The expression of tumor-associated antigens (TAAs) might play a critical role in the control of minimal residual disease (MRD) in acute myeloid leukemia (AML), and therefore might be associated with clinical outcome in AML. In a DNA microarray analysis of 116 AML samples, we found a significant correlation between high mRNA levels of G250/CA9 and longer overall survival (P = .022), a similar trend with high mRNA levels of PRAME (P = .103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1, we found no correlation with clinical outcome. High expression of at least 1 of the 3 TAAs, RHAMM/HMMR, PRAME, or G250/CA9, provided the strongest favorable prognostic effect (P = .005). Specific T-cell responses were detected in 8 (47%) of 17 patients with AML in complete remission for RHAMM/HMMR-R3 peptide, in 7 (70%) of 10 for PRAME-P3 peptide, and in 6 (60%) of 10 for newly characterized G250/CA9-G2 peptide, a significant increased immune response compared with patients with AML patients who had refractory disease (P < .001). Furthermore, we could demonstrate specific lysis of T2 cells presenting these epitope peptides. In conclusion, expression of the TAAs RHAMM/HMMR, PRAME, and G250/CA9 can induce strong antileukemic immune responses, possibly enabling MRD control. Thus, these TAAs represent interesting targets for polyvalent immunotherapeutic approaches in AML.

Mutations in the nucleophosmin gene (NPM1^mut) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1^mut. In the present study, we were able to demonstrate both CD4⁺ and CD8⁺ T-cell responses against NPM1^mut. Ten peptides derived from wild-type NPM1 and NPM1^mut were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr⁵¹-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR–binding epitopes were used to test the role of CD4⁺ T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1^mut induced CD8⁺ T-cell responses. A total of 33% of the NPM1^mut AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4⁺ T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8⁺ and CD4⁺ T cells. The results of the present study show that NPM1^mut induces specific T-cell responses of CD4⁺ and CD8⁺ T cells and therefore is a promising target for specific immunotherapies in AML.

Background Recently, we demonstrated immunological and clinical responses to a RHAMM-R3 peptide vaccine in patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma. To improve the outcome of the vaccine, a second cohort was vaccinated with a higher dose of 1,000 μg peptide. Design and Methods Nine patients received four vaccinations subcutaneously at a biweekly interval. Immunomonitoring of cytotoxic CD8⁺ as well as regulatory CD4⁺ T cells was performed by flow cytometry as well as by enzyme-linked immunospot (ELISpot) assays. Parameters of clinical response were assessed. Results In 4 of 9 patients (44%) we detected positive immunological responses. These patients showed an increase of CD8⁺RHAMM-R3_tetramer⁺/CD45RA⁺/CCR7⁻/CD27⁻/CD28⁻ effector T cells and an increase of R3-specific CD8+ T cells. Two of these patients showed a significant decrease of regulatory T cells (Tregs). In one patient without response Tregs frequency increased from 5 to 16%. Three patients showed clinical effects: one patient with myelodysplastic syndrome RAEB-1 showed a reduction of leukemic blasts in the bone marrow, another myelodysplastic syndrome patient an improvement of peripheral blood counts and one patient with multiple myeloma a reduction of free light chains. Clinical and immunological reactions were lower in this cohort than in the 300 μg cohort. Conclusions High-dose RHAMM-R3 peptide vaccination induced immunological responses and positive clinical effects. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematologic malignancies. However, higher doses of peptide did not improve the frequency and intensity of immune responses in this trial. (This study is registered at http://ISRCTN.org as ISRCTN32763606)

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. With intensive induction therapy, most patients younger than 60 years achieve complete remission. However, even if these younger patients were treated intensively, more than 50% will relapse. Clinical results of patients older than 60 years are more unfavorable. Therefore, in all patients with AML, the overall survival is still low. In the past decade, several leukemia-associated antigens (LAA) have been identified in patients with acute myeloid leukemia. BAGE, BCL-2, OFA-iLRP, FLT3-ITD, G250, hTERT, PRAME, proteinase 3, RHAMM, survivin, and WT-1 are all LAAs that have been shown to induce CD8⁺ T-cell recognition and for some antigens also humoral immune responses. Interestingly, most of these LAAs are linked to cell cycle or proliferation. This article discusses the balance between LAA-driven leukemia cell expansion and the elimination of these cells through attacks on LAAs by the immune system. Current knowledge of the function and CD8⁺ T-cell recognition of LAAs is reviewed and an outlook is given on how to improve T-cell responses to LAAs in acute myeloid leukemia cells.