or expressing either RHOA WT or G17V mutant and CD28 WT or T195P mutant were generated by lentiviral infection and positive FACS sorting for GFP (RHOA selection) and CD28 expression at the plasma membrane. We sought for RHOA G17V partners performing co‐immunoprecipitation experiments with Jurkat cells expressing empty vector or different RHOA variants (WT, G17V and K18N) followed by mass spectrometry. Results: The presence of RHOA G17V and CD28 T195P mutants induced significantly higher levels of IL‐2 secretion and IL‐2 transcription than either mutant alone upon CD3/CD28 stimulation, suggesting a synergistic effect. First, we sought for changes in phosphorylation of key TCR cytoplasmic transductors upon CD3/ CD28 stimulation. Similar phosphorylation levels were found in cells harboring both mutants versus CD28 mutant alone. Then we addressed the activity of NF‐kB, NFAT and AP1 transcription factors. Luciferase assays showed a significant increase of the transcriptional activity of NFAT and, to a lesser extent, AP1 factors, in cells harboring both mutants versus either mutant alone. Thus, in this model the cooperation of RHOA and CD28 mutants on increased IL2 secretion does not involve increased phosphorylation of TCR signaling molecules, but an increased NFAT and AP1 transcription activity. We then searched for RHOA G17V partners that could explain our observations. Co‐immunoprecipitation analysis identified about 20 interactors specifically associating to RHOA G17V, among which those most abundant were VAV1 and the acetyl transferase P300. NFAT luciferase experiments were conducted with either VAV1 siRNA or P300‐dependent NFAT transactivation inhibition by expressing the E1A viral protein. Both VAV1 siRNA and P300 inhibition induced a decreased NFAT transactivation in cells expressing either mutants alone or both. Moreover, P300 inhibition by the small molecule A‐485 abolished the increased IL‐2 secretion induced by CD3/CD28 stimulation in cells transfected with RHOA G17V, CD28 T195P, or both. Conclusion: Our results suggest that RHOA G17V expression does not involve increased phosphorylation of TCR signaling molecules but rather potentiates CD28 T195P‐increased NFAT transactivation through VAV1 and P300 activities modulation, leading to a synergistic IL2 secretion.

by the small molecule A ‐ 485 abolished the increased IL ‐ 2 secretion induced by CD3/CD28 stimulation in cells transfected with RHOA G17V, CD28 T195P, or both. Conclusion: Our results suggest that RHOA G17V expression does not involve increased phosphorylation of TCR signaling molecules but rather potentiates CD28 T195P ‐ increased NFAT transactivation through VAV1 and P300 activities modulation, leading to a synergistic IL2 secretion. procedures. Here, we explored the value of ctDNA in PCNSL patients for disease classification, MRD detection, and early prediction of relapse. patient. Pretreatment plasma ctDNA was detectable in 82% of patients and in 100% of CSF samples (Fig. 1A), with ctDNA concentrations ranging from 0.013 ‐ 1038 hGE/mL (median: 0.97) in plasma and 0.043 ‐ 4342 hGE/mL (median: 3.55) in CSF (Fig. 1B). While ctDNA levels were significantly correlated with total tumor volume in MRI (p = 0.004, Fig. 1C), we did not observe significant associations between ctDNA levels and MSKCC score or concur-rent steroid treatment (Fig. 1D ‐ E). Pretreatment ctDNA was significantly associated with PFS (p = 0.0005, HR 3.6) and OS (p = 0.019, HR 3.1), both as continuous and binary variable (Fig. 1F). Furthermore, ctDNA positivity during curative intent induction therapy accurately predicted clinical outcomes (Fig. 1G). Finally, we applied our novel machine learning classifier to 129 specimens from an independent validation cohort. We observed high specificity (100%) and positive predictive value (100%) for noninvasive diagnosis of CNSL, with moderate sensitivity (50% for CSF, 20% for plasma), suggesting that a significant subset of CNSL patients might be able to forego invasive biopsies. Conclusions: We demonstrate robust and ultrasensitive detection of ctDNA at various disease milestones in PCNSL. Our findings suggest that ctDNA could serve as a valuable clinical biomarker for tumor burden assessment, outcome prediction, and biopsy ‐ free lymphoma classification. We envision an important future role of ctDNA for personalized risk stratification and guiding therapies in clinical trials and in routine PCNSL management. Biomarkers, Liquid biopsy, Extranodal non ‐ Hodgkin lymphoma