Place de l’ ADN tumoral circulant dans la prise en charge des patients atteints d’une tumeur pulmonaire : impact pronostique et suivi dynamique de l’évolution des altérations génétiques au cours du traitement

L’incidence globale et la mortalité dues au cancer bronchique font de cette pathologie un problème majeur de santé publique. Avec plus de 30 000 nouveaux cas par an en France, il représente la première cause de mortalité par cancer chez l’homme et la troisième chez la femme. Le pronostic de ces patients demeure sombre : la survie relative à 5 ans est estimée à 15% tous stades confondus.La prise en charge des patients atteints d’un cancer au stade métastatique passe aujourd’hui par une caractérisation des altérations somatiques tumorales à la recherche d’une cible thérapeutique potentielle.L'isolement et l'analyse de l’ADN tumoral circulant (ADNtc) issu de plasma est susceptible d’être utilisé comme approche non invasive pour détecter ces altérations et les suivre tout au long du traitement. Les résultats analytiques et la sensibilité « individuelle » sont dépendants de l’étape d’extraction qui doit permettre d’isoler efficacement des quantités souvent faibles de matériel. Le choix de la technique d’extraction employée est crucial. Nous avons vu lors de nos tests que plusieurs kits d’extraction ont des performances analytiques identiques mais qu’il était plus ou moins compliqué de mettre en place ces différentes méthodes en routine hospitalière. L’optimisation des techniques d’analyse de l’ADNtc est également indispensable : il faut une méthode sensible et robuste, l’ADNtc étant largement dilué dans l’ADN libre circulant particulièrement dans le cas des cancers pulmonaires. L’utilisation clinique sans a priori de l’ADNtc pour caractériser les altérations génétiques de la tumeur exige le développement et la validation de nouvelles méthodes basées sur le séquençage nouvelle génération (NGS). Nous avons mis en place une nouvelle approche statistique d’analyse de ces données basée sur la quantification du nombre d’erreur retrouvé sur chacune des bases séquencées (PER-Position Error Rate). Sa sensibilité de détection sur les échantillons cliniques d’ADNtc est de 0,3% pour les variations de nucléotides simples et de 0,1% pour les insertion / délétion de plus de trois paires de bases.Nous avons mis en pratique notre méthode d’analyse BPER sur une cohorte de patients avec un cancer pulmonaire de stade IV. Cette étude prospective a montré que l’ADNtc est un outil d’évaluation précoce de la réponse tumorale au traitement dans les CBNPC chez les patients métastatiques. Le NGS ciblé et la méthode d’analyse statistique BPER sont aujourd’hui prêts et validés pour une utilisation en clinique précise : au moment du diagnostic pour la recherche d’une cible thérapeutique en l’absence de tissu et dans le cadre du suivi pour identifier une mutation de résistance secondaire aux anti-EGFR

Epigenetic Mechanisms of Resistance to Immune Checkpoint Inhibitors

International audienceImmune checkpoint inhibitors (ICIs) have demonstrated to be highly efficient in treating solid tumors; however, many patients have limited benefits in terms of response and survival. This rapidly led to the investigation of combination therapies to enhance response rates. Moreover, predictive biomarkers were assessed to better select patients. Although PD-L1 expression remains the only validated marker in clinics, molecular profiling has brought valuable information, showing that the tumor mutation load and microsatellite instability (MSI) status were associated to higher response rates in nearly all cancer types. Moreover, in lung cancer, EGFR and MET mutations, oncogene fusions or STK11 inactivating mutations were associated with low response rates. Cancer progression towards invasive phenotypes that impede immune surveillance relies on complex regulatory networks and cell interactions within the tumor microenvironment. Epigenetic modifications, such as the alteration of histone patterns, chromatin structure, DNA methylation status at specific promoters and changes in microRNA levels, may alter the cell phenotype and reshape the tumor microenvironment, allowing cells to grow and escape from immune surveillance. The objective of this review is to make an update on the identified epigenetic changes that target immune surveillance and, ultimately, ICI responses, such as histone marks, DNA methylation and miR signatures. Translational studies or clinical trials, when available, and potential epigenetic biomarkers will be discussed as perspectives in the context of combination treatment strategies to enhance ICI responses in patients with solid tumors

Role of Circulating Tumor DNA in Gastrointestinal Cancers: Current Knowledge and Perspectives

International audienceGastrointestinal (GI) cancers are major health burdens worldwide and biomarkers are needed to improve the management of these diseases along their evolution. Circulating tumor DNA (ctDNA) is a promising non-invasive blood and other bodily-fluid-based biomarker in cancer management that can help clinicians in various cases for the detection, diagnosis, prognosis, monitoring and personalization of treatment in digestive oncology. In addition to the well-studied prognostic role of ctDNA, the main real-world applications appear to be the assessment of minimal residual disease to further guide adjuvant therapy and predict relapse, but also the monitoring of clonal evolution to tailor treatments in metastatic setting. Other challenges such as predicting response to treatment including immune checkpoint inhibitors could also be among the potential applications of ctDNA. Although the level of advancement of ctDNA development in the different tumor localizations is still inhomogeneous, it might be now reliable enough to be soon used in clinical routine for colorectal cancers and shows promising results in other GI cancers

Role of circulating tumor DNA in the management of patients with colorectal cancer

International audienceColorectal cancer is a major health burden with a prognosis that has been improved with the progresses in diagnosis and the advance of chemotherapy and personalized medicine. However, because of intra-tumor heterogeneity, clonal evolution and selection, tumors often develop resistance to treatments. "Liquid biopsy" is a minimally invasive method, based on analysis of tumor-specific material in peripheral blood samples of patients. Analysis of tumor specific genetic or epigenetic alterations in cell-free circulating nucleic acids may reflect the molecular heterogeneity of the underlying disease process and serial testing could allow to monitor its temporal genomic changing without using re-biopsy. In this review, we focused on the role of circulating tumor DNA (ctDNA) as a biomarker in the management of patients with colorectal cancer at early and advanced stages. Through recent studies, we described its promising clinical applications for diagnosis, detection of recurrence after surgery and monitoring for tumor response or therapeutic resistance in metastatic setting. Such recent developments offer new perspectives for personalized medicine in colorectal cancer but still needs some standardized detection methods and further studies to validate its use in clinical routine

Multiplex Picoliter-Droplet Digital PCR for Quantitative Assessment of DNA Integrity in Clinical Samples

International audienceBACKGROUND:Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA.METHODS:Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues.RESULTS:One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants.CONCLUSIONS:The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients

Epithelial-to-Mesenchymal Transition and MicroRNAs in Lung Cancer

Despite major advances, non-small cell lung cancer (NSCLC) remains the major cause of cancer-related death in developed countries. Metastasis and drug resistance are the main factors contributing to relapse and death. Epithelial-to-mesenchymal transition (EMT) is a complex molecular and cellular process involved in tissue remodelling that was extensively studied as an actor of tumour progression, metastasis and drug resistance in many cancer types and in lung cancers. Here we described with an emphasis on NSCLC how the changes in signalling pathways, transcription factors expression or microRNAs that occur in cancer promote EMT. Understanding the biology of EMT will help to define reversing process and treatment strategies. We will see that this complex mechanism is related to inflammation, cell mobility and stem cell features and that it is a dynamic process. The existence of intermediate phenotypes and tumour heterogeneity may be debated in the literature concerning EMT markers, EMT signatures and clinical consequences in NSCLC. However, given the role of EMT in metastasis and in drug resistance the development of EMT inhibitors is an interesting approach to counteract tumour progression and drug resistance. This review describes EMT involvement in cancer with an emphasis on NSCLC and microRNA regulation

Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study

International audienceBACKGROUND:Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive "real-time" biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC).METHODS AND FINDINGS:We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01-3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30-3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66-6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations.CONCLUSIONS:In this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies

Analysis of Base-Position Error Rate of Next-Generation Sequencing to Detect Tumor Mutations in Circulating DNA

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ANNALS OF ONCOLOGY Volume: 30 Supplement: 5 Pages: 236-236 Meeting Abstract: 623P Published: OCT 2019

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