PMC9584643_01
Clinical course. (A) Clinical timeline with immunohistochemical evaluation of lymph node biopsy at pre-bispecific (anti-CD20Xanti-CD3) antibody stage, that shows CD19 and CD20 positivity, CD30 negativity, the expression of PD-L1 in the neoplastic population and PD-1 in the tumor microenvironment. Relevant PET/TC scans at: (B) pre-CAR-T cell therapy, (C) day +60 after CAR-T cell infusion. (D) Immunohistochemical evaluation of lymph node biopsy at: pre-PBZ administration stage, that shows CD19 and CD20 negativity, CD30 positivity, the expression of PD-L1 in the neoplastic population and PD-1 in the tumor microenvironment. Relevant PET/TC scans: (E) at day +90 after CAR-T cell therapy, (F) at day +42 following PBZ treatment, (G) after the 4th cycle of Brentuximab.
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A post-therapy lymph node biopsy revealed a diagnosis of DLBCL, germinal center B-cell (GCB) like, with the expression of CD19 and CD20 pan-B-cell antigens ( Figure 1A ). Tisagenlecleucel therapy. The fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT scan at screening showed multiple supra and sub diaphragmatic adenopathies and an extensive skeletal involvement ( Figure 1B ). The PET/CT scan at day+60 ( Figure 1C ) revealed a stable disease with a response in some lymph nodes, but higher FDG-avidity in others, in comparison to the PET/CT performed at day+30. At day +75 after CAR-T cell infusion, the patient underwent a PET-guided biopsy of the abdominal lymph nodes ( Figure 1D ). The diagnosis was of Hodgkin-like B cell lymphoma, with a defective B-phenotype due to the lack of CD20 and CD19 expression ( Figure 1D ). Moreover, a population of atypical CD30 positive lymphocytes, a strong PD-L1 expression in tumor cells, concomitant to the up-regulation of PD-1 expression in the tumor microenvironment were observed ( Figure 1D , see  Figure 1A  for comparison). The PET/CT scan at day +90 ( Figure 1E ) revealed a progression disease and at day +100, the patient received the first dose of PBZ (200 mg flat dose) showing a rapid resolution of symptoms the patient complained before starting therapy, such as abdominal pain, serotine fever and fatigue. Nevertheless, two months later, the patient experienced a clinical progression confirmed by PET/CT scan that showed FDG-uptake in multiple lymph nodes, right anonymous vein thrombosis and bilateral pleural effusion ( Figure 1F ). The PET/CT scan performed after the 4th cycle of therapy in October 2021 showed a complete response ( Figure 1G ). However, the percentage of non-CAR CD8+PD-1+ T cells increased again at day+42 ( Figure 2F ), when PET/CT scans showed signs of disease progression (see  Figure 1E ).
Flow cytometric analysis of peripheral blood CAR+ and CAR-non expressing T cell compartment. (A) Gating strategy used to identify the senescent/clonal expanded and exhausted CD8+ T cells in the CAR+ compartment. Pie charts report the percentage of exhausted and senescent/clonal expanded CAR+CD8+ T cells at (B) pre-PBZ and (C) D+21 and D+42 after PBZ. (D) Gating strategy used to identify the senescent/clonal expanded and exhausted CD8+ T cells in the non-CAR CD8+ T cell compartment. Pie charts report the percentage of (E) exhausted and senescent/clonal expanded CAR-CD8+ T cells at pre-PBZ, (F) exhausted and (G) senescent/clonal expanded CAR-CD8+ T cells at D+21 and D+42 after PBZ. (H) Identification of pathogenetic genetic alterations in the ccfDNA at LD (blu bars, n=16) and at day +42 (orange bars, n=18) and relative VAF. (I) Dot plots display CD3+ and CD3+CD19 CAR+ T cell populations before and after PBZ.
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Before PBZ treatment, the entire compartment of circulating CAR+ T cells was set up by a population of terminally differentiated (CD45RA+ CD62L-), senescent (CD28-CD57+), exhausted (PD-1+) CD8+ cells ( Figures 2A, B ). Notably, PBZ treatment did not modify the percentages of neither exhausted nor senescent CAR+T cells ( Figure 2C ). In the non-CAR cell compartment, a remarkably high percentage of CD8+ T cells showed exhausted (69%) and senescent (40%) phenotype at pre-PBZ time point ( Figures 2D, E ). However, the percentage of non-CAR CD8+PD-1+ T cells increased again at day+42 ( Figure 2F ), when PET/CT scans showed signs of disease progression (see  Figure 1E ). Figure 2G ). Finally, out of a total of 18 mutations detected in the circulating cell free (ccf) DNA at day +42, at least 4 mutations (in KDR, TP53, ARID1A and KMT2C loci) were almost undetectable in the ccfDNA at the LD timepoint, when a total of 16 mutations were detectable ( Figure 2H ).