Acute myeloid leukemia with mutated nucleophosmin 1: an immunogenic acute myeloid leukemia subtype and potential candidate for immune checkpoint inhibition

Clinical and preclinical data suggest that acute myeloid leukemia (AML) with mutated nucleophosmin 1 (NPM1) might constitute an immunogenic leukemia subtype. In general, AML with NPM1 correlates with better prognosis, but the underlying mechanisms still remain to be elucidated. Our group previously described specific immune responses against epitopes derived from the mutational region of NPM1.1 Furthermore, in a smaller cohort of AML patients we found a significantly better overall survival for patients with specific cytotoxic T-lymphocyte (CTL) response against NPM1-derived immunogenic epitopes,2 when compared to NPM1 AML patients. This suggests that immune responses against NPM1 might in part contribute to the more favorable outcome in AML patients. Recently, checkpoint inhibition targeting targeting Programmed cell death protein 1 (PD-1)/Programmed cell death 1 ligand 1 (PD-L1) has been proven to be an effective novel immunotherapeutic approach in cancer, including hematological malignancies.43 Therefore, we questioned whether NPM1 AML patients might be candidates for PD-1/PD-L1-directed immune checkpoint inhibition. Thus, we performed flow cytometry and microarray analyses to assess PD-L1 (CD274) expression in leukemic cells, including leukemic progenitor/stem cell compartments, of AML patients with mutant versus wild-type (WT) NPM1.

In total 30 AML patient samples (15 AML NPM1 and 15 AML NPM1) were evaluated, with the informed consent of the patients involved. The patient characteristics are described in Table 1A. At diagnosis, peripheral blood mononuclear cells (PBMC) were separated from blood samples by Ficoll (Pan Biotech, Germany) density gradient centrifugation. Samples were stained with CD34/CD38/CD274 and cell-surface expression was evaluated by flow cytometry using a fluorescence-activated cell sorting (FACS) Aria flow cytometer (BD Biosciences). An NPM1 specific antibody for intracellular immunostaining (NPM1 hu fluorescein isothiocyanate (FITC), specific for the mutant form of nucleophosmin; Bio-Techne) was used to demonstrate the presence of NPM1 in the leukemic progenitor/stem cell compartment of NPM1 AML cases. Moreover, for gene expression analysis of the CD34CD38 compartment enriched for leukemia stem cells (LSC) and progenitor cells, patient samples were sorted using a FACS Aria flow cytometer based on four quadrants (CD34CD38, CD34CD38, CD34CD38 and CD34CD38). PD-L1 expression in CD34CD38 cells was then assessed by Affymetrix U133plus2.0 microarray analysis in comparison to the other cell compartments, designated “AML bulk cells”.

Table 1A.Summary of patient characteristics and PD-L1 (CD274) positive cells.

Via FACS analysis, we observed that many AML cases had relevant expression of PD-L1, and that bulk AML cells of NPM1 AML showed a significantly higher PD-L1 expression in comparison to NPM1 AML patients (median of 1.4% positive cells, range 0.0–8.5%, versus median of 0.3% positive cells, range 0.1–1.1%, respectively; P<0.0001; Table 1B). Importantly, PD-L1 expression was detected at a higher percentage of leukemic progenitor/stem cells (CD34CD38) in NPM1 than in that of NPM1 AML (median of 3.6% positive cells, range 0.0–17.2%, versus median of 0.3% positive cells, range 0.0–3.0%, respectively; P<0.0001; Table 1B and Figure 1A). Results for non-LSC are shown in Figure 1B. In general, the LSC fraction showed a higher PD-L1 expression than the non-LSC bulk AML cells (P=0.0013; Figure 1C). Figures 1D–F display the FACS analysis data of representative patient samples. In Figure 1D, the data of an NPM1 AML case with a relatively high percentage of PD-L1 expressing cells (3.98%) in the LSC fraction is shown. In comparison, Figure 1E shows data for an NPM1 AML patient with lower expression (0.76%), and Figure 1F an NPM1 AML patient with no PD-L1 expression. In order to demonstrate that in NPM1 AML cases the LSC enriched compartment cells also carry the NPM1, intracellular immunostaining was performed with an NPM1 specific antibody. This analysis showed that PD-L1 positive progenitor/leukemic stem cells of NPM1 AML cases showed significant expression of mutant NPM1, thereby indicating that the PD-L1 positive cells are part of the malignant clone (Figure 1G). By microarray analysis, we detected CD274 expression in both AML bulk and in leukemic progenitor/stem cells, however, expression was not significantly different with regard to the ribonucleic acid (RNA) level (Figure 1H), in contrast to FACS analysis. Nevertheless, microarray analysis comparing enriched LSC populations of NPM1 and NPM1 AML patients underlined the potential relevance of the immune system in NPM1 AML patients, as highly immunological regulatory pathways are differentially regulated in NPM1 compared to NPM1 samples.5 However, aside from the PD-L1 expression in this patient population, a high number of other relevant factors e.g., other molecular markers, different characteristics of subclones, the microenvironment and niche of leukemic and normal stem cells, and other immune checkpoint molecules may influence immune reactions against NPM1 AML cells and hence, the outcome of the patients.

Figure 1.Flow cytometry and microarray based analysis of CD274 expression in AML. (A) More LSC compartment cells of NPM1^mut AML patients showed PD-L1 expression compared to NPM1^wt cases (P<0.0001). (B) Similarly, more non-LSC cells of NPM1^mut AML patients showed PD-L1 expression compared to NPM1^wt cases (P<0.0001). (C) The expression analysis (PD-L1 in AML bulk cells (all cells) versus LSC demonstrated that more LSCs express PD-L1 than bulk AML cells) (P=0.0013). (D) – (F) Typical examples of FACS analysis of PD-L1 expression in LSC-enriched cells of two NPM1^mut patients and one NPM1^wt AML patient. (D) Shows an NPM1^mut AML patient with a relatively high percentage (3.98%) of PD-L1 expressing cells in the LSC fraction compared to (E) another NPM1^wt AML patient with lower expression (0.76%) in LSC. (F) Demonstrates an exemplary NPM1^wt AML patient without relevant PD-L1 expression. (G) FACS staining of LSC (CD34⁺CD38⁻) separated cells in NPM1^mut AML samples. These LSC enriched cells harbor cytoplasmic NPM1, thus indicating that these cells belong to the leukemic clone. (G1) Shows a representative dot plot of one NPM1^mut patient, gate R0 represents the blast population. In (G2) the LSC population in gate R1 (R1: 9.73%) is displayed. (G3) Shows the percentage of the NPM1^mut population (99.9%) within the LSC gate R1. In (G4) the percentage of the PD-L1 (CD274) positive cells in gates R0 and R1 (6.19%) is exhibited and (G5) shows the percentage of the NPM1^mut population within the PD-L1 (CD274) positive cells (using gates R0⁺R1⁺R2 consecutively, 100%). (G6) Represents the negative control (CD34/CD38/CD274/no NPM1^mut antibody). (H) Affymetrix gene expression analysis of CD274 expression in LSC (CD34+CD38− enriched cells) versus AML patient blasts. No significant difference in PD-L1 expression was detected in LSC compared to non-LSC cells. LSC: leukemia stem cells; FSC: forward-scattered light; FITC: fluorescein isothiocyanate.

Table 1B.

LSC might be the source of leukemic disease relapse following treatment, making them a critical target for further therapeutic options. Immunotherapy in cancer treatment has experienced a breakthrough in recent years. Consequently, in addition to allogenic stem cell transplantation, T-cell activating immunotherapeutic approaches like immune checkpoint inhibition, chimeric antigen receptor T cells (CARs) or bi-specific T-cell activating antibodies are becoming increasingly important treatment strategies.763 Genetic instability of tumors increases the neoantigen load and might therefore be a prerequisite for a broader spectrum of anti-tumor immune responses.8 However, mechanisms of immune responses and responsible antigen structures have to be further investigated. In AML, the mutational load is lower compared to most solid tumor entities,8 but genetic instability and neoantigen load might depend on the AML subtype. The expression of PD-L1 (CD274) seems to be a predictive marker for the response to PD-1/PD-L1-directed immunotherapies in different solid and hematological malignancies,9 however, there are further challenges to face and overcome in order to clarify the role of PD-L1 expression for immune checkpoint inhibition. In accordance, our findings underline that NPM1 patients might be better candidates for immune checkpoint PD-1/PD-L1-driven immunogenic approaches than other AML subtypes. For example, immune checkpoint inhibition utilized as a maintenance therapy following chemotherapy might be able to prevent relapse, especially as the immune response against leukemic cells is stronger in NPM1 compared to NPM1 AML patients.2 Thus, PD-1/PD-L1 inhibition in NPM1 AML might further boost immune responses, which are possibly responsible for the high cure rate in this AML cohort.

Moreover, immune responses induced by immune checkpoint molecules may result in the activated effector T cells being able to lyse not only NPM1-positive, but also NPM1-negative subclones, independent from their PD-1/PD-L1 status, due to a broader stimulation of these T cells. Markedly, genes with immunological functions seem to play an important role in the NPM1 AML subtype,5 and as such could also induce immune responses against subclones without NPM1 mutation. These facts might explain why the existence of NPM1 clones could mirror the overall immunogenicity of a genetically unstable underlying disease.

In patients relapsing after allogeneic hematopoietic stem cell transplantation, reactivating the immune response via immune checkpoint has been demonstrated to produce a response,10 as has treatment with hypomethylating agents which have the ability to enhance the expression of PD-1/PD-L1.11 Thus, the combination of immunotherapies holds great promise to further improve the efficacy of T cells against cancer cells.127 A high number of immunotherapies are currently under investigation as part of combination therapies, e.g., in combination with vaccination strategies. As mutated NPM1 is an immunogenic neoantigen with epitopes derived from the mutational region of NPM1 inducing specific immune responses in over 75% of patients,1 it is an interesting immunogenic target structure, and NPM1 -derived peptides might be used in combination with immune checkpoint inhibition to prevent AML relapse. Moreover, polyvalent immune responses of CTL against NPM1 specific and known leukemia-associated antigens in NPM1 AML might also be further exploited to achieve negativity for NPM1 MRD.13

Today, due to the good prognosis of most NPM1 AML cases, the majority of NPM1 patients do not receive an allogeneic stem cell transplantation in first complete remission. However, Röllig and colleagues demonstrated a very favorable clinical outcome for NPM1 patients undergoing upfront allogeneic stem cell transplantation.14 This approach further supports the positive effects of the immune system in NPM1 leukemia eradication. In addition, Kuželová and colleagues noted that AML patients expressing certain groups of human leukocyte antigen alleles are predisposed to develop an efficient anti-AML immune response against the cytoplasmatic located NPM1 protein.15

In summary, we detected higher PD-L1 expression in NPM1 patients, especially in the leukemic progenitor/stem cell compartment of NPM1 AML patients. This observation further supports the hypothesis that NPM1-directed immune responses might play an important role in tumor cell rejection, which tumor cells try to escape via the expression of PD-L1. Therefore, in NPM1 AML cases the immunogenicity of neoantigens derived from the NPM1 mutation and the higher CD274 expression constitute promising target structures for individualized immunotherapeutic approaches. PD-1/PD-L1-directed immune checkpoint inhibition approaches to enhance NPM1 specific T-cell responses might be combined with antigen-directed immunotherapies e.g., peptide vaccination against immunogenic NPM1 epitopes, in order to eradicate persisting MRD following conventional chemotherapy.