Epigenetic changes in human model KMT2A leukemias highlight early events during leukemogenesis.

Chromosomal translocations involving KMT2A gene are one of the most common genetic alterations found in pediatric acute myeloid leukemias (AML) although the molecular mechanisms that initiate the disease remain incompletely defined. To elucidate these initiating events we have used a human model system of AML driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observe the development of a profound hypomethylation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem cell associated genes along with skewed expression in other genes such as S100A8/9 implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-seq data showed that there were relatively few leukemiaspecific changes and the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed the adenylate cyclase gene ADCY9 as an essential gene in KM3-AML, and suggest the potential for autocrine signalling through the chemokine receptor CCR1 and CCL23 ligand. Together, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.

Dosimetric impact of mechanical movements of the Linac gantry during treatments with small fields.

Objective: Current accepted linac Quality Assurance (QA) guidelines used for Volumetric Modulated Arc Therapy (VMAT) suggest a mechanical isocentre tolerance level of 1 mm. However, this tolerance level has not been well-established for the specific case of small field stereotactic VMAT. This study aims to evaluate the clinical impact of mechanical uncertainty on this treatment modality by modelling systematic gantry sag derived isocentre variance in the Treatment Planning System (TPS). Approach: A previously reported dataset of gantry sag values in the literature served as a starting point for this study. Using an in-house developed VMAT arc splitting algorithm, isocentre shifts were applied at a Control Point (CP) level to DICOM-RT treatment plans. Dose distributions for varying isocentre shift magnitudes were calculated for a set of 29 stereotactic VMAT plans using the Eclipse Acuros XB dose algorithm. These plans had a range of Planning Target Volume (PTV) sizes. A quantitative comparison of each plan was conducted by evaluating five Dose Volume Histogram (DVH)-derived plan quality metrics. Results: All metrics exhibited a deterioration in plan quality with increasing magnitudes of isocentre shift. At small PTV sizes, these effects were amplified, exhibiting significant changes at 1 mm of average shift when typical targets and tolerances were considered. For plans with PTVs between 0 and 5 cm3, a 1 mm shift reduced PTV coverage by 6.6 ± 2.2% and caused a 12.1 ± 3.8% deterioration in the conformity index. Based on the results of this study, the prevalent tolerance of 1 mm may not be suitable for treatments of small PTVs with small fields. Significance: In contrast to commonly accepted values, an absolute mechanical isocentre of 0.5 mm with action level at 0.75 mm is recommended for stereotactic VMAT of PTV sizes below 10 cm3.

Evaluation of the Impact of the Linac MLC and Gantry Sag in volumetric modulated arc therapy.

PURPOSE: Mechanical sag in the radiotherapy linear accelerator gantry and multi-leaf collimator (MLC) carriage effectively causes systematic deviations in the isocenter with respect to gantry angle. To minimize the impact of this error on treatment, a tolerance value of a 1-mm mechanical isocenter shift is commonly accepted for intensity-modulated radiation therapy quality assurance (QA). However, this tolerance value has not been firmly established for volumetric modulated arc therapy (VMAT) treatments. The purpose of this study is therefore to evaluate the impact of gantry and MLC carriage sag on VMAT clinical performance. METHODS: A published dataset of Elekta and Varian sag measurements served as a starting point for the investigation. Typical sag profiles were chosen and modeled as continuous isocenter deviations in three dimensions. The data were then incorporated into existing Digital Imaging and Communications in Medicine protocol, extended for radiotherapy plans via a "beam-splitting" algorithm. Three treatment sites were investigated in parallel: head and neck, prostate, and prostate with surrounding lymph nodes. Monte Carlo-simulated dose distributions were obtained for varying magnifications of the modeled sag. The resulting dose distributions, including that for no error, were compared qualitatively and quantitatively, against multiple metrics. RESULTS: The dose-volume histograms (DVHs) for all plans exhibited a decrease in planning target volume (PTV) dose uniformity with increasing sag magnification, whereas dose to organs at risk exhibited no coherent trend. The prostate plan was shown to be the most vulnerable to mechanical sag across all considered metrics. However, all plans with peak isocenter deviation less than 1 mm were well within typical cutoff points for each metric. CONCLUSIONS: All avenues of investigation presented substantiate the commonly accepted tolerance value of a 1-mm peak isocenter shift in annual linac QA.

Pediatric leukemia: Moving toward more accurate models.

Leukemia is a complex genetic disease caused by errors in differentiation, growth, and apoptosis of hematopoietic cells in either lymphoid or myeloid lineages. Large-scale genomic characterization of thousands of leukemia patients has produced a tremendous amount of data that have enabled a better understanding of the differences between adult and pediatric patients. For instance, although phenotypically similar, pediatric and adult myeloid leukemia patients differ in their mutational profiles, typically involving either chromosomal translocations or recurrent single-base-pair mutations, respectively. To elucidate the molecular mechanisms underlying the biology of this cancer, continual efforts have been made to develop more contextually and biologically relevant experimental models. Leukemic cell lines, for example, provide an inexpensive and tractable model but often fail to recapitulate critical aspects of tumor biology. Likewise, murine leukemia models of leukemia have been highly informative but also do not entirely reproduce the human disease. More recent advances in the development of patient-derived xenografts (PDXs) or human models of leukemias are poised to provide a more comprehensive, and biologically relevant, approach to directly assess the impact of the in vivo environment on human samples. In this review, the advantages and limitations of the various current models used to functionally define the genetic requirements of leukemogenesis are discussed.

Human models of NUP98-KDM5A megakaryocytic leukemia in mice contribute to uncovering new biomarkers and therapeutic vulnerabilities.

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.

Mining Cancer Transcriptomes: Bioinformatic Tools and the Remaining Challenges.

The development of next-generation sequencing technologies has had a profound impact on the field of cancer genomics. With the enormous quantities of data being generated from tumor samples, researchers have had to rapidly adapt tools or develop new ones to analyse the raw data to maximize its value. While much of this effort has been focused on improving specific algorithms to get faster and more precise results, the accessibility of the final data for the research community remains a significant problem. Large amounts of data exist but are not easily available to researchers who lack the resources and experience to download and reanalyze them. In this article, we focus on RNA-seq analysis in the context of cancer genomics and discuss the bioinformatic tools available to explore these data. We also highlight the importance of developing new and more intuitive tools to provide easier access to public data and discuss the related issues of data sharing and patient privacy.