BACKGROUND:Circulating tumour DNA (ctDNA) testing by liquid biopsy has become a cornerstone of precision oncology, yet technical heterogeneity across laboratories continues to hamper reproducibility and clinical reliability. METHODS:To address this challenge, the Groupe Français de Cytogénétique Oncologique (GFCO) conducted the largest European Delphi consensus initiative to date on technical standardization of ctDNA analysis, involving 39 high-volume molecular platforms (71 % response rate). RESULTS:Using iterative anonymous surveys and live voting, we established 37 recommendations (≥80 % agreement) spanning pre-analytical, analytical, bioinformatics, and post-analytical phases. Key consensus points include mandatory use of unique molecular identifiers (UMIs) with preference for ligation-based incorporation (95-100 %), double centrifugation with high-speed second spin, systematic positive controls mimicking ctDNA characteristics, leukocyte aliquot preservation for clonal haematopoiesis evaluation, and explicit reporting of variant allele frequency, CHIP risk, and limitations of negative results. These recommendations demonstrate more than 90 % alignment with recent ESMO (2022), ELBS (2025), ISLB (2025), and AMP/CAP guidelines while providing more prescriptive, operationally oriented specifications on critical steps (e.g., UMI implementation, plasma storage, centrifugation parameters) that address real-world gaps repeatedly identified in European external quality assessment schemes. CONCLUSIONS:By offering a robust, ready-to-implement framework that complements and refines existing international standards, this consensus paves the way for broader harmonization of ctDNA testing across Europe and beyond, ultimately strengthening confidence in liquid biopsy results for routine diagnostic and theragnostic applications in oncology.

Introduction L’analyse de l’ADN tumoral circulant (ADNtc) par biopsie liquide est une avancée majeure dans la prise en charge des patients atteints de cancer, offrant une méthode peu invasive pour la détection des altérations génétiques tumorales et permettant un suivi personnalisé. Cette approche est essentielle pour guider l’introduction d’inhibiteurs de kinase spécifiques ou d’immunothérapies. Face aux défis technologiques complexes et à la nécessité d’une validation rigoureuse, le Groupe français de cytogénétique oncologique (GFCO) a établi des recommandations consensuelles nationales afin d’accompagner les laboratoires de biologie moléculaire sur les enjeux techniques de l’analyse de l’ADNtc. Méthodes Ces recommandations, destinées à encadrer le testing somatique de l’ADNtc, ont été élaborées selon la méthodologie structurée du consensus DELPHI. Un questionnaire élaboré par un groupe de travail de huit biologistes et deux bio-informaticiens a été soumis à 55 plateformes françaises spécialisées dans la biopsie liquide. Un taux de participation notable de 71 % (39 réponses) a été atteint. Le consensus a été défini par un seuil d’accord d’au moins 80 % des participants pour chaque proposition. Une session de discussion et de vote en direct a été organisée lors des Journées du GFCO 2024 pour les points n’ayant pas atteint initialement ce seuil. Résultats Les recommandations validées couvrent les phases pré-analytique (collecte, conservation), analytique (NGS, UMIs, profondeur de séquençage) et post-analytique (bio-informatique, compte-rendu). Les points de consensus forts incluent la nécessité d’utiliser des tubes avec stabilisateurs si le traitement n’est pas possible dans les 3–4heures ou pendant le week-end (97,8 % d’accord). La double centrifugation des échantillons est requise pour minimiser la contamination par les débris cellulaires et les leucocytes (87,37 %). En phase analytique, l’utilisation d’identifiants moléculaires uniques (UMIs) est unanimement recommandée (100 %) pour améliorer la sensibilité et réduire les biais. L’objectif de sensibilité est d’au moins 0,5 % pour le testing ADNtc, nécessitant une profondeur d’environ 10 000 x avant déduplication. En phase post-analytique, l’évaluation de l’hématopoïèse clonale (CHIP) est jugée essentielle pour fiabiliser le résultat (100 %). Conclusion Cette mise à jour des recommandations du GFCO fournit un cadre standardisé pour l’utilisation de la biopsie liquide en oncologie en France. Des mises à jour régulières seront nécessaires pour s’adapter à l’évolution rapide de la technologie.

INTRODUCTION:The analysis of circulating tumor DNA (ctDNA) by liquid biopsy is a major advance in the management of patients with cancer, offering a minimally invasive method for detecting tumor genetic alterations and enabling personalized monitoring. Faced with complex technological challenges and the need for rigorous validation, the French-Speaking Group for Oncological Cytogenomics (GFCO) has established national consensus guidelines. METHODS:These guidelines, designed to govern ctDNA analysis, were developed using the Delphi consensus methodology. A questionnaire developed by a working group of eight biologists and two bioinformaticians was submitted to 55 French platforms specializing in liquid biopsy in oncology. A participation rate of 71% was achieved. Consensus was defined by an agreement threshold of at least 80%. A live discussion and voting session was held during the GFCO 2024 Days for items that did not initially reach this threshold. RESULTS:The validated recommendations cover the pre-analytical, analytical, and post-analytical phases. In total, 37 recommendations with a consensus exceeding 80% were adopted. CONCLUSION:These GFCO recommendations provide a standardized framework for the use of liquid biopsy in oncology in France. Regular updates will be necessary to adapt to the rapid evolution of the technology.

prospective collection used to train GIScar (n = 250)

Kaplan–Meier estimates of PFS stratified on Giscar HRD status among inconclusive status of MGMC (n = 43).

List of BRCA variants detected by both GIScar tests and MGMC tests

PFS in patients according to GIScar (left panel) and MGMC scores (right panel).

MGMC and GIScar scores among concordant and discordant HRD classification on the clinical collection (n = 469)

Panel of gene used for the GIScar calculation (n =127)

Kaplan–Meier estimates of PFS stratified on treatment arm (n = 469).

SUMMARY:Functional testing of RNA using minigene splicing assays is increasingly being realized to demonstrate the effects of variants on splicing. In complex cases, variant pathogenicity is assessed by Sanger sequencing, which can be time consuming and may be replaced by short read sequencing. Moreover, strategies based on long read sequencing of the amplified minigene construct are promising and allow the isoforms to be fully characterized. We introduce MAGIC, a user-friendly tool that first generates the artificial construction genome files required to then perform alignment, assembly and annotation of the isoforms obtained by either short or long read minigene splicing assay sequencing. AVAILABILITY AND IMPLEMENTATION:MAGIC is available at https://github.com/LBGC-CFB/MAGIC. Zenodo DOI: 10.5281/zenodo.17052752.

Clinical collection used to validate GIScar (n = 469)

Histologic type of tumors used in this study (n = 719)

L’insuffisance ovarienne prématurée (IOP) peut être due à l’altération de différentes voies biologiques nécessaires à la mise en place de la fonction ovarienne. Plusieurs classifications des gènes responsables d’IOP en sous-catégories fonctionnelles ont été proposées, avec des groupes plus ou moins détaillés. Afin de proposer une catégorisation adaptée à l’application clinique, nous avons repris les données génomiques d’une cohorte de plus de 150 patientes avec IOP analysées par séquençage haut débit. Trois catégories de gènes ont particulièrement retenu notre attention : les gènes méiotiques ou de réparation de l’ADN, les gènes mitochondriaux et les gènes codant pour les protéines se liant à l’ARNm.Nous rapportons par la description d’un nouveau gène, SWSAP1, formant avec ZSWIM7 le complexe Shu intervenant lors de la recombinaison homologue. Nous avons également mis en évidence des variants dans des gènes de prédisposition à certaines formes de cancers tels que RAD51D, NBN ou BRIP1 chez des patientes sans histoire familiale ou personnelle de cancer. Nous identifions également des variants dans des gènes de pathologies mitochondriales comme TUFM, MIGA2 ou encore MFN1 pour lesquels le phénotype d’IOP n’est que peu ou pas rapporté. Nous avons enfin mis en évidence des variants d’intérêt dans des gènes responsables de défauts de maturation ovocytaire (PANX1) ou de fausses couches à répétition (SYCP3), chez des patientes avec IOP. Enfin, les processus de maturation de l’ovocyte sont tributaires du contrôle des taux d’ARNm dès le stade de follicule primordial jusqu’aux premiers stades de l’embryogenèse. Nous avons identifié deux candidats majeurs : ELALV2 interrompu au niveau d’un point de cassure de remaniement chromosomique, caractérisé par séquençage de génome short-read et longread, et LSM14B. Ce travail permet de mieux caractériser les groupes de gènes impliqués dans l’IOP et pourra être complété par le développement d’un outil de priorisation grâce aux outils de machine-learning.

BACKGROUND:Solving the structure of mRNA transcripts is a major challenge for both research and molecular diagnostic purposes. Current approaches based on short-read RNA sequencing and RT-PCR techniques cannot fully explore the complexity of transcript structure. The emergence of third-generation long-read sequencing addresses this problem by solving this sequence directly. However, genes with low expression levels are difficult to study with the whole transcriptome sequencing approach. To fix this technical limitation, we propose a novel method to capture transcripts of a gene panel using a targeted enrichment approach suitable for Pacific Biosciences and Oxford Nanopore Technologies platforms. RESULTS:We designed a set of probes to capture transcripts of a panel of genes involved in hereditary breast and ovarian cancer syndrome. We present SOSTAR (iSofOrmS annoTAtoR), a versatile pipeline to assemble, quantify and annotate isoforms from long read sequencing using a new tool specially designed for this application. The significant enrichment of transcripts by our capture protocol, together with the SOSTAR annotation, allowed the identification of 1,231 unique transcripts within the gene panel from the eight patients sequenced. The structure of these transcripts was annotated with a resolution of one base relative to a reference transcript. All major alternative splicing events of the BRCA1 and BRCA2 genes described in the literature were found. Complex splicing events such as pseudoexons were correctly annotated. SOSTAR enabled the identification of abnormal transcripts in the positive controls. In addition, a case of unexplained inheritance in a family with a history of breast and ovarian cancer was solved by identifying an SVA retrotransposon in intron 13 of the BRCA1 gene. CONCLUSIONS:We have validated a new protocol for the enrichment of transcripts of interest using probes adapted to the ONT and PacBio platforms. This protocol allows a complete description of the alternative structures of transcripts, the estimation of their expression and the identification of aberrant transcripts in a single experiment. This proof-of-concept opens new possibilities for RNA structure exploration in both research and molecular diagnostics.

Introduction: DNA damage repair genes are altered in 20–35% of metastatic castration-resistant prostate cancer (mCRPC). Poly-ADP (Adénosine Diphosphate)-ribose polymerase inhibitors (PARPi) showed significant activity for these selected tumors, especially with homologous recombination repair (HRR) deficiency. These alterations could also predict platinum sensitivity. Although carboplatin was inconclusive in unselected mCRPC, the literature suggests an anti-tumoral activity in mCRPC with HHR gene alterations. We aimed to assess the efficacy of carboplatin monotherapy in mCRPC patients with HRR deficiency. Methods: This prospective multicenter single-arm two-stage phase II addressed mCRPC men with HRR somatic and/or germline alterations, pretreated with ⩾2 taxane chemotherapy regimens and one androgen receptor pathway inhibitor. Prior PARPi treatment was allowed. Enrolled patients received intravenous carboplatin (AUC5) every 21 days for 6–9 cycles. The primary endpoint was the best response rate according to adapted PCWG3 guidelines: radiological response (RECIST 1.1 criteria) and/or biological response [⩾50% prostate-specific antigen (PSA) decline]. Results: A total of 15 out of 16 enrolled patients started carboplatin treatment. Genomic alterations were identified for BRCA2 ( n = 5), CDK12 ( n = 3), ATM ( n = 3) CHEK2 ( n = 2), CHEK1 ( n = 1), and BRCA1 ( n = 1) genes. Objective response (partial biological response + stable radiological response) was achieved in one patient (6.7%), carrying a BRCA2 mutation and not pre-treated with PARPi; stable disease was observed for five patients (33.5%). Among seven patients (46.7%) with previous PARPi treatment, four patients (57.1%) had a stable disease. The median progression-free and overall survivals were 1.9 [95% confidence interval (95% CI), 1.8–9.5] and 8.6 months (95% CI, 4.3–19.5), respectively. The most common severe (grade 3–4) treatment-related toxicities were thrombocytopenia (66.7%), anemia (66.7%), and nausea (60%). Overall, 8 (53.3%) patients experienced a severe hematological event. Conclusion: The study was prematurely stopped as pre-planned considering the limited activity of carboplatin monotherapy in heavily pre-treated, HHR-deficient mCRPC patients. Larger experience is needed in mCRPC with BRCA alterations. Trial registration: NCT03652493, EudraCT ID number 2017-004764-35.