During the last few years, detection of epidermal growth-factor–receptor (EGFR)-activating mutations has become a routine part of clinical practice because of their importance in choosing the optimal ...treatment strategy for non-small-cell lung cancers (NSCLCs). The emergence of third-generation EGFR–tyrosine-kinase inhibitors required the implementation of sensitive methods to detect the subclonal EGFRT790M mutation. Clinical implications make it essential to rapidly search for the T790M mutation, which is a real challenge for laboratories. The aim of this study was to compare performances of next-generation sequencing (NGS), one of the most frequently used molecular biology methods, and Idylla EGFR-Mutation Assay (henceforth Idylla), a fully automated real-time polymerase chain reaction (PCR) that is increasingly used in pathology laboratories, to detect the EGFRT790M mutation using DNA.
This retrospective study used 47 DNA samples extracted from NSCLC biopsies that previous NGS identified as: 29 harboring EGFR and T790M resistance mutations, 11 EGFR-activating mutation without T790 M and 7 wild-type EGFR. EGFRT790M limit-of-detection (LOD) experiments used a commercial DNA known to harbor that mutation.
Idylla detected primary EGFR-activating mutations and the T790 M mutation in 97.5 % and 65.5 % of the cases, respectively. The results of this retrospective analysis and LOD experiments showed that the Idylla should only be used to detect EGFR mutations in samples with > 25 ng of DNA and > 10 % tumor cells.
Idylla was able to rapidly detect EGFR-activating mutations but detecting subclone mutations, like T790M, with < 25 ng of good-quality DNA or < 10 % tumor cells (variant allele frequency below the assay’s validated LOD) was not always reliable.
During the last few years, detection of epidermal growth-factor-receptor (EGFR)-activating mutations has become a routine part of clinical practice because of their importance in choosing the optimal ...treatment strategy for non-small-cell lung cancers (NSCLCs). The emergence of third-generation EGFR-tyrosine-kinase inhibitors required the implementation of sensitive methods to detect the subclonal EGFR
mutation. Clinical implications make it essential to rapidly search for the T790M mutation, which is a real challenge for laboratories. The aim of this study was to compare performances of next-generation sequencing (NGS), one of the most frequently used molecular biology methods, and Idylla EGFR-Mutation Assay (henceforth Idylla), a fully automated real-time polymerase chain reaction (PCR) that is increasingly used in pathology laboratories, to detect the EGFR
mutation using DNA.
This retrospective study used 47 DNA samples extracted from NSCLC biopsies that previous NGS identified as: 29 harboring EGFR and T790M resistance mutations, 11 EGFR-activating mutation without T790 M and 7 wild-type EGFR. EGFR
limit-of-detection (LOD) experiments used a commercial DNA known to harbor that mutation.
Idylla detected primary EGFR-activating mutations and the T790 M mutation in 97.5 % and 65.5 % of the cases, respectively. The results of this retrospective analysis and LOD experiments showed that the Idylla should only be used to detect EGFR mutations in samples with > 25 ng of DNA and > 10 % tumor cells.
Idylla was able to rapidly detect EGFR-activating mutations but detecting subclone mutations, like T790M, with < 25 ng of good-quality DNA or < 10 % tumor cells (variant allele frequency below the assay's validated LOD) was not always reliable.
Anaplastic lymphoma kinase (
ALK
) rearrangement is reported in 3% to 8% of patients with lung adenocarcinoma and can be detected by fluorescent
in situ
hybridization (FISH) or indirectly by ...immunohistochemistry. In FISH assay, isolated 5′ signal (loss of 3′ signal) is usually considered negative. We report three young nonsmoking patients with stage IV lung adenocarcinoma. Strong ALK expression in tumor cells detected by immunohistochemistry was observed in all cases, but FISH revealed an isolated 5′ signal pattern. Massive parallel “next-generation” sequencing was performed in two patients and confirmed
ALK
rearrangement. The three patients were treated and responded to crizotinib after 14, 10, and 31 months.
Résumé en français : Notre équipe étudie les mécanismes de l’apoptose induite par la nutline-3, une molécule capable de se fixer sur MDM2 et d’activer la p53, dans différents types de lymphomes ...associés au virus d’Epstein-Barr (EBV) comme le lymphome de Burkitt (LB) ou syndromes lymphoprolifératifs post-transplantation (PTLD). Nos résultats montrent que la nutline-3 induit l’apoptose des cellules de LB EBV (-) alors que les cellules EBV (+) en latence de type III sont résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans ce phénomène de résistance afin de mettre en place des stratégies pour les contourner. Une première étude initiée par les résultats d’une analyse transcriptomique, effectuée après traitement avec la nutline-3 de deux lignées qui ne diffèrent que par leur statut EBV, nous a permis de montrer que : 1) l’autophagie est induite en réponse au traitement dans les cellules EBV (+) en latence de type III ; 2) ces cellules expriment fortement Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie contribue à la résistance de ces cellules à l’apoptose. Par ailleurs, nos résultats indiquent que la protéine anti-apoptotique Bcl-2 est également impliquée dans la résistance de ces cellules et que l’utilisation d’ABT-737, un inhibiteur de Bcl-2, restaure leur sensibilité à la nutline-3. L’efficacité de ce composé a donc été évaluée in vivo, seul ou en combinaison avec des traitements conventionnels (Cyclophosphamide pour le LB et Rituximab pour les PTLD). Les résultats obtenus lors de ces études pré-cliniques montrent que : 1) ABT-737 réduit considérablement la croissance tumorale et augmente la survie de souris xénogreffées avec des cellules d’une lignée lymphoblastoïde (LCL, utilisées comme modèle pour les PTLD) alors qu’il n’a pas d’effets chez les souris xénogreffées avec une lignée de LB ; 2) la combinaison BT-737/Cyclophosphamide permet de limiter la croissance tumorale durant le traitement mais n’améliore pas la survie des souris xénogreffées avec une lignée de LB ; 3) l’association ABT-737/Rituximab est très efficace et induit une rémission complète chez 70% des souris xénogreffées avec la lignée de LCL
- Résumé en anglais : Our team is working on the mechanisms of apoptosis induced by nutlin-3, a small molecule which binds to MDM2 and activates p53, in different lymphomas associated with Epstein-Barr virus such as Burkitt lymphoma (BL) or Post-transplant lymphoproliferative disorder (PTLD). Our results show that nutlin-3 strongly induce apoptosis in EBV (-) cells whereas EBV (+) latency III cells are much more resistant. The aim of my PhD project was to study the mechanisms involved in the resistance of EBV (+) latency III cells to apoptosis and to develop new therapeutic strategies to bypass these mechanisms. A transcriptomic analysis was realized after treatment with nutlin-3 of two cell lines which only differs by their EBV status. Based on the results obtained, a study was performed which allow us to show that: 1) autophagy is induced after nutlin-3 treatment in EBV (+) latency III cells; 2) these cells strongly expressed beclin-1 and present a constitutively high level of autophagy; 3) autophagy is involved in the resistance of apoptosis observed in these cells. Furthermore, our results demonstrate that Bcl-2 also contributes to the resistance of EBV (+) latency III cells and that treatment with ABT-737, a Bcl-2 inhibitor, restores their susceptibility to nutlin-3 treatment. We thus assessed the efficiency of this compound in vivo, in monotherapy or associated with conventional treatments (Cyclophosphamide for BL and Rituximab for PTLD). Results obtained during these pre-clinical studies show that: 1) ABT-737 reduces tumor growth and increase the overall survival of mice xenografted with a lymphoblastoïd cell line (LCL, used as a model for PTLD studies) but has no effects on mice xenografed with BL cell lines; 2) the association ABT-737/Cyclophosphamide reduces tumor growth during treatment but doesn’t improve the overall survival of mice xenografed with BL cell lines; 3) the association ABT-737/Rituximab is very efficient and induces 70% of complete remission in mice xenografted with LCL.
Oncogenic isocitrate dehydrogenase (IDH)1 and IDH2 mutations at three hotspot arginine residues cause an enzymatic gain of function that leads to the production and accumulation of the metabolite ...2-hydroxyglutarate (2HG), which contributes to the development of a number of malignancies. In the hematopoietic system, mutations in IDH1 at arginine (R) 132 and in IDH2 at R140 and R172 are commonly observed in acute myeloid leukemia, and elevated 2HG is observed in cells and serum. However, in angioimmunoblastic T-cell lymphoma (AITL), mutations are almost exclusively restricted to IDH2 R172, and levels of 2HG have not been comprehensively measured. In this study, we investigate the expression pattern of mutant IDH2 in the AITL tumor microenvironment and measure levels of 2HG in tissue and serum of AITL patients. We find that mutant IDH2 expression is restricted to the malignant T-cell component of AITL, and that 2HG is elevated in tumor tissue and serum of patients. We also investigate the differences between the three hotspot mutation sites in IDH1 and IDH2 using conditional knock-in mouse models. These studies show that in the lymphoid system, mutations in IDH2 at R172 produce high levels of 2HG compared with mutations at the other two sites and that lymphoid development is impaired in these animals. These data provide evidence that IDH2 R172 mutations may be the only variants present in AITL because of their capacity to produce significant amounts of the oncometabolite 2HG in the cell of origin of this disease.
Primary liver cancer arises either from hepatocytic or biliary lineage cells, giving rise to hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICCA). Combined hepatocellular- ...cholangiocarcinomas (cHCC-CCA) exhibit equivocal or mixed features of both, causing diagnostic uncertainty and difficulty in determining proper management. Here, we perform a comprehensive deep learning-based phenotyping of multiple cohorts of patients. We show that deep learning can reproduce the diagnosis of HCC vs. CCA with a high performance. We analyze a series of 405 cHCC-CCA patients and demonstrate that the model can reclassify the tumors as HCC or ICCA, and that the predictions are consistent with clinical outcomes, genetic alterations and in situ spatial gene expression profiling. This type of approach could improve treatment decisions and ultimately clinical outcome for patients with rare and biphenotypic cancers such as cHCC-CCA.
PDF
