Aniek O. de Graaf

Expression of C‐IAP1, C‐IAP2 and SURVIVIN discriminates different types of lymphoid malignancies

(De‐)regulation of apoptosis plays an important role in normal and malignant lymphopoiesis. Apoptosis‐regulating genes of the BCL‐2 family and the recently identified inhibitors of apoptosis (IAP) family have been implicated in different types of non‐Hodgkin lymphoma (NHL). To investigate whether expression of specific apoptosis‐regulating genes correlated with different types of lymphoid malignancies, we measured the expression of five BCL‐2 family genes, four IAP family genes and SMAC by real‐time quantitative polymerase chain reaction in patient samples. In total, 137 samples from B‐ and T‐cell acute lymphoblastic leukaemia (ALL), B‐cell chronic lymphocytic leukaemia (CLL), six different NHL types and three control tissue types were analysed. The data were further analysed using cluster and discriminant analysis. Three specific expression patterns were identified for CLL, ALL and NHL respectively. CLL samples, as well as B‐ALL and follicular lymphoma samples showed high similarity in the expression of these apoptosis‐regulating genes and could be distinguished from each other and other diseases and controls. Discriminant analysis identified three members of the IAP family, C‐IAP1, C‐IAP2 and SURVIVIN, as the most informative genes to discriminate between these lymphoid malignancies.

Expression of P2X5 in lymphoid malignancies results in LRH-1-specific cytotoxic T-cell-mediated lysis

Minor histocompatibility antigens (MiHA) selectively expressed by haematopoietic cells are attractive targets for specific immunotherapy after allogeneic stem cell transplantation (SCT). Previously, we described LRH‐1 as a haematopoietic‐lineage restricted MiHA that is capable of eliciting an allogeneic cytotoxic T‐lymphocyte (CTL) response after SCT and donor lymphocyte infusion. Importantly, the gene encoding LRH‐1, P2X5, is not expressed in prominent graft‐versus‐host‐disease target tissues such as skin, liver and gut. Here, we investigate whether LRH‐1‐specific immunotherapy may be exploited for the treatment of lymphoid malignancies. We examined P2X5 mRNA expression in a large panel of patient samples and cell lines from different types of lymphoid malignancies by real‐time quantitative reverse transcription polymerase chain reaction. P2X5 mRNA was highly expressed in malignant cells from all stages of lymphoid development. Furthermore, all LRH‐1‐positive lymphoid tumour cell lines were susceptible to LRH‐1 CTL‐mediated lysis in flow cytometry‐based cytotoxicity assays. However, interferon‐γ production was low or absent after stimulation with some cell lines, possibly due to differences in activation thresholds for CTL effector functions. Importantly, primary cells from patients with lymphoid malignancies were effectively lysed by LRH‐1‐specific CTL. These findings indicate that MiHA LRH‐1 is a potential therapeutic target for cellular immunotherapy of lymphoid malignancies.

Bcl-2 protects against apoptosis induced by antimycin A and bongkrekic acid without restoring cellular ATP levels.

Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production using antimycin A (AA) (complex III inhibition) combined with inhibition of ADP/ATP exchange by bongkrekic acid (BA) (adenine nucleotide translocator (ANT) inhibition) induced a sharp decrease in total cellular ATP in FL5.12 parental cells (to 35% of untreated controls after 24 h of incubation). Within 24 and 48 h, 38% and 75% of the cells had died, respectively. However, in stably transfected FL5.12 Bcl-2 subclones, no cell death occurred under these experimental conditions. Similar results were obtained with Jurkat and Bcl-2 overexpressing Jurkat cells. Total cellular ATP levels were equally affected in FL5.12 Bcl-2 overexpressing cells and FL5.12 parental cells. This indicates that Bcl-2 overexpressing cells are able to survive with very low cellular ATP content. Furthermore, Bcl-2 did not protect against cell death by restoring ATP levels. This suggests that, under these conditions, Bcl-2 acts by inhibiting the signalling cascade triggered by the inhibitors that would normally lead to apoptosis.

Clonal evolution in myelodysplastic syndromes

Cancer development is a dynamic process during which the successive accumulation of mutations results in cells with increasingly malignant characteristics. Here, we show the clonal evolution pattern in myelodysplastic syndrome (MDS) patients receiving supportive care, with or without lenalidomide (follow-up 2.5–11 years). Whole-exome and targeted deep sequencing at multiple time points during the disease course reveals that both linear and branched evolutionary patterns occur with and without disease-modifying treatment. The application of disease-modifying therapy may create an evolutionary bottleneck after which more complex MDS, but also unrelated clones of haematopoietic cells, may emerge. In addition, subclones that acquired an additional mutation associated with treatment resistance (TP53) or disease progression (NRAS, KRAS) may be detected months before clinical changes become apparent. Monitoring the genetic landscape during the disease may help to guide treatment decisions.

Successful Transfer of Umbilical Cord Blood CD34+ Hematopoietic Stem and Progenitor-derived NK Cells in Older Acute Myeloid Leukemia Patients

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units. Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting. Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 104 T cells/kg and <3 × 105 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months. Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential “off-the-shelf” translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107–18. ©2017 AACR.

Platelet CD34 expression and α/δ-granule abnormalities in GFI1B - and RUNX1 -related familial bleeding disorders

To the editor: Mutations in the transcription factor Growth Factor Independent 1B ( GFI1B ) are causal to an autosomal dominant inherited bleeding disorder characterized by macrothrombocytopenia and reduced platelet α-granule numbers (platelet-type bleeding disorder-17, Online Mendelian

Early detection and evolution of preleukemic clones in therapy-related myeloid neoplasms following autologous SCT

Therapy-related myeloid neoplasms (tMNs) are severe adverse events that can occur after treatment with autologous hematopoietic stem cell transplantation (ASCT). This study aimed to investigate the development of tMNs following ASCT at the molecular level by whole-exome sequencing (WES) and targeted deep sequencing (TDS) in sequential (pre-) tMN samples. WES identified a significantly higher number of mutations in tMNs as compared with de novo myelodysplastic syndrome (MDS) (median 27 vs 12 mutations; P = .001). The mutations found in tMNs did not carry a clear aging-signature, unlike the mutations found in de novo MDS, indicating a different mutational mechanism. In some patients, tMN mutations were identified in both myeloid and T cells, suggesting that tMNs may originate from early hematopoietic stem cells (HSCs). However, the mutational spectra of tMNs and the preceding malignancies did not overlap, excluding common ancestry for these malignancies. By use of TDS, tMN mutations were identified at low variant allele frequencies (VAFs) in transplant material in 70% of the patients with tMNs. Reconstruction of clonal patterns based on VAFs revealed that premalignant clones can be present more than 7 years preceding a tMN diagnosis, a finding that was confirmed by immunohistochemistry on bone marrow biopsies. Our results indicate that tMN development after ASCT originates in HSCs bearing (pre-)tMN mutations that are present years before disease onset and that post-ASCT treatment can influence the selection of these clones. Early detection of premalignant clones and monitoring of their evolutionary trajectory may help to predict the development of tMNs and guide early intervention in the future.

appreci8: a pipeline for precise variant calling integrating 8 tools

Motivation: The application of next‐generation sequencing in research and particularly in clinical routine requires valid variant calling results. However, evaluation of several commonly used tools has pointed out that not a single tool meets this requirement. False positive as well as false negative calls necessitate additional experiments and extensive manual work. Intelligent combination and output filtration of different tools could significantly improve the current situation. Results: We developed appreci8, an automatic variant calling pipeline for calling single nucleotide variants and short indels by combining and filtering the output of eight open‐source variant calling tools, based on a novel artifact‐ and polymorphism score. Appreci8 was trained on two data sets from patients with myelodysplastic syndrome, covering 165 Illumina samples. Subsequently, appreci8s performance was tested on five independent data sets, covering 513 samples. Variation in sequencing platform, target region and disease entity was considered. All calls were validated by re‐sequencing on the same platform, a different platform or expert‐based review. Sensitivity of appreci8 ranged between 0.93 and 1.00, while positive predictive value ranged between 0.65 and 1.00. In all cases, appreci8 showed superior performance compared to any evaluated alternative approach. Availability and implementation: Appreci8 is freely available at https://hub.docker.com/r/wwuimi/appreci8/. Sequencing data (BAM files) of the 678 patients analyzed with appreci8 have been deposited into the NCBI Sequence Read Archive (BioProjectID: 388411; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA388411). Supplementary information: Supplementary data are available at Bioinformatics online.

BBCAnalyzer: a visual approach to facilitate variant calling

AbstractBackgroundDeriving valid variant calling results from raw next-generation sequencing data is a particularly challenging task, especially with respect to clinical diagnostics and personalized medicine. However, when using classic variant calling software, the user usually obtains nothing more than a list of variants that pass the corresponding caller’s internal filters. Any expected mutations (e.g. hotspot mutations), that have not been called by the software, need to be investigated manually.ResultsBBCAnalyzer (Bases By CIGAR Analyzer) provides a novel visual approach to facilitate this step of time-consuming, manual inspection of common mutation sites. BBCAnalyzer is able to visualize base counts at predefined positions or regions in any sequence alignment data that are available as BAM files. Thereby, the tool provides a straightforward solution for evaluating any list of expected mutations like hotspot mutations, or even whole regions of interest. In addition to an ordinary textual report, BBCAnalyzer reports highly customizable plots. Information on the counted number of bases, the reference bases, known mutations or polymorphisms, called mutations and base qualities is summarized in a single plot. By uniting this information in a graphical way, the user may easily decide on a variant being present or not – completely independent of any internal filters or frequency thresholds.ConclusionsBBCAnalyzer provides a unique, novel approach to facilitate variant calling where classical tools frequently fail to call. The R package is freely available at http://bioconductor.org. The local web application is available at Additional file 2. A documentation of the R package (Additional file 1) as well as the web application (Additional file 2) with detailed descriptions, examples of all input- and output elements, exemplary code as well as exemplary data are included. A video demonstrates the exemplary usage of the local web application (Additional file 3). Additional file 3: Supplement_3. Video demonstrating the exemplary usage of the web application “BBCAnalyzer”. (MP4 11571 kb)