Introduction: Human leukocyte antigen (HLA) molecules are membrane-bound transporters carrying peptides from the cytoplasm to the cell surface where they become exposed to T lymphocytes. Soluble HLA molecules (sHLA) bound to their specific peptides are present in human plasma. Patient-based evidence that most of these peptides are derived from cancerous cells may lead to the identification of cancer-related sHLA peptidomes in the disease state. Leukemic cells in the bone marrow (BM) are usually composed of several malignant subclones even within the same patient. These leukemic subclones may vary in their sensitivity to chemotherapy. During the first days of chemotherapy, a dynamic change in the blast mass and subclonal cell composition is observed. We have thus hypothesized that the repertoire and amount of peptides bound to sHLA molecules derived from the BM plasma of acute myeloid leukemia (AML) patients will be changing during induction. Our study has aimed at identifying peptides derived from resistant subclones, assuming that they could represent potential immunogenic targets specific to these leukemic cells. Particular focus has been made on the peptides common among multiple AML patients.Methods: Mononuclear cells and plasma fractions obtained from AML patients at diagnosis (day 1), during induction chemotherapy (day 5) and on day 14 of induction were studied. HLA molecules loaded with peptides were purified using pretreated pan HLA-A, B and C monoclonal antibody W6/32 TopTip column, and sHLA class I molecules with their bound peptides were eluted with tri-fluoracetic acid. The eluted fraction containing sHLA was separated from its peptides on C18 Micro TipColumn with acetonitrile. The peptide fraction was then examined using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Based on the LC-MS analysis, we excluded peptides longer than 15 amino-acids and shorter than 8 amino-acids, peptides known as contaminators and peptides highly expressed by healthy donors. Immunogenicity of the identified candidate peptides was evaluated by ɣ-IFN Elispot assay.Results: The total amount of sHLA-bound peptides in sequential BM plasma samples of 11 patients was significantly decreased at nadir (day 14) compared to diagnosis or day 5 samples (3 and 1.33 fold, respectively). About 17 percent (16.9%) of the peptides identified on day 5 of induction were not present at diagnosis. These peptides originate from the proteins known to be involved in cell proliferation, migration, death, cycle control, metastasis promotion, DNA damage, or mitochondria processes, and could thus reflect chemotherapy-induced effects. On day 14, 75% of identified peptides were similar to those observed on days 1 and 5, representing cancer-derived peptides expressed by leukemic cells that survived the first days of therapy.Analysis of whole peptidome sequences was performed in the plasma specimens obtained from 8 healthy donors (HLA A02) and collected at the above 3 time points from 11 patients (4 HLA A02, 5 HLA A01, 2 HLA A68). Three major peptides were found to be common across the tested patient samples. The peptide originating from GUCY1B3 protein was solely detected in 10 patients and in none of the healthy donors; one peptide derived from ETS1 protein and one peptide cut from STAG1 protein were identified in 7 and 6 patient samples, respectively, as well as in one healthy donor. Immunogenicity of the 3 peptides was examined against T lymphocytes derived from 5 AML patients (HLA 02) in remission. High T cell reactivity was confirmed in the peptide derived from GUCY1B3 protein. Additionally, similarities between membranal HLA (mHLA) and plasma sHLA peptidomes derived from the same patient were observed (87.7%) (n=3, HLA A01).Conclusions: The requirement of a large number of cells, limiting the application of membranal HLA peptidome analysis could be overcome using sHLA-bound peptides from the BM plasma. Plasma sHLA-originating peptides, detectable by a simple blood test, may serve as potential biomarkers of response to treatment or as targets for immunotherapy. Such peptides derived from resistant subclones during induction chemotherapy could be used to stimulate T cell clones in a peptide-restricted HLA manner and thus act as a personalized immunotherapy adjuvant in AML. DisclosuresOfran:Novartis: Other: Served on a Novartis advisory board.