PMC6901822_01
Morphologic examination of leukemic cells obtained from bone marrow aspirates exhibit (A) Leukemic cells with nucleonic vacuolation. (B) Megakaryocyte dysplasia (C) hemophagocytosis (D) cytoplasmic vacuolation. (E) G-banded bone marrow karyogram showing 46,XY,t(4;8)(q28;q24.1), t(16;21)(p11.2;q22). Black arrows indicate abnormal karyotypes.
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F1
The morphology examination exhibited megakaryocyte dysplasia, erythrophagocytosis, vacuolation in both cytoplasm and nucleus in leukemia cells in BM (Figures 1A-D). HLA-DR, CD13, CD33, CD15, CD64, CD11b, CD56, CD117, CD123, MPO, and CyCD3 in BM. His karyotype showed 46, XX, t(4;8) (q28;q24.1,t(16;21) (p11.2;q22) /46, XY (Figure 1E).
Evaluation of clinical response and hemogram changes during treatment. (A) Changes in tumor burden and donor chimerism during treatment in BM. * data from day 56 were obtained from PB samples because the BM samples were unobtainable. (B) Hemogram changes during treatment. (C) Process of donor-derived CART123 infusion and haplo-HSCT. The patient received a RIC regimen of TVFB and CART123 infusion. Given that the patient possibly developed an intense reaction during ATG infusion, basiliximab was administered. CsA/MTX/MMF was administered for GVHD prophylaxis after PBSC. Thus, PBSC was infused on day 0 and 1. Due to poor implantation, the patient received infusions of PBSC again on days 18 and 19. CART123, CD123-targeted chimeric antigen receptor (CAR) T cell; BM, bone marrow; FCM, flow cytometry; PB, peripheral blood; CRi, complete remission (CR) with incomplete blood count recovery; PLT, platelet; Hb, hemoglobin. DA, daunorubicin, cytarabine; MA, mitoxantrone, cytarabine; IDA, idarubicin, cytarabine; allo-HSCT, allogeneic hematopoietic stem cell transplantation; DCAG, decitabine, cytarabine, aclacinomycin, G-CSF; DMA, decitabine, mitoxantrone, Ara-c; CLAG, cladribine, Ara-c, G-CSF; DLI, donor lymphocyte infusion; TVFB, therarubicin, teniposide, fludarabine, busulfan; RIC, reduced-intensity conditioning; THP, therarubicin; FLU, fludarabine; VM-26, teniposide; Bu, busulfan; CsA, Cyclosporine A; MMF, mycophenolate mofetil; MTX, Methotrexate; Cy, Cyclophosphamide; haplo-HSCT, haploidentical hematopoietic stem cell transplantation; PBSC, peripheral blood stem cell; ATG, anti-thymocyte globulin; GVHD, graft-vs-host disease.
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CLAG + DLI. CNSL was controlled, but the disease progressed (Figure 2A). CD34+ 4.8 x 106/Kg, CD3+ 4.1 x 108/Kg) 6 days after CART123 infusion. Considering the potential hematopoietic toxicity of CART123, the second infusion of PBMC will be performed to promote implantation if the hematopoietic system remains unrecovered within about 14 days (Figure 2C). The patient achieved myeloid implantation on day 42 but was not weaned from platelet (PLT) and red blood cell (RBC) transfusion (Figures 2A,B).
CAR design and expression; the expansion of CART123. (A) The structure of retroviral vectors encoding anti-CD123 CARs. The 41BB CAR consisted of anti-CD123 scFv FMC32716, a CD8a hinge region, CD8 transmembrane and cytoplasmic regions, and a CD3zeta cytoplasmic region. tEGFR was connected with the anti-CD123 CAR via the P2A peptide in this investigation. (B) CAR expression and CD3, CD4, CD8, CD45RO, and CD62L on T cells was detected by flow cytometry. CAR expression on T cells was detected by anti-EGFR antibodies. (C) Changes in the proportion of T lymphocytes in PB after CART123 infusion. (D) Changes in CAR copies in PB after CART123 infusion by qPCR. CAR, chimeric antigen receptor; CART123, CD123-targeted CAR T cell; PB, peripheral blood; qPCR, quantitative polymerase chain reaction; EGFR, human Epidermal Growth Factor Receptor.
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Truncated human Epidermal Growth Factor Receptor (EGFR) polypeptide (tEGFR) was integrated with CAR gene through a P2A peptide (Figure 3A). The viability was 89.0%, and the CD4+/CD8+ ratio was 1.81. Of the infused cells, 98.2% were CD3+ cells principally composed of the CD8+ subset (30.2%) and CD4+ subset (54.7%), and 8.43% and 90% of CAR+ cells were characterized with the central memory phenotype (CD45RO+/CD62L+) and effect memory phenotype (CD45RO+/CD62L-), respectively (Figure 3B). T lymphocytes (CD3+, CD4+, and CD8+) in PBMC was significantly increased after CART123 infusion. Then, T cells were sharply reduced after the administration of medications, such as methylprednisolone, ATG, and basiliximab (Figure 3C). Direct evidence of CART123 amplification was detected by qPCR (Figure 3D).
Trends of serum cytokines, body temperature, and major blood biochemical indexes after CART123 infusion. (A) Cytokines changed after CART123 infusion. Serum cytokine levels were measured at the indicated time points before or after CART123 and PBSC infusions. (B) Changes in body temperature after CART123 infusion. (C) Changes in CRP and LDH levels after G-PBSC infusion. (D) Changes in Cre, TBiL, DBiL, and ALT levels after G-PBSC infusion. CART123, CD123-targeted chimeric antigen receptor (CAR) T cell; PBMC, peripheral blood mononuclear cell; IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; CRP, C-reactive protein; ALT, Alanine transaminase; aGVHD, acute graft-vs-host disease; CsA, Cyclosporine A; MMF, mycophenolate mofetil; GC, glucocorticoids; UCB-MSC, umbilical cord blood mesenchymal stem cells; DIC, disseminated intravascular coagulation; LDH, lactate dehydrogenase; Cre, creatinine; TBiL, total bilirubin; DBiL, direct bilirubin.
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Considering that tocilizumab on days -5 (240 mg) and -3 (400 mg) was invalid, methylprednisolone was administered from days -2 to 8 (day 4-7: 2 mg/kg for the first dose, 1 mg/kg q12h; d8-10: 2 mg/kg q12h) and the dose was gradually decreased. CRS was rapidly controlled after the infusion of methylprednisolone and ATG, with the decline of CRP, LDH, body temperature, and IFN-gamma (Figures 4A-C). On day 28, he developed disseminated intravascular coagulation (DIC) due to infection and was controlled by the symptomatic treatment (Figure 4C). Unfortunately, the patient died of aGVHD, severe pneumonia, intestinal obstruction, and multiple organ failure on day 56 (Figure 4D).