CD9 in acute myeloid leukemia: Prognostic role and usefulness to target leukemic stem cells

Abstract CD9 is a cell surface protein and belongs to the tetraspanin family. Its role in carcinomagenesis has been widely studied in solid tumors but remains controversial, depending on the cancer type. Although CD9 seems to be associated with unfavorable outcome and disease progression in acute lymphoblastic leukemia (ALL), this marker has not yet been studied in acute myeloid leukemia (AML). First, we explored its prognostic role and its association with biological factors in a cohort of 112 AML patients treated with intensive chemotherapy. CD9 was expressed in 40% of AML and was associated with a favorable outcome (event‐free survival and relapse‐free survival) in univariate (P = 0.009 and P = 0.048, respectively) and multivariate (P = 0.004 and P = 0.039, respectively) analyses. Interestingly, CD9 expression was different between the more immature physiologic and AML cells (CD34+CD38−) as it was also expressed in AML on putative leukemic stem cells (LSCs) but not on hematopoietic stem cells (HSCs). Hence, CD9 could be a very relevant marker for minimal residual disease (MRD) monitoring in AML based on LSC targeting.


| INTRODUCTION
Acute myeloid leukemia (AML) is the most common cause of leukemia-related mortality in adult patients with a 5-year overall survival (OS) around 40%. 1 Initially, AML is a clonal disorder of hematopoietic stem cells (HSCs) characterized by an arrest of differentiation with associated proliferation, a subsequent accumulation of blast cells at various stages of incomplete maturation, and a reduced production of healthy hematopoietic elements. Furthermore, AML is a heterogeneous disease at both the phenotypic and molecular levels with an accumulation of successive genetic defects and coexisting clones. 2 This heterogeneity extends to the leukemic stem cells (LSCs), which are thought to be resistant to intensive chemotherapy based on anthracycline and cytarabine and therefore, mediate disease relapse. 3 The frequency of these cells at diagnosis is associated with worse outcome in AML. 4 LSCs are enriched in the CD34+/CD38− cell compartment and these cells are able to reproduce human AML in NOD/ SCID mice. 5 These LSCs are found in this compartment even in NPM1-mutated AML patients which are associated with a CD34-negative phenotype. 6 Over the past few years, various markers have been described to better characterize LSC or blast cells from AML with normal cytogenetics (CN-AML) for minimal residual disease (MRD) monitoring. [7][8][9][10] Furthermore, targeting MRD by multiparametric flow cytometry (MFC) to monitor treatment efficiency is one of the greatest challenges in the treatment of AML. Recently, we reported a reliable method by MFC to quantify three hematopoietic progenitor populations and the putative LSC compartment by using a combination of antibodies at diagnosis and follow-up. 11 However, these markers are not always enough to target LSCs and new strategies are needed, even in conventional MRD by MFC based on leukemia-associated immunophenotypes (LAIP).
CD9 antigen belongs to the tetraspanin family, which are cell surface proteins clustering in membrane entities called tetraspanin-enriched microdomains (TEMs). 12 Tetraspanins contribute to numerous cellular process as cell adhesion, cell activation, or proliferation and have been implicated in several carcinogenous processes such as angiogenesis or metastasis in human cancers. 13 Among them, CD9 was first known as a tumor suppressor but several studies showed its oncogenic and prometastatic functions, [14][15][16] suggesting that the different partners of CD9 in TEMs could explain these different roles. In hematological malignancies, CD9 has been mainly studied in acute lymphoblastic leukemia (ALL) where it might promote cancer stem cell-like properties and dissemination of the disease with CXCR4-mediated migration. 17,18 Its role in AML remains controversial:a few studies suggested a pejorative role of CD9 with a decreased OS 19 and a negative association with t(8;21)(q22;q22) 20 while another study showed a correlation between CD9 and mutated NPM1 AML 21 which are associated with a favorable prognosis. Here, we analyzed the expression of CD9 on AML primary cells and physiologic progenitors, the prognostic role of CD9 on survival in AML patients treated with intensive chemotherapy, its association with classical biological factors and its usefulness to discriminate LSCs from HSCs.

| Patients
One hundred and twelve patients with AML de novo diagnosed between 2009 and 2016 and treated with intensive chemotherapy were included in this study. Patients with acute promyelocytic leukemia were excluded. Patients were treated with induction chemotherapy (continuous infusion of cytarabine for 7 days with daunorubicin or idarubicine for 3 days) and at least two consolidation courses after complete remission (CR). High-risk cases (unfavorable cytogenetic or combined genetic risk, patients with early relapse…) underwent hematopoietic stem cell transplantation (HSCT). All patients were treated in the department of Hematology of Lille hospital. Signed informed consent was obtained from each patient in accordance with the declaration of Helsinki. Cytogenetic and molecular risks and CR criteria were determined according to European Leukemia Net recommendations. 22

| Multiparametric flow cytometry
Diagnostic blast cells were obtained from cryopreserved bone marrow (BM) aspirates. MFC results were not different between fresh and frozen cells for three patients (data not shown). Each sample was washed twice in RPMI with 10% fetal bovine serum (FBS) at 37°C then stained for 30 minutes at room temperature with the following antibody panel The gating strategy for flow cytometry analysis was then performed as described previously. 11 Briefly, blast cells were gated as CD45dim/SSClow population and hematogones (CD38++CD19+ phenotype) were excluded from this gate using these two antibodies. CD34 and CD38 positivity were preset on this population and then, P6 (CD34+CD38−), P7 (CD34+CD38dim), and P8 (CD34+CD38+) populations were determined within blast cells. Finally, from the P6 gated cells, the different progenitor populations were determined using CD90 and CD45RA expression.

| Statistical analysis
Comparison of CD9 expression and CD9 mean fluorescence intensity (MFI) between AML bone marrows, normal bone marrows, and hematogones was assessed by Kruskal-Wallis nonparametric test. Differences between the patients negative and positive for CD9 (ie, less or more than 20% of expression on blast cells) on quantitative variables were assessed by Student t test and qualitative variables were compared using chi-square test.
Quantitative variables associated with either OS, event-free survival (EFS), and relapse-free survival (RFS) were tested | 1281 with the Cox model. OS, EFS, and RFS were then described by the Kaplan-Meier method. For patients who underwent BM transplantation, survival was censored at the date of transplantation and for patients alive, survival was censored at the date of last known alive. Multivariate analysis was performed with a Cox method:hazard ratios were adjusted on variables with significant pronostic value (P < 0.05) for EFS, OS, and RFS.
A P-value < 0.05 (two-tailed) was considered statistically significant. Statistical analysis was performed using SPSS 22.0 software (IBM, New York, USA).

| CD9 expression on normal and leukemic cells
CD9 expression was observed on monocytes on normal BM and normal peripheral blood, and absence of CD9 expression was observed on granulocytes as previously reported ( Figure 1A). 23 We analyzed CD9 expression on normal hematogones (HTG) (CD38++ CD19+ CD45 dim), on physiologic myeloblasts gated as CD45dim/SSClow cells after exclusion of hematogones from 25 normal BM and on blast cells from 112 AML patients with the same gating. CD9 expression was strong and homogeneous on normal HTG (MFI median = 125, range 81.7 to 256.3), heterogeneous on blast cells (median of expression = 29.8%, range = 0 to 99%, MFI median = 66, ranging from 2.5 to 294), and weak and homogeneous on physiologic myeloblast compartment (median of expression = 6.4%, range = 1.3 to 16.6%, MFI median = 12.6, range = 2.2 to 30.1, Figure 1B). A significant higher expression of CD9 was found on malignant blast cells compared to physiologic myeloblasts considering percentages of expression and MFI levels ( Figure 1C, Kruskal-Wallis nonparametric test P < 0.001). Thus, we could consider expression of CD9 as a frequent leukemia-associated immunophenotype (LAIP) in our study.

| CD9 and prognostic factors in AML
One hundred and twelve patients were included in our study with ages ranging from 21 to 78 years. First, we observed three types of CD9 expression profile:weak or negative (Figure 2A), intermediate with heterogeneous positive expression ( Figure 2B) and strong and homogenous expression ( Figure 2C). Then, we compared patient characteristics according to CD9 expression ( Table 1). Expression of CD9 was found in 45 of 112 patients (40%) and there was no association with sex, age, white blood count (WBC), FAB type, hemoglobin level, platelet count, peripheral blood (PB) blast cells percentage, bone marrow (BM) blast cells percentage, cytogenetic risk, FLT3-ITD and NPM1 mutations. CD9-positive AML tended to include more AML with NPM1 mutation (P = 0.09). There was no association of CD9 expression with

| Prognostic role of CD9 in AML
We used univariate analysis to evaluate the following parameters for EFS, OS, and RFS: age, WBC count, hemoglobin level, platelet count, cytogenetic risk group, FLT3-ITD status, NPM1 mutational status, and CD9 expression. A multivariate model for EFS, OS, and RFS was used for all variables with a P-value < 0.05. All the results are summarized in Table 2. As expected, cytogenetics was the most powerful variable (P < 0.001) for EFS, OS, and RFS.
For OS, in univariate analysis, CD9 expression on blast cells had no significant impact (P = 0.16) ( Figure 3B).
Then, percentage of CD9-positive cells was associated in this study with significant better EFS and RFS in univariate and multivariate analyses.

| CD9 is expressed on LSC and not on the more immature progenitors
We then investigated CD9 expression on normal hematopoietic progenitors (HSCs) and on their leukemic counterparts (LSCs) to use CD9 as a marker of MRD in the most immature compartment. We studied progenitor cell populations  10 These phenotypic definitions of progenitor cells have been previously verified in functional assays. 24 CD9 expression on these populations was studied in 17 normal BM and 17 BM from CD9+ AML patients. Interestingly, the expression of CD9 was always positive on LSCs and always negative on HSCs from normal BM (Figures 4 and 5A). We observed a similar CD9 MFI level on LSC compared to AML blast cells (MFI on LSCs, median = 76 and MFI on blast cells, median = 66, P = 0.11, Figure 5A). On normal bone marrow progenitors, CD9 appeared at the MPP stage  and was observed on LMPP with similar MFI values ( Figure  5A). Nevertheless, the percentage of CD9-positive cells was higher at the LMPP stage ( Figure 5B). On the contrary, CD9 expression and CD9 MFI were steady in all AML subcompartments such as CD34+CD38− (P6), CD34+CD38dim (P7), and CD34+CD38+ (P8) AML cells ( Figure 5C,D).

| DISCUSSION
Acute myeloid leukemia is an aggressive condition and chemotherapy regimens need to be adapted to the patient's disease. New prognostic tools are then important, especially for CN-AML patients, to refine the prognosis. One of the most interesting measurement of treatment response is MRD monitoring. Detection by MFC of markers expressed in blast cells is one of the most studied field over the past few years but identification of a cell antigen only expressed in AML (and not in physiologic myeloblasts) has not been successful so far. In our study, CD9 is expressed in 40% of AML and, considering its expression at a significant level on LSCs, it should be investigated when these cells are studied by the MRD strategy that we described previously. 11 In ALL, CD9 seems to be associated with cancer stem cell properties too and is involved in leukemic progression. 25 Interestingly, CD9 antigen level of expression is identical between LSCs and AML blast cells and is not expressed on normal HSCs. These findings are particularly relevant as the majority of antigens associated with LSCs (ie, TIM3, CLL1, and CD244) are less expressed on these cells compared to bulk cells and are frequently coexpressed on normal HSCs and progenitors. 26 Recently, Coustan-Smith et al published a study on MRD monitoring in AML by MFC and, interestingly, they found an overexpression of CD9 on blast cells (in 30% of AML cases) compared to physiologic myeloblasts. 27 Furthermore, they showed that CD9 was abnormally overexpressed on CD34+CD38− AML cells at diagnosis and relapse of 10 paired samples, thus corroborating the MFC strategy on LSCs that we used in this study. We investigated CD9 expression on progenitors from 17 normal BM and 17 AML BM so to strengthen the conclusions on LSCs, more AML, and normal samples, need to be studied.
With these first results, targeting CD9 in AML seems to be an interesting approach to eliminate LSCs and prevent relapses with monoclonal antibodies or even chimeric antigen receptor (CAR) T-cell therapy. Anti-CD9 has already been used in vitro and could induce apoptosis of Jurkat cells and inhibition of proliferation of B-ALL cells. 25,28 CD9 is also widely expressed on monocytes and depletion of these cells could be interesting in AML as they are implicated in disease progression by producing interleukin-1. 29 In our study, we demonstrated a favorable role of CD9 on AML prognosis, especially on EFS and RFS in univariate and multivariate analyses. This favorable role was related to the percentage of CD9+ blast cells but was not highlighted according to the MFI values. Some of the patients in this