A 35-year-old female patient was admitted with major joint pain and fatigue for 1 month. There were 74.55% blasts in the bone marrow (BM) detected by BM aspiration, and 70.71% abnormal B lymphocytes in the BM measured by flow cytometry (FCM). The patient's leukemia cells expressed CD19+CD22+CD10+CD38+CD20- (Fig. 1A). She was diagnosed with B-cell ALL. Cytogenetic studies showed a complex karyotype: 47, XX, +5, t(12; 17) (p13, q21) [4]/47, idem, add (1) (p36.1), add (18) (q23), inc [cp6]/46, XX[15]. No genetic mutation was found in the leukemia cells. After two courses of induction therapy with vincristine, daunorubicin, cyclophosphamide, and prednisolone (VDCP), 1.98% leukemia blasts in the BM measured by FCM were revealed, which indicates there was no remission of B-cell acute lymphoblastic leukemia (B-ALL). Then a lumbar puncture and intrathecal injection of cytarabine, methotrexate, and dexamethasone were performed. After a following two courses of Hyper-CVAD B (methotrexate, leucovorin, and cytarabine) and one cycle of inter-median dose cytarabine chemotherapy, the patient never achieved complete remission (CR), and the leukemia cells in the BM reached 50.45% measured by FCM.
The patient was enrolled in a clinical trial of anti-CD19-chimeric antigen receptor (CAR) T-cell therapy (ChiCTR1800019622) as a treatment for refractory B-ALL in our hospital. The expression of leukemia cells measured by FCM was consistent with the initial diagnosis of leukemia. After her autogenous peripheral blood mononuclear cells (PBMCs) were collected  , she received lymphodepleting chemotherapy with fludarabine (30 mg/m2) and cytarabine (1,000 mg/m2) from day -12 to day -8 before anti-CD19 CAR T-cell infusion. Autologous anti-CD19-CAR T cells were infused on day 0 (1 x 106 cells/kg). During anti-CD19-CAR T-cell therapy, adverse events (AEs) manifested by fever with chills, fatigue, weakness, headache, dizziness, edema, increased transaminas, cough, and tachycardia started 3 days after CAR T-cell infusion. The highest temperature was 39.2 C, recorded on day 7 after the infusion. The patient was diagnosed with grade 2 cytokine release syndrome (CRS) according to the National Cancer Institute Common Terminology Criteria for AE v4.03   and grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS)  . Antipyretic drugs, methylprednisolone, and symptomatic treatment were used to overcome these AEs. The amplification of anti-CD19-CAR T cells in CD3+ T cells in the peripheral blood was detected on days 0, 4, 7, 14, and 28 after infusion measured by FCM. The anti-CD19-CAR T-cell peak reached 35.24% on day 7. DNA levels of the anti-CD19-CAR gene were detected by quantitative polymerase chain reaction (qPCR) and reached their peak of 24,500 copies/ng gDNA on day 14. The cytokines, including interleukin-6 (IL-6), IL-2R, and tumor necrosis factor-alpha (TNF-alpha) in peripheral blood were detected by enzyme linked immunosorbent assay (ELISA) and reached their peaks on day 7 after infusion (134 pg/mL, 6,800 U/mL, and 56.3 pg/mL, respectively). The patient achieved minimal residual disease (MRD)-positive (0.03%) CR with incomplete count recovery (Cri) measured by FCM at 14 days after anti-CD19-CAR T-cell infusion with no evidence of leukemic cells in cerebrospinal fluid (CSF) analysis.
At 28 days after anti-CD19-CAR T-cell therapy, the patient received a bridging therapy of haplo-SCT. Before the transplantation, computed tomography (CT) of the head, chest, abdomen, and pelvis showed no abnormalities. The patient had no extramedullary disease before haplo-SCT. DNA copy numbers of EBV and cytomegalovirus (CMV) measured by real-time qPCR were negative. The patient underwent haplo-SCT from a haploid donor (daughter, 5/10 HLA match). Prior to haplo-SCT treatment, the patient underwent regimens including total body irradiation (TBI, 3 Gy/day, -9 to -7 day), cyclophosphamide (Cy, 50 mg/kg/day, -6, and -5 day), fludarabine (Flu, 30 mg/m2/day, -4 to -2 days), and cytarabine (Ara-C, 3.0 g/m2/day, -4 to -2 days). Graft-versus-host disease (GVHD) prophylaxis consisted of ATG, cyclosporin A (CsA), and mycophenolate mofetil (MMF). The dose of CD34+ cells from PBMCs from her donor was 4.28 x 106 cells/kg. The patient's neutrophils and platelets were engrafted on 12 and 25 days after haplo-SCT, respectively. She achieved MRD-negative Cri determined by FCM at 14 days after haplo-SCT, and full donor chimerism according to short-tandem repeat (STR) at 28 days after haplo-SCT. Grade 1 aGVHD of the skin occurred 32 days after haplo-SCT and was in remission after short course of glucocorticoids and CsA therapy. EBV and CMV DNA copies in the blood were measured by qPCR and monitored weekly after haplo-SCT. The patient maintained an MRD-negative in the BM, full donor chimerism in the BM, and negative DNA copy numbers of EBV and CMV in peripheral blood. The patient received intrathecal chemotherapy monthly after haplo-SCT to prevent central nervous system leukemia.
At 91 days after haplo-SCT, the patient presented with dizziness and fatigue, without fever, nausea, vomiting, and lymphadenopathy. After admission to our center, the patient was MRD-negative in the BM (Fig. 1B), and donor chimerism in the BM was at 100%. The DNA copies of EBV and CMV in the peripheral blood were negative. Lumbar puncture revealed that the intracranial pressure exceeded 280 mmH2O, and protein in the CSF was 130.60 mg/dk. No leukemia cells or abnormal expression cells were detected in the CSF by FCM. The patient received methotrexate (MTX, 10 mg) combined with Ara-C (25 mg) by intrathecal injection. CT of the chest, abdomen, and pelvis showed no abnormalities. Magnetic resonance imaging (MRI) demonstrated multiple abnormal hyperintensity lesions surrounded by edema in the cerebellar vermis and the left cerebellar hemisphere (Fig. 1C). Dynamic enhanced MRI demonstrated intracranial neoplasm surrounded by edema in the cerebellar vermis and the left cerebellar hemisphere, which was considered to be an intracranial invasion of leukemia (Fig. 1D, E). At this time, the patient was temporarily diagnosed with IEMR after haplo-SC. Therefore, CsA administration (50 mg/day) was discontinued immediately and symptomatic dehydration treatment was started. The patient enrolled in another clinical trial of anti-CD22-CAR T-cell therapy (ChiCTR1800019298) as a relapsed or refractory (R/R) B-ALL in our center. Her donor provided the PBMCs for anti-CD22-CAR T-cell therapy, but the symptoms of dizziness and fatigue were not relieved, there was still no fever, nausea, vomiting, or lymphadenopathy.
A second lumbar puncture and intrathecal injection were performed. The intracranial pressure was 280 mm H2O, and protein in the CSF was 142.80 mg/dk. No leukemia cells were detected in the CSF by FCM. Next-generation sequencing (NGS) of the CSF was performed to rule out a diagnosis of infectious disease of the CNS. The DNA analysis showed 2,782 EBV sequences, and RNA analysis showed 174 EBV sequences in the CSF measured by NGS methods. We clarified the diagnosis of IEMR after haplo-SCT. Although the patient was EBV-DNA negative in peripheral blood, she did have EBV-DNA in her CSF, suggesting a diagnosis of PTLD. The patient underwent a cerebellar lesion biopsy and a left ventricular external drainage via robotic surgical assistant (ROSA, Medtech, Montpellier, France) using the assisted stereostatic method. The external ventricular drainage tube was kept open intermittently after surgery. The symptoms of dizziness and fatigue were slightly relieved after the surgery. The biopsy of the brain tissue showed diffuse infiltration of small round cells with deep staining (Fig. 1F, G). The tumor cells stained positive for CD19, CD20, Ki-67 (approximately 75%) (Fig. 1H-J), and C-myc (approximately 25%). Although the result of EBV-encoded RNA (EBER) in situ hybridization was sparsely positive (Fig. 1K), NGS of the brain tissue was positive for EBV. The brain tissue DNA analysis showed 3,815 EBV sequences, while the brain tissue RNA analysis showed 3,140 EBV sequences. The brain tissue and the BM specimens showed full donor chimerism and were of great diagnostic value. Based on these results, we confirmed the diagnosis of CNS-PTLD and identified monomorphic small B-cell lymphoma with EBV infection. A ventriculoperitoneal shunt was placed first before an anti-CD22-CAR T-cell Zanubrutinib combination therapy. Unfortunately, the patient suddenly developed drowsiness and cognitive impairment and died during the combination therapy.