ALBERT

Description: Introduction: Aberrant DNA methylation is frequently found in hematologic malignancies where it is associated with altered gene expression. DNA hypomethylating agents (DNMTi), e.g. 5-aza-2'-deoxycytidine (DAC), are used for both global and gene-specific in vivo demethylation and offer a therapeutic option in myelodysplastic syndromes (MDS) and AML. DNMTi have already been utilized to upregulate suppressed fetal hemoglobin (HbF) in adult patients (pts) suffering from hemoglobinopathies. Here we systematically investigated the potential of DAC for in vitro induction of erythroid differentiation as well as HbF expression in the bipotent myeloid leukemia cell line K562 and in vivo in a clinical treatment situation in MDS pts. Methods and Results: We treated K562 cells with non-toxic concentrations of DAC (100 nM, three 24 hour pulses), hemin (50 nM) and phorbol myristate acetate (PMA, 5 nM). DAC treatment led to morphological changes indicating erythroid but not megakaryocytic differentiation. This was confirmed by benzidine staining where DAC (13% positive cells) and hemin (58%) but not PMA treated cells (0%) became positive for hemoglobin synthesis. Lack of CD41 detection by FACS analysis for DAC and hemin indicated absence of megakaryocytic differentiation. Transcriptome profiling by mRNA expression arrays (Affymetrix GeneChip® HG U133 Plus 2.0) revealed highest similarity between hemin and DAC treatment by unsupervised hierarchical clustering, followed by vehicle control and untreated cells. The transcriptome of PMA treated cells clustered most distantly to all other treatments. Both, DAC and hemin induced moderately balanced up- and downregulation of transcripts to an almost identical extent. 1414 transcripts were 〉2 fold upregulated and 1505 were 〉2 fold downregulated upon DAC treatment, whereas 1548 were up- and 2404 were downregulated in hemin treated cells, respectively. The extent of transcriptome dynamics was considerably stronger upon PMA treatment, where 4196 and 3780 transcripts were up- and downregulated, respectively. When intersecting transcriptome changes between the 3 drug treatments (Fig. 1), 368 out of 1548 (23.7%) upregulated transcripts in hemin treated cells were concordantly upregulated upon DAC treatment. The overlap of upregulated transcript was lower compared to PMA treated cells (14.9%). GO analyses of upregulated transcripts identified terms related to erythropoesis and iron metabolism among the top regulated groups of transcripts in DAC treated cells whereas terms related to megakaryocytic differentiation did not show significance. Particularly strong differences of transcripts were observed for a1-, a2-, Ag-, e- and z-globin expression upon DAC and hemin treatment, whereas b- and d-globin were expressed at low levels. These changes were not observed for PMA treated cells. Induction of a- and Ag-globin on mRNA level resulted in enrichment of a- and Ag-globin protein to 15.8% of total cellular protein amount, and consequently in HbF formation in K562 cells as assessed by reversed phase and anion exchange chromatography. HbF levels in peripheral blood were measured from 16 MDS pts, median age 74 years (range 66-78) also treated with a 3-day DAC schedule. Median HbF fraction at baseline was 0.4% (0.1-3.9%) of total hemoglobin with 6 pts (37.5%) exhibiting increased HbF levels (〉1%) already before treatment. In 13/16 (81%) pts, increase of HbF with a median increment of 1.2% (range 0.3-3.7%) was observed. In 3 pts, HbF decreased over the treatment course. Median number of courses until maximum increment was 3 (2-6). HbF levels in 2 pts with AML and 1 with pancreatic cancer treated with nucleoside analogues without demethylating activity (cytosine arabinoside and gemcitabine, respectively) according to standard chemotherapy protocols served as control group and did not show comparable increments. Conclusions: We describe an erythroid differentiation program, from transcriptome level to HbF protein formation, induced by the hypomethylating agent DAC in the bipotent cell line K562. This DAC-mediated differentiation process is specific for erythropoesis but not megakaryopoesis. This is substantiated by in vivo upregulation of HbF upon DAC adminstration in MDS pts. Therefore, we propose to utilize HbF expression as potential biomarker during DAC treatment. Figure 1. Intersection of 〉2 fold upregulated transcripts in K562 cells upon drug treatment. Figure 1. Intersection of 〉2 fold upregulated transcripts in K562 cells upon drug treatment. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION: We recently described the first case of the evolution of inv(16) AML on the background of a clonal hematopoiesis due to a germline CBL mutation (defining the CBL syndrome), and we identified possibly cooperating mutations by exome sequencing (Becker et al. Blood 2014;123:1883-6). Among the mutated genes was PTPRT, encoding a protein tyrosine phosphatase that inhibits STAT3 activity and is commonly mutated in cancer (reviewed by Zhao et al. Oncogene 2015;34:3885-94). Here, we investigated the co-occurrence of mutated PTPRT with other mutated genes by single cell genotyping in order gain insights into the clonal architecture and sequence of mutation acquisition. METHODS: Exome sequencing of the bulk specimens was previously described; germline or somatic origin of mutations was verified in skin fibroblasts (Becker et al. Blood 2014;123:1883-6). For single cell genotyping, Ficoll-enriched bone marrow aspirates were DAPI stained, and single cells were placed into each well of a PCR plate using a MoFlo high speed cell sorter (Beckman Coulter). Genomic DNA was amplified by whole genome amplification (WGA) using the REPLI-g Mini Kit (Qiagen) according to a modified protocol, and subjected to PCR and Sanger sequencing of the respective mutation loci. As WGA can lead to allele dropout (ADO), we also sequenced single nucleotide polymorphisms (SNPs), that were identified by CytoScan HD array (Affymetrix) to be heterozygous in the sample and that were located nearby the respective mutation loci. RESULTS: Exome sequencing allows prediction of a possible clonal architecture based on the variant allele frequencies (VAFs). VAFs of the mutations identified in the AML were as follows: KIF14 p.V341I (VAF 51%), TMEM125 p.D113N (51%), MIOX p.W225R (46%), CAND1 p.E584* (39%), NID2 p.D319N (38%), ARF3 p.N101S (36%), PRSS16 p.R491C (36%), PTPRT p.T844M (33%), DOCK6 p.R1872_K1873insP (33%), ADAM12 p.A222V (21%), CMIP p.T323M (15%) and MYOCD p.D283N (7%); due to its germline nature, all leukemic cells harbored the CBL p.D390V mutation. In order to verify the co-occurrence of mutations in a clone and thus the clonal architecture, we performed single cell genotyping of the mutations in PTPRT as well as CAND1 and DOCK6. CAND1 and DOCK6 were selected in addition to PTPRT since their comparable VAFs did not allow identifying the sequence of acquisition. Moreover, CAND1 and DOCK6 were affected by likely deleterious mutations and were previously found mutated in AML. To control for ADO, we included the heterozygous SNPs rs2867061 (PTPRT), rs1252402 (C AND1), and rs12980863 (DOCK6) in our analyses. We analyzed 19 single cells for the 6 mutations and SNPs. This resulted in 102 successful sequencing reactions, and yielded informative results for at least 2 mutations in 12 cells and for all 3 mutations in 5 cells. Based on the concurrent presence of the wild-type allele at the mutation locus and ADO at the SNP site, 18 mutation analyses were judged to be inconclusive. Overall, our analyses confirmed that the mutations in CAND1, PTPRT and DOCK6 occurred together in the same clone. Moreover, based on the identification of cells with the presence of both CAND1 and DOCK6 mutations but presence or absence of PTPRT mutations, respectively, we concluded that PTPRT mutations were acquired after the mutations in DOCK6 and CAND1. CONCLUSION: Single cell genotyping verified the co-occurrence of PTPRT, CAND1 and DOCK6 mutations in the same AML clone and revealed a clonal hierarchy, as the PTPRT mutation was acquired after the mutations in CAND1 and DOCK6. These insights into the clonal architecture and evolution had not been possible solely based on exome sequencing and suggest that the sequential expression of mutated PTPRT may cooperate with mutated CBL and inv(16) at a late stage of AML development. Disclosures No relevant conflicts of interest to declare.

Description: Key Points We provide a functional analysis of IGF1R expression in primary human B-CLL. Sorafenib reduces IGF1R expression in B-CLL.

Description: Key Points The CBL syndrome may predispose to myeloid neoplasias other than juvenile myelomonocytic leukemia. Whole-exome sequencing identifies mutations that possibly cooperate with mutant CBL in AML development.

Description: Mutual interactions of the neoplastic clone with the non-neoplastic immune system may influence immune function and the clinical behaviour of lymphoma. Individuals with immunodeficiency or autoimmune diseases have an increased risk for lymphoma development. The immune microenvironment appears to have a major influence on the prognosis of indolent lymphomas. Conversely, leukemic lymphomas may also cause immunodeficiency: In CLL, direct lymphoma-T cell interactions, which may occur ubiquitously, induce defects in T cell functions (Görgün et al., 2005). We demonstrate here a systemic perturbance of cellular immunity in a prospective study in patients with untreated de novo, limited-stage, non-leukemic indolent B cell lymphomas. Calibrated, quantitative flow cytometry showed a significant reduction of circulating T helper (TH) cells in follicular (FL; n=11; p

Description: Key Points IL-33 and ST2 expression are increased post-conditioning and with GVHD, resulting in increased T-cell activation via the IL-33/ST2 axis. Infusion of ST2-Fc protein exploits sST2’s function as a negative regulator of acute GVHD inhibiting pro-inflammatory cytokines.

Description: Epidemiologic data show that the immune system may control or promote the emergence and growth of neoplastic lymphomatous clones. Conversely, systemic lymphomas, especially myeloma and chronic lymphocytic leukemia (CLL), are associated with clinical immunodeficiency. This prospective controlled study demonstrates substantially reduced circulating T helper cells, predominantly naive CD4+ cells, in patients with nonleukemic follicular lymphoma and extranodal marginal zone lymphoma, but not in monoclonal gammopathy and early CLL. These changes were correlated with a preactivated phenotype, hyperreactivity in vitro, presenescence, and a T helper 2 shift of peripheral T helper cells. No prominent alterations existed in the regulatory T-cell compartment. Gene expression profiling of in vitro–stimulated CD4+ cells revealed an independent second alteration of T helper cell physiology, which was most pronounced in early CLL but also detectable in follicular lymphoma/extranodal marginal zone lymphoma. This pattern consisted of down-regulation of T-cell receptor signaling cascades and globally reduced cytokine secretion. Both types of T-cell dysfunction may contribute to significant immunodeficiency in nonleukemic indolent B-cell lymphomas as demonstrated by unresponsiveness to hepatitis B vaccination. The precise definition of systemic T-cell dysfunction serves as the basis to study its prognostic impact, its relationship to the established influence of the lymphoma microenvironment, and its therapeutic manipulation.

Description: Abstract 692 Congenital neutropenia syndromes comprise a heterogeneous group of disorders, whose genetic etiology remains often unknown. We describe a consanguineous pedigree with several affected individuals characterized by predisposition to recurrent and chronic bacterial and viral infections. Affected patients had chronic bronchitis/bronchiectasis, recurrent bacterial and herpes simplex skin infections, and disseminated warts associated with human papillomavirus and molluscum contagiosum virus. One patient developed a lymphoproliferative disorder associated with EBV-infection. Patients had congenital neutropenia with fluctuating absolute neutrophil granulocyte counts (180-4000/μl), yet no evidence of cyclic neutropenia. Immunophenotyping of peripheral blood revealed a paucity of peripheral T- and B-cells. Interestingly all patients showed evidences of autoimmunity. In addition all affected patients had subtle and hemodynamically not relevant cardiac defects such as ASD-II (P1), patent foramen ovale (P2) and patent foramen ovale associated with mitral, tricuspid and pulmonary insufficiency (P3). Genome-wide genotyping and linkage analysis of the index family yielded a LOD score of 4.3 on a linkage interval on chromosome 20. Candidate gene sequencing revealed a homozygous nonsense mutation in exon 7 of the gene STK4 (formerly MST1). STK4 is the human ortholog of Drosophila Hippo, the central constituent of a highly conserved pathway controlling cell growth and apoptosis. Isolated STK4-deficient lymphocytes and neutrophils of these patients exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis in response to various proapoptotic stimuli. Lymphopenia and congenital neutropenia may therefore be a consequence of increased loss of peripheral lymphocytes and neutrophils, similar to other well defined monogenetic diseases of the immune system. STK4 deficiency is a novel human primary immunodeficiency syndrome and highlights the role of the HIPPO pathway for the development of the human immune and cardiac systems. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3564 Poster Board III-501 Regulatory T cells (Treg) have been shown previously to reduce graft-versus-host disease (GvHD) but allow for graft-versus-leukemia (GvL) effects. It is still unclear why the adoptive Treg transfer does not paralyze every immune response to the same extent. In that context Treg recruitment and localization may account for differential immune regulation. Therefore, we studied the impact of lymphoma and inflammation derived chemoattractive signals on Treg recruitment. Luciferase transgenic Treg accumulated in B cell lymphoma (BCL) tissue after allogeneic hematopoietic cell transplantation (alloHCT) when no conventional T cells were given. Microarray based analysis of the BCL tissue revealed increased expression of the CXC chemokine ligands (CXCL) 9, 10, 12 and CCL22. In vivo antibody based neutralization or interference with receptor signaling identified CCL22 and CXCL12 as critical chemokines for Treg attraction towards BCL tissue after alloHCT. Conversely, the CXCL9/10-CXCR3 axis was not critical for Treg recruitment. Depletion of antigen presenting cells (APC) from BCL tissue abrogated CCL22/CXCL12 secretion. CD11c-DTR recipients displayed reduced Treg recruitment towards BCL, indicative for a major role of host APC in the process of Treg recruitment. Strong local inflammatory stimuli caused by subcutaneous complete Freund's adjuvant injection caused local Treg accumulation and reduced recruitment to the BCL. Infusion of conventional T cells that caused subacute GvHD related inflammation redirected Treg towards secondary lymphoid organs and abdominal GvHD target organs. In conclusion our study demonstrates the following novel findings: First Treg are recruited towards BCL infiltrated tissues after alloHCT and this process is dependent on CCL22 and CXCL12 production. Secondly, BCL infiltrating host type APC are the major source for CCL22 and CXCL12 and their targeted deletion impacts Treg recruitment towards BCL tissue. Thirdly, strong local or systemic inflammation can redirect Treg trafficking in the presence of BCL and affect the accumulation of this cell population at the tumor site which may explain why Treg preferentially regulate GvHD reactions post allografting. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Azanucleoside DNA demethylating agents upregulate large sets of genes in vitro. Cancer testis antigens (CTAs) are a growing group of immunogenic proteins that provide attractive targets for cancer-specific immunotherapy in solid tumors. In contrast, only a very limited number of studies has been performed on epigenetic regulation of CTAs in myeloid leukemia. A single report suggested specific in vivo induction of CTA mRNAs in blasts from AML and MDS pts treated with intermediate-dose Decitabine (DAC; Sigalotti et al., Blood101:4644–6, 2003). We wished to extend this finding by examining kinetics and spectrum of CTA and leukemia-associated antigen (LAA) regulation by DAC in myeloid cell lines and primary AML blasts. Materials and Methods: Myeloid cell lines were treated over 72 hrs with 50–200 nM DAC. RNA was isolated from peripheral blood blasts of AML pts treated with DAC (135mg/m2 over 72 hours within the 00331 phase II trial) before and during early phase of the treatment (= days 2, 5 from start of DAC) when circulating blasts were still 〉40% (mean 75%). Expression analyses were performed by Western Blot (NY-ESO-1), qPCR (NY-ESO-1, MAGEA1, MAGEA3, Myeloblastin), and by HG-U133plus 2.0 mRNA microarray. Results: The five myeloid cell lines Kasumi-1, U937, HL60, K562 and NB4 did not express NY-ESO-1 protein. Upon treatment with DAC, NY-ESO-1 was markedly de-repressed in 3/5 cell lines (Kasumi-1, U937, HL60). Induction of expression was dose- and time-dependent, with maximum induction at day 6, and reversible with prolonged culturing up to day 21. No derepression was seen when U-937 and HL-60 were treated with equitoxic low concentrations of cytarabine (not a Dnmt inhibitor). MAGEA1, MAGEA3 and the LAA Myeloblastin (MBN) were also induced by DAC in Kasumi-1, with the strongest effect observed for NY-ESO-1 and the least for MBN (high baseline expression). Expression analyses were extended to all 67 CTAs present on the U133plus array (45 located on the X-chromosome, 22 autosomally), of which 11/67 (17%) were induced 〉2 fold in Kasumi-1. Notably, all 11 CTA genes were X-chromosomal, whereas 0/22 autosomal CTAs showed reinduction (p= 0.008 by Fisher-Exact test). 2/9 LAA genes (22%) were also reinduced. In primary AML blasts from 9 pts, DAC treatment resulted in 〉2fold induction of 3 CTAs and 5 LAAs. qPCR demonstrated NY-ESO-1 upregulation in 2/5 patients, with a maximum at day 5 from start of treatment. Conclusions: Marked derepression of NY-ESO-1 and other CTAs by treatment of myeloid cell lines with an azanucleoside DNA demethylating agent occurred preferentially with genes residing on the X chromosome, in line with a major role of DNA methylation for their regulation. Derepression by DAC was also observed in vivo, albeit less marked and occurring early during treatment. One of the therapeutic effects of low-dose DNA demethylating agents on leukemic myeloid blasts may be mediated via upregulation of antigens rendering the malignant cells more immunogenic.