A 25-year-old male was diagnosed with AML-M2 1 year ago, according to the French-American-British classification. Bone marrow (BM) morphology revealed 62.2% blasts, and peripheral blood (PB) was manually sorted by 39% blasts. The morphology examination exhibited megakaryocyte dysplasia, erythrophagocytosis, vacuolation in both cytoplasm and nucleus in leukemia cells in BM (Figures 1A-D).
Immunophenotyping by flow cytometry (FCM) analysis revealed positive results for CD34, CD38, 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). The FUS-ERG fusion gene was positive at 21.96% quantitatively in BM. He received induction chemotherapy DA (daunorubicin, cytarabine) and reinduction MA (mitoxantrone, cytarabine) and achieved CR. Then, he received two cycles of chemotherapy MA and IDA (idarubicin, cytarabine) and achieved minimal residual disease (MRD) negative by FCM. He received a human leukocyte antigen (HLA)-matched unrelated donor allo-HSCT after cyclophosphamide and total body irradiation (TBI) as preconditioning followed by Cyclosporine A (CsA), mycophenolate mofetil (MMF), basiliximab and short-term Methotrexate (MTX) for prophylaxis of graft-vs-host disease (GVHD). He achieved MRD-negative CR 1 month after HSCT but relapsed 2 months later.
Then, he successively received DCAG (decitabine, cytarabine, aclacinomycin, G-CSF), DMA (decitabine, mitoxantrone, Ara-c), and CLAG (cladribine, Ara-c, G-CSF) combined with donor lymphocyte infusion (DLI) and achieved transient CR with MRD positive. He developed an anal fissure and perianal abscess, and the infection was controlled by anti-infective therapy. He subsequently relapsed 1 month later with central nervous system leukemia (CNSL) and was administered four cycles of Ara-c, MTX, and DXM by intrathecal injection and CLAG + DLI. CNSL was controlled, but the disease progressed (Figure 2A).
The patient received reduced-intensity conditioning (RIC) regimen of TVFB (therarubicin 60 mg on d1 and 40 mg on d2-3; teniposide 200 mg on d1 and 150 mg on d3 and d5; fludarabine 50 mg on d1-5; and busulfan 3 mg/kg on d6-8) and CART123 1 day after preconditioning. The total infused CART123 was 1.1 x 108 cells, and 9 x 107 cells (CAR+ 80.2%) were CAR+ cells (1 x 106/kg). This second-generation CAR consisted of anti-CD123 single chain fragment variable (scFv), CD8a hinge region, CD8 transmembrane domain, 41BB costimulatory domain, and CD3zeta cytoplasmic region. 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). Both the stem cells and the CAR T cells were from his father, who exhibited a 5/10 HLA loci matching and ABO incompatibility with the patient. Subsequently, 4 days after CART123 infusion, anti-thymocyte globulin (ATG; 2.5 mg/kg/d 3d) was administrated for prophylaxis GVHD. However, during the second infusion of ATG, the patient developed tachypnea, tachycardia, and persistent hypoxemia. Given these serious side effects, the third-day infusion of ATG was canceled. Instead, the prophylactic regimen is adjusted to basiliximab (20 mg/d; days 0, 4, and 8), CsA, MTX (0.33g d1 0.02g d3, 6), mycophenolate mofetil (MMF; 1.8g 1/day 1.5g 1/night), and ATG (2.5 mg/kg/d 2d). Granulocyte colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSC) was infused (mononuclear cells 11.77 x 108/Kg, 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 blasts in BM decreased from 40.8 to 10.3% by FCM 6 days after CART123 infusion and decreased from 38 to 8% by morphology 14 days after CART123 infusion. On day 18, the second donor engraftment achieved 97.7% in BM. Although G-CSF was administrated to promote implantation from day 6, the hematopoietic system remains unrecovered until day 16. Thus, cyclophosphamide (4150mg) was administered as conditioning regimen and G-PBSC was infused again on day 18 and day 19 (mononuclear cells 14.28 x 108/Kg, CD34+ 4.74 x 106/Kg, CD3+ 4.44 x 108/Kg). On day 32, blasts in BM were 0.5, 0.05, 0.042, and 0.02% by morphology, FCM, Wilms tumor-1 (WT1) and FUS-ERG detection, respectively. Compared to the first allo-HSCT, the second allo-HSCT was conducted in non-remission status using a RIC regimen, indicating the anti-leukemic activity of CART123. The patient achieved myeloid implantation on day 42 but was not weaned from platelet (PLT) and red blood cell (RBC) transfusion (Figures 2A,B).
The proportion of 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).
The patient developed a fever (>39 C), hypotension (92/58 mmHg) and pneumonia within 24 h after infusion, and these effects were evaluated as grade 3 CRS. He was immediately administered tocilizumab, a pressor agent and empirical anti-infective therapy. Assessment of cytokines in serum revealed an increasing trend for IL-6 and IFN-gamma, and the effects in IL-6 was most obvious. Four days later, dyspnea, progressive pneumonia, and fever persisted (up to 41 C), and these features were evaluated as grade 4 CRS. The changing trend of C-reactive protein (CRP), lactate dehydrogenase (LDH), and body temperature was consistent with the level of cytokines and the clinical symptoms of the patient. 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).
The patient has an anal fissure before transplantation, and then it progressed to anal fistula with perianal infection after transplantation. However, the perianal infection caused repeated sepsis and pneumonia. Intermittent fevers occurred and were accompanied by sharp elevations in CRP and LDH after allo-HSCT. Repeated anti-infective, symptomatic and supportive treatment was administered to the patient and exhibited effective results. On day 28, he developed disseminated intravascular coagulation (DIC) due to infection and was controlled by the symptomatic treatment (Figure 4C).
On day 32, after CRi was achieved, he soon developed fever, vomit, stomachache, and severe diarrhea. Total bilirubin (TBiL) progressively increased, mainly direct bilirubin (DBiL). He was diagnosed with aGVHD and administered by CsA, glucocorticoids (GC), MMF, basiliximab, tacrolimus, and maraviroc were successively for the treatment of aGVHD. On day 48, a total number of 7 x 107 umbilical cord blood mesenchymal stem cells (UCB-MSC) were administered for the treatment of aGVHD. Finally, he was diagnosed with grade IV aGVHD involving liver and gut. In the final stage, creatinine increased progressively, reflecting the deterioration of renal function. Unfortunately, the patient died of aGVHD, severe pneumonia, intestinal obstruction, and multiple organ failure on day 56 (Figure 4D).