Monitoring of post-transplant MLL-PTD as minimal residual disease can predict relapse after allogeneic HSCT in patients with acute myeloid leukemia and myelodysplastic syndrome

The mixed-lineage leukemia (MLL) gene is located on chromosome 11q23. The MLL gene can be rearranged to generate partial tandem duplications (MLL-PTD), which occurs in about 5-10% of acute myeloid leukemia (AML) with a normal karyotype and in 5-6% of myelodysplastic syndrome (MDS) patients. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently one of the curative therapies available for AML and MDS with excess blasts (MDS-EB). However, how the prognosis of patients with high levels of MLL-PTD after allo-HSCT, and whether MLL-PTD could be used as a reliable indicator for minimal residual disease (MRD) monitoring in transplant patients remains unknown. Our study purposed to analyze the dynamic changes of MLL-PTD peri-transplantation and the best threshold for predicting relapse after transplantation. We retrospectively collected the clinical data of 48 patients with MLL-PTD AML or MDS-EB who underwent allo-HSCT in Peking University People’s Hospital. The MLL-PTD was examined by real-time quantitative polymerase chain reaction (RQ-PCR) at the diagnosis, before transplantation and the fixed time points after transplantation. Detectable MLL-PTD/ABL > 0.08% was defined as MLL-PTD positive in this study. The 48 patients included 33 AML patients and 15 MDS-EB patients. The median follow-up time was 26(0.7-56) months after HSCT. In AML patients, 7 patients (21.2%) died of treatment-related mortality (TRM), 6 patients (18.2%) underwent hematological relapse and died ultimately. Of the 15 patients with MDS-EB, 2 patients (13.3%) died of infection. The 3-year cumulative incidence of relapse (CIR), overall survival (OS), disease-free survival (DFS) and TRM were 13.7 ± 5.2, 67.8 ± 6.9, 68.1 ± 6.8 and 20.3% ± 6.1%, respectively. ROC curve showed that post-transplant MLL-PTD ≥ 1.0% was the optimal cut-off value for predicting hematological relapse after allo-HSCT. There was statistical difference between post-transplant MLL-PTD ≥ 1.0% and MLL-PTD < 1.0% groups (3-year CIR: 75% ± 15.3% vs. 0%, P < 0.001; 3-year OS: 25.0 ± 15.3% vs. 80.7% ± 6.6%, P < 0.001; 3-year DFS: 25.0 ± 15.3% vs. 80.7 ± 6.6%, P < 0.001; 3-year TRM: 0 vs. 19.3 ± 6.6%, P = 0.277). However, whether MLL-PTD ≥ 1% or MLL-PTD < 1% before transplantation has no significant difference on the prognosis. Our study indicated that MLL-PTD had a certain stability and could effectively reflect the change of tumor burden. The expression level of MLL-PTD after transplantation can serve as an effective indicator for predicting relapse.


Background
Acute myeloid leukemia (AML) is a highly malignant hematopoietic system disease and myelodysplastic syndrome (MDS) is a type of heterogeneous myeloid malignancies and frequently progress to AML [1][2][3][4]. In previous studies, molecular genetic aberrations have become important approaches for minimal residual disease (MRD) detection for AML and MDS. Especially, the polymerase chain reaction (PCR)-based gene detection has been proven to be an effective MRD monitoring method for AML patients [5][6][7]. However, more than half of AML cases still lack effective specific MRD molecular markers [5].
The mixed-lineage leukemia (MLL) gene, also named lysine (K)-specific methyltransferase 2A (KMT2A), is located on chromosome 11q23. Genetic alterations of the MLL gene are usually associated with the development of acute leukemia [8]. The MLL gene may be rearranged to generate partial tandem duplications (MLL-PTD), which usually spans exons 2 to 6, 2 to 7, and 2 to 8, or exons 3-9, exons 3-10, exons 3-11, or exons 3-10 and exons 3-11 at the molecular level [8][9][10][11]. MLL-PTD has been detected in approximately 5-10% of AML and 5-6% of MDS patients [12][13][14]. Low level of MLL-PTD (< 0.08%) may also be present in the blood and bone marrow of healthy individuals [5]. Previous reports support that polymerase chain reaction (PCR)-based MLL-PTD is a reliable MRD marker and is associated with poor prognosis [5,[12][13][14][15]. For chemotherapy patients, a higher MLL-PTD level at initial diagnosis predicts a lower incidence of chemotherapy complete remission (CR) and a lower survival rate [13]. The dynamic changes of chemotherapy patients also show that MLL-PTD levels within the first 6 months after the start of therapy are useful for early risk assessment of AML patients, and that a reduction of MLL-PTD level ≥ 2 log is a good prognostic factor for overall survival [5]. Furthermore, compared with healthy donors, MLL-PTD level have no difference from that of non-transplanted patients in continuous CR, while was significantly higher than that of transplanted patients in continuous CR [15]. Taken together, these findings support that MLL-PTD is a specific clinical prognostic marker in the initial diagnosis and chemotherapy for AML patients. However, there are few reports on the dynamics of MLL-PTD peri-transplantation, especially after transplantation. Thus, whether MLL-PTD could be used as a stable and reliable MRD marker in the process of transplantation and whether there is an optimal value of MLL-PTD to predict relapse after transplantation will be explored for the first time in our study.
In this study, we investigated a consecutive cohort of 33 AML and 15 MDS patients with MLL-PTD who received allo-HSCT at our institute. Most MLL-PTD MDS cases are classified as MDS with excess blasts (MDS-EB) [16]. Our study purposed to analyze the dynamic changes of MLL-PTD peri-transplantation and the best threshold for predicting relapse after transplantation.

Patients
The consecutive patients diagnosed with MLL-PTD expression> 0.08% AML or MDS undergoing allo-HSCT between January 2015 and March 2019 at the Peking University People's Hospital, Institute of Hematology were enrolled in this study. The patients' data were updated until September 30, 2020. The institutional review board at the hospital approved the protocol, and all patients or their guardians signed consent forms approved by the institutional review board.

Donor Lymphocyte Infusion (DLI)
Prophylactic DLI was administered for patients in relapse or no remission (NR) state before transplantation. The indications for DLI included hematological leukemia relapse, receiving chemotherapy followed by DLI, or positive MRD detection as previously described [19].

Detection of MRD
In this study, MRD was evaluated by Flow Cytometry (FCM) [20], the expression level of WT1 and MLL-PTD determined by RQ-PCR. The pre-transplant FCM, MLL-PTD and WT1 were performed using bone marrow (BM) samples within a month before the transplant as a routine. The post-transplant scheduled time points were + 1, + 2, + 3, + 4.5, + 6, + 9, and + 12 months post-HSCT and every 6 months thereafter.
The patients were analyzed for the presence of MLL-PTD at the MLL gene locus, as described previously [13,15]. Briefly, MLL primers and hybridization probes were placed in exons 8-10 and 3 of the MLL gene, allowing for detection of MLL-PTD with exon 8/exon 3 fusion, exon 9/exon 3 fusion, or exon 10/exon 3 fusion. The transcript level was calculated as target transcript copies/ABL copies in percentages. Detectable MLL-PTD/ABL > 0.08% was defined as MLL-PTD positive [13]. The WT1 was detected as described previously and a WT1 transcript level less than 0.60% was defined as negative [21].

Definitions and assessments
The day of neutrophil engraftment was defined as the first day of 3 consecutive post-transplantation days on which the absolute neutrophil count (ANC) exceeded 500/μL. Patients who survived at least 28 days were considered to have had successful engraftment. The criteria for grading acute graft versus host disease (aGVHD) have been previously published [22,23]. CR was defined as hematological CR that is, < 5% BM blasts, the absence of blasts in peripheral blood, the absence of extramedullary disease, an ANC > 1.0 × 10 9 /L, and a platelet count > 100 × 10 9 /L with no red cell transfusions. Hematological relapse was defined by morphologic evidence of disease in the peripheral blood, marrow, or extramedullary sites.

Statistical analysis
The primary study end point was the cumulative incidence of relapse (CIR). The secondary end points were the OS, disease-free survival (DFS) and treatment-related mortality (TRM). CIR, OS, DFS and TRM were defined as previously described [24]. Summary statistics, such as proportions, medians and ranges, were used to describe the patient characteristics and outcomes. The associations between MLL-PTD expression and post-transplantation outcomes were analyzed by the Kaplan-Meier method. Differences in CIR, DFS, OS and TRM between groups were calculated using the log-rank test. A two-sided P value of 0.05 was considered statistically significant. The independence of categorical parameters was calculated using the chi-square test or Fisher exact test, and the distribution of continuous variables was calculated using the Mann-Whitney U-test. All statistical analyses were performed using SPSS 23.0 (Chicago, IL, USA).

Patients characteristics
A total of 33 AML patients included 13 males and 20 females, with a median age of 42 years (10-57 years) and 15 MDS-EB patients included 11 males and 4 females, with a median age of 51 years (4-60 years). The median follow-up time was 26 (0.7-56) months after HSCT. Patient characteristics are shown in Table 1. Of these 33 AML patients, 31 patients had gotten CR after chemotherapy, and 2 patients had gotten NR after 3 courses of chemotherapy. And 5 MDS-EB patients receiving chemotherapy including decitabine had gotten CR pretransplantation. All patients had neutrophil engraftment, and 39 patients had platelet engraftment. Of the 33 patients with AML, 7 patients (21.2%) died of TRM and 6 patients (18.2%) underwent hematological relapse who died ultimately. The median hematological relapse time was 4.8 months (range 4-9 months) after HSCT in 6 relapsed patients. Of the 15 patients with MDS-EB, 2 patients (13.3%) died of infection. In addition, all enrolled patients had a 3-year CIR of 13.7% ± 5.2%, 3-year OS of 67.8% ± 6.9%, 3-year DFS of 68.1% ± 6.8% and 3-year TRM of 20.3% ± 6.1% (Fig. 1).

Dynamic changes of MLL-PTD before and after transplantation
Observing the changes in the expression level of MLL-PTD at different time points peri-transplantation is helpful to analyze the stability of MLL-PTD as an MRD indicator in the transplantation system. Our results showed that the MLL-PTD level before transplantation was significantly lower than that at the initial diagnosis, but there were still 37 cases were MLL-PTD positive before transplantation, and 33 of 37 cases became negative within post-transplant 1 month. However, during our follow-up period, 25 cases eventually occurred posttransplant MLL-PTD positive. The median MLL-PTD level in all enrolled patients was decreased by around 35 folds after transplantation compared with that of pre-transplant CR status and was similar to the healthy controls (Table 2). Furthermore, among the 6 relapsed patients after transplantation, 3 of them maintained MLL-PTD at the healthy level (< 0.08%) within a month after transplantation. But before relapse, the MLL-PTD level of these 3 patients gradually increased (> 0.08%) and reached the highest level at the time of relapse. The MLL-PTD level of the other 3 relapsed patients continuously remained > 0.08% after transplantation, and the MLL-PTD levels of these 3 patients suddenly increased by hundreds of times before relapse.

The effect of MLL-PTD level before and after transplantation on prognosis
Having analyzed the dynamic changes above which peri-transplant MLL-PTD can stably reflect the disease   state we next studied the optimal threshold of posttransplant MLL-PTD for relapse. Our previous study shows that patients with MLL-PTD/ABL ≥ 1% based on initial diagnosis have a poor clinical prognosis [13]. In order to explore whether MLL-PTD could be used as a MRD marker after transplantation, we performed a receiver operating characteristic (ROC) with the highest expression level of post-transplant MLL-PTD before hematological relapse in all patients to determine the optimal cut-off value to predict relapse. The area under the ROC curve value was 0.977 (P < 0.001, Fig. 2A). The optimal cut-off value was MLL-PTD/ABL = 1.0%. And as shown in Fig. 2B, most post-transplant patients with MLL-PTD maintained a low level of expression, only 8 patients had MLL-PTD ≥ 1%, and 6 of the 8 patients eventually relapsed, which also implied the importance of MLL-PTD ≥ 1% in predicting relapse after transplantation. Based on the optimal cut-off value, we 80.7% ± 6.6%, P < 0.001, Fig. 3B) and 3-year DFS (25.0 ± 15.3% vs. 80.7 ± 6.6%, P < 0.001, Fig. 3C) compared with that of group of MLL-PTD/ABL < 1%. However, there was no statistical difference between the two groups in TRM (P > 0.05, Fig. 3D). Both at the initial diagnosis and post-transplantation, it was analyzed that MLL-PTD = 1% was the optimal cut-off value, which implied that MLL-PTD/ABL = 1% was of important value in predicting prognosis. Therefore, we further analyzed whether MLL-PTD/ABL ≥ 1% before transplantation also indicated a poor prognosis after transplantation. However, our results showed that there was no statistical difference in prognosis between the MLL-PTD/ABL ≥ 1% and MLL-PTD/ABL < 1% group based on the level of MLL-PTD before transplantation (All P > 0.05, Fig. 4A-D), but the group of MLL-PTD/ABL ≥ 1% tended to have lower OS (P = 0.202, Fig. 4B), DFS (P = 0.202, Fig. 4C), and have a higher TRM(P = 0.105, Fig. 4D) compared with that of MLL-PTD/ABL < 1% group .

Factors affecting the prognosis of transplant patients with MLL-PTD
Factors affecting the prognosis were analyzed, including transplantation age, gender, disease type, donor type, blood type compatibility (Table 3). There was no statistical difference in TRM (P = 0.675), CIR (P = 0.115), DFS (P = 0.151) and OS (P = 0.157) between AML and MDS-EB. Among the 12 patients who received MSDT, 2 (16.7%) patients underwent hematological relapse both at 5 months after HSCT, and 1 patient died of pneumonia at 5.5 months. Among 36 patients who received haplo-HSCT, 4 patients (11.1%) underwent hematological relapse at a median of 4.5 months (range, 4-9 months) after HSCT, and 8 patients (22.2%) died due to TRM at a median of 5.3 months (range, 0.7-17.5 months). Based on the results of the analysis, it seemed that patients who The factor analysis of MLL-PTD level before and after transplantation showed that there was no statistical difference in pre-transplant MLL-PTD level. And posttransplant group of MLL-PTD/ABL ≥ 1% had a higher CIR, a lower OS and a lower DFS than that of group of MLL-PTD/ABL < 1% (all P < 0.001). In addition, other factors such as age, pre-transplant FCM, WT1 status and prophylactic DLI have no significant impact on prognosis. The ABO blood type and FLT3-ITD mutation at first diagnosis were important risk factors of CIR and OS after transplantation, respectively. Incompatible ABO blood type indicated a higher CIR than that of compatible ABO blood type, and patients with FLT3-ITD mutation had a low OS than that of without FLT3-ITD (Table 3).

Comparison of MLL-PTD and other MRD parameters
After transplantation, 8 patients were detected MLL-PTD/ABL ≥ 1.0% at a median of 3 months. Of the 8 patients, 7 patients were simultaneously (5 patients) or subsequently (2 patients) MRD positive detected by FCM at a median of 4.25 months (range,3-12 months), and 6 patients ultimately progressed to hematological relapse at a median of 2 months (range, 0.25-6 months) from the first time MLL-PTD/ABL ≥ 1.0%, half of whom receiving chemotherapy plus DLI. Finally, 2 patients receiving chemotherapy plus DLI became MRD negative gradually.
WT1 has been confirmed in previous studies to be an effective indicator of MRD monitoring and implementing interventions [21]. In order to analyze the specificity and sensitivity of MLL-PTD compared with WT1, we showed in Table 2 the dynamic changes of expression of MLL-PTD and WT1 at the initial diagnosis and different time points before and after transplantation. All 6 relapsed patients were detected MLL-PTD positive prior to relapse, while only 4 patients were detected positive for WT1. As shown in Table 2, the expression levels of MLL-PTD and WT1 both changed with the tumor burden. However, within post-transplant 3 months, MLL-PTD seemed be more sensitive than WT1 for MRD

Discussion
MLL-PTD is a special MLL rearrangement gene. No report had focused on the predictive significance of peritransplant MLL-PTD expression on leukemia relapse after transplantation. In our retrospective study, results showed dynamic changes of MLL-PTD peri-transplantation, and the post-transplant MLL-PTD level is related to the prognosis of patients.
Previous reports have established the best threshold of MLL-PTD at the initial diagnosis for predicting the CR or relapse in AML patients [13,15]. However, the AML patients with MLL-PTD analyzed in above reports included both non-transplanted patients and transplanted patients. Since different treatments (chemotherapy and transplantation) have a great impact on the prognosis of AML patients, they also have a certain impact on the accuracy of the MLL-PTD threshold for predicting relapse. Allo-HSCT is one of the curative therapies currently available for AML and MDS-EB, so it is very necessary to establish an optimal threshold of post-transplant MLL-PTD for relapse in transplanted AML patients. In the analysis of the post-transplant best cut-off value, we found that MLL-PTD/ABL = 1% can be used as the threshold for predicting relapse. Based on this result, physicians could need to pay more attention to the occurrence of relapse for post-transplant patients with MLL-PTD/ABL ≥ 1%. Under this condition, it is also necessary to shorten the MRD monitoring interval, or give appropriate relapse preventive interventions in combination with the clinical condition.
A stable and reliable MRD marker whose expression level needs to vary with the tumor burden. Our data showed that MLL-PTD levels in relapsed patients were significantly increased before relapse. Importantly, there was no occurrence of MLL-PTD turning negative or losing before relapse, which indicated that MLL-PTD had a certain stability and could effectively reflect the change of tumor burden. As expected, MLL-PTD was available prior to hematological relapse, but the relapse after MLL-PTD positive occurred at different rates. One of the explanations may be due to the patient's combination of additional mutations such as FLT3-ITD. Previous report confirms that MLL-PTD positive relapses harboring an additional FLT3-ITD mutation to relapse faster than other patients with MLL-PTD alone [15]. In our study, the initial diagnosis of 2 relapsed patients was accompanied by FLT3-ITD mutation. They respectively relapsed at 12 days and 35 days after post-transplant MLL-PTD/ ABL ≥ 1%, and the relapse was significantly faster than that of other relapsed patients. These data suggested MLL-PTD patients with other mutations such as FLT3-ITD may need to be shortened intervals of MRD monitoring after transplantation. Of course, a larger sample size or data is needed in the future to further support the above result.
The timely monitoring of MRD in the early stage after transplantation was beneficial to guide early clinical intervention to improve the prognosis of patients. Some studies have confirmed that the WT1 expression level is an independent prognostic indicator that can predict clinical outcome and combined use of WT1 and flow cytometry monitoring can promote sensitivity of predicting relapse after allo-HSCT [21,25]. For AML and MDS lacking specific markers, we usually need to combine FCM and WT1 to evaluate MRD status. In the study, MLL-PTD became positive before relapse and prior to flow cytometry results. Thus, in contrast to FCM, PCR-based MLL-PTD detection have higher sensitivity. Our data showed that MLL-PTD seemed to be more sensitive than WT1 in early MRD monitoring after transplantation. Furthermore, in contrast to WT1, MLL-PTD is more specific for the type of MLL-PTD positive AML and MDS. However, for post-transplant patients with MLL-PTD, in order to monitor MRD more effectively and accurately, there may not be a better way than monitoring FCM, WT1 and MLL-PTD at the same time.
AML with MLL-PTD is a type of leukemia with a relatively poor prognosis compared with the standard-risk AML [13,14]. In standard-risk AML, the post-transplant overall CIR and OS are around 15-20% and 60-70% at our institute, respectively [26,27]. Our present results showed that the overall prognosis of post-transplant MLL-PTD patients (3-year OS: 67.8%; 3-year CIR: 13.7%) was similar to that of standard-risk patients. In addition, the other MLL rearrangement study about the transplant-related prognosis found that allo-HSCT would have a lower relapse risk and a higher survival probability compared to the results obtained from patients with chemotherapy alone [28]. The outcomes of patients with MLL-PTD are similar to the above results. The posttransplant OS in our study was significantly better than that of receiving chemotherapy alone (3-year OS< 40%) in previous study [5]. These data supported that allo-HSCT could achieve good therapeutic effect in patients with MLL-PTD at our institute. Furthermore, haplo-HSCT could achieve the similar therapeutic effect to the MSDT in patients with MLL-PTD. Therefore, our institution's transplant and relapse prevention system may be effective for MLL-PTD patients.

Conclusions
In conclusion, MLL-PTD expression is a sensitive and specific MRD marker for the MLL-PTD patients received allo-HSCT. MLL-PTD expression level higher than 1.0% suggested a high risk of hematological relapse and tended to have a worse prognosis. Furthermore, allo-HSCT could achieve good therapeutic effect in patients with MLL-PTD AML and MDS-EB. Of course, due to the limited number of patients with MLL-PTD patients, we still need to continue research to accumulate more cases to further confirm the significance of MLL-PTD for MRD monitoring around transplantation.