CAR-T therapy followed by allogeneic hematopoietic stem cell transplantation for refractory/relapsed acute B lymphocytic leukemia: Long-term follow-up results

Background Patients with refractory/relapsed (r/r) acute B lymphocytic leukemia (B-ALL) can achieve complete response (CR) after chimeric antigen receptor T-cell (CAR-T) therapy, but recurrence occurs in the short term. To reduce recurrence and improve survival, CAR-T therapy followed by transplantation is a feasible option. We analyzed the long-term follow-up outcomes and the risk factors for allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CR by CAR-T therapy in this study. Methods A total of 144 patients who underwent allo-HSCT after CAR-T therapy in our hospital were enrolled in this study. Target gene analysis was performed in 137 r/r B-ALL patients receiving allo-HSCT after CR by CAR-T therapy. Among the 137 patients, 87 were evaluated for germline predisposition gene mutations, and 92 were evaluated for tumor somatic gene mutations using NGS. The clinical factors, germline predisposition gene and somatic gene mutations associated with the prognosis of patients receiving transplantation after CAR-T therapy were analyzed using univariate Cox regression. Factors related to disease-free survival (DFS) and overall survival (OS) were analyzed using multivariate Cox regression analysis. Results In 137 r/r B-ALL patients, the 2-year cumulative incidence of recurrence (CIR), OS and DFS in patients receiving allo-HSCT after CAR-T therapy was 31.5%, 71.4%, and 60.5%, respectively. The 2-year OS and DFS in MRD-negative patients were 80.9% and 69.3%, respectively. Univariate Cox analysis showed that pretransplant MRD positivity, fungal infection, germline EP300 mutation and somatic TP53 mutation were associated with a poor prognosis after transplantation; a TBI-based regimen was a protective factor for survival and recurrence after transplantation. Multivariate Cox regression analysis showed that the TBI-based regimen was an independent protective factor for DFS, fungal infection and MRD positivity were independent risk factors for DFS, and tumor somatic TP53 mutation and germline EP300 mutation were independent risk factors for DFS and OS. Conclusion Germline EP300 mutation and tumor somatic TP53 mutation are poor prognostic factors for posttransplant recurrence and survival in r/r B-ALL patients achieving CR after CAR-T therapy. The prognostic risk factors should be considered in adjusting treatment strategies to improve the efficacy of clinical diagnosis and treatment.


Introduction
Patients with refractory/relapsed acute B-lymphocytic leukemia (r/r B-ALL) usually have a poor prognosis even if they receive chemotherapy and salvage allogeneic hematopoietic stem cell transplantation (allo-HSCT). The 1-year disease-free survival (DFS) in patients with r/r B-ALL after salvage allo-HSCT is 20%-30%, and the 5-year overall survival (OS) is approximately 30% and decreases to 10% in patients with a second recurrence (1). In recent years, chimeric antigen receptor T-cell (CAR-T) therapy, as a new immunotherapy method, has increased the complete response (CR) rate of r/r B-ALL to 70%-93% (2)(3)(4)(5); however, 50%-67.9% of patients experience recurrence within one year (6,7). Our previous study showed that CD19/CD22 CAR-T therapy for r/r B-ALL followed by allo-HSCT significantly improved patient survival, with a 1-year OS of 87.7% and 1-year event-free survival (EFS) of 73% (8).
Although allo-HSCT after CAR-T therapy brings more survival benefits to patients with r/r B-ALL, some patients still have a recurrence within one year after transplantation. A clinical study showed that patients with minimal residual disease (MRD) positivity (MRD+) before transplantation had a significantly lower DFS than MRD-negative (MRD-) patients at 2-years after transplantation (27.6% vs. 76.1%) (9).
In addition to the impact of MRD on the prognosis of transplantation, an increasing number of studies have shown that predisposition gene mutations and somatic gene mutations in tumors play a vital role in the occurrence and development of hematological malignancies. The germline predisposition of myeloid tumors was taken into account in classifying myeloid tumors and acute leukemia by the WHO in 2016 (10). Recently, researchers have found that in patients with r/r B-ALL who received CD19 CAR-T therapy, TP53 mutation (TP53-mut) in somatic cells was significantly associated with a poor prognosis after CAR-T therapy (11). The 6-month survival in patients with TP53-mut was 51.9%, and the leukemia-free survival was only 42.4%, which was significantly lower than that in patients with TP53 wild-type (TP53-wt) (11). The abovementioned data indicate the value of gene mutation in predicting the prognosis of patients receiving CAR-T therapy.
This study analyzed the prognostic factors of patients with r/ r B-ALL who received allo-HSCT after achieving CR by CAR-T therapy. We systematically explored germline predisposition gene and somatic gene variation profiles and their effect on the prognosis of patients after transplantation to provide a reference for clarifying the mechanism of r/r B-ALL and developing treatment strategies to improve patient survival.

Methods Patients
In total, 144 consecutive r/r B-ALL patients who underwent allo-HSCT after CD19/CD22 CAR-T therapy in our hospital from June 2017 to June 2021 were enrolled in this study, and 7 patients who did not achieve CR after CAR-T therapy were excluded from the subsequent analyses. A long-term follow-up and statistical analysis were performed on 137 r/r B-ALL patients who achieved CR after CD19/CD22 CAR-T therapy and underwent allo-HSCT (Figure 1), including 133 patients in our hospital (n=133) and 11 patients in the Seventh Medical Center of PLA General Hospital (n=11) (allo-HSCT was performed under the same protocol). All subjects or their guardians signed an informed consent form. This study was approved by the Medical Ethics Committees of Beijing Boren Hospital and the Seventh Medical Center of PLA General Hospital, in accordance with the Declaration of Helsinki.

CAR-T therapy
The data were obtained from clinical trials on immunotherapy conducted in Beijing Boren Hospital (registered at www.chictr.org #ChiCTR-OIC-17013623, #ChiCTR-OIC-17013523). Lentiviruses (provided by Shanghai Yake Biotechnology Co., Ltd.) were used to construct CD19-or CD22-directed chimeric antigen receptors (CARs) with a 4-1BB costimulatory domain and CD3f signaling domain. Activation, transduction and expansion of CAR-T cells were performed in the Cell Processing Laboratory of Beijing Boren Hospital. All patients underwent lymphocyte-depleting chemotherapy with fludarabine (30 mg/m2 per day, Days 5 to 3) and cyclophosphamide (250 mg/m2 per day, Days 5 to 3) and then received CD19/CD22 CAR-T infusion. Then, CAR-T-cell targets were selected according to the fluorescent intensity of antigen expression of leukemia cells through flow cytometry. In total, 102 patients received CD19 CAR-T cells, 17 received CD22 CAR-T cells, and 18 patients received CD19 CART and CD22 CART twice before transplantation. Because they did not get remission after receiving CART treatment for the first time, so they had to do CART for the second time to achieve remission.

Detection of germline predisposition gene and somatic gene mutations
Before transplantation, the peripheral blood mononuclear cells (PBMCs) of patients were collected to detect predisposition gene mutations related to hematologic malignancies or immune system diseases. The sample of bone marrow (BM) of patients were collected to analysis somatic gene mutations related to hematologic malignancies. Genomes were extracted from PBMCs and BM samples of patients before CAR-T therapy using the TIANamp blood DNA kit (Tiangen Biotechnology Co.). The extraction were performed according to the manufacturer's instructions. The extracted DNA concentration was determined using a Nanodrop 2000 (Thermo Fisher). The genomic DNA library was constructed using KAPA HyperPlus Kits (Roche). Library concentration was measured using the Qubit 4.0 Fluorometer (Thermo Fisher). The constructed DNA library was captured using the NimbleGen SeqCap EZ Library SR (Roche) kit, and the probes contained the exon regions of 339 genes relevant to hematologic malignancies. The captured library was sequenced using the NextSeq550 (Illumina) sequencer, and the sequencing was performed using the PE150 program according to the manufacturer's instructions. For the somatic gene mutations average sequencing depth of gene target regions was 3000×. For WES average sequencing depth was 100×.

Bioinformatics analysis
Germline variant calling was performed with the Broad Institute's Genome Analysis Toolkit (GATK). The variant list was further filtered to select variants of approximately 700 genes that were associated with hematological tumors and immunodeficiency. Germline variants with an allele frequency >15% in our samples and with a predicted moderate or high impact on protein function were further filtered to select variants according to their frequency in the genomAD, ExAC and 1000G databases (minor allele frequency (MAF) < 5%). Integrative Genomics Viewer (IGV) software was used to manually inspect sequencing results. The candidate genetic risk variants were validated in family members of patients by Sanger sequencing. And the interpretation of variants was based on the HGMD and ACMG guideline (12,13).
Somatic variant calling was performed with the Broad Institute's Genome Analysis Toolkit (GATK) and Vardict.  Somatic variants were filtered to select rare variants according to their frequency in the genomAD, ExAC, and 1000G databases (MAF< 1%) and with an allele frequency >5%, but if the variant was a hotspot and the allele frequency was >1%, Integrative Genomics Viewer (IGV) software was used for the manual inspection of sequencing results.
Cyclosporine, mycophenolate mofetil (MMF), and shortcourse methotrexate (MTX) were used for GVHD prophylaxis. Intravenous injection of cyclosporine (1.25 mg/kg, Q12 h) started on day -9, and oral administration was given one month later if no diarrhea occurred. MTX (15 mg/m 2 ) was administered intravenously on Day 1 and then 10 mg/m 2 on Days 3, 6 and 11. MMF (7.5 mg/kg twice per day) was given on day -2, was tapered upon neutrophil engraftment and stopped on Day 30. Acute graft-versus-host disease (aGVHD) was evaluated using the Glucksberg scale modified by Przepiorka et al. (1995), and grades III and IV should be regarded as severe aGVHD (15). Chronic graft-versus-host disease (cGVHD) was assessed using the consensus criteria by the U.S. National Institutes of Health (16).
Prophylaxis against fungi, herpesviruses, and pneumocystis pneumonia (PCP) was routinely given. Echinocandins or triazoles were used for primary or secondary prevention of fungal infections based on pretransplant medical history. Acyclovir and trimethoprim-sulfamethoxazole were used to prevent herpesvirus infection and PCP. Plasma cytomegalovirus (CMV) DNA and Epstein-Barr virus (EBV) DNA were routinely monitored by PCR 1-2 times a week after transplantation. If urinary irritation symptoms occurred, urine BKV and JCV were detected by PCR.

Efficacy assessment
Complete response (CR), CRi, and no response (NR) are defined according to the U.S. National Comprehensive Cancer Network (NCCN) guidelines (17). According to NCCN guidelines, recurrence is defined as reappearance of blasts in the BM or peripheral blood (>5%) or any extramedullary diseases (EMDs) after CR. Refractory disease was defined as failure after 2 standard treatment regimens (18). EMDs were assessed using imaging techniques, including computed tomography (CT), positron emission tomography-computed tomography (PET-CT), and magnetic resonance imaging (MRI), on Days 15 and 30 after CAR-T infusion and 2 months after HSCT. Afterward, the patient was evaluated every 1-2 months until CR was observed. A negative status for MRD was defined as less than 0.01% BM blasts as determined by multiparameter flow cytometry (FCM, FACSCalibur, BD, USA) and/or less than 0.01% frequency of reverse transcription (RT)-PCR if the patients had leukemia-specific fusion genes or mutations. OS was defined as the interval from Day 0 (the day of transplantation) to the date of death or the last follow-up. DFS was defined as the interval from Day 0 (the day of transplantation) to the date of recurrence, death, or the last follow-up. All patients were followed up until December 31, 2021.

Statistical analysis
The Kaplan-Meier method was used to calculate DFS and OS, the cumulative incidence of recurrence (CIR), DFS, and OS at corresponding time points (one-year, two-year). The difference in survival curves was assessed using the log-rank test. Treatmentrelated mortality (TRM) and CIR were assessed using a competing risk model. Recurrence mortality was defined as a competing risk of TRM. Death without recurrence was defined as a competing risk of CIR. Hazard ratios (HRs) and their 95% confidence intervals were estimated by Cox proportional hazards models. Factors with a significance of P< 0.1 in the univariate Cox regression analysis were included in the multivariate Cox regression model. For clinical characteristics, measurement data were compared by t test, and categorical data were compared by chi-square test or Fisher's exact test. The significance level a was set as 0.05 for both sides. Statistical analysis was performed using IBM SPSS statistics 25 and R v3.6.2.

Results
Clinical characteristics of patients before transplantation All patients were r/r B-ALL and heavily treated. Their clinical characteristics before transplantation are shown in Table 1 and Figure 1A. The median age was 9.7 years (range: <1-56 years), and the median duration of follow-up was 22.5 months (95% CI: 20.2-24.8 months). At enrollment, 95 patients (66.0%) had recurrence, and 49 (34.0%) had refractory diseases. A total of 144 patients received CAR-T therapy, among them 137 patients achieved CR after CAR-T therapy, and 7 patients who did not achieve CR were excluded from the subsequent analysis.

Relationship between pretransplant MRD and the prognosis of patients receiving CAR-T therapy followed by transplantation
Univariate Cox analysis showed that pretransplant MRD was associated with DFS and OS ( Table 2)

Relationship between germline predisposition genes of hematologic malignancies and somatic gene variation and posttransplant prognosis
In all 137 patients in this cohort, 87 patients were evaluated for germline predisposition variants, and 92 patients were evaluated for somatic gene variants. Sixty-seven patients underwent both germline predisposition gene and somatic gene variation sequencing ( Figure 1B). The inclusion and exclusion criteria and the status of molecular testing are shown in Figures 1A, B.
The analysis of germline predisposition gene variants showed that genes with the highest mutation frequency were as follows: BLTA, F7, KIT, SERPINE1, TP53, DPYD, MLH1, TNFAIP3, YARS2, HIFIA, ATM, EP300, and ADAMTS13 ( Figure 3A). Univariate Cox regression analysis of DFS and OS on most frequency mutant genes (Table S1) showed that EP300-mut patients had a significantly lower DFS and OS than EP300-wt patients (EP300-mut: mDFS=3.13 months; EP300-wt: A B C FIGURE 3 Germline EP300 variation associated with poor prognosis. (A) Germline predisposition genes variation of leukemia. (B, C) DFS (B) and OS (C) analysis for patients with mutated or wild-type EP300 in r/r B-ALL patients treated with CAR-T therapy followed by allo-HSCT using Kaplan-Meier curves.
We further performed analysis on conditioning regimens for patients with TP53-mut and TP53-wt. The results showed that for TP53-mut patients, TBI-based or BU-based regimens showed no significant improvement in DFS and OS (p> 0.05, Figures S2A, B); for TP53-wt patients, the TBI-based conditioning regimen significantly increased the DFS of patients (p value = 0.025; Figure S2A).
In conclusion, univariate Cox regression analysis of germline predisposition gene and somatic gene mutations showed that germline EP300 mutations and somatic TP53 mutations were associated with a high risk of recurrence and death after allo-HSCT.

Multivariate Cox regression analysis of the relationship between clinical characteristics, gene mutation profile and prognosis
Multivariate Cox regression analysis was used to further investigate the relationship of germline predisposition gene and somatic gene mutations with DFS and OS in patients receiving allo-HSCT after CAR-T therapy. The factors significantly related to prognosis in univariate Cox regression (clinical parameters and germline predisposition gene and somatic gene mutations) were included in multivariate analysis. The results showed that MRD before transplantation, somatic TP53 mutations and germline EP300 mutations were independent risk factors for DFS (DFS: global log-rank p value < 0.0001, C-index = 0.75, MRD+: HR = 2.3, p -value = 0.033; somatic TP53-mut: HR = 2.9, p value = 0.009; germline EP300-mut: HR = 6.4, p value = 0.008; Figure 5A). Somatic TP53 mutations and germline EP300 mutations were independent risk factors for OS (OS: global log-rank p value = 0.0038, C-index = 0.73, somatic TP53-mut: HR = 2.67, p value = 0.028; germline EP300-mut: HR = 6.74, p value = 0.01; Figure 5B).

Discussion
Patients with r/r B-ALL usually have a poor prognosis. CAR-T therapy, as a novel immunotherapy in recent years, has dramatically increased the CR rates of such patients (19)(20)(21); however, more than 50% of CR patients who only received CAR-T therapy relapse within one year (6,19,20,22,23). CAR-T therapy followed by allo-HSCT may be a valuable treatment strategy to further improve the prognosis of patients with r/r B-ALL. This study reported the follow-up results of patients with r/ r B-ALL who received allo-HSCT after CR by CAR-T therapy in our hospital since 2017 and analyzed the characteristics that affected the prognosis of patients after transplantation.
In our cohort, the 2-year CIR, DFS and OS in patients who received allo-HSCT after CR by CAR-T therapy were 31.5%, 60.5% and 71.4%, respectively, similar to previous studies in China and other centers in other countries (8,9,11,24,25). Moreover, the recurrence rate and mortality rate were lower than those in patients who only received CAR-T therapy reported in the literature, indicating that the strategy of allo-HSCT after CAR-T therapy can improve the prognosis of patients with r/r B-ALL. In addition, compared with allo-HSCT patients who had not received CAR-T therapy, our patients showed no increase in transplant-related toxicity, consistent with the literature results (9). Our study showed that conditioning regimens and posttransplant fungal infection were associated with a poor prognosis. The patients receiving the BU-based regimen had a lower DFS than the patients receiving the TBI-based regimen, consistent with previous studies in ALL patients (26,27), suggesting that the TBI-based conditioning regimen should be the first choice for allo-HSCT in r/r B-ALL patients. In the stratified analysis, TBI-based regimens improved DFS and OS in MRD-positive patients. At the same time, there was no significant difference in recurrence and survival of MRDnegative patients regardless of which regimen was used. For patients with somatic TP53 mutations, the TBI-based regimen did not improve their prognosis compared with the BU-based regimen; for patients with wild-type TP53, the DFS of patients who received the TBI-based regimen was superior to that of patients receiving the BU-based regimen. In conclusion, TBIbased conditioning regimens have an overall survival advantage in patients with r/r B-ALL who undergo allo-HSCT after CR by CAR-T therapy.
There is a consensus that MRD is an independent prognostic indicator for B-ALL patients (28). Among the patients with CR after CAR-T therapy, MRD-positive patients usually have a higher risk of recurrence (29). In our study, FCM and RT-PCR were used to detect MRD. Univariate analysis showed that MRD-positive patients had significantly lower DFS and OS than MRD-negative patients. The 2-year recurrence rate, DFS and OS in MRD-negative patients in our cohort were 27.2%, 69.3% and 80.9%, respectively, consistent with previous reports (9,11,30). Zhao et al. (9) found that in a cohort of Chinese r/r B-ALL patients who were MRD-negative, the 2-year DFS and OS after CAR-T therapy followed by allo-HSCT were 65.6% and 77.0%, respectively, which were similar to our data. In multivariate analysis, MRD-positivity remained a significant independent risk factor for DFS. Therefore, MRD-positivity is significantly associated with a poor prognosis in r/r B-ALL patients receiving allo-HSCT after CAR-T therapy. Our study suggests that a TBIbased regimen can improve DFS and OS in MRDpositive patients.
In recent years, it has been reported that germline and tumor somatic gene variation affects the treatment response and prognosis of patients with hematological tumors (31)(32)(33). In this study, we applied NGS to systematically analysis germline predisposition gene variants and somatic gene variants in r/r B-ALL patients who presented a higher frequency of mutations and analyzed the effects of relevant germline and somatic gene variants on the prognosis of patients receiving allo-HSCT after CAR-T therapy. EP300 p. I997 V is a deleterious inactivating mutation in patients with hematologic malignancies undergoing allo-SCT (34). Somatic EP300 mutation is primarily seen in A B FIGURE 5 Multivariate cox regression analysis between clinical characteristics, gene mutation profile and prognosis. (A, B) Forest plot of hazard ratios for conditioning regimen, fungal infection, MRD before allo-HSCT, somatic TP53 mutations and germline EP300 mutations associated with DFS (A) and OS (B). Two-sided Wald-test p-value are reported. *p<0.05, **p<0.01.
diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), resulting in abnormal acetylation of p53 and BCL6 and thereby promoting tumorigenesis (35). Li, et al. (36) reported that the EP300 p. I997 V inactivating mutation is an indicating factor for poor prognosis in DLBCL patients treated with R-CHOP. Germline EP300 mutations can cause autosomal dominant Rubinstein-Taybi syndrome (RSTS) and are associated with predisposition to lymphoma (37). However, our study revealed the relationship between germline EP300 mutations and the prognosis of r/r B-ALL patients for the first time. Germline EP300 p. I997 V was detected in 5 patients in our cohort, an independent risk factor for DFS and OS. Those patients were prone to EMDs and a poor prognosis after allo-HSCT. This suggests that this variation should be considered in donor selection.
TP53 is the most frequently mutated gene in human cancers. Its translation product p53 suppresses tumorigenesis through transcriptional regulation of a network of target genes that play a role in various cellular processes (38). Somatic TP53 mutations are associated with treatment resistance and poor prognosis of different cancers, which has also been reported in patients receiving CAR-T therapy (39-43). In our study, in patients with TP53 mutations after CAR-T therapy and allo-HSCT, the 1-year CIR was 60%, twice as high as that in wild-type patients; the 1-year DFS and OS rates were 38.4% and 57.2%, respectively, which was much lower than those in wild-type patients. Multivariate analysis also showed that TP53 mutation was a significant independent risk factor for DFS and OS. Zhang et al. found similar result in their research study using multivariable analysis, and the existence of TP53 mutations is related to poor OS and LFS. Bridging allogeneic HSCT after CAR T cell therapy is associated with improved OS and LFS (11). However, some studieshave shown that CD19-22 CART sequential therapy is safe and effective in r/r B-ALL (21).
With the current treatment strategy for r/r B-ALL patients, other factors (age, sex, unfavorable fusion gene and/or chromosomal abnormalities, EMDs, different donor types, different blood types of donors/patients, aGVHD and cGVHD) showed no significant effect on the prognosis of patients after transplantation.

Conclusion
CAR-T therapy followed by allo-HSCT can improve survival in patients with r/r B-ALL. The 2-year DFS and OS in MRDnegative patients in our cohort were 69.3% and 80.9%, respectively. TBI-based conditioning regimens can significantly improve the prognosis of patients; fungal infection is significantly related to the risk of recurrence and death after transplantation. MRD positivity remains an independent risk factor for the prognosis of patients receiving CAR-T therapy and allo-HSCT. Germline EP300 mutations and somatic TP53 mutations are significantly associated with a poor prognosis after allo-HSCT. For patients with r/r B-ALL, it is necessary to provide an individualized and precise conditioning regimen and posttransplant maintenance therapy based on MRD status and molecular variation characteristics.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Ethics statement
The studies involving human participants were reviewed and approved by the Medical Ethics Committees of Beijing Boren Hospital and the Seventh Medical Center of PLA General Hospital. The patients/participants provided their written informed consent to participate in this study.

SUPPLEMENTARY TABLE 2
Univariate cox regression analysis of high population mutation frequency of somatic genes. (Mutation n>=5).

SUPPLEMENTARY FIGURE 1
Survival analysis of conditioning regimen and MRD status before allo-HSCT.

SUPPLEMENTARY FIGURE 2
S u r v i v a l a n a l y s i s o f s o m a t i c T P 5 3 m u t a t i o n s t a t u s a n d conditioning regimen.

SUPPLEMENTARY FIGURE 3
Multi-cox regression in each MRD before allo-HSCT subgroup. (A, B) Forest plot of hazard ratios for conditioning regimen, fungal infection, somatic TP53 mutations and germline EP300 mutations associated with DFS (A) and OS (B). Two-sided Wald-test p-value is reported.