PMC9330448_01
(A) FDG-PET/CT coronal maximum intensity projection (MIP) image before sTLI showing avid uptake of BM, multiple lymph nodes and iliopsoas muscle. (B, C) sTLI dose distribution color wash (B), coronal view (C), sagittal view. (D) Massive BM involvement by DLBCL. The asterisk * indicates a large area of necrosis. T indicates a BM trabecula (Hematoxylin-Eosin; x 100). (E) An area from the same section as (D) showing infiltration by low grade B-cell lymphoma and occasional large cells (arrows) (Hematoxylin-Eosin; x400). (F) The same section as (D) showing another area infiltrated by DLBCL cells (Hematoxylin-Eosin; x400), that express the CD19 molecule (G) (Leica immunoperoxidase staining; x400). (H, I) Total marrow irradiation (TMI) dose distribution color wash (H), coronal view (I), axial and sagittal view.
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After a FEAM conditioning regimen (fotemustine, etoposide, cytarabine, and melphalan), she underwent an autologous hematopoietic stem cell transplantation (auto-HSCT) without significant response (Figure 1A). In particular, 20 Gy was delivered in 10 fractions over 5 days in all PET/TC positive tumor sites (i.e., the left iliopsoas muscle and all the main nodal stations, minus the mediastinum) except for the spleen, which received 11.5 Gy (Figures 1B, C). A BM biopsy showed massive involvement by DLBCL expressing CD19 (Figures 1D-G). For this reason, the patient received TMI (18 Gy; 1.8 Gy x 2/die for 5 days) (Figures 1H, I), followed by thiotepa (5 mg/kg) and an infusion of residual, previously collected autologous CD34+ hematopoietic stem cells (5.5 x 106/kg).
(A-C) FDG-PET/CT coronal maximum intensity projection (MIP) image after sTLI/TMI and before CART cells; white arrow in (A) indicates metabolic uptake in the iliopsoas muscle (A) FDG-PET/CT after CD19-directed CAR-T-cell therapy showing metabolic complete response at 6 months (B) and DLBCL relapse at 11 months (C). (D) Imprint of latero-cervical lymph node at relapse immunostained for the CD19 CAR target (detected in red). Almost all large lymphoma cells appear CD19-negative (single arrows); the double arrow points to a CD19-negative tumor cell in mitosis. The red arrow indicates a CD19 positive (red) large tumor cell while the arrowhead indicates a CD19 positive (red) normal small B lymphocyte (Alkaline Phosphatase Anti-Alkaline Phosphatase (APAAP) technique; x400). Negativity of >95% of tumor cells for CD19 was also confirmed in frozen and paraffin sections of the lymph node (not shown). (E) BM biopsy taken 11 months after CAR-T cell infusion showing a markedly hypocellular marrow without lymphoma infiltration. T indicated BM trabecula. (Hematoxylin-Eosin; x400). (F) Flow cytometry plots showing CAR T cells detected in the CD3+ T-cell subsets in the peripheral blood every week the first month after CAR-T cell infusion and in bone marrow aspirate at day 28-disease assessment (top). Comparison of CAR-T cell absolute count expansion between the patient in subject and mean of the other treated patients in our center (n = 12) at indicated time points after CAR T-cell infusion (bottom). CAR T cells were detected staining anti-CD19 CARs by the biotinylated CD19 CAR detection reagent (Miltenyi) together with anti-biotin-APC. (G) Immunofluorescence image stained by biotinylated CD19 CAR detection reagent (Miltenyi) together with anti-FC FITCH conjugated secondary antibody (Thermo-Fisher, green) and DAPI (for cell nuclei, blue) performed on cytospin preparation from the peripheral blood of the patient obtained 14 days after CAR T-cell infusion. The white arrow indicates a CAR-T cell probably embracing a leukemic B cell.
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TMI therapy, the PET/CT showed the disappearance of all metabolic positive lymphadenopathy but persistence of uptake in the left iliopsoas muscle (Figure 2A), while BM biopsy revealed about a 50% reduction of tumor cells. Indeed, PET/CT scans performed 1, 3, and 6 months after tisagenlecleucel showed CR (Figure 2B). CAR-T-cell therapy. CAR T-cell expansion, monitored by flow cytometry every week after CAR T infusion, showed high CAR T cell levels in peripheral blood 14 days after infusion (276 CAR T positive cells/microliter) (Figures 2F, G). BM biopsy showed a markedly hypocellular marrow without infiltration by lymphoma (Figure 2E). Because of her young age and good performance status, she is now being considered for haploidentical HSCT (Figure 3, timeline of events).
Timelines of events.
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BM biopsy showed a markedly hypocellular marrow without infiltration by lymphoma (Figure 2E). Because of her young age and good performance status, she is now being considered for haploidentical HSCT (Figure 3, timeline of events).