case_images: PMC10574963_01
case_images: (A) Timeline illustrating the therapies carried out and the outcomes. R-COMP: Prednisone, Cyclophosphamide, Vincristine, Liposomal Doxorubicin and Rituximab; R-DHAP, Rituximab, Dexamethasone, Cytarabine and Cisplatin; R-IEV, Rituximab, Ifosfamide, Epirubicin and Etoposide. (B) FDG-PET/CT image pre-CAR-T infusion revealed a hypermetabolic activity in the right lung and mediastinum (SUV max 18.4) with necrosis of the mediastinal mass. (C) FDG-PET/CT image at +30 days after CAR-T infusion showed partial response (PR) to therapy according to Lugano and Lyric criteria (SUVmax 4.9) with a new area of hypermetabolic activity in right lung and bronchus of infectious origin. (D) FDG-PET/CT image at +15 months demonstrated complete response (CR) to therapy according to Lugano and Lyric criteria. No hypermetabolic activity of infectious origin.
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case_images: Status score of 1 and no organ dysfunctions including normal left-ventricular ejection fraction. She was not candidate for an autologous stem cell transplant because of refractory disease and she was referred for treatment with CD19 CAR-T cell therapy using axicabtagene ciloleucel ( Figure 1A ). PET/CT scan revealed a hypermetabolic activity in the right lung and within the mediastinum ( Figure 1B ). PET/CT performed on day +30 showed partial response (PR) to CAR-T treatment ( Figure 1C ). TIM3 and LAG3) examined in all CAR-T cell subsets up to day +50 showed a constant expression over the time of the PD1 with a reduction of the expression of TIM3 and LAG3 in comparison with infused cells ( Figure 3C ). The patient is now in Complete Remission (CR) at last PET/TC scan (+ 15 months) ( Figure 1D ). The patient is now enjoying her life and reports well-being.
case_images: Monitoring of diagnostic CRS parameters including ferritin (A), IL-6 (B) and CRP (C).
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case_images: On day +1, the patient developed fever with unstable cardiopulmonary function and concomitant IL-6 arising ( Figure 2 ), which was classified as CRS grading 3 according to ASCT consensus grading.
case_images: Monitoring of infused CART+CD3+ cells. (A) Analysis of CAR T+CD3+ cells from patient-derived peripheral blood (PB) samples collected at different timepoints after infusion using both ddPCR and Flow Cytometry (expressed as CAR-T cells/muL). (A) For Flow Cytometry absolute count, PB-samples were stained with the following antibodies, as recommended by manufacturer's instructions: anti-CD45 (clone: 2D1), anti-CD3 (clone: SK7), anti-CD4 (clone: RPA-T4), anti-CD8 (clone: RPA-T8), anti-CD19 (clone: SJ25C1) and anti-CD16/CD56 (clone: 3G8/NCAM16.2) from BD Biosciences (BD Biosciences, La Jolla, CA); anti-CD19CAR (130-129-550) from Miltenyi Biotec (Miltenyi Biotech, Bergisch Gladbach Germany). Fluorescence Minus One (FMO) controls were used to set the background signals and to identify and gate the positive CD19CAR+ cells population on total CD3+T cells. Data acquisition was performed using BD FACS Lyric  instrument (BD Biosciences) and data were analyzed with the BD FACSuite  software version 1.4 with predefined and optimized settings for CAR T-cell analysis. A total of 300 ng of DNA extracted from peripheral blood was used for digital droplet PCR (ddPCR) analysis for CAR-T+ cell detection. The CAR-T CD-19 test was used according to the specific protocol (dEXD88164642 Bio-Rad, Hercules, CA). (B, C) Flow cytometry analysis of CART+CD3+ cells Memory Compartment and Exhaustion markers from post-infusion bag to day+50 collected samples. Briefly, cells were labeled with the following set of antibodies: anti-CD19CAR (130-129-550), anti-PD-1 (clone: REA802) and anti-TIM-3 (clone: F38-2E2) from Miltenyi Biotec; anti-CD45 (clone: 2D1), anti-CD3 (clone: SK7), anti-CD4 (clone: RPA-T4), anti-CD8 (clone: RPA-T8), anti-CD197 (clone: 150503), 7-AAD, anti-CD45RA (clone: HI100), anti-LAG-3 (clone: T47-530), anti-CD25 (clone: 2A3) and anti-HLA-DR (clone: G46-6) from BD Biosciences. Data acquisition was performed using BD FACS ARIA III  instrument (BD Biosciences) and data were analyzed with the BD FACSDiva  software version 9.0.1.
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case_images: Monitoring showed negativization of CD3/CAR-T on day +170 by flow cytometry, when there was still positivity in ddPCR (5.9 cell/microl of gDNA) ( Figure 3A ). T cells ( Figure 3B ). Exhaustion markers (PD1, TIM3 and LAG3) examined in all CAR-T cell subsets up to day +50 showed a constant expression over the time of the PD1 with a reduction of the expression of TIM3 and LAG3 in comparison with infused cells ( Figure 3C ). The patient is now in Complete Remission (CR) at last PET/TC scan (+ 15 months) ( Figure 1D ). The patient is now enjoying her life and reports well-being.