Comparison of characteristics and outcomes on ETP-ALL/LBL and non-ETP ALL patients receiving allogeneic hematopoietic stem cell transplantation

Objective This study aims to compare the characteristics of early T-cell precursor acute lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) and non-ETP ALL patients and the outcomes of these patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Method A total of 57 patients with T-cell acute lymphoblastic leukemia/lymphoma receiving allo-HSCT at our center between January 2016 and March 2022 were enrolled in the study. Twenty-eight patients were diagnosed as ETP-ALL/LBL (28/57, 49.12%) in the cohort. Results The baseline characteristic was not significantly different between the two groups. The median time for myeloid engraftment was 14 days (ranged from 11 to 21) versus 14 days (ranged from 10 to 20) (P = 0.067) and 18 days (ranged from 12 to 27) versus 15.5 days (ranged from 12 to 72) (P = 0.183) for platelet engraftment in the ETP-ALL/LBL and non-ETP ALL groups, respectively. There was no significant difference in 5-year overall survival (54.74% ± 10.33% vs. 64.20% ± 10.30%, P = 0.786), relapse-free survival (56.22% ± 10.11% vs. 57.17% ± 12.71%, P = 0.841), cumulative incidence of relapse (30.14% ± 9.85% vs. 22.79% ± 8.24%, P = 0.774), and non-relapse mortality (19.52% ± 8.99% vs. 25.95% ± 14.44%, P = 0.967) between the two groups. The incidence of acute graft versus host disease (aGVHD) (P = 0.922), II–IV aGVHD (P = 0.940), III–IV aGVHD (P = 0.664), cytomegalovirus infection (P = 0.862), Epstein–Barr virus infection (P = 0.610), and severe bacterial infection (P = 0.145) was also similar. Conclusion The prognosis of patients with ETP-ALL/LBL was similar to non-ETP ALL patients when they received allo-HSCT.

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy which accounts for 15% and 25% of childhood and adult ALL cases, respectively (1).
Early T-cell precursor lymphoblastic leukemia/lymphoma (ETP-ALL/LBL) is a special subtype of T-ALL first recognized in 2009 (2), which is characterized by arrested early T-cell differentiation, with some myeloid and stem cell characteristics remaining at the immunophenotypic and also genetic levels (3,4). The incidence of ETP-ALL reported in previous studies is 11%-16% of T-ALL cases in children and 7.4%-32% in adults (5)(6)(7), respectively. In a large cohort study in Chinese adult T-ALL (n = 112), ETP-ALL accounts for 47.3% of all patients (8). Some studies suggested that the prognosis of ETP-ALL/LBL was worse than that of typical T-ALL (2, 5,[9][10][11]. However, other studies have found that the prognosis of ETP-ALL and non-ETP was not significantly different (8,(12)(13)(14).
Although many efforts have been made to uncover the genetic aberrations and molecular pathogenesis of ETP-ALL (15)(16)(17)(18), the management of ETP-ALL is still challenging. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important potentially curative treatment for ETP-ALL/LBL. In this study, we aim to assess the efficacy of allo-HSCT on ETP-ALL/LBL patients and compare the outcomes of ETP-ALL/LBL and non-ETP patients.

Patients and definitions
We retrospectively analyzed the data of 57 patients who received allo-HSCT in our center from January 2016 to March 2022. The final date of follow-up was June 30, 2022 for patients without events. Of the 57 patients, 28 were diagnosed as ETP-ALL/LBL (one patient was diagnosed as ETP-LBL) according to the diagnosis criteria. ETP was diagnosed by the immunophenotype of the positive expression of CD7 but lack of CD1a and CD8, weak expression of CD5 (with <75% positive blasts), and positive expression of one or more stem cell or myeloid markers including CD117, HLA-DR, CD13, CD33, CD11b, or CD65 (3). The initial induction chemotherapy was VDCLP or Hyper-CVAD. After complete remission, we conducted three to six courses of consolidation chemotherapy before allo-HSCT. Minimal residual disease (MRD) analysis was detected by flow cytometry, and MRD <0.01% (1 * 10 -4 ) of nucleated cells was defined as negative. All patients and donors provided written informed consent for this protocol. For patients younger than 18 years old in the cohort, the consent was carried out by their parents. This study was approved by the Ethics Review Committee of the Institute of Hematology, Chinese Academy of Medical Science and Peking Union Medical College, and was in compliance with the Declaration of Helsinki.
Graft versus host disease (GVHD) prophylaxis and supportive care were as described previously (19).

Criteria of outcomes
Engraftment was defined as absolute neutrophil counts (ANC) ≥0.5 × 10 9 /L for 3 consecutive days and platelet count ≥20 × 10 9 /L without transfusion for seven consecutive days. The Mount Sinai Acute GVHD International Consortium criteria were used to diagnose and grade acute GVHD (aGVHD) (20). Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viremia was defined as before (19). Severe bacterial infection referred to bacteremia or severe tissue infections. Complete remission (CR) referred to no blasts in blood, ANC >1.0 × 10 9 /L, platelets >100 × 10 9 /L, <5% bone marrow blasts, and no extramedullary leukemia. Overall survival (OS) was calculated from HSCT to death of any cause or last follow-up. Relapse-free survival (RFS) was defined as the time from HSCT to relapse, censoring at death or last follow-up. Cumulative incidence of relapse (CIR) was defined as relapse after HSCT.

Statistical analysis
The data were analyzed by the software GraphPad Prism 8 (version 8, supplied by GraphPad Software, Inc.) and IBM SPSS statistics 25 (version 25, supplied by IBM). The descriptive statistics for continuous variables and chi-square test and Fisher's exact test for categorical variables were used to compare incidence in univariate analysis. The Kaplan-Meier method was used to estimate the cumulative survival/incidence, and differences were compared by the log-rank/Wilcoxon test. A two-sided P-value <0.05 was considered as statistically significant.

Characteristics of patients
There are 28 and 29 patients in the ETP group and non-ETP group, respectively. The baseline characteristics of patients in the two groups are listed in Table 1. Gender, age, WBC/HB/PLT at diagnosis, BM blast, chromosome karyotype, and interval from diagnosis to HSCT did not differ among the two groups. There are 26 and 27 patients in the ETP group and non-ETP group who underwent next-generation sequencing. The top mutated gene in both groups was NOTCH1 (12/26, 46.2% in the ETP group and 15/27, 55.6% in the non-ETP group), followed by NRAS, JAK3, WT1, EZH2 in the ETP group and FBXW7, NRAS, DNMT3A, and PHF6 in the non-ETP group ( Figure 1).

Transplantation details
The transplantation-associated details including donor type, chemotherapy before CR, MRD status before transplantation, GVHD prophylaxis, and dose of MNC and CD34 + cells between

Engraftment
All patients had ANC engraftment, whereas 25 patients (89.3%) in the ETP group and 28 patients (96.6%) in the non-ETP group had platelet engraftment in 100 days posttransplantation. The median time of ANC recovery in the ETP group and non-ETP group was 14 days (ranged from 11 to 21) and 14 days (ranged from 10 to 20), respectively. For platelet  Gene mutation of early T-cell precursor (ETP) and non-ETP patients.
recovery, the median time was 18 days (ranged from 12 to 27) and 15.5 days (ranged from 12 to 72), respectively.

Infection and GVHD
The incidence of CMV viremia and EBV viremia was not significantly different in the ETP group and the non-ETP group (64.3% vs. 62.1%, P = 0.862; 10.7% vs. 6.9%, P = 0.610, respectively). In total, 14 patients in the ETP group and nine patients in the non-ETP group developed severe infection (50.0% vs. 31.0%, P = 0.431). The incidence of I-IV, II-IV, and III-IV aGVHD was similar in the two groups (P = 0.922; P = 0.940; P = 0.664).

Deaths and survival
The median time from HSCT to death or last follow-up was 424 days (ranged from 46 to 1841). The estimated 5-year OS of the total cohort was 55.40% ± 7.90% (Figure 2A). Until the last follow-up, there were 11 patients who died in the ETP group, seven had a relapse, two had infection or aGVHD, and one had graft failure. A total of 10 patients died in the non-ETP group, six had a relapse and four had infection or aGVHD. In the ETP group, at a median follow-up of 435 days (ranges from 93 to 1,841), 17 patients survived, and the 5-year OS was 54.74% ± 10.33%. In the non-ETP group, at a median follow-up of 419 days (ranged from 46 to 1,434), 19 patients survived, and the 5year OS was 64.20% ± 10.30%. There was no significant difference in terms of the 5-year OS between the two groups (P = 0.786), and so were the 5-year RFS, CIR, and non-relapse mortality (NRM) (P = 0.841; P = 0.774; P = 0.697) ( Table 3 and Figures 2B-E). Moreover, we compared the survival of MRDpositive and MRD-negative patients. Patients who were MRDnegative before transplantation had a higher 5-year OS than the MRD-positive patients (59.79% ± 9.04% vs. 43.08% ± 14.67%, P = 0.048) (Figure 3).

Discussion
ETP-ALL/LBL was first recognized in 2009 and defined by World Health Organization classification 2016 version as a distinct subtype of ALL due to its unique immunophenotypic and genomic profile (2, 3). Since then, many clinicians and researchers started to pay attention to the subtype. ETP-ALL is characterized by early differentiation arrest and distinct genetic and transcriptional features and thought to be a high-risk subgroup of ALL. ETP-ALL tends to be resistant to chemotherapy, and novel agents such as BCL-2 inhibitors have shown a good response for this disease. Another important and potentially curative treatment is allo-HSCT (21,22). In this study, we summarized the characteristics of 57 T-ALL patients who received allo-HSCT at our center and compared the heterogeneity between ETP and non-ETP.
A study conducted by St. Jude Children's Research Hospital demonstrated that, with standard intensive chemotherapy, the 10-year overall survival for patients with ETP-ALL was significantly lower than for the non-ETP patients (19% vs. 84%, P < 0.0001) (2). Other two studies by MD Anderson Cancer Center and Pediatric Blood Diseases Center in our hospital also indicated inferior prognosis of ETP-ALL (5,26). However, the Group for Research on Adult Acute Lymphoblastic Leukemia-2003 and -2005 studies showed that the 5-year overall survival for patients with ETP-ALL was not inferior to that of the non-ETP-ALL group (59.6%, 95% CI: 44.2% to 72.0% vs. 66.5%, 95% CI: 58.7% to 73.2%; P = 0.33) (14). A recent study in Chinese ALL patients also suggested a similar 2-year overall survival between the ETP and non-ETP patients (40.7% ± 8.2% vs. 37.9% ± 7.0%, P > 0.05) (8). A proportion of patients in the latter two studies received allo-HSCT other than chemotherapy alone, indicating that allo-HSCT could overcome the poor prognosis of ETP patients. In this study, we focused on ALL patients undergoing allo-HSCT and found that the 5-year OS, RFS, CIR, and NRM were not significantly different between the ETP and non-ETP patients (54.74% ± 10.33% vs. 64.20% ± 10.30%, hazard ratio (HR): 1.125, P = 0.786; 56.22% ± 10.11% vs. 57.17% ± 12.71%, HR: 1.091, P = 0.841; 30.14% ± 9.85% vs. 22.79% ± 8.24%, HR: 1.173, P = 0.774; 19.52% ± 8.99% vs. 25.95% ± 14.44%, HR: 0.971, P = 0.967). The survival of ETP-ALL/LBL patients was similar with or superior to other studies. Moreover, the OS in our study was calculated from HSCT, while in most of the previous studies it was calculated from diagnosis. Thus, our survival data was better than that of the previous studies as the median interval from diagnosis to HSCT was approximately 200 days, suggesting that allo-HSCT was an effective treatment for these patients and should be considered. However, due to the retrospective origin and small sample size, future prospective, large-scaled clinical trials are needed to investigate and confirm the results. Furthermore, MRD status was associated with the prognosis reported by many studies (7,12,27). In the study, we also compared the survival of MRD-positive and MRD-negative patients and found that the 5-year OS was significantly lower in the MRD-positive patients (43.08% ± 14.67% vs. 59.79% ± 9.04%, P = 0.048).
In conclusion, in the setting of allo-HSCT, ETP-ALL/LBL and non-ETP patients could achieve similar survival. Moreover, MRDnegativity before transplantation was associated with better prognosis. Allo-HSCT should be considered for ETP patients and novel treatment strategies (such BCL-2 inhibitors, etc.) to eliminate MRD before transplantation could further improve the efficacy.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.

Ethics statement
This study was approved by the Ethics Review Committee of the Institute of Hematology, Chinese Academy of Medical Science and Peking Union Medical College and was in compliance with the Declaration of Helsinki. All patients and donors provided written informed consent for this protocol. For patients younger than 18 years old in the cohort, the consent was carried out by their parents.