Genetic landscape of a large cohort of Primary Ovarian Insufficiency : New genes and pathways and implications for personalized medicine

Background Primary Ovarian Insufficiency (POI), a public health problem, affects 1-3.7% of women under 40 yield-ing infertility and a shorter lifespan. Most causes are unknown. Recently, genetic causes were identified, mostly in single families. We studied an unprecedented large cohort of POI to unravel its molecular pathophysiology.Methods 375 patients with 70 families were studied using targeted (88 genes) or whole exome sequencing with pathogenic/likely-pathogenic variant selection. Mitomycin-induced chromosome breakages were studied in patients' lymphocytes if necessary. Findings A high-yield of 29.3% supports a clinical genetic diagnosis of POI. In addition, we found strong evidence of pathogenicity for nine genes not previously related to a Mendelian phenotype or POI: ELAVL2, NLRP11, CENPE, SPATA33, CCDC150, CCDC185, including DNA repair genes: C17orf53(HROB), HELQ, SWI5 yielding high chromo-somal fragility. We confirmed the causal role of BRCA2, FANCM, BNC1, ERCC6, MSH4, BMPR1A, BMPR1B, BMPR2, ESR2, CAV1, SPIDR, RCBTB1 and ATG7 previously reported in isolated patients/families. In 8.5% of cases, POI is the only symptom of a multi-organ genetic disease. New pathways were identified: NF-kB, post-translational regulation, and mitophagy (mitochondrial autophagy), providing future therapeutic targets. Three new genes have been shown to affect the age of natural menopause supporting a genetic link.Interpretation We have developed high-performance genetic diagnostic of POI, dissecting the molecular pathogene-sis of POI and enabling personalized medicine to i) prevent/cure comorbidities for tumour/cancer susceptibility genes that could affect life-expectancy (37.4% of cases), or for genetically-revealed syndromic POI (8.5% of cases), ii) predict residual ovarian reserve (60.5% of cases). Genetic diagnosis could help to identify patients who may benefit from the promising in vitro activation-IVA technique in the near future, greatly improving its success in treating infertility.Funding Universite? Paris Saclay, Agence Nationale de Biome?decine.Copyright (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)Peer reviewe

Analyse moléculaire, enjeux et limites des thérapies ciblées en oncologie : extension des sensibilités aux anti-PARP dans les cancers ovariens par caractérisation de variants non annotés et nouveaux mécanismes de résistance dans les cancers bronchiques.

Despite significant clinical benefit from the consideration of molecular context, targeted therapies are still challenging. First part of this work focused on tyrosine kinase inhibitors targeting EGFR in non small cell lung cancers. Thus, improvement of biomarkers detection methods was completed by in vitro characterization of an unreported mechanism of acquired resistance. Briefly, pulmonary cells were exposed to a mutagen agent and a selection pressure was applied with EGFR inhibitors allowing the detection of TBK1 signature. Finally, synergic effect of that co-inhibition was highlighted. Now essentials in gynaecological cancers management, PARP inhibitors represent the second part of that work. Those targeted therapies are based on synthetic lethality. Consequently, BRCA1/2 pathogenic mutations are required for their administration, illustrating the issue of variants of uncertain significance. Toward their functional characterization necessity, a transcriptional analysis of splicing variant was first conducted on mRNA extracted from FFPE samples. Then, to evaluate functional signification of all types of variants, genomic edition was developed. Editing efficiencies of the unknown variant and a silent control one were compared in a haploid model where those genes are essentials. Functional signification of BRCA1/2 variants, and thereby mutations from all essential tumor suppressor genes in our model, can be evaluated in three weeks which is compatible with clinical management.Malgré les bénéficies cliniques considérables de la prise en considération du contexte moléculaire tumoral, les thérapies ciblées présentent certaines limitations majeures. La première partie de ces travaux se concentre sur l’étude des inhibiteurs de tyrosine kinase ciblant l’EGFR dans les cancers bronchiques non à petites cellules. L’amélioration des méthodes de détection des biomarqueurs a été complétée par la caractérisation in vitro d’un mécanisme de résistance acquise non précédemment identifié. L’exposition de cellules pulmonaires à un agent mutagène puis une pression de sélection a ainsi mis en évidence la signature TBK1 et l’effet synergique de la co-inhibition s’est révélé intéressant. Le deuxième aspect concernent les inhibiteurs de PARP, incontournables dans la prise en charge des cancers de la sphère gynécologique. Ces thérapies reposant sur le principe de létalité synthétique, la présence de mutations pathogènes de BRCA1/2 est un pré-requis, illustrant la problématique des variants de signification inconnue. Face à la nécessité de leur caractérisation fonctionnelle, une étude transcriptionnelle des variants d’épissage par extraction de l’ARNm depuis les prélèvements FFPE a d’abord été réalisée. Puis, afin d’évaluer tous les variants, l’édition génomique a été développée comparant les efficacités d’édition d’un variant d’intérêt et d’un variant silencieux dans un contexte haploïde où ces gènes sont essentiels. La signification biologique de variants de BRCA1/2, et la généralisation de notre approche à l’ensemble des gènes suppresseurs de tumeur essentiels de nos cellules, peut ainsi être évaluée en 3 semaines, délai compatible avec les impératifs cliniques

Molecular analysis, advantages and challenges of targeted therapies in oncology : increasing sensitivity to PARP inhibitors in ovarian cancer by characterization of unknown variants and new acquired resistance mechanism in lung cancers.

Malgré les bénéficies cliniques considérables de la prise en considération du contexte moléculaire tumoral, les thérapies ciblées présentent certaines limitations majeures. La première partie de ces travaux se concentre sur l’étude des inhibiteurs de tyrosine kinase ciblant l’EGFR dans les cancers bronchiques non à petites cellules. L’amélioration des méthodes de détection des biomarqueurs a été complétée par la caractérisation in vitro d’un mécanisme de résistance acquise non précédemment identifié. L’exposition de cellules pulmonaires à un agent mutagène puis une pression de sélection a ainsi mis en évidence la signature TBK1 et l’effet synergique de la co-inhibition s’est révélé intéressant. Le deuxième aspect concernent les inhibiteurs de PARP, incontournables dans la prise en charge des cancers de la sphère gynécologique. Ces thérapies reposant sur le principe de létalité synthétique, la présence de mutations pathogènes de BRCA1/2 est un pré-requis, illustrant la problématique des variants de signification inconnue. Face à la nécessité de leur caractérisation fonctionnelle, une étude transcriptionnelle des variants d’épissage par extraction de l’ARNm depuis les prélèvements FFPE a d’abord été réalisée. Puis, afin d’évaluer tous les variants, l’édition génomique a été développée comparant les efficacités d’édition d’un variant d’intérêt et d’un variant silencieux dans un contexte haploïde où ces gènes sont essentiels. La signification biologique de variants de BRCA1/2, et la généralisation de notre approche à l’ensemble des gènes suppresseurs de tumeur essentiels de nos cellules, peut ainsi être évaluée en 3 semaines, délai compatible avec les impératifs cliniques.Despite significant clinical benefit from the consideration of molecular context, targeted therapies are still challenging. First part of this work focused on tyrosine kinase inhibitors targeting EGFR in non small cell lung cancers. Thus, improvement of biomarkers detection methods was completed by in vitro characterization of an unreported mechanism of acquired resistance. Briefly, pulmonary cells were exposed to a mutagen agent and a selection pressure was applied with EGFR inhibitors allowing the detection of TBK1 signature. Finally, synergic effect of that co-inhibition was highlighted. Now essentials in gynaecological cancers management, PARP inhibitors represent the second part of that work. Those targeted therapies are based on synthetic lethality. Consequently, BRCA1/2 pathogenic mutations are required for their administration, illustrating the issue of variants of uncertain significance. Toward their functional characterization necessity, a transcriptional analysis of splicing variant was first conducted on mRNA extracted from FFPE samples. Then, to evaluate functional signification of all types of variants, genomic edition was developed. Editing efficiencies of the unknown variant and a silent control one were compared in a haploid model where those genes are essentials. Functional signification of BRCA1/2 variants, and thereby mutations from all essential tumor suppressor genes in our model, can be evaluated in three weeks which is compatible with clinical management

Non-Small-Cell Lung Cancer-Sensitive Detection of the p.Thr790Met EGFR Alteration by Preamplification before PNA-Mediated PCR Clamping and Pyrosequencing

International audienceTargeted therapies and, more precisely, EGFR tyrosine kinase inhibitors (TKIs) have been a major improvement in the therapeutic management of EGFR-mutated non-small-cell lung cancers (NSCLCs). Earlier administration of these TKIs throughout tumor progression is imperative to improve patient outcomes. Consequently, studies have focused on refining the characterization of biomarkers, especially concerning the resistance mutation p.Thr790Met of EGFR. Herein, we developed peptide nucleic acid (PNA)-mediated PCR clamping followed by pyrosequencing, favoring enrichment of the mutated fraction. A preamplification step was first added to increase the amplifiable DNA fraction. Throughout the application of our method on DNA extracted from FFPE samples of 46 patients with NSCLC who had relapsed under first-generation EGFR TKI, we evaluated a sensitivity of 93.3% and a specificity of 100%. All 19 patients who were positive for the p.Thr790Met mutation with NGS were also found to be positive with our protocol. The only discordant case was a sample with no mutation detected with NGS, but which was positive with PNA. This protocol allows for the detection of the p.Thr790Met mutation with a sensitivity of 0.5% which will permit earlier detection and an improvement of therapeutic management

L’instabilité génomique, paramètre limitant l’efficacité des thérapies ciblées en oncologie

International audienceGenomic instability is one of the main properties of tumour development, promoting first the acquisition of genetic alterations and thus carcinogenesis. Then, the chronic and anarchic proliferation of cancer cells also supports and contributes to this instability allowing a continuous evolution of the tumour. The accumulation of mutations resulting from that instability contributes to tumour heterogeneity that occurs in a specific environment. The resulting diversity of oncogenic drivers further complicates the characterization of the origin of cancer cells dysfunction and consequently therapeutic decision. However, the consideration of the molecular context in oncology has initiated the development of targeted therapies. Based on the concept of oncogene addiction and synthetic lethality, these new drugs require the characterization and identification of specific tumour biomarkers. Targeted therapies have thus considerably optimized patient management, improving efficiency and quality of life while limiting the side effects observed with conventional chemotherapies. However, despite significant clinical benefits, some major limitations to their administration remain. The study of the current issues related to these new therapeutic molecules is becoming crucial for patient management towards an improvement of personalized medicine

Somatic mRNA Analysis of BRCA1 Splice Variants Provides a Direct Theranostic Impact on PARP Inhibitors

International audienceBACKGROUND:The identification of pretherapeutic somatic BRCA variants can have considerable clinical impact given that they affect response to the new poly (ADP-ribose) polymerase (PARP)-targeted therapy. One major issue with this type of testing is the identification of splicing variants of uncertain significance (VUS) on degraded somatic messenger RNA. It is therefore important to be able to quickly characterize these splice variants.OBJECTIVE:As part of PARP inhibitor targeted therapy, we have investigated a method for the direct confirmation of potential pathogenic somatic splice variants of BRCA1 found in fixed tumor samples. Previously these VUS have commonly only been tested by in silico analysis.METHODS:Five BRCA1 variants affecting splicing were characterized from formalin-fixed, paraffin-embedded (FFPE) ovarian carcinoma tissues by next-generation sequencing (NGS). Three patient samples had already been functionally characterized and were used as controls. Total somatic RNA from samples was extracted, reverse-transcribed, and amplified with several primer pairs encompassing the target exon. The polymerase chain reaction (PCR) products were analyzed by capillary gel electrophoresis to assess possible changes in size due to splicing alterations. Finally, we confirmed our results by cloning, followed by Sanger sequencing, and analyzed the expression of the aberrant forms.RESULTS:Our molecular approach made it possible to visualize the splicing outcomes of three variants (c.5194-2A>G, c.5434C>G, and c.547+1G>A) already identified and present in databases and/or identified with prediction tools (ClinVar, UMD, ARUP Utah database, and Human Splice Finder splices sites prediction) and to confirm their exon skipping consequences, their expression in tumors, and thus their pathogenicity. The c.4484+5G>A variant was not found in databases and was predicted to have no impact on splicing, but was found to display altered processing in tumor tissue. This variant also had a major detrimental impact on transcriptional expression.CONCLUSION:In a break from purely in silico approaches, we propose a simple and rapid pretherapeutic functional analysis of somatic BRCA1 variants potentially involved in splicing alterations. This approach will allow more ovarian cancer patients to benefit from new therapies targeting PARP

Caractérisation moléculaire de l'EGFR dans les cancers bronchiques non à petites cellules : étude prospective comparative des technologies NGS et automate Idylla

International audienceIntroduction: Detection of genetic alterations in the EGFR tyrosine kinase domain is a major concern in the management of non-small cell lung cancer because it conditions access to tyrosine kinase inhibitors. In practice, it is possible to characterize only well-documented mutations or to sequence all relevant EGFR exons and also other targets of theranostic interest. This prospective study compares the targeted EGFR characterization on Idylla platform (Biocartis) and a more extensive one by next generation sequencing using Ion Torrent technology.Material and methods: A total of 100 formalin-fixed paraffin-embedded tumour samples were tested simultaneously by both techniques under the conditions recommended by the suppliers. The comparison covered all technical and practical aspects of the laboratory.Results: At least one EGFR mutation of interest for tyrosine kinase inhibitors for 9 and 7 samples was detected respectively by sequencing and by the Idylla system. For three samples, EGFR sensitive mutations to tyrosine kinase inhibitors were detected only by next-generation sequencing. In addition, for 37 samples, mutations of clinical interest outside EGFR were characterized by sequencing and communicated to the prescriber.Conclusion: Idylla technology allows the rapid characterization of a majority of EGFR variants. The result can be optimized by careful analysis of the amplification curves with the Idylla Explore tool or by increasing the amount of initial material. A complementary new generation sequencing analysis for non-contributory results by Idylla should also be recommended

Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

International audienceBackground: Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using BRCA1 and BRCA2 variants, which have consequences on PARP inhibitor sensitivity, and POLE variants, potential biomarkers of immunotherapy response, we developed a rapid functional assay based on CRISPR-Cas9 genome editing to determine the functional consequences of these variants having potentially direct implications on patients' access to targeted therapies.Methods: We first evaluated the functional impact of 26 BRCA1 and 7 BRCA2 variants by editing and comparing NGS results between the variant of interest and a silent control variant. Ten of these variants had already been classified as benign or pathogenic and were used as controls. Finally, we extended this method to the characterization of POLE VUS.Results: For the 23 variants that were unclassified or for which conflicting interpretations had been reported, 15 were classified as functionally normal and 6 as functionally abnormal. Another two variants were found to have intermediate consequences, both with potential impacts on splicing. We then compared these scores to the patients' responses to PARP inhibitors when possible. Finally, to prove the application of our method to the classification of variants from other tumor suppressor genes, we exemplified with three POLE VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four POLE variants previously classified in databases were also evaluated. However, we found evidence of a discordance with the classification, variant p.Leu424Val being found here functionally normal.Conclusions: Our new rapid functional assay can be used to characterize the functional implication of BRCA1 and BRCA2 variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients' responses to PARP inhibitors, where accessible, was consistent with our functional score evaluation and confirmed the accuracy of our protocol. This method could potentially be extended to the classification of VUS from all essential tumor suppressor genes and can be performed within a timeframe compatible with clinical applications, thereby having a direct theranostic impact