIENTS AND METHODS Patients with advanced ALK/ROS1-positive NSCLC were prospectively enrolled in the study by researchers at eight French institutions. Plasma samples were analyzed using InVisionFirst-Lung and correlated with clinical outcomes. RESULTS Of the 128 patients included in the study, 101 were positive for ALK and 27 for ROS1 alterations. Blood samples (N = 405) were collected from 29 patients naïve for treatment with tyrosine kinase inhibitors (TKI) or from 375 patients under treatment, including 105 samples collected at disease progression (PD). Sensitivity was 67% (n = 18 of 27) for ALK/ROS1 fusion detection. Higher detection was observed for ALK fusions at TKI failure (n = 33 of 74; 46%) versus in patients with therapeutic response (n = 12 of 109; 11%). ALK-resistance mutations were detected in 22% patients (n = 16 of 74) overall; 43% of the total ALK-resistance mutations identified occurred after next-generation TKI therapy. ALK G1202R was the most common mutation detected (n = 7 of 16). Heterogeneity of resistance was observed. ROS1 G2032R resistance was detected in 30% (n = 3 of 10). The absence of circulating tumor DNA mutations at TKI failure was associated with prolonged median overall survival (105.7 months). Complex ALK-resistance mutations correlated with poor overall survival (median, 26.9 months v NR for single mutation; P = .003) and progression-free survival to subsequent therapy (median 1.7 v 6.3 months; P = .003). CONCLUSION Next-generation, targeted, amplicon-based sequencing for liquid biopsy specimen profiling provides clinically relevant detection of ALK/ROS1 fusions in TKI-naïve patients and allows for the identification of resistance mutations in patients treated with TKIs. Liquid biopsy specimens from patients treated with TKIs may affect clinical outcomes and capture heterogeneity of TKI resistance, supporting their role in selecting sequential therapy.

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The advent of molecular targets for novel therapeutics in oncology, notably for non–small cell lung carcinoma (NSCLC), led the French National Cancer Institute (INCa) to establish a national network of 28 hospital Molecular Genetics Centers for Cancer (MGCC) in 2007. In each University in France, laboratories were established to develop molecular biology testing to evaluate a few genomic alterations, initially a selection of genes, by using specific targeted polymerase chain reaction (PCR) assays. In a second phase, the number of studied genes was increased. In 2015, the MGCC benefited from an additional dedicated budget from the INCa to develop next‐generation sequencing (NGS) technology. In the meantime, a new financial regulation for innovative testing has been established for the acts out of nomenclature. Consequently, all private and public laboratories in France have access to funding for molecular biology testing in oncology. The gene‐based PCR assays or NGS tests have benefitted from reimbursement of cost testing by the INCa. Today, the laboratories consider this reimbursement to be only partial, and its use to be complex. In 2018, a strategic plan for medical genomic analyses (France Médecine Génomique 2025) was implemented to introduce more systematic sequencing into the health care pathway and oncology practice. The large panel of molecular tests should be centralized to a limited number of molecular genetic centers. This review describes the evolution of the different stages of implementation of molecular pathology testing for NSCLC patients over the last few years in France.

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Background: While studies have shown feasibility and reported preliminary evidence of utility, there is no evidence that multigene sequencing improves outcome in patients with metastatic cancer. The aim of the present study was to assess the clinical utility of multigene sequencing and DNA copy number analyses.. Methods: In SAFIR02-BREAST (NCT: 02299999) and SAFIR-PI3K (NCT: 03386162), open-label multicentric phase II randomized trials, patients were selected if they had a Her2-negative metastatic breast cancer eligible to 1st or 2nd line chemotherapy. Patients underwent a pre-treatment biopsy of metastatic disease when feasible, followed by genomic analysis by next generation sequencing and SNParray. After 6 to 8 cycles of induction chemotherapy, patients without progressive disease and presenting an actionable genomic alteration, were randomized between targeted therapies matched to genomic alterations or maintenance chemotherapy. The primary objective was to evaluate whether targeted therapies guided by genomics improves progression-free survival (PFS) as compared to maintenance chemotherapy, in a pooled analyses of SAFIR02-BREAST and SAFIR-PI3K populations. A hierarchical testing was applied. The efficacy of targeted therapies matched to genomic alterations was first tested in patients presenting an ESCAT I/II alteration (ESMO Scale of Actionability of Molecular Targets). If a p value <0.1 was observed in the first step, analyses were then performed in the Intent-to-treat population. Results: Out of the 1462 patients included, 238 (16%) were subsequently randomized between maintenance chemotherapy (n=81) and targeted therapy (n=157). In 115 patients presenting an ESCAT I/II genomic alteration, the median PFS was 9.1 months (90%CI: 7.1-9.8) and 2.8 (90%CI: 2.1-4.8) in matched targeted therapy and maintenance chemotherapy arms respectively (adjusted HR for stratification factors =0.41;90%CI: 0.27-0.61, p<0.001). In the overall population, there was no significant difference in the duration of PFS between the two arms (adjusted HR: 0.77 (95%CI: 0.56- 1.06, p=0.109). ESCAT classification was highly predictive for the benefit of targeted therapies matched to genomic alterations (interaction test, p= 0.004). Targeted therapies matched to genomic alterations were not effective in patients without ESCAT I/II alteration (HR: 1.15, 95%CI: 0.76-1.75). The SNP array analyses (n=926) identified 21 genes altered more frequently in metastases as compared to primary tumors (TCGA+ METABRIC). Of these, focal TERT amplifications were associated with a poor outcome. Focal CDK4 amplifications were observed after resistance to CDK4 inhibitors. Finally, high HRD was associated with longer PFS in patients with BRCA mutation treated with olaparib (HR: 0.32 [95%CI: 0.12;0.83], p=0.013).. Conclusion: SAFIR02/PI3K trials report that the clinical use of multigene sequencing must be driven by a framework of actionability, and identifies new genomic alterations associated with metastatic evolution and drug resistance or sensitivity. Citation Format: Fabrice André, Anthony Gonçalves, Thomas Filleron, Florence Dalenc, Amélie Lusque, Mario Campone, Marie-Paule Sablin, Hervé Bonnefoi, Ivan Bieche, Ludovic Lacroix, Alicia Tran-Dien, Marta Jimenez, Alexandra Jacquet, Qing Wang, Etienne Rouleau, David Gentien, Isabelle Soubeyran, Alain Morel, Monica Arnedos, Thomas Bachelot. Clinical utility of molecular tumor profiling: Results from the randomized trial SAFIR02-BREAST [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-10.