P.A.C. ’t Hoen

The value of data

Data citation and the derivation of semantic constructs directly from datasets have now both found their place in scientific communication. The social challenge facing us is to maintain the value of traditional narrative publications and their relationship to the datasets they report upon while at the same time developing appropriate metrics for citation of data and data constructs.

Gene-expression and in vitro function of mesenchymal stromal cells are affected in Juvenile Myelomonocytic Leukemia

An aberrant interaction between hematopoietic stem cells and mesenchymal stromal cells has been linked to disease and shown to contribute to the pathophysiology of hematologic malignancies in murine models. Juvenile myelomonocytic leukemia is an aggressive malignant disease affecting young infants. Here we investigated the impact of juvenile myelomonocytic leukemia on mesenchymal stromal cells. Mesenchymal stromal cells were expanded from bone marrow samples of patients at diagnosis (n=9) and after hematopoietic stem cell transplantation (n=7; from 5 patients) and from healthy children (n=10). Cells were characterized by phenotyping, differentiation, gene expression analysis (of controls and samples obtained at diagnosis) and in vitro functional studies assessing immunomodulation and hematopoietic support. Mesenchymal stromal cells from patients did not differ from controls in differentiation capacity nor did they differ in their capacity to support in vitro hematopoiesis. Deep-SAGE sequencing revealed differential mRNA expression in patient-derived samples, including genes encoding proteins involved in immunomodulation and cell-cell interaction. Selected gene expression normalized during remission after successful hematopoietic stem cell transplantation. Whereas natural killer cell activation and peripheral blood mononuclear cell proliferation were not differentially affected, the suppressive effect on monocyte to dendritic cell differentiation was increased by mesenchymal stromal cells obtained at diagnosis, but not at time of remission. This study shows that active juvenile myelomonocytic leukemia affects the immune response-related gene expression and function of mesenchymal stromal cells. In contrast, the differential gene expression of hematopoiesis-related genes could not be supported by functional data. Decreased immune surveillance might contribute to the therapy resistance and progression in juvenile myelomonocytic leukemia.

Integrated whole genome and transcriptome analysis identified a therapeutic minor histocompatibility antigen encoded by an alternative ITGB2 transcript

Patients with hematological malignancies can be successfully treated with allogeneic stem cell transplantation (alloSCT). In HLA-matched alloSCT, donor T cells can mediate desired anti-tumor immunity as well as undesired side effects by recognizing minor histocompatibility antigens (MiHA) on patient cells. MiHA are polymorphic peptides with amino acid changes that are created by genetic variants and recognized by specific T cells. MiHA with hematopoiesis restricted expression are relevant targets for immunotherapy to augment anti-tumor immunity after alloSCT without side effects. For high-throughput discovery of MiHA, we previously developed whole genome association scanning (WGAs) in which T cell recognition of a panel of EBV-B cell lines is investigated for association with single nucleotide polymorphisms (SNPs) to identify the genomic region that encodes the antigen. Although WGAs is an efficient strategy for MiHA discovery, strong association with SNPs in introns or other regions outside coding exons can be found for approximately one third of the T cell clones, whereas no SNP disparity in the primary transcript is present between patient and donor, suggesting that the antigen may be encoded by an alternative transcript. To identify MiHA encoded by alternative transcripts, we developed an integrated approach in which WGAs is combined with whole transcriptome data from the GEUVADIS project. By performing WGAs, we identified associating SNPs for two HLA-B*15:01-restricted MiHA that were targeted by donor CD8 T cells in a patient with strong anti-tumor immunity after HLA-matched alloSCT. One antigen (LB-GLE1-1V) was encoded by an associating SNP in exon 6 of the GLE1 gene. For the other antigen, an associating SNP in intron 3 of the ITGB2 gene was found, but no SNP disparity was present in the normal ITGB2 transcript between patient and donor. Therefore, we investigated whether the antigen may be encoded by an alternative transcript. Using RNA-sequence data, we identified an alternative ITGB2 transcript in which part of intron 3 was retained. Q-PCR analysis showed that expression of this transcript was restricted to hematopoietic cells from SNP-positive individuals. In silico translation of the transcript revealed a peptide with strong predicted binding to HLA-B*15:01 (LB-ITGB2-1), which was recognized by specific T cells. T cells for LB-ITGB2-1 also recognized and lysed leukemic cells of different origins, while no reactivity against patient fibroblasts could be detected. In summary, RNA-sequence analysis enabled discovery of LB-ITGB2-1 as immune epitope encoded by an alternative gene transcript. Our data support the therapeutic relevance of LB-ITGB2-1 as target for T cell therapy to stimulate anti-tumor immunity after alloSCT.

T.P.26 Low dystrophin levels increase survival and improve pathology and motor function in dystrophin/utrophin double knockout mice

Abstract In Duchenne Muscular Dystrophy (DMD) patients muscle fibers are susceptible to exercise-induced injury due to absence of functional dystrophin. No cure is available, but in the last decade major progress has been made in the challenge to restore dystrophin expression in DMD patients. It is unknown how much dystrophin is needed to slow or prevent disease progression. To elucidate this, we generated mdx-Xist Δhs utrn − / − mice in which skewed X-inactivation results in expression of variable, low dystrophin levels in a utrophin negative background. These mice ( n =20) underwent a 12week functional test regime after which histopathology was assessed. Dystrophin levels of 3–10% already significantly improved performance of two and four limb hanging wire tests and histopathology, while 10–17% further normalized this towards wild type. For improvement in grip strength higher dystrophin levels are needed. Most striking was the effect of already very modest dystrophin levels in maintenance of basic muscle function and protection against death from overall weakness. Whereas mdx/utrn − / − mice did not live beyond 12weeks, 62% of the mice expressing 3–10% dystrophin and all mice expressing 10–17% dystrophin survived 16weeks. A survival study in 42 mdx-Xist Δhs utrn − / − mice assessing skeletal muscle function and histopathology showed a median survival extension to 26weeks in mice with 3–10% dystrophin, while mice with 10–30% lived even longer. Biomarkers, skeletal muscle and heart function, and histopathology were significantly improved in mice with 3–10% dystrophin and further improvement was achieved with 10–30% dystrophin. These results suggest that even very low dystrophin levels already may have beneficial effects, and that survival and improvement of endurance efforts may be amongst the early effects of treatment. This underscores the urgency to develop better clinical readouts for the non-ambulatory phase.

P1.52 BIO-NMD: Discovery and validation of biomarkers for neuromuscular diseases (NMDs) – An EU funded FP7 project

www.bio-nmd.eu Volker Straub1, Annemieke Aartsma-Rus2, Cristina Al-Khalili Szigyarto3, Christophe Beroud4, Paolo Bonaldo5, Paola Borgiani6, Paola Braghetta5, Amina Chaouch1, Sebahattin Cirak7, Roseline Favresse8, Nikolai Daraselia9, Cecilia Gelfi10, Peter A.C. ’t Hoen2, Ekaterina Kotelnikova9, Yannick Le Priol9, Hanns Lochmuller1, Jenny Morgan7, Francesco Muntoni7, Giuseppe Novelli6, Nicoletta Paolillo6, Raimo Tanzi11, Cathy Turner1, Mathias Uhlen3, Alessandra Ferlini12 On behalf of the BIO-NMD consortium

P1.44 Development of heart failure in mice with low dystrophin levels

MR data acquisition is performed on a Philips 3T Achieva, Siemens Verio, or Siemens TIM Trio system. Reproducibility of the MR measures have been evaluated for the soleus using MRI-T2 (Day 1: 43 ± 9 ms, Day 2: 44 ± 9 ms; CV 2.2 ± 1.9%), T2 of H2O from spectroscopic relaxometry (Day 1: 30.3 ± 2.6 ms, Day 2: 30.4 ± 2.9 ms; CV 2.1 ± 1.5%), and the ratio of lipid/(lipid + water) using H-spectroscopy (Day 1: 0.32 ± 0.23 ms, Day 2: 0.32 ± 0.22 ms; CV 4.8 ± 2.8%). In summary, the MR measures implemented in this multisite study are highly reproducible in children with DMD and controls. These noninvasive measures show promise for evaluating disease progression and treatment in DMD subjects, and are continuing to be evaluated in this multi-center study.