Successful therapy of chimeric antigen receptor T cells for isolated extramedullary acute lymphoblastic leukemia

Despite many clinical trials of CD19-targeted CAR T cells for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) having been reported, few studies of CD19 CAR T therapy for isolated refrac-tory/relapsed (r/r) extramedullary ALL (EM-ALL) patients have been published. Extramedullary involvement is considered an unfavorable prognostic factor, which often reduces leukemia response to induction chemotherapy and is usually associated with shorter progression-free survival (PFS) and overall survival (OS) [1, 2]. There is still no standard guideline on the treatment of isolated EM-ALL [3]. Considering the absence of leukemia blasts in the peripheral blood and bone marrow in isolated EM-ALL patients, the questions of whether CAR T cells can successfully expand in these patients and whether these patients can benefit from CD19 CAR T therapy remain unanswered. In this study, we evaluated the safety and efficacy of CD19 CAR T cells for isolated r/r EM-ALL patients (ChiCTR2000038532).

published. Extramedullary involvement is considered an unfavorable prognostic factor, which often reduces leukemia response to induction chemotherapy and is usually associated with shorter progression-free survival (PFS) and overall survival (OS) [1,2]. There is still no standard guideline on the treatment of isolated EM-ALL [3]. Considering the absence of leukemia blasts in the peripheral blood and bone marrow in isolated EM-ALL patients, the questions of whether CAR T cells can successfully expand in these patients and whether these patients can benefit from CD19 CAR T therapy remain unanswered. In this study, we evaluated the safety and efficacy of CD19 CAR T cells for isolated r/r EM-ALL patients (ChiCTR2000038532).
Our cohort enrolled nine refractory or relapsed isolated EM-ALL three consecutive days before the CD19 CAR T cell infusion. As for the patients with CNS infiltration, intrathecal chemotherapy was performed to reduce blasts in the cerebrospinal fluid (CSF) before CAR-T cell infusion. On day 0, all patients received CAR T cell infusion (3 × 10 5 to 10 × 10 5 per kg body weight). The doses were determined based on our previous studies and are described in Table 1. Once complete remission (CR) was achieved, some patients received consolidative hematopoietic stem cell transplantation (allo-HSCT).
Robust expansion and persistence of genetically modified T cells in vivo are critical for durable clinical remissions in patients with hematologic malignancies [4]. Previous studies report that exposure to CD19 protein in vivo can trigger the proliferation of CD19 CAR T cells [5]. Therefore, we first evaluated the expansion and persistence of infused CAR-T cells in the blood by flow cytometry (FCM). As shown in Figure 1A, infused CAR-T cell expansion peaked from day 7 to 17 in peripheral blood (PB). The median peak of the CAR T cells as a percentage of total lymphocytes was in the range of 3%-33.29%, and a rapid decline followed. The results indicated that CD19 CAR T cells had successfully expanded in most of the isolated EM-ALL patients, although at a comparatively lower level than those with leukemia blasts in bone marrow (BM) or PB or both (data not shown).
As for the persistence, dynamic FCM analysis showed that CAR T cells in PB persisted for ≤20 days and the percentage of CD19 CAR T cells of total lymphocytes decreased rapidly in most of the isolated EM-ALL patients ( Figure 1A, usually more rapidly than those with leukemia blasts in BM or PB or both based on our other studies, in the paper). CAR T cells in patient 6 were barely detectable on day 14 after infusion. CAR T cell percentage of total lymphocytes in the other eight patients also dropped below 2% on day 20 after infusion. This short duration in PB might be due to the lack of CD19 stimulation in PB or BM, further confirming that CD19 positive blasts in BM and PB not only can trigger CD19 CAR T cell proliferation but also help maintain its persistence, which is consistent with the previous view that circulating antigen is an important factor in the proliferation and persistence of CAR T cells in vivo.

The trafficking of CAR T cells to tumor sites is necessary to their
antitumor activity [6,7]. As shown in Figure 1B There is still controversy about whether allo-HSCT is necessary for r/r B-ALL patients after CAR T therapy. [8] Some findings have indicated no difference in leukemia-free survival (LFS) between the patients that underwent allo-HSCT after CAR T therapy and those who did not, [9] while other studies observed that bridging into allo-HSCT after CAR T therapy could improve LFS in r/r B-ALL patients. [10,11] In this study, as shown in Table S1, once achieved CR after CAR T-cell treatment, 6/9 of the patients proceeded to consolidative allo- In summary, based on this study, we envision that CAR T-cell therapy combining allo-HSCT is a safe and feasible regimen and offers new hope for these patients. However, only a small number of patients were enrolled in this study, and more cases and further studies are needed to verify these findings.

ACKNOWLEDGMENTS
We thank the patients who participated in this study and their families, the staff in the laboratory for immunotherapy from the Lu Daopei Hospital, the physicians in the Hematology Department for providing clinical consultation and care, and all the nurses for their devotion and care.
This study was funded by Kecellitics Biotech Company, a subsidiary of the Daopei Medical Group.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

AUTHOR CONTRIBUTIONS
Peihua Lu, Bo Chen, and Xiangqun Li designed the clinical trial. Kylan Chen, Xian Zhang, Junfang Yang, Jianwei Zheng, Fei Dong, Yongbo Zhu, and Jiao Yu executed the clinical trial and collected the data. Xiangqun Li analyzed the data and wrote the paper.

ETHICAL APPROVAL
This study was conducted according to the principles of the Declaration of Helsinki and with the approval of the Ethics Committee of Daopei Hospital.
All the enrolled patients or their families provided informed consent.