BACKGROUND:The implementation of pharmacogenetics in clinical practice increasingly relies on multigene panels. OBJECTIVES:The objective of this study is to develop harmonized recommendations for the design and analytical implementation of multigene pharmacogenetic panels, defining clinically relevant genes and associated regions of interest (ROIs) based on evidence strength, therapeutic applicability, and compatibility with genotyping or sequencing technologies. METHODS:The French-Speaking Network of Pharmacogenetics (RNPGx) evaluated 81 candidate genes across five therapeutic domains (i.e., oncology and supportive care, anesthesia and pain management, cardiology, neurology and psychiatry and immunology and infectious diseases) using a structured, evidence-based scoring system. Each gene was evaluated using a 25-point scoring system integrating pharmacogenetic importance, regulatory and professional society recommendations, and expert consensus. For the genes ultimately selected for the core panel, clinically relevant regions of interest were defined and assigned to one of three analytical classes. Class 1 includes variants with established clinical actionability; Class 2 adds optional variants suitable for extended testing in specialized settings; and Class 3 covers broader genomic regions mainly intended for rare variant or structural analyses. RESULTS:A 28-gene core panel was retained. Class 1 included 76 prioritized variants (including CYP2D6 CNV variants), and Class 2 comprised 62 additional variants (with extended analysis for CYP2D6). Class 3 eligibility was retained for 18 genes. CONCLUSION:The RNPGx recommendations offer a harmonized and flexible framework for pharmacogenetic panel design and for the extraction and interpretation of pharmacogenetic data from whole-exome or whole-genome sequencing.

TP53 is included in most cancer predisposition multigene panels, especially those exploring Hereditary Breast and Ovarian Cancer (HBOC) predisposition. The purpose of this study was to define the contribution of TP53 pathogenic variants (PV) to the HBOC phenotype by collecting genotypes and phenotypes of 398 patients harboring a TP53 variant identified by 53,085 HBOC panel sequencing in 15 French laboratories. Heterozygous TP53 variants were identified in 0.44% of HBOC panels, evenly distributed between PV and VUS. Breast cancers associated with TP53 were predominantly triple positive, particularly Her2+ breast cancer, in situ cancer, or phyllodes tumors ( p < 0.0001 for both). Interestingly, TP53 PV were identified across all ages in breast cancer patients, with enrichment before 36y. We demonstrated that null variants were linked with the HBOC phenotype, and missense variants, especially with a dominant negative effect, with the LFS phenotype ( p = 0.0096). Patients with breast cancer harboring null variants displayed an earlier age of onset compared to missense ( p = 0.0030). Surprisingly, we identified, in late‐onset cancer patients, TP53 hotspot PV usually identified in classic LFS, which underlines variable penetrance. Thus, this study suggests the existence of two phenotypic entities associated with TP53 PV: clinical LFS and TP53 ‐related breast cancer. The type of TP53 variant, as well as modifying factors reflected in family history, may influence these phenotypes, and both should be considered to define the clinical follow‐up of patients and relatives.

The enzyme dihydropyrimidine dehydrogenase (DPD) is the primary catabolic pathway of fluoropyrimidines including 5 fluorouracil (5FU) and capecitabine. Cases of lethal toxicity have been reported in cancer patients with complete DPD deficiency receiving standard dose of 5FU or capecitabine. DPD is encoded by the pharmacogene DPYD in which more than 200 variants have been identified. Different approaches have been developed for screening DPD-deficiency, including DPYD genotyping and phenotyping. Plasma uracil ([U]) and dihydrouracil ([UH2]) concentrations are routinely used as surrogate markers for systemic DPD activity: [U] ≥ 16 ng/ml and < 150 ng/ml, and [U] ≥ 150 ng/mL indicate partial and complete DPD deficient phenotype, respectively, while values of 5 or 10 for [UH2]/([U] ratio are often cited. Four clinically relevant DPYD defective variants (DPYD*13, DPYD*2A, p.Asp949Val and haplotype B3), are targeted in genetic testing via PCR. In practice, pretreatment [U], alone or combined with these 4 recommended DPYD alleles guides individual dosage selection, though this approach has limitations. This is illustrated by two cases showing discrepancy between DPD deficient phenotype and normal standard genotype. In these two cases, DPYD exome sequencing with Next Generation Sequencing identified rare inactive variants, establishing concordance between phenotype and genotype. In patient 1, [U] levels of 21.1 and 25.5 ng/mL, indicated partial deficiency though the targeted genotype was normal and 5FU dose was adjusted based on the phenotype. In patient 2, [U] levels of 16.2 and 15.2 ng/mL were near the 16 ng/ml threshold. With a normal genotype, he as considered non-deficient as targeted genotype was normal and the standard dose was administered. These two cases underscore the need to pair DPD phenotyping with whole DPYD gene sequencing, due to the frequent discrepancies between these pharmacogenetic tools, the burden of rare variants and ethnic differences in variant frequencies.

Mavacamten, the first drug in the class of β-cardiac myosin modulator, is used for the treatment of patients with hypertrophic cardiomyopathy. This orally administered drug demonstrates wide interpatient variability in pharmacokinetics parameters, due in part to variant CYP2C19 alleles. Individuals who are CYP2C19 poor metabolizers have increased exposure and are at increased risk of reduced cardiac hypercontractility. To ensure the safety of all patients, European Medicines Agency recommends CYP2C19 preemptive genotyping, and consecutively, to adapt maintenance and initial mavacamten doses, and to manage drug-drug interactions, according to CYP2C19 phenotype. In this article, we summarize evidence from the literature supporting the association between CYP2C19 phenotype and pharmacological features of mavacamten and provide, beyond biologic guidelines, therapeutic recommendations for the use of mavacamten based on CYP2C19 and CYP3A4/CYP3A5 genotype.

The role of the focal adhesion protein kindlin-3 as a tumor suppressor and its interaction mechanisms with extracellular matrix constitute a major field of investigation to better decipher tumor progression. Besides the well-described role of kindlin-3 in integrin activation, evidence regarding modulatory functions between melanoma cells and tumor microenvironment are lacking and data are needed to understand mechanisms driven by kindlin-3 inactivation. Here, we show that kindlin-3 inactivation through knockdown or somatic mutations increases BRAFV600mut melanoma cells oncogenic properties via collagen-related signaling by decreasing cell adhesion and enhancing proliferation and migration in vitro, and by promoting tumor growth in mice. Mechanistic analysis reveals that kindlin-3 interacts with the collagen-activated tyrosine kinase receptor DDR1 (Discoidin domain receptor 1) modulating its expression and its interaction with β1-integrin. Kindlin-3 knockdown or mutational inactivation disrupt DDR1/β1-integrin complex in vitro and in vivo and its loss improves the anti-proliferative effect of DDR1 inhibition. In agreement, kindlin-3 downregulation is associated with DDR1 over-expression in situ and linked to worse melanoma prognosis. Our study reveals a unique mechanism of action of kindlin-3 in the regulation of tumorigenesis mediated by the collagen-activated tyrosine kinase receptor DDR1 thus paving the way for innovative therapeutic targeting approaches in melanoma.

Figure S1. (A) RNA expression and copy number variations in A375 melanoma cell lines resistant to vemurafenib (A375R). RNA expression was normalized according to PPIA, B2M and ACTB gene expression (siRNA: small interfering RNA). (B) Inhibition of CHEK2 at both the mRNA and protein levels in A375 melanoma cell lines resistant to vemurafenib (A375R) transfected with siRNA CHEK2 (50 nM and 100 nM) compared to those transfected with siRNA control (siRNA: small interfering RNA).

Figure S2. (A) Change in the tumor burden from baseline over time according to RECIST for all the included patients. The tumor burden was measured as the sum of the longest diameters of target lesions. Each line represents a patient. (B) Kaplan-Meier plot of the overall survival for all the included patients (N =18). (C) Kaplan-Meier plot of the progression-free survival for all the included patients (N =18).

Supplementary Materials and Methods

Figure S3. DNA alterations (mutations and copy number alterations) uncovered in all samples collected before and during treatment (Baseline, after cycle 2 and end of treatment). * indicates samples processed only in mRNA expression and copy number analysis (no NGS data available).

Supplementary Tables S1-S4. Table S1. Description of primers used in this study. Table S2. Variants selected in BRCA2 exon 12 and its flanking intronic regions. Table S3. Overview of bioinformatics predictions and experimental data obtained for the 40 selected BRCA2 exon 12 variants. Table S4. Clinical and family data of patients carrying BRCA2 exon 12 spliceogenic variants.

Fig. S1 to S10. Figure S1. Variant selection from human variation databases; Figure S2. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of presumed LoF variants; Figure S3. Bioinformatics predictions of 3'/5' ss alterations for variants located at position IVS{plus minus}1/2 of BRCA2 exon 12; Figure S4. Bioinformatics analysis of variants predicted to alter 3'/5' ss of BRCA2 exon 12 (â^†MES {less than or equal to} -15%); Figure S5. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of variants predicted to alter 3'/5' ss (A) or ESR (B); Figure S6. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in control and patient lymphoblastoid cell lines; Figure S7. RT-PCR analysis of BRCA2 exon 12 splicing patterns in puromycin- or mock-treated lymphoblastoid cell lines of control individuals and patients carrying the c.6844G>T or c.6901G>T nonsense variants; Figure S8. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in variant-expressing mESC; Figure S9. Quantitation of BRCA2 protein expression in mESC; Figure S10. Sensitivity of BRCA2 variants to cisplatin and PARP inhibitors.

Figure S4. Differential gene expression analysis in responders vs nonresponders conducted on baseline samples. Three hundred fifty-eight genes were screened. Genes with FDR P-value <0.05 are labeled and in blue.

The discovery of molecular alterations involved in oncogenesis is evolving rapidly and has led to the development of new innovative targeted therapies in oncology. High-throughput sequencing techniques help to identify genomic targets and to provide predictive molecular biomarkers of response to guide alternative therapeutic strategies. Besides the emergence of these theranostic markers for the new targeted treatments, pharmacogenetic markers (corresponding to genetic variants existing in the constitutional DNA, i.e., the host genome) can help to optimize the use of chemotherapy. In this review, we present the current clinical applications of constitutional PG and the recent concepts and advances in pharmacogenomics, a rapidly evolving field that focuses on various molecular alterations identified on constitutional or somatic (tumor) genome.

Assessment of age-dependent cancer risk for carriers of a predicted pathogenic variant (PPV) is often hampered by biases in data collection, with a frequent under-representation of cancer-free PPV carriers. TUMOSPEC was designed to estimate the cumulative risk of cancer for carriers of a PPV in a gene that is usually tested in a hereditary breast and ovarian cancer context. Index cases are enrolled consecutively among patients who undergo genetic testing as part of their care plan in France. First- and second-degree relatives and cousins of PPV carriers are invited to participate whether they are affected by cancer or not, and genotyped for the familial PPV. Clinical, family and epidemiological data are collected, and all data including sequencing data are centralized at the coordinating centre. The three-year feasibility study included 4431 prospective index cases, with 19.1% of them carrying a PPV. When invited by the coordinating centre, 65.3% of the relatives of index cases (5.7 relatives per family, on average) accepted the invitation to participate. The study logistics were well adapted to clinical and laboratory constraints, and collaboration between partners (clinicians, biologists, coordinating centre and participants) was smooth. Hence, TUMOSPEC is being pursued, with the aim of optimizing clinical management guidelines specific to each gene.

Up to 80% of BRCA1 and BRCA2 genetic variants remain of uncertain clinical significance (VUSs). Only variants classified as pathogenic or likely pathogenic can guide breast and ovarian cancer prevention measures and treatment by PARP inhibitors. We report the first results of the ongoing French national COVAR (cosegregation variant) study, the aim of which is to classify BRCA1/2 VUSs. The classification method was a multifactorial model combining different associations between VUSs and cancer, including cosegregation data. At this time, among the 653 variants selected, 101 (15%) distinct variants shared by 1,624 families were classified as pathogenic/likely pathogenic or benign/likely benign by the COVAR study. Sixty-six of the 101 (65%) variants classified by COVAR would have remained VUSs without cosegregation data. Of note, among the 34 variants classified as pathogenic by COVAR, 16 remained VUSs or likely pathogenic when following the ACMG/AMP variant classification guidelines. Although the initiation and organization of cosegregation analyses require a considerable effort, the growing number of available genetic tests results in an increasing number of families sharing a particular variant, and thereby increases the power of such analyses. Here we demonstrate that variant cosegregation analyses are a powerful tool for the classification of variants in the BRCA1/2 breast-ovarian cancer predisposition genes.

Abstract Purpose: In BRAFV600MUT metastatic melanoma, cyclin D–CDK4/6–INK4–Rb pathway alterations are involved in resistance to MAPK inhibitors, suggesting a clinical benefit of cyclin-dependent kinase 4 (CDK4) inhibitors. In this phase I–II study, we aimed to establish the MTD of palbociclib when added to vemurafenib. Patients and Methods: Patients with BRAFV600E/KMUT metastatic melanoma harboring CDKN2A loss and RB1 expression were included and stratified into two groups according to previous BRAF inhibitor treatment (no:strata 1; yes:strata 2). Treatment comprised palbociclib once daily for 14 days followed by a 7-day break + continuous dosing of vemurafenib. The primary endpoint was the occurrence of dose-limiting toxicity (DLT), and the secondary endpoints included the best response, survival, pharmacokinetics, and tumor molecular profiling. Results: Eighteen patients were enrolled, with 15 in strata 2. Characteristics at inclusion were American Joint Committee on Cancer stage IVM1c (N = 16; 88.9%), high lactate dehydrogenase (N = 9; 50.0%), and median number of previous treatments of 2. One and 5 patients experienced DLT in strata 1 and 2, respectively, defining the MTD at palbociclib 25 mg and vemurafenib 960 mg in strata 2. No significant evidence for drug–drug interactions was highlighted. The median progression-free survival was 2.8 months, and 5 (27.8%) patients showed a clinical response. The baseline differential mRNA expression analysis and in vitro data revealed the role of CHEK2 in the response to palbociclib. Conclusions: Although the combination of palbociclib + fixed-dose vemurafenib did not allow an increased palbociclib dosage above 25 mg, a significant clinical benefit was achieved in pretreated patients with melanoma. An association between the transcriptomic data and clinical response was highlighted.

Amyotrophic lateral sclerosis (ALS) is the most common and severe adult-onset motoneuron disease and has currently no effective therapy. Approximately 20% of familial ALS cases are caused by dominantly-inherited mutations in the gene encoding Cu/Zn superoxide dismutase ( SOD1 ), which represents one of the most frequent genetic cause of ALS. Despite the overwhelming majority of ALS-causing missense mutations in SOD1 , a minority of premature termination codons (PTCs) have been identified. mRNA harboring PTCs are known to be rapidly degraded by nonsense-mediated mRNA decay (NMD), which limits the production of truncated proteins. The rules of NMD surveillance varying with PTC location in mRNA, we analyzed the localization of PTCs in SOD1 mRNA to evaluate whether or not those PTCs can be triggered to degradation by the NMD pathway. Our study shows that all pathogenic PTCs described in SOD1 so far can theoretically escape the NMD, resulting in the production of truncated protein. This finding supports the hypothesis that haploinsufficiency is not an underlying mechanism of SOD1 mutant-associated ALS and suggests that PTCs found in the regions that trigger NMD are not pathogenic. Such a consideration is particularly important since the availability of SOD1 antisense strategies, in view of variant treatment assignment.

AbstractGermline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell–based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications.Significance:This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.