An increased cytokine response may be driving early cytopenia in myelodysplastic syndromes

Immunotherapy is being thoroughly tested for hematological malignancies. In myelodysplastic syndromes (MDS), greatest focus has been towards vaccines and checkpoint inhibitors (CI). The cancer types, where CI has shown the greatest clinical benefit, are the ones that carry a high mutational burden. Mutations create neoantigens, that can be presented on HLA molecules, and are suggested to be the main mediator of immunogenic cancer cell elimination. MDS carry comparably few mutations in their malignant cells. Therefore, it remains doubtful whether the malignant cells in MDS can be sufficiently immunogenic to generate an adequate immune response when treated with CI. In this study we investigated T cell responses to personal neoantigens in patients with MDS. We ran DNA and RNA sequencing data, from mesenchymal stem cells and CD34+ cells in 15 patients with MDS, through an MHC binding prediction algorithm, to create personal libraries of neo-peptides corresponding to the individual patients’ mutations. Bone marrow samples from the patients were screened for T cell binding to peptide-MHC-multimers (pMHC) coupled with a DNA-barcode, allowing us to look for T cell reactivity against more than 1000 pMHC in parallel. T cell responses were then validated using functional analysis. Current results indicate that several neoepitopes in MDS are indeed immunogenic and are recognized by cytotoxic T cells in the bone marrow. Interestingly, many of the same neoepitopes are also recognized by T cells in healthy donors, but in contrary to the patient samples, neoepitope binding T cells from healthy donors are not functionally active. To our knowledge, this is the first time neoantigens have been detected and functionally verified in the context of MDS. These findings could lead to further explorations into personal neoepitope vaccines, currently tested in other malignancies, and suggests a role for CI in MDS, possibly combined with specific neoepitope targeting. Copy Number Variations Predict Poor Survival in Patients with Idiopathic Cytopenia of Undetermined Significance (ICUS) and are Associated with Macrocytosis SU Mikkelsen,1,2 S Safavi,3 JW Hansen,1,2,4 K Dimopoulos,5 C O’Rourke,2 MK Andersen,3 JB Andersen,2 and K Grønbæk1,2,4 1Department of Hematology, Rigshospitalet; 2BRIC, University of Copenhagen; 3Department of Clinical Genetics, Rigshospitalet; 4DanStem, University of Copenhagen; 5Department of Clinical Biochemistry, Rigshospitalet Approximately half of ICUS patients have somatic mutations associated with increased risk of progression. The cause of cytopenia in the remaining ICUS patients is unknown. We hypothesize that uniparental disomy (UPD) and submicroscopic copy number variations (CNVs; gains and deletions) are present in ICUS patients and may be associated with prognosis. Patients (n=154) referred with ICUS (2008-2017) were included if cytopenia persisted for >6 months, cytogenetics was normal and BM morphology was not diagnostic. SNP-A was performed on DNA from MNCs or granulocytes using Illumina Infinium-CytoSNP-850K and analyzed with GenomeStudio-v1.1 (Illumina). Only dels>30 markers, gains>90 markers and UPD>5Mb were reported. A total of 33 CNVs/UPDs (excluding delY) were detected in 27/154 patients (18%). Mutations were present in 12/27 patients (44%) with CNVs/UPDs. Twenty-six deletions or UPDs (del/UPD) were detected in 21/154 patients (14%) of whom 11/21 (52%) harbored mutations. After a median follow-up of 25 months (range, 2-114), median OS was 67 months (95%CI:19-not reached) in patients with del/UPD and not reached in patients without (p=0.003), Fig.1. The association with poor OS was also significant when including gains (p=0.02), however, the impact seemed driven by del/UPD, and here results with del/UPD are presented. In multivariate analysis, del/UPD (HR=2.7, 95%CI:1.22-5.9, p=0.014) and deep anemia (hgb<6.2) (HR=2.2, 95%CI:1.104.4, p=0.025) were independent adverse prognostic factors for OS, Fig.2. Interestingly, an almost identical pattern was observed between macrocytosis (MCV>100fL) and del/UPD indicating a linked impact on survival. Patients with del/UPD had significantly higher MCV (p=0.005) and Pferritin (p=0.03). Importantly, macrocytosis was not associated with deep anemia (p=0.317). To our knowledge, this is the first study investigating CNVs/UPDs in ICUS patients. Our results suggest that SNP-A can aid the prognostics in ICUS by identifying submicroscopic structural variations as risk markers for poor OS. A possible role of macrocytosis as a surrogate marker should be explored. Figure 2. Forest plot with hazard ratios (HR). Deletions or UPD (Deletions_UPD) and macrocytosis were not included in the same model due to multicollinearity. Cox proportional hazard regression models were used to estimate HRs and associated 95% CIs. Anemia: hgb < 6.2 mM; Mutations: Somatic mutations with a VAF ≥ 5%. Figure 1. Kaplan-Meier survival curves for patients with deletions or UPD (del/UPD) and patients without del/UPD. Median overall survival (OS) was compared using a stratified log-rank test. X axis: Time from first visit (study inclusion) in months. Oral vitamin C supplementation to myeloid cancer patients on azacitidine treatment: Normalization of plasma vitamin C induces epigenetic changes L.Gillberg,1,2 A.D.Ørskov,1,2 A.Nasif,3 H.Ohtani,4 Z.Madaj,4 J.W.Hansen,1,2,7 N.Rapin,2 J.B. Mogensen,1,2 M.Liu,4 I.H.Dufva,5 J.Lykkesfeldt,6 P.Hajkova,3 P.A.Jones,4 K.Grønbæk1,2,7 1Dept. of Haematology, Rigshospitalet. 2BRIC, University of Copenhagen; 3MRC London Institute of Medical Sciences (LMS), Imperial College, London, UK; 4Van Andel Research Institute, Grand Rapids, Michigan, USA; 5Department of Haematology, Herlev University Hospital; 6Department of Veterinary and Animal Sciences, University of Copenhagen; and 7The Danish Stem Cell Center (Danstem), University of Copenhagen, Denmark. Abstract Purpose: Hematological cancer patients are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC); the first step in active DNA demethylation. Here, we investigated whether oral vitamin C supplementation can correct vitamin C deficiency, enhance the 5hmC/5mC ratio and upregulate expression of viral defense genes in myeloid cancer patients treated with DNA methyltransferase inhibitors (DNMTis). Experimental design: A randomized, placebo-controlled clinical trial in myeloid cancer patients performed during 3 cycles of DNMTi-treatment (5-azacitidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n=10) or placebo (n=10) daily during the last 2 cycles (Figure 1). Results: Fourteen patients were deficient in plasma vitamin C (<23 μM) and four of the remaining six patients were taking vitamin supplement at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (<11.4 μM; P=0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (P=0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in patients receiving vitamin C compared to placebo (P=0.041). Additionally, preliminary data suggest that vitamin C supplement may increase the upregulation of viral defense genes in DNMTi naïve patients. Conclusions: Normalization of plasma vitamin C by oral supplementation may enhance the biological effects of DNMTis in patients and prompts the investigation of the clinical efficacy of vitamin C supplementation to DNMTis in a large randomized, placebocontrolled trial.Purpose: Hematological cancer patients are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC); the first step in active DNA demethylation. Here, we investigated whether oral vitamin C supplementation can correct vitamin C deficiency, enhance the 5hmC/5mC ratio and upregulate expression of viral defense genes in myeloid cancer patients treated with DNA methyltransferase inhibitors (DNMTis). Experimental design: A randomized, placebo-controlled clinical trial in myeloid cancer patients performed during 3 cycles of DNMTi-treatment (5-azacitidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n=10) or placebo (n=10) daily during the last 2 cycles (Figure 1). Results: Fourteen patients were deficient in plasma vitamin C (<23 μM) and four of the remaining six patients were taking vitamin supplement at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (<11.4 μM; P=0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (P=0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in patients receiving vitamin C compared to placebo (P=0.041). Additionally, preliminary data suggest that vitamin C supplement may increase the upregulation of viral defense genes in DNMTi naïve patients. Conclusions: Normalization of plasma vitamin C by oral supplementation may enhance the biological effects of DNMTis in patients and prompts the investigation of the clinical efficacy of vitamin C supplementation to DNMTis in a large randomized, placebocontrolled trial. Figure 1. Study design. Days 1, 5, and 28: before 500 mg vitamin C/placebo exposure. Day 32: after short-term vitamin C/placebo exposure. Days 56, 60, and 84: after longerterm vitamin C/placebo exposure. DEPRESSION AND ANXIETY IN HODGKIN LYMPHOMA SURVIVORS: A DANISH NATIONWIDE COHORT STUDY OF 896 PATIENTS Andreas Kiesbye Øvlisen1,2, Lasse Hjort Jakobsen1,2, Kristian Hay Kragholm3, Martin Hutchings4, Christian Bjørn Poulsen5, Henrik Frederiksen6, Danny Stoltenberg7, Martin Bøgsted1,2, Lene Sofie Granfelt Østgård8, Marianne Tang Severinsen1,2,3, Tarec Christoffer El-Galaly1,2,3. 1. De