ALBERT

Description: Background HD is a B-lineage lymphoma characterized, depending on subtype, by a prominent inflammatory infiltrate and fibrosis. Clinically, inflammatory symptoms like fever, weight loss and night sweats (B-symptoms) and increased blood biomarkers of inflammation, including ESR and CRP, are characteristic of more advanced disease. The clinical trial EURONET-PHL-C2 (Second International Inter-Group Study for Classical Hodgkin's Lymphoma in Children and Adolescents) is a randomized, prospective trial that compares chemo- and radiotherapy treatment concepts of different intensities in patients with intermediate and advanced HD. Patients are stratified by risk into 3 therapy groups (TL-1 to TL-3). An ESR〉 30mm/h had been a risk factor for relapse in previous studies and leads to upstaging from the lowest (TL-1, Ann Arbor stage I and IIa without additional risk factors) to the intermediate risk group TL-2 in the current study. This addon pilot study tested urine proteomic patterns from pediatric patients with HD at diagnosis and compared them to the patterns of normal children. The questions were: Is there a HD-specific pattern, a pattern that identifies high risk or a pattern that correlates with inflammatory markers? Patients and methods Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to compare the peptide profiles in the mass range of 0.8 to 20 kDa of urine samples (N=34) from 16 children with pediatric Hodgkin lymphoma (PHL) as case and 32 age-matched children with no evidence of a disease (N=28) or with urinary tract infection (N=4) as control groups. Marker selection was based on a two-step strategy. First, a group-wise comparison of rank sum differences was performed on a set of 2418 annotated peptides with distribution frequencies above 30% in at least one of the groups with subsequent adjustment for multiple testing by the method of Bonferroni. In the second step marker candidates were further restricted to those demonstrating a significant positive or negative Spearman rho correlation coefficient (≥0.34 or ≤-0.34) to the Ann-Arbor classification criteria. From the resulting peptides a multivariate peptide marker classifier was established by support vector machine modeling and applied to an independent confirmation set of PHL (N=16, 31 urine samples) and control (N=18, 18 urine samples) patients to determine classification accuracy in receiver operating characteristics (ROC) analysis. Peptides included in the PHL classifier were resolved in their amino acid sequence by tandem mass spectrometry to identify the proteins from which the peptide markers are derived. Results The established multivariate peptide marker model consisting of 40 naturally occurring urinary peptides enabled absolute differentiation between PHL patients and children without signs of disease or urinary tract infection in independent validation as revealed by an area under the ROC curve value of 1.0 (95% confidence interval: 0.93 to 1.00, p

Description: MLL-fusion is the most common genetic abnormality in acute lymphoblastic leukemia (ALL) of infancy and occurs in approximately 80% of the cases. Infant ALL represents a biologically distinctive entity with a highly immature pro-B immunophenotype associated with a particularly unfavorable prognosis due to a high proportion of early relapses. This may be due to the survival of dormant residual disease protected by the bone marrow niche. We have characterized a pair of monozygotic twin sisters diagnosed with ALL in early infancy. Tumor cells from both children carried the t(11;19) translocation (MLL-ENL fusion) and both patients were treated according to the ALL-BFM 2000 protocol. Despite good initial treatment responses in both twins, one sister developed very early relapse (twin A) and succumbed to the disease. The other went into continuous complete remission (twin B) and, as of today, is alive after 9 years. The clinical data of the 2 patients are presented in Table 1. Diagnostic bone marrow (BM) aspirates of both sisters were injected into the femoral bones of NOD SCID gamma (NSG) mice. Survival of xenografted mice bearing twin A was significantly longer due to a lower proliferative capacity of twin A as compared to twin B cells. In vivo Bromodeoxyuridine (BrdU) assays revealed that twin B cells were proliferating equally fast in BM and spleen, while in twin A cells, the BM markedly suppressed entry into S-phase. Flow cytometry from xenograft BM identified a large CD34+ population in twin A cells that was almost absent in twin B cells. Furthermore, BM of twin A xenografts showed a CD34+/CD38-/CD19- stem cell like population undetectable in twin B animals. Most interestingly, injection of minimal residual disease (MRD) negative remission bone marrow (PCR for Ig-gene rearrangements and the MLL-fusion gene) of both sisters into NSG mice resulted in a full-blown xenograft leukemia in animals bearing twin A but not twin B. Taken together, these data suggest that fatal relapse in twin A may be due to a quiescent stem cell like population kept in check by unknown mechanisms. Next, we performed gene expression analyses on xenografted leukemias from twin A (initial, leukemia amplified from remission BM, relapse) and from twin B (initial). STRING analysis of gene expression differences revealed that, amongst other findings related to cell cycle regulation and DNA-repair, twin A cells downregulated genes indicating a reduced interferon pathway activity (CXCL10, IFI30, TRAIL, STAT1, OAS1, MX1) and several genes connected to TYRO protein kinase binding protein (TYROBP) shown previously to regulate the activity of natural killer (NK) cells. This suggests that twin A cells may evade immunosurveillance as a mechanism of disease persistence. 51Chromium-release assays with IL-2 stimulated allogeneic NK cells from two healthy donors and the xenografted twin cells showed that twin A cells were significantly less sensitive to NK cell mediated lysis as compared to twin B cells. Whether the tumor cells also display differential susceptibility to antibody-dependent cell-mediated cytotoxicity is currently investigated. While the extensive genomic characterization of the twin leukemias is under way, we show evidence from xenograft experiments, gene expression profiles and functional in vitro experiments that dormant residual cells in MLL-rearranged ALL can evade immunosurveillance. These findings serve as a rationale to employ immunotherapeutic approaches in infant ALL patients in order to improve their dismal prognosis. Finally, we report the first monozygotic twin pair with MLL-rearranged ALL and discordant clinical outcomes. This provides a unique opportunity and a model to gain insight into the clonal composition of infant leukemia and the origins of leukemia relapse. Table 1: Patient characteristics of the MLL-ENL positive twin pair. WBC, white blood cells; BM, bone marrow; CNS, central nervous system; MRD, minimal residual disease. Parameter Twin A Twin B Age at diagnosis (days) 98 147 WBC (initial)/µl 334.000 103.000 Blasts (Peripheral Blood) % 97 91 Blasts (BM) % 98 97 CNS involvement Not determined Negative Immunophenotype Pro-B Pro-B Cytogenetics t(11;19) t(11;19) Prednisone-Response Poor Good Blasts (BM), day 15 % 32 1 MRD day 33 Negative Negative MRD day 78 Negative 9 years Time to relapse 227 days n/a Overall survival 390 days 〉9 years *low positive, not quantifiable Disclosures No relevant conflicts of interest to declare.

Description: Diamond-Blackfan anemia (DBA) is a prototypic ribosomopathy and remains the most common cause of congenital pure red cell aplasia (PRCA). In 2/3 of patients, ribosomal protein haploinsufficiency is disease-causing, while in remaining 1/3 the genetic etiology is unknown. Recently, deficiency of ADA2 (DADA2) due to biallelic CECR1 -mutations was reported in patients with systemic autoinflammatory disease presenting with early onset vasculopathy, strokes, antibody deficiency, and in some cases variable cytopenias. Based on the clinical findings in an ADA2-deficient patient with PRCA resembling DBA, we aimed to define the prevalence and clinical picture of DADA2 within DBA patient cohorts. Patients enrolled in the national observational DBA registry in Germany were evaluated for the presence of mutations in CECR1 gene; additional nonconsecutive patients from the French and Turkish registries within the European DBA (EuroDBA) consortium were part of this study. Functional studies included profiling of polysomes and pre-rRNAs in patient-derived EBV-cell lines, CECR1 RT-PCR, measurements of autophagy and apoptosis, and analysis of erythropoiesis in zebrafish embryos. Systematic mutational and copy number analysis had identified typical ribosomal haploinsufficiency in 169/242 patients (70%). Out of 73 remaining patients, full CECR1 -sequencing was accomplished in 68 cases, of which 4 (6%) carried biallelic CECR1 -mutations. Additional 3 patients with biallelic CECR1 -mutations and DBA phenotype were referred from Germany (the index PRCA case), France and Turkey. In contrast to typical autoinflammatory DADA2 (caused by missense biallelic CECR1 -mutations) all patients studied here had at least one CECR1 -allele affected by truncating/stop-gain/deletion mutation leading to mRNA degradation in patient cells. Low or missing ADA2 enzyme activity in plasma confirmed DADA2, while erythrocyte ADA (eADA) levels and MCV were normal. Transfusion-dependent hypoproliferative anemia developed at a median age of 5 weeks (birth-14 years), while hypogammaglobulinemia developed in all cases either initially or during disease course. Notably, a transient hematologic response to steroids was achieved in 5/7 patients, but no improvement was observed in 2 patients treated with TNF-inhibitor; all patients at one point became heavily transfusion-dependent. Systemic vasculitis or cerebral complications were not observed in our cohort. At the last follow-up, 6/7 patients were alive; 3 had successfully undergone hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning and 1 patient had died due to septic shock. Next, we addressed the question if ribosome biogenesis is affected in ADA2-deficient patient cells. Using pre-rRNA maturation assays, polysome profiling and Western blots we established that ribosome biogenesis is normal in DADA2-related PRCA and there is no increase of TP53 stabilization over basal levels in patient LCLs. Analysis of CECR1 -morpholino zebrafish embryos revealed early anemia with lethal phenotype. Although there was no evidence for extrinsic (e.g. immune-mediated) pathomechanisms in our patients, it remains to be answered if CECR1 loss directly affects erythroid development. Finally, the association between elevated levels of eADA (=ADA1) specific to classical DBA and decreased ADA2 enzyme levels in DADA2-related PRCA remains obscure. In summary, DADA2 can phenotypically mimic DBA and thus extends the spectrum of congenital PRCA. Ribosome synthesis seems not to be affected by CECR1 mutations. DADA2 should be considered in patients with DBA-like phenotype but with normal eADA/MCV and hypogammaglobulinemia, allowing for early stratification aimed at HSCT in affected individuals. Disclosures Grosse: Addmedica: Membership on an entity's Board of Directors or advisory committees.