The effect of haploidentical hematopoietic stem cell transplantation on comutations based on next‐generation sequencing in adult acute myeloid leukemia patients with the FLT3‐ITD mutation

According to the 2022 European LeukemiaNet, all acute myeloid leukemia (AML) cases with FLT3‐ITD mutations are now categorized as intermediate risk irrespective of the FLT3‐ITD allelic ratio or concurrent presence of NPM1 mutation. However, whether other next‐generation sequencing (NGS) comutation genes can add layers to FLT3‐ITD and whether the poor outcomes of FLT3‐ITD comutations can be overcome by haploidentical hematopoietic stem cell transplantation (haplo‐HSCT) are unclear. This study aimed to investigate which comutations based on NGS at diagnosis affect the clinical prognosis of de novo AML patients with FLT3‐ITD mutations and the effect of haplo‐HSCT on comutations. We analyzed 95 de novo AML patients with FLT3‐ITD mutations from January 2018 to August 2021 based on the NGS 99‐gene platform. Forty‐one other types of molecular mutations were detected. The most common cooccurring mutations were NPM1 (n = 43, 45.3%) and DNMT3A (n = 21, 22.1%). NPM1 mutation did not affect the clinical outcomes. Acute myeloid leukemia patients with FLT3‐ITD and DNMT3A comutations had significantly worse 3‐year Disease‐free survival (DFS) (49.5% vs. 69.3%, P = 0.01) and Overall survival (OS) rates (61.1% vs. 69.8%, P = 0.54) than those without DNMT3A mutations, and survival was significantly more favorable after haplo‐HSCT than that after chemotherapy (3‐year DFS, 85.7% vs. 30.8%, P = 0.006; 3‐year OS, 85.7% vs. 43.1%, p = 0.08). In multivariate analysis, DNMT3A mutation was a risk factor for DFS, while haplo‐HSCT was a protective factor. In conclusion, DNMT3A mutation might be a poor prognostic factor in adult AML patients with FLT3‐ITD mutations, and haplo‐HSCT could overcome the poor prognosis of DNMT3A comutation.

In the 2022 European LeukemiaNet (ELN), all AML cases with FLT3-ITD mutations are now categorized as intermediate risk irrespective of FLT3-ITD allelic ratio (AR) or concurrent presence of NPM1 mutation, which is one of the most important changes made to the previous risk classification including the 2017 ELN and 2022   National Comprehensive Cancer Network (NCCN). 11,12This suggests that widely accepted prognostic factors predicting the clinical outcome of newly diagnosed FLT3-ITD-positive AML are still lacking.
Hence, the molecular characteristics of FLT3-ITD need to be studied, and the assessment of additional potential prognostic markers is urgently needed.
Next-generation sequencing (NGS) is widely used in the clinic to help diagnose AML and stratify prognosis and has the advantages of high throughput, high sensitivity, and high stability.The most common FLT3-ITD comutation is NPM1 mutation, which is no longer a factor in FLT3-ITD-mutated AML per the 2022 ELN but still helps to stratify the prognosis in the 2022 NCCN. 11,12Additionally, whether other NGS comutation genes can add layers to FLT3-ITD is unclear.4][15] Hence, in the present study, we focused on the detailed comutations of FLT3-ITD based on NGS and analyzed the effect of haplo-HSCT on FLT3-ITD mutations in AML patients with comutations.

| Patients
Among 436 newly diagnosed patients with AML (other than acute promyelocytic leukemia, APL), a total of 109 (25%) consecutive de novo AML patients with FLT3-ITD mutations were diagnosed based on the NGS 99-gene platform and treated at the Peking University Institute of Hematology from January 2018 to August 2021.Four patients who died early during induction were excluded from the analysis.Ten patients received matched sibling donor (MSD) transplantation, and no other types of transplants were performed during the period.Owing to the small number of MSD transplantations and to analyze the effect of haplo-HSCT on FLT3-ITD mutation AML patients, MSD transplantations were excluded from the analysis.We also excluded 14 patients who did not achieve CR to control bias when comparing the outcomes of patients who receiving haplo-HSCT (n = 40) or chemotherapy (n = 41).Acute myeloid leukemia was diagnosed according to the 2016 revision World Health Organization classification of myeloid neoplasms and acute leukemia. 16The last follow-up date was 31 March 2023.Informed consent was obtained from all patients by inpatient medical records.This study was performed in accordance with the Declaration of Helsinki and was approved by the ethics committee of Peking University People's Hospital.

| Detection of FLT3-ITD mutations
The FLT3-ITD assay was performed using fluorescent polymerase chain reaction (PCR) with primers and amplification conditions as described previously. 17The FLT3-ITD AR was calculated as the ratio of the area under the curve of mutant to wild-type alleles (FLT3-ITDmut/FLT3wt). Next-generation sequencing was used to detect comutations.In addition, three main mutants of the NPM1 gene, A, B and D, were detected by real-time fluorescence quantitative PCR. 18

| Induction therapy
According to the Chinese guidelines for the diagnosis and treatment of adult AML (not APL) (2021), 19 the induction regimens of patients fit for intensive therapy were as follows: IA, idarubicin 8-10 mg/m 2 /day d1-3 and cytarabine 100 mg/m 2 /day d1-7; HAA, homoharringtonine 2 mg/ m 2 /day d1-7, aclacinomycin 20 mg/m 2 /day d1-7 and cytarabine 100 mg/m 2 /day d1-7; and CAG, aclacinomycin 20 mg/day d1-4, cytarabine 10 mg/m 2 every 12 h d1-14, and granulocyte colonystimulating factor, 300 μg/day d1-14.One of these regimens could be chosen after discussion between doctors and patients, with the patient's family financial situation and tolerance to drugs also considered.The use of sorafenib during induction therapy was allowed but not mandatory because sorafenib was not recommended for FLT3-ITD AML in the Chinese guidelines. 19The induction regimens of patients unfit for intensive therapy were venetoclax and azacitidine (VEN+AZA) with or without sorafenib as follows: oral venetoclax 100 mg d1, 200 mg d2, 400 mg d3-28.If a strong CYP3A inhibitor was required after dose escalation, venetoclax was decreased to 100 mg; azacitidine 75 mg/m 2 was administered IV or subcutaneously on d1-7; and sorafenib 400 mg Bid was or was not administered on days 1-28.Additionally, all patients received cyclosporine, mycophenolate mofetil, and short-term methotrexate for GVHD prophylaxis. 20,21rafenib maintenance (400 mg orally twice daily) was recommended after discussion between doctors and patients, with the patient's family financial situation and tolerance to drugs also considered when hematopoietic recovery occurred within 60 days posttransplantation.Sorafenib was administered at 31-60 days posttransplantation and continued until at least day 180 and up to day 365.The dose modifications and use time of sorafenib were based on adverse events and tolerance.An alternative FLT3 inhibitor could also be started posttransplantation as maintenance after allo-HSCT.

| NGS
Next-generation sequencing was performed in all 95 patients using a panel of 99 genes, which covered all the mutation hotspots of acute leukemia, myelodysplastic syndrome and myeloproliferative neoplasms.Bone marrow samples from patients were collected.
GDNA was extracted with the QIAsymphony SP nucleic acid extraction and purification analyzer (product of Kaijie company in Germany).An Illumina standard library was constructed.Ninetynine genes related to myeloid tumors (Supplementary Table 1) were captured using the target sequence of a hematological malignancy customized probe (product of twist Bioscience, USA), and pe150 was sequenced on a Nova SEQ (Illumina, USA).The analysis included the determination of point mutations (SNVs), insertions and deletions (indels), ITDs and partial tandem duplications (PTDs).The algorithm independently developed by Guangzhou Jinyu company is used to detect the variation in SNV, INDEL, ITD and PTD.The variation detection results were annotated with software such as ANNOVAR.To ensure the accuracy of variation, the original variation detection results were filtered: the average effective depth of each sample capture target area was ≥2000�.The read ratio mass and base mass values supporting the mutant were higher than 30 and had both positive and negative chain support.

| Definition of clinical end points
Composite complete remission (cCR), comprised complete remission (CR), complete remission with incomplete platelet recovery (CRp), and complete remission with incomplete hematological recovery (CRi).CR was defined as bone marrow blasts <5%, no circulating blasts and blasts with Auer rods, the absence of extramedullary disease, an absolute neutrophil count ≥1.0 � 10⁹/L and platelets ≥100 �10⁹/L.CRp met all criteria for CR except that for platelets (platelets <100 � 10⁹/ L), and CRi met all criteria for CR except that for residual neutropenia (absolute neutrophil count <1.0 � 10⁹/L), thrombocytopenia (platelets <100 � 10⁹/L) or both.Relapse was defined as blasts >5% in bone marrow, reappearance of blasts in peripheral blood, or extramedullary leukemia in patients with previously documented CR.Overall survival (OS) was calculated from diagnosis to death or last follow-up.Diseasefree survival (DFS) was calculated from the date of CR to relapse or death, and patients who did not achieve CR were excluded.

| Statistical analysis
Mann-Whitney test, independent sample t test, and chi-square test/ Fisher's exact test were used for comparisons between groups.
Competing risk analysis was used to calculate cumulative rates of relapse and NRM.The DFS and OS rates were estimated using the Kaplan-Meier method with the log-rank test.Cox regression was used for the prognosis analysis.Variables in the Cox regression were as follows: patient age (<55 years vs. ≥ 55 years), NPM1 mutation (yes vs. no), DNMT3A gene mutation (yes vs. no), haplo-HSCT (yes vs. no), cytogenetic risk (intermediate-adverse risk vs. favorable risk), FLT3 inhibitors (sorafenib and gilteritinib) (yes vs. no).Multivariate analysis was performed for risk factors with a P < 0.1 by univariate analysis.P values < 0.05 were considered statistically significant.Hazard ratios (HRs) were calculated with their 95% confidence intervals.The Statistical Package for the Social Sciences (SPSS), version 13.0 (SPSS, Inc., Chicago, IL, USA), was used for data analysis.

| Clinical features
The baseline characteristics of patients are listed in Table 1.The median (range) follow-up for all survivors was 30.9 (20.6-70.

T A B L E 1
The baseline characteristics of the acute myeloid leukemia (AML) patients with FLT3-ITD mutation.

| Impact of NPM1 mutation
In NPM1+FLT3-ITD+AML group and NPM1-FLT3-ITD+AML group, the 3-year DFS and OS were 55.3% versus 72.7% (p = 0.03, Figure 3A) and 66.7% versus 67.9% (p = 0.99, Figure 3B), respectively.The 3-year DFS and OS between haplo-HSCT and chemotherapy in NPM1+FLT3-ITD+AML group were 65.5% versus 49.7% (p = 0.13, Figure 3C) and 80.0% versus 59.4% (p = 0.20, Figure 3D), respectively.The 3-year DFS and OS between haplo-HSCT and chemotherapy in NPM1-FLT3-ITD+AML group were 74.0%The reason that our study found no difference might be due to sorafenib use and active measurable residual disease monitoring in treatment decisions. 25It should be noted that the OS and DFS after haplo-HSCT in our study were up to approximately 80%.Several reasons may contribute to our superior outcomes.First, the high rate (72.5%) of maintenance treatment with sorafenib after transplantation might further contribute to the improvement in survival after haplo-HSCT, which was confirmed in previous studies. 26Second, the median age of patients undergoing haplo-HSCT in our study (30 years) was younger than that of patients receiving chemotherapy (50 years).

| Impact of other cooccurring mutations
Third, patients receiving haplo-HSCT in our study all received MAC.
Compared to a reduced-intensity conditioning regimen or non-MAC, the relapse rate can be reduced by enhancing the condition intensity. 21 further found that DNMT3A comutations in FLT3-ITD mutation AML conferred a worse outcome in this study, which is in accordance with previous studies. 27,28Whether the poor outcomes of FLT3-ITD and DNMT3A comutations can be overcome by haplo-HSCT has not been reported.Although Tang et al reported that FLT3-ITD with DNMT3A comutation is a poor prognostic factor in Chinese AML patients after allo-HSCT, the effect of haplo-HSCT on FLT3-ITD with DNMT3A mutation was not analyzed separately. 28In this study, AML patients with FLT3-ITD and DNMT3A comutations achieved superior survival after haplo-HSCT.These results suggested that adult AML patients with FLT3-ITD mutations and DNMT3A mutations might benefit from haplo-HSCT.Transplantation is strongly recommended for AML patients with FLT3-ITD mutations and DNMT3A mutations.
This study had several limitations.First, it was a single-center, retrospective study.Second, it must be stated that patients were not For the ELN favorable-risk group, consolidation chemotherapy involved 6-7 cycles, including four courses of intermediate-dose734-TANG ET AL.cytarabine (IDAC, 2 g/m 2 /q12 h for three days) and 2-3 courses of '3 + 7' regimens (three days of anthracycline and seven days of cytarabine).For the ELN intermediate-risk and adverse-risk groups, patients received 2-3 cycles of consolidation chemotherapy of intermediate-dose cytarabine or '3 + 7' regimens, and allo-HSCT was then performed.2.3.3 | Haplo-HSCTPatients received a myeloablative conditioning regimen (MAC) without in vitro T-cell depletion.The conditioning therapy was modified BUCY2 plus ATG (thymoglobulin).20,21All patients received G-CSF-mobilized, fresh, and unmanipulated G-CSF-primed bone marrow cells plus G-CSF-primed peripheral blood stem cells.

F I G U R E 1
Patients with FLT3-ITD+ DNMT3A+ had significantly worse outcomes than those with FLT3-ITD+ DNMT3A-.The 3-year DFS and OS were 49.5% versus 69.3% (p = 0.01, Figure4A) and 61.1% versus 69.8% (p = 0.54, Figure4B), respectively.Patients with FLT3-ITD + DNMT3A+ who underwent haplo-HSCT had significantly more favorable outcomes than those received chemotherapy.The 3-year DFS and OS were 85.7% versus 30.8% (P = 0.006, Figure4C) and 85.7% versus 43.1% (p = 0.08, Figure4D), respectively.Other common cooccurring mutations, such as NRAS (DFS, p = 0.90; OS, Mutational landscape in 95 patients with FLT3-ITD-positive acute myeloid leukemia (AML).TANG ET AL.To our knowledge, this is the first study to analyze the effect of haplo-HSCT on FLT3-ITD-mutated AML comutations at diagnosis based on NGS with a detailed subgroup analysis.Our analysis demonstrated that the most common comutations were NPM1 and DNMT3A.NPM1 mutation did not affect the clinical outcomes of FLT3-ITD-mutated AML patients.DNMT3A mutation was a poor prognostic factor in FLT3-ITD-mutated AML patients, and haplo-HSCT might overcome the adverse influence of DNMT3A mutation.Genetic mutations cooccurring with FLT3-ITD mutations have been evaluated in recent years as pretreatment parameters. 13Fortyone types of other molecular comutations based on NGS were detected in 86 (90.5%) adult patients with FLT3-ITD mutations.NPM1 and DNMT3A were more frequently identified in this study, while mutations in TP53, CSF3R, ASXL1 and GATA2 were rare.The incidence and distribution of FLT3-ITD comutations was similar to other studies about genomic characteristic in adult de novo AML F I G U R E 2 Impact of halpo-HSCT and allelic ratio (AR) (the cut off value is 0.5) on FLT3-ITD-positive acute myeloid leukemia (AML).(A) Disease-free survival (DFS) between haploidentical hematopoietic stem cell transplantation (haplo-HSCT) and chemotherapy; (B) Overall survival (OS) between haplo-HSCT and chemotherapy; (C) DFS between FLT3-ITD AR low (<0.5) and FLT3 AR high (≥0.5);(D) OS between FLT3-ITD AR low (<0.5) and FLT3 AR high (≥0.5).738 -TANG ET AL.patients.13,[22][23][24]Genes involved in DNA methylation (such as TET2 and DNMT3A) were frequently mutated in FLT3-ITD-mutated AML, but TET2 comutations did not influence the survival of FLT3-ITD-mutated AML patients.Prospective large samples are needed to confirm the incidence of comutations and the most common comutation gene types in FLT3-ITD-mutated AML.

F I G U R E 3
Impact of NPM1 mutation on FLT3-ITD-positive acute myeloid leukemia (AML).(A) Disease-free survival (DFS) between NPM1 mutation and NPM1 wild; (B) Overall survival (OS) between NPM1 mutation and NPM1 wild; (C) DFS in NPM1+FLT3-ITD+ between haploidentical hematopoietic stem cell transplantation (haplo-HSCT) and chemotherapy; (D) OS in NPM1+FLT3-ITD+ between haplo-HSCT and chemotherapy; (E) DFS in NPM1-FLT3-ITD+ between haplo-HSCT and chemotherapy; (F) OS in NPM1-FLT3-ITD+ between haplo-HSCT and chemotherapy.TANG ET AL.In this study, the outcomes of FLT3-ITD patients, irrespective of NPM1 mutation and FLT3-ITD AR, who underwent haplo-HSCT were significantly more favorable than those who received only chemotherapy.NPM1 mutation and FLT3-ITD AR did not affect the clinical outcomes of adult AML patients with FLT3-ITD mutation in our study, which is in accordance with the 2022 ELN, is one of the most important changes in the 2022 ELN and different from the 2022 NCCN and 2017 ELN.

F I G U R E 4
Impact of DNMT3A mutation on FLT3-ITD-positive acute myeloid leukemia (AML).(A) Disease-free survival (DFS) between DNMT3A comutation and without DNMT3A mutation; (B) Overall survival (OS) between DNMT3A comutation and without DNMT3A mutation; (C) DFS in FLT3-ITD+ DNMT3A+ between haploidentical hematopoietic stem cell transplantation (haplo-HSCT) and chemotherapy; (D) OS in FLT3-ITD+ DNMT3A+ between haplo-HSCT and chemotherapy.740 -TANG AL. randomized to receive chemotherapy or haplo-HSCT.Finally, there was a significant difference in age between two groups: the transplant cohort was younger than the nontransplant cohort.In conclusion, this study firstly analyzed the effect of haplo-HSCT on other comutations at diagnosis based on NGS in FLT3-ITDmutated AML patients.Our analysis demonstrated that the most common comutations are NPM1 and DNMT3A.DNMT3A mutation might be a poor prognostic factor in adult AML patients with FLT3-ITD mutations.Haplo-HSCT might overcome the adverse influence of DNMT3A mutation.Further well-designed, randomized trials are needed to confirm these conclusions.