CD7-positive leukemic blasts with DNMT3A mutations predict poor prognosis in patients with acute myeloid leukemia

Background DNMT3A mutations can be detected in premalignant hematopoietic stem cells and are primarily associated with clonal hematopoiesis of indeterminate potential; however, current evidence does not support assigning them to a distinct European Leukemia Net (ELN) prognostic risk stratification. CD7 is a lymphoid antigen expressed on blasts in approximately 30% of acute myeloid leukemia (AML), and its role in AML remains unclear and depends on subgroup evaluation. This study investigated the prognostic value of DNMT3A mutation combined with CD7 expression in AML. Methods We retrospectively analyzed the clinical data of 297 newly diagnosed non-M3 AML patients. According to the DNMT3A mutation and CD7 expression in AML cells, patients were divided into the DNMT3A-mutated/CD7-positive (CD7+), DNMT3A-mutated/CD7-negative (CD7-), DNMT3A-wild-type/CD7+, and DNMT3A-wild-type/CD7- groups. Results The DNMT3A-mutated/CD7+ group had lower complete remission rates and higher relapse rates. Importantly, these patients had significantly shorter overall survival (OS) and relapse-free survival (RFS). Furthermore, multivariate analysis showed that CD7+ with DNMT3A mutation was an independent risk factor for OS and RFS. Conclusion CD7+ with DNMT3A mutation predicts a poor prognosis in AML patients, and the immunophenotype combined with molecular genetic markers can help to further refine the current risk stratification of AML.


Introduction
Acute myeloid leukemia (AML) is a hematologic malignancy caused by abnormal clonal proliferation of hematopoietic stem and progenitor cells, and its clinical manifestations and prognosis are highly heterogeneous (1).Currently, the prognosis of AML patients is based mainly on cytogenetic and molecular genetic alterations (2).Although the relationship between some genetic mutations and the prognosis of AML has been clarified, the relationship between other genetic alterations and AML prognosis is not yet fully understood (3).DNA methyltransferase 3A (DNMT3A) is an epigenetic protease that, along with DNMT3B, is responsible for de novo DNA methylation (4,5).At present, there is still some controversy about the prognostic significance of DNMT3A mutations.Most investigators contend that DNMT3A mutations are related to the poor prognosis of AML patients (6, 7).Moreover, some studies suggest that the poor prognostic value of DNMT3A mutations may be limited to specific types of AML patients (5,(8)(9)(10)(11).However, the 2022 European Leukemia Net (ELN) guidelines state that current evidence is insufficient to classify DNMT3A mutations into a clear prognostic group (3).Therefore, more prognostic markers must be combined as a supplement to assess the prognostic value of DNMT3A mutations.
The prognostic significance of DNMT3A mutation and CD7 expression in AML is still inconclusive, and there are currently no studies exploring the clinical significance of DNMT3A mutation combined with CD7 expression in AML patients.Therefore, we conducted a retrospective study to evaluate the prognostic value of DNMT3A mutation combined with CD7 expression in AML patients.

Patients
In this retrospective analysis, 297 patients with de novo AML were enrolled.Samples were collected at Zhengzhou University People's Hospital and the First Affiliated Hospital of Zhengzhou University from January 2017 to July 2021.The last date of follow-up was July 30, 2022.Patients with a history of hematologic disease, therapy-related AML or other malignancies, or acute promyelocytic leukemia (FAB-M3) were excluded.All baseline data were obtained from medical records, including age, sex, white blood cell count (WBC), hemoglobin (HB), platelet (PLT) count, lactate dehydrogenase (LDH), peripheral blood (PB) blast percentage, bone marrow (BM) blast percentage, immunophenotype, karyotype, gene mutations, and French-American-British classification (FAB) subtypes as well as the induction therapy regimen and treatment response.All patients signed an informed con-sent form at the time of treatment, and this study complied with the requirements of our institutional ethics committee (2021-140-02).

Immunophenotypic analysis
Before initial treatment, 2-3 ml of BM aspiration fluid was collected and placed in an EDTA anticoagulant tube.The Navios flow cytometer from Beckman Coulter Company in the United States was used for a 4-color immunolabeling method to detect the expression levels of CD2, CD4, CD7, CD34, CD56, CD38, CD117, CD33, CD13, HLA-DR, and others.CD45/SSC was utilized to set the gate, and the Kaluza software was employed to perform immunophenotypic analysis of BM blasts.Antigen expression was defined as positive when the amount of antigen expression was ≥20%.Representative flow cytometry plots of bone marrow blasts from AML patients in the DNMT3A-mutated/CD7-positive (CD7+) group, the DNMT3A-mutated/CD7-negative (CD7-) group, the DNMT3A-wild-type/CD7+ group, and the DNMT3Awild-type/CD7-group are displayed in Supplementary Figure 1.

Definitions of treatment response and survival time
CR was defined as <5% blasts in BM, no blasts with Auer bodies, no blasts in peripheral blood, no extramedullary leukemia, neutrophil count ≥1.0×10 9 /L, and PLT count ≥100× 10 9 /L.CR with incomplete hematologic recovery (CRi) was defined as meeting all CR criteria, except residual neutropenia (<1.0×10 9 /L) or thrombocytopenia (<100×10 9 /L).PR was defined as the proportion of BM blasts between 5% and 25%, which should be at least 50% lower than before therapy, and the blood cell count must be within the range of CR.NR was defined as failure to achieve CR, CRi or PR after chemotherapy.The primary study endpoints were overall survival (OS), measured as the time from diagnosis to the date of death from any cause or the date of the last follow-up, and relapse-free survival (RFS), measured as the time from the date of CR or CRi to the date of hematologic relapse, death from any cause, or the date of the last follow-up (3).

Statistical analysis
The comparisons between groups were made using Kruskal-Wallis rank-sum test and one-way ANOVA for continuous variables, which are presented as medians (ranges).Fisher's exact test or the chi-square test was used to compare categorical variables.The Kaplan−Meier method was used to examine OS and RFS, and the log rank test was used to compare group differences.To examine the parameters influencing OS and RFS, univariate and multivariate Cox proportional hazards regression were performed.All statistical analyses were carried out using SPSS software version 21.0, and graphs were created using GraphPad Prism version 9.0.Two-sided p values <0.05 were considered statistically significant.Additionally, OncoPrint was utilized to identify and visualize gene mutations in AML patients.

Correlation between CD7 expression and other immunophenotypes
Our results showed that the positive expression proportions of CD34, CD117 and CD123 were significantly higher in the CD7+ group than in the CD7-group (p<0.05,Table 2); the positive expression proportions of CD4, CD56 and CD19 were significantly lower in the CD7+ group than in the CD7-group (p<0.05;Table 2).The differences in the proportions of CD38, HLA-DR, CD15, CD33, CD2, CD64 and CD71 expression between the two groups were compared separately, and the results were not significantly different (p>0.05,Table 2).

Correlation of DNMT3A mutation with other molecular genetics
According to the 2022 ELN genetic risk stratification, we divided AML patients into favorable (n=36), intermediate (n=242), and adverse (n=19) karyotype groups.Within the intermediate karyotype group, the CEBPA bZIP mutation rate  was significantly lower in the DNMT3A-mutated group (0.0%) than in the DNMT3A-wild-type group (12.0%); the FLT3-ITD mutation rate (32.2%),NPM1 mutation rate (47.5%) and IDH1/2 mutation rate (28.8%) were significantly higher in the DNMT3A-mutated group than in the DNMT3A-wild-type group (11.5%, 13.1%, 10.9%), the differences were statistically significant (p<0.05,Table 3).However, no differences in gene mutation rates were observed between the DNMT3A-mutated group and the DNMT3Awild-type group in the favorable and adverse karyotype groups (Supplementary Table 2).Our further analysis revealed that the DNMT3A-mutated/CD7+ group still exhibited a higher FLT3-ITD mutation rate compared with the non-DNMT3Amut/CD7+ group (Supplementary Table 3).In addition, we generated an oncoprint to display the distribution of gene mutations in AML patients (Figure 1).

Prognostic impact of DNMT3A mutation and CD7 expression on AML patients respectively
AML patients were divided into two groups based on their DNMT3A mutation status; the median OS time was 22 months for DNMT3A-mutated patients and 32 months for DNMT3A-wild-type patients, with no statistically significant difference (p>0.05, Figure 2A); similarly, the median RFS of the DNMT3A-mutated group was not significantly different from that of the DNMT3Awild-type group (10 months vs. 22 months, p>0.05, Figure 2B).AML patients were divided into two groups according to CD7 expression; the median OS time was 24 months in the CD7+ group and 51 months in the CD7-group, and the difference in OS between the two groups was not significantly different (p>0.05, Figure 2C); the median RFS time was 20 months and 19 months in the CD7+ and CD7-groups, respectively, and the difference was also not significantly different (p>0.05, Figure 2D).

Prognostic impact of CD7+ with DNMT3A mutation in AML patients
The median OS time for patients in the DNMT3A-mutated/ CD7+ group was 6 months, whereas that in the DNMT3A-mutated/ CD7-group was 51 months (p=0.003, Figure 2E); similarly, the median OS in the DNMT3A-mutated/CD7+ group was significantly shorter than that in the DNMT3A-wild-type/CD7+ and DNMT3A- The OncoPrint of genes in AML.Mutations were colored as green.The upper barplot indicates the number of genetic mutation per patient, while the right barplot shows the proportion of genetic mutations per gene.The group, sex, age, 2022-ELN risk stratification and induction chemotherapy regimen were added as annotations for the patients.wild-type/CD7-groups (6 months vs. 36 months, p=0.001; 6 months vs. 32 months, p=0.001, Figure 2E).In addition, the median RFS was significantly shorter in the DNMT3A-mutated/ CD7+ group than in the other three groups (3 months vs. 42 months, p=0.003; 3 months vs. 28 months, p<0.001; 3 months vs. 19 months, p<0.001, Figure 2F).Therefore, we reclassified AML patients into the DNMT3A-mutated/CD7+ group (n=21) and the other (non-DNMT3Amut/CD7+) group (n=276) based on whether they had concomitant DNMT3A mutation and positive CD7 expression.Comparing the OS and RFS of patients in the two groups separately, we found that the DNMT3A-mutated/CD7+ group had significantly shorter OS and RFS (p<0.001,Figures 2G, H).

Effect of CD7+ with DNMT3A mutation on the initial treatment response in AML patients
Among the entire cohort of 297 patients, CR (CR/CRi) was achieved in 233 patients (78.5%) after two cycles of induction chemotherapy.
CR was achieved in 12 patients (57.1%) in the DNMT3Amutated/CD7+ group compared to 221 patients (80.1%) in the non-DNMT3Amut/CD7+ group, and the CR rate was significantly lower in the DNMT3A-mutated/CD7+ group than in the non-DNMT3Amut/CD7+ group (p=0.003).Of the 233 patients with CR, 97 experienced disease recurrence, including 9 (75.0%) in the Kaplan-Meier curves of overall survival (OS) and relapse-free survival (RFS) in AML patients according to DNMT3A mutation and CD7 expression.4).Consistent with this, we found that DNMT3A-mutated/ CD7+ patients still showed a low CR rate and a high relapse rate among patients who received intensive induction therapy (Supplementary Table 4).

Univariate and multivariable analyses of clinical prognostic factors
We analyzed various prognostic factors affecting OS and RFS in AML patients.Univariate Cox regression analysis showed that age >60 years, WBC count ≥100 × 10 9 /L, no HSCT, and DNMT3A-mutated/CD7+ were risk factors for poorer OS and RFS in patients with newly diagnosed AML.Multifactorial Cox regression analysis showed that DNMT3A-mutated/CD7+ remained significantly associated with shorter OS and RFS (Table 5).In addition, we further analyzed the factors affecting prognosis in patients who did not undergo HSCT and those who received intensive induction therapy, and multifactorial Cox regression analysis still suggested that DNMT3A-mutated/CD7+ was an independent risk factor for AML patients (Supplementary Table 5).

Discussion
In this study, the importance of DNMT3A mutation combined with CD7 expression for the precise stratification of AML patients was evaluated.Our results showed that CD7+ AML patients with DNMT3A mutations had lower CR rates and higher relapse rates.More importantly, our data showed for the first time that OS and RFS were significantly shorter in AML patients with DNMT3A mutations combined with CD7 expression, whereas DNMT3A mutations or CD7-positive expression alone had no significant effect on prognosis.
The CD7 antigen is a T-cell-related antigen that is expressed on thymocytes, T cells, and natural killer cells as well as lymphocytes and myeloid progenitors in healthy individuals (18), whereas 30% of AML patients exhibit positive CD7 expression (16-18).There is still controversy about whether CD7+ indicates poor prognosis of AML.Our data showed that there was no significant difference in OS and RFS between CD7+ AML patients and CD7-AML patients, which was similar to that reported by Fang et al. (23), suggesting that CD7 expression may not be related to the prognosis of AML.However, Ogata et al. found differences in the prognostic impact of CD7+ in AML patients under different cytogenetic conditions (18).Therefore, the prognostic significance of CD7 positivity may need further subgroup analysis.
Some studies have shown that DNMT3A gene mutations precede the AML stage (26, 27).Therefore, the impact of DNMT3A gene mutations on the prognosis of AML patients should be given sufficient attention.In our analysis, there was no significant difference in OS and RFS between the DNMT3Amutated group and the DNMT3A-wild-type group.Univariate and multivariate analysis showed that DNMT3A mutation was also unrelated with OS and EFS.The present study is consistent with previous studies showing that DNMT3A mutation cannot be used as an independent prognostic marker for AML patients (28,29).However, some studies have found that DNMT3A mutations in AML patients affect patient prognosis and are negatively associated with patient prognosis (6, 7), and this adverse prognostic effect may be limited to specific types of AML groups (8)(9)(10)(11).Therefore, we further analyzed their prognostic impact in combination with specific immunophenotypes.
Interestingly, when we combined DNMT3A mutation with CD7-positive expression, we found that patients in the DNMT3Amutated/CD7+ group had significantly shorter OS and RFS compared with patients in the other three groups.However, no significant differences were observed in OS and RFS among the remaining three groups.Therefore, we reclassified AML patients into DNMT3A-mutated/CD7+ and non-DNMT3Amut/CD7+ groups.Notably, we found that patients in the DNMT3Amutated/CD7+ group had lower CR rates, higher relapse rates, and shorter OS and RFS.Multivariate regression analysis also showed that CD7+ with DNMT3A mutation was a Powerful Predictor for poor prognosis in AML patients.This result has not yet been reported in the literature.
The reason for the poorer prognosis of DNMT3A-mutated/ CD7+ patients is currently unknown.We hypothesize that it is related to the following factors.First, we observed higher WBC counts at the initial diagnosis in patients with DNMT3A-mutated/ CD7+, potentially explaining their poorer prognosis.In addition, our data showed that AML patients with DNMT3A mutations frequently carry NPM1 and FLT3-ITD mutations, which is consistent with previous studies (9,11,29,30).Studies have shown that AML patients with mutations in all three DNMT3A/ FLT3/NPM1 genes have a higher disease load and lower cumulative overall survival (31)(32)(33).Our data also showed that the DNMT3A mutation group was associated with lower CEBPA bZIP mutation  [34][35][36].Therefore, we infer this is related to the poor prognosis of DNMT3A-mutated/ CD7+ patients.Furthermore, we also found that the positive expression proportion of CD34, CD117, and CD123 was significantly higher in the CD7+ AML group than in the CD7-AML group, and the proportion of positive expression of CD4-was lower than in the CD7-AML group.CD34 and CD117 are precursor markers, and previous studies have shown that CD34positive expression is associated with lower CR rates (17, 37-39).
CD123 is a specific antigen on the surface of leukemia stem cells (LSCs) (40), and several studies have found that CD123 is an independent risk factor for CR and OS in AML patients (40,41).
Relevant literature shows that AML leukemia cells with CD4 expression originate from a relatively mature stage (42).Our study showed that CD7+ was associated with high expression of immature phenotype and low expression of mature phenotype.This association may contribute to the poorer clinical outcomes of AML patients with DNMT3A-mutated/CD7+.However, the underlying mechanism of this association requires further exploratory studies.
As this study was a retrospective study, there may be a small number of cases in some groups after grouping, which may have biased the data.In addition, the heterogeneity of treatment regimens may confound the results of this study.The data we collected showed no patients in the DNMT3A-mutated/CD7+ group who underwent HSCT.We consider that the reason for this phenomenon may be related to the small sample size in this subgroup analysis.Furthermore, not undergoing HSCT may also be influenced by the patient's financial status, HLA matching, and complications.Therefore, large-scale, multicenter prospective studies are needed to further validate the prognostic value of CD7+ with DNMT3A mutation.

Conclusions
In conclusion, we found that CD7+ with DNMT3A mutation is an effective prognostic marker in AML patients and has a negative impact on the OS and RFS of these patients.Our results reveal the clinical value of immunophenotyping for the precise molecular genetic stratification of AML.

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

TABLE 1
Clinical characteristics of patients with AML classified according to the status of DNMT3A mutation and CD7 expression.

TABLE 3
Correlation of DNMT3A mutation status with other molecular genetic mutations in intermediate karyotype.

TABLE 4
Treatment response to the first and second cycles of induction chemotherapy between the two groups.

TABLE 5
Univariate and multivariate analysis of OS and RFS in AML patients.