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International audience; Abstract Background: Resistance to PD1/L1 immune checkpoint inhibitors (ICIs) in advanced NSCLC patients is observed in about 80% of individuals with no robust predictive biomarker yet. The PIONeeR trial (NCT03493581) aims to predict such resistances through a comprehensive multiparametric biomarkers analysis. Methodology: Among the >300 advanced NSCLC patients (pts) recruited in PIONeeR, we focused on the first 137 ≥2nd line ECOG PS0-1 pts treated with single-agent nivolumab, pembrolizumab or atezolizumab. Tumor tissue was collected at baseline and pts were re-biopsied at 6 weeks, and blood-sampled every cycle throughout the 24 weeks post C1D1. Response to PD1/L1 ICIs was assessed by RECIST 1.1 every 6 weeks. Immune contexture was characterized in tumor & blood of each pt through FACS for circulating immune cell subtypes quantification and endothelial activation, blood soluble factors dosage, dual- & multiplex IHC/digital pathology to quantify immune cells infiltrating the tumor, WES for TMB & ICI plasma dosage, leading to 331 measured biomarkers in addition to routine clinical parameters. Multivariable (MV) logistic regression was used to examine the association of each biomarker (controlled by sex, age, smoking status, histological type & PDL1+ Tumor Cells) with the risk of Early Progression (EP), i.e. within 3.5 months of treatment. Multivariable Cox regression analysis was conducted for association with PFS and OS. Results: Overall, the 137 pts were mainly male (64%), smokers (92%) and 1% PDL1+ TC in 36% of the cases, and 21% of pts were still on treatment at data cut-off. Archived samples were available for 80% of pts at inclusion and re-biopsy was available in 52.9% of these cases. The median follow up was 19.8 months, 22.5% of pts did not progress at data cut-off while 62% presented EP. Tumor Cytotoxic T-cells density, especially PD1+ were lower in EP (MV OR=0.45, p=0.022); conversely, higher proportions of circulating cytotoxic T-cells and activated T-cells (HLA-DR+) were observed in EP (MV OR=3.8, p65% inter-pt variability was observed in plasma exposures for all ICIs, with 8-10% of pts displaying trough levels below the target engagement threshold. Data will be presented through unsupervised clustering algorithms & multi-modal supervised learning methods. Changes after 6 weeks of treatment will be analyzed to further investigate drugs mechanisms of action. Conclusion: The PIONeeR trial provides with the 1st comprehensive biomarkers’ analysis to establish predictive models of resistance in advanced NSCLC pts treated with PD1/L1 ICIs and highlights how tumor and circulating biomarkers are complementary.

Background: Different liquid chromatography tandem mass spectrometry (LC–MS/MS) methods have been published for quantification of monoclonal antibodies (mAbs) in plasma but thus far none allowed the simultaneous quantification of several mAbs, including immune checkpoint inhibitors. We developed and validated an original multiplex LC–MS/MS method using a ready-to-use kit to simultaneously assay 7 mAbs (i.e., bevacizumab, cetuximab, ipilimumab, nivolumab, pembrolizumab, rituximab and trastuzumab) in plasma. This method was next cross-validated with respective reference methods (ELISA or LC–MS/MS). Methods: The mAbXmise kit was used for mAb extraction and full-length stable-isotope-labeled antibodies as internal standards. The LC–MS/MS method was fully validated following current EMA guidelines. Each cross validation between reference methods and ours included 16–28 plasma samples from cancer patients. Results: The method was linear from 2 to 100 µg/mL for all mAbs. Inter- and intra-assay precision was &lt, 14.6% and accuracy was 90.1–111.1%. The mean absolute bias of measured concentrations between multiplex and reference methods was 10.6% (range 3.0–19.9%). Conclusions: We developed and cross-validated a simple, accurate and precise method that allows the assay of up to 7 mAbs. Furthermore, the present method is the first to offer a simultaneous quantification of three immune checkpoint inhibitors likely to be associated in patients.

La prise en charge chimiothérapeutique de nombreuses tumeurs solides repose sur l’administration du 5-fluororacile (5-FU), qui est connu pour régulièrement induire des toxicités sévères et dans de rares cas des décès toxiques. Ces toxicités sont dues à l’importante variabilité interindividuelle de l’étape de détoxication hépatique du 5-FU par la Dihydro Pyrimidine Déhydrogénase (DPD). Cette variabilité interindividuelle s’explique en partie par des facteurs génétiques. Ainsi, toute diminution importante de l’activité de la DPD va se traduire, chez un patient recevant une dose standard de 5-FU, par un surdosage pouvant entrainer des toxicités, notamment hématologiques et digestives. Différentes méthodes ont été développées pour déterminer le statut DPD parmi lesquelles : le génotypage du gène DPYD, la mesure de l’uracilémie (U) associée ou non à la mesure de la dihydrouracilémie (UH2) permettant de calculer le ratio UH2/U. En février 2018, l’Agence Nationale de Sécurité du Médicament (ANSM) a préconisé la recherche d’un déficit en DPD pour tout patient concerné par une chimiothérapie intégrant des fluoropyrimidines sans préciser la méthode de cette recherche. Depuis décembre 2018 il est recommandé par l’Institut National du Cancer (INCa), et la Haute Autorité de Santé (HAS) de déterminer le statut de la DPD par phénotypage à l’aide de la mesure de l’uracilémie. Ces recommandations ont eu pour conséquences une importante augmentation de l’activité de phénotypage de la DPD au sein des laboratoires réalisant cette analyse. Ainsi, pour pouvoir absorber cette augmentation des demandes de phénotypage de la DPD, proposer une technique de dosage de l’uracilémie aisément transposable à tous les laboratoires, robuste, spécifique, et rapide semble être une démarche intéressante.