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Leukemic Stem Cells of Acute Myeloid Leukemia Patients Carrying NPM1 Mutation Are Candidates for Targeted Immunotherapy

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Leukemic Stem Cells of Acute Myeloid Leukemia Patients Carrying NPM1 Mutation Are Candidates for Targeted Immunotherapy Letters to the Editor 1759 Leukemic stem cells of acute myeloid leukemia patients carrying NPM1 mutation are candidates for targeted immunotherapy Leukemia (2014) 28, 1759–1762; doi:10.1038/leu.2014.116 prognostic and predictive maker.7 AML patients with normal karyotype carrying NPM1mut have a significant better overall survival compared with wild-type patients without FLT3-ITD.8 However, the underlying mechanisms are still not clear. These Targeting minimal residual disease (MRD) to prevent relapse is mutations cause an abnormal shift of the NPM1 protein from the one of the major challenges in treatment of acute leukemias. nucleus to the cytoplasm, a mechanism first described by the group Leukemic stem cells (LSCs) seem to host biological properties that of Falini. LSC of NPM1mut patients might be the source for leukemic render them resistant to chemotherapy and thus might be self-renewal and might account to disease relapse after treatment. responsible for MRD.1 Because traditional chemotherapy often We were interested in the expression differences of the LSC- fails to eliminate LSC, development of novel therapeutics is enriched cell fraction descendent of NPM1mut and NPM1wt primary essential for the eradication of the complete leukemic cell fraction. AML patients. We screened our data with regard to new potential An immunotherapeutic approach could be one strategy to therapeutic structures to target LSC of NPM1mut patients. Our eliminate residual leukemic cells. focus was on targets for immunotherapeutic approaches that are Leukemia-associated antigens (LAAs) constitute targets for potential candidates for treatment of MRD. Functional immuno- immunotherapeutic approaches as they are involved in cell logical assays indicate the inhibition of CD34 þ CD38 À cells by differentiation and proliferation mechanisms.2 LAAs like RHAMM, immunotargeting strategies. proteinase 3 and Wilms’ tumor antigen 1 (WT1) were tested in We chose CD34 þ and CD38 À as surface markers for patients clinical peptide vaccination trials revealing immunological and and healthy volunteers to find access to the LSC-enriched cell clinical responses in patients with different hematological population. First, we screened our data to define expression malignancies.3 differences in the two AML groups NPM1mut and NPM1wt (Table 1). Recently, we described specific T-cell responses against Although most cells in NPM1mut AML are CD33 þ and CD34 À , the epitopes derived of the mutated region of nucleophosmin 1 CD34 þ CD38 À cell fraction harbors LSC within the NPM1mut (NPM1mut).4 Immune responses against the mutated protein of leukemic cells. Several groups demonstrated the presence of NPM1 might contribute to the favorable prognosis of acute NPM1mut in LSC and showed an adverse outcome in patients not myeloid leukemia (AML) patients carrying NPM1mut. We detected a carrying the NPM1mut confirming the prognostic value of mutated better overall survival in a smaller cohort of NPM1mut AML patients NPM1.9,10 Differentially expressed genes might influence showing immune responses against NPM1.5 We demonstrated molecularly and immunologically relevant pathways that could polyspecific cytotoxic T-cell (CTL) responses against several known have an impact on the better overall survival of the mutated LAAs but also NPM1#3 after preemptive donor lymphocyte patients. Therefore, we enriched the CD34 þ CD38 À cell infusion in an AML patient with NPM1mut and molecular population of 21 patient samples by fluorescence-activated relapse.6 cell sorting using 5 Â 107–2 Â 108 cells, depending on the cell Mutations in NPM1 are one of the most frequent molecular frequency (Figure 1a). CD34 þ CD38 À cell frequency in NPM1mut alterations in AML and therefore NPM1mut became an important AML patients (n ¼ 9) ranged from 0.63 to 6.8% (mean 3.87%) Table 1. Gene expression level analysis of the enriched LSC-containing fraction of NPM1mut, NPM1wt AML patients and HSC of healthy volunteers Parametric LSC LSC HSC Fold- Fold-change Symbol Function P-value NPM1mut NPM1wt change HSC patients NPM1mut 0.0074418 249.32 25.71 14.6 9.7 17.05 SERPINA1 A serine protease inhibitor whose targets include elastase, plasmin, thrombin, trypsin, chymotrypsin and plasminogen activator 0.00005 205.93 30.08 27.7 6.85 7.43 H19 Encodes for noncoding RNA, may act as a tumor suppressor 0.0003405 92.67 14.89 — 6.22 — IGSF10 Cell adhesion, protein amino-acid phosphorylation 0.0016458 299.88 64.33 33.2 4.66 9.03 OSCAR Member of the leukocyte receptor complex protein family that has critical roles in the regulation of innate and adaptive immune responses 0.0035087 178.44 44.36 28.87 4.02 6.1 SUCNR1 Hematopoietic progenitor cell development 0.0005275 214.17 55.49 34.38 3.86 6.22 CD96 Member of the immunoglobulin superfamily; has a role in adhesive interactions of activated T and NK cells; functions in antigen presentation 0.0028565 146.39 56.39 — 2.6 — SH2D1A Has a role in the bidirectional stimulation of T and B cells 0.000834 70.45 37.78 24.28 1.87 2.9 IL12RB1 Lack associated with immunodeficiency Abbreviations: AML, acute myeloid leukemia; HSC, hematopoietic stem cell; LSC, leukemic stem cell; NPM1, nucleophosmin 1. Analysis of gene expression differences of both patient groups compared with the healthy HSC using BRB-ArrayTools, version 4.2.1 (Bethesda, MD, USA). Description of the gene function mentioned in the table refers to: http://www.ncbi.nlm.nih.gov/gene. Expression profiles of the two AML patient groups were compared with the expression levels in the HSC segment. We found several differentially expressed genes that are of immunological relevance. CD96 and IL12RB1 seem to be interesting targets for an immunotherapeutic approach, as they are overexpressed in enriched LSC fractions of NPM1mut AML patients. Accepted article preview online 28 March 2014; advance online publication, 25 April 2014 & 2014 Macmillan Publishers Limited Leukemia (2014) 1736 – 1764 Letters to the Editor 1760 Patient A Patient B negative control mut OCI AML 2 NPM1wt OCI AML 3 NPM1 all cells CD34+ cells *** *** ** ** ** p=0.0005 * * * * * * * * * * Figure 1. (a) CD34/CD38 cell sorting of NPM1mut and NPM1wt. Frequency of CD34 þ CD38 À cells in NPM1mut patients before sorting is lower than that in NPM1wt patients. There is a trend in the difference of frequency (P ¼ 0.07). (b) Immunocytochemistry staining of the enriched LSC fraction for cytoplasmic NPM1. Enriched CD34 þ CD38 À cells of two representative AML patients (denominated A/B) with NPM1mut show cytoplasmic nucleophosmin stained in brownish color. The control samples (HV) shows no staining for NPM1 in the cytoplasm, but as expected NPM1 is detectable in the nucleus. (c) IL-12 influence on immunogenetic primed effector cells. Analyses of the immune-stimulating effect of IL-12 on activated CTL and their lyses potential on target cells. Cell lines OCI AML 2 (NPM1wt) and OCI AML 3 (NPM1mut) were used as target cells. There is no effect on adding IL-12 to target cells only. Lysis rate increases when effector cells are added. IL-12 seems to have an influence on the lysis potential of CTLs, the biggest effect was seen after 48 h (OCI2 P ¼ 0.0017; OCI3 P ¼ 0.0001). No significant difference could be seen between the two cell lines. (d) Flow cytometric CD96 expression analysis of 18 patients. The graph shows the mean CD96 expression of either all cells or CD34 þ cells only. Left: NPM1mut patients show a mean expression of CD96 on the cell surface of all cells of 24.35% compared with a mean expression of 7.2% in NPM1wt. Right: The mean expression of CD96 on the cell surface of CD34 þ cells of NPM1mut patients is 54% compared with 4.4% in NPM1wt patients. Expression differences in CD34 þ cells are highly significant (P ¼ 0.0005) comparing NPM1mut with NPM1wt patients. (e) CFU blockage by anti-CD96. CFU assays were performed using two AML patient samples with high blast counts. Addition of anti-CD96 antibody to blasts and effectors was inhibiting the growth of CFU comparable to the growth of blasts and effectors only (Pp0.05). Lysis through effector cells was most effective using 0.5 mg anti-CD96 and 0.05 mg human anti-mouse antibody (HAMA) (Pp0.05) with a mean difference of 82% in colony number. Anti-CD96 antibody or HAMA only had no effect on the colony formation (*P ¼ 0.05, **P ¼ 0.01, ***Pp0.001). before sorting, after sorting purity increased to 91–97.6% (mean CD34 þ CD38 À cells in NPM1mut AML samples harbor cytoplasmic 93%). In NPM1wt patients (n ¼ 12), CD34 þ CD38 À frequency NPM1 demonstrated via immunocytochemical staining ranged between 0.78 and 86.2% (mean 33.08%) before sorting (Figure 1b), which indicates that these cells belong to the and after sorting from 91.3 to 98.4% (mean 95.2%). Enriched leukemic clone. We examined the expression of potentially Leukemia (2014) 1736 – 1764 & 2014 Macmillan Publishers Limited Letters to the Editor 1761 relevant genes in LSC-enriched CD34 þ CD38 À cell fractions of LSC marker. IL-12 and anti-CD96 are also potential candidates NPM1mut compared with NPM1wt AML primary patients (Table 1). combining immunotherapeutic approaches—for example, with Interestingly, the differences are accentuated on genes that are vaccination strategies targeting NPM1, RHAMM, WT1 or Proteinase involved in T-cell immunity. Stronger immune responses in 3 or with other newly discovered T-cell-activating antibodies NPM1mut AML patients against leukemic cells and especially the mentioned above. The microarray analysis comparing enriched LSC fraction could be a factor for the better overall survival of LSC populations of NPM1wt and NPM1mut AML patients underlines NPM1mut patients compared with NPM1wt patients. Some of the the importance of the immune system in NPM1mut AML patients as detected genes are candidates for immunotherapeutic purposes, highly immunological regulatory pathways are differentially and we further analyzed their relevance in CD34 þ CD38 À cells regulated in NPM1mut compared with NPM1wt patients: For targeted by functional assays.
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