3 Results
Patient characteristics were shown in TABLE 1 . Briefly, a total of 8 extramedullary relapsed childhood B-ALL patients with the average age of 7.9 years old (range, 4.3-13.0) were enrolled in this study and treated at Shanghai Children’s Medical Center (6 patients) and Children’s Hospital of Soochow University (2 patients). There were 4 patients infiltrated with CNS, 1 with testis, 2 with subcutaneous tumor and 1 with both CNS and testis. All patients were all relapsed. The mean time follow-up time was 21.9 months (5.93 to 39.97 months). In addition, 5 of 8 patients (39.4%) relapsed more than once in which merely patient S003 relapsed after HSCT. Furthermore, all patients received fresh CAR-T product and the average infusion dose was 9.9×106 per kilogram of body weight (range, 1.8–15.0).
All 8 patients achieved complete remission (CR), in which 5 patients are continuous CR, and no CAR-T related death happened. Patients S001 was bridged to HSCT after the first CR of CAR-T. Patient-level clinical response outcomes are exhibited in Fig. 1A . The estimated EFS of patients with 19CAR-T cells was approximately 68.57% at 12 months and decreased to 51.43% at 24 months. Patients S002, S004 and S006 were relapsed with CD19+ and CD22+ blasts after prior exposure to CAR-T cells. Therefore, they sequentially accepted combined CD19 and CD22 CAR-T cells as a second CAR-T therapy at dose of 1.5×107, 4.26×106 and 1.2107 per kilogram of body weight, respectively (TABLE 2 ). Then, all of them achieved CR again (CR2). To date, none of them chose bridging to HSCT after the second CAR-T.
Except patient S006, CRS was observed in all patients. 3 in 8 patients were grade 3~4. CAR-T related encephalopathy syndrome (CRES) was observed in 6 patients (75%) and no grade 3~4 CRES was identified. 4 patients received the tocilizumab, in which 2 patients also received glucocorticoids simultaneously. Only patient S004 received glucocorticoids intrathecal injection alone. No patient died of severe CRS or CRES (TABLE 2 ). Interleukin 6 (IL-6) and interferon γ (IFNγ) concentrations are main biomarkers of CRS severity. Similar patterns were observed in both IL-6 and IFNγ (Fig. 2A ), the highest level of IL-6 was 60618.45 pg/ml detected in patient S003 (TABLE 2 ). At day 7, transient increases in levels of serum IL-6 and IFNγ occurred in most patients during CRS after CAR-T administration. We also observed that a drop of IL-6 was always followed with a lift of lymphocytes in peripheral blood (Fig. 2B ), which was temporally in accordance with the resolution of CRS.
Different from the symptoms of testicular infiltration after chemotherapy, patients had fever and the affected testis showed progressive redness and swelling 3~4 days after CAR-T infusion, and patient may have a little pain. Although the testicle was swollen, it feels soft by physical palpation. The swelling testis reached the most serious situation in about a week, then began to decrease and continue to soften gradually until it turned close to normal size.
Patients with CNS involvement had more severe symptoms of encephalopathy manifested as high fever, headache, frequent vomiting, drowsiness, unconsciousness, hypotension or hypertension, limb trembling, convulsions. For patient with convulsions, lumber puncture was required, and the cerebrospinal fluid (CSF) protein increased significantly, meanwhile, the IL-6 concentration in CSF was much higher than that in peripheral blood. For example, on day 6, patient S004 suffered 1272.27 pg/ml of IL-6 in CSF whereas 26.62 pg/ml in peripheral blood. We also detected higher level of CAR gene in CSF compared with peripheral blood. In patient S005, 465.026×103 copies CAR per ug DNA was detected, which was over 10 times more than that detected in peripheral blood on day 3 (Fig. 2C ). Additionally, CAR-T cells expansion were determined by real-time qPCR and the result showed that the peak CAR-T expansion was variable in different EM relapsed patients but not observed after Day 7 (Fig. 2D ).