ALBERT

Beschreibung: Background: Sickle cell disease (SCD) and its complications are associated with frequent hospital visits and treatment often requires venous access for administration of fluids, medications and blood transfusions. Due to frequent use, peripheral venous access can become difficult over time because of venous scarring. Moreover, certain interventions such as chronic simple or exchange transfusions require reliable venous access for prolonged periods of time. Implantable central venous devices such as ports offer definitive access and mitigate the need for frequent peripheral access attempts. However, existing knowledge on the use of these devices in pediatric patients with SCD is limited. Due to possible increased risks of thrombosis and mechanical occlusion from hypercoagulable state, risk of infectious complications and need for surgical placement, ports are often not used routinely. We review the indications and complications associated with placement of ports in the pediatric cohort of patients with SCD - to better define the scope of port placement in this group. Methods: We performed an IRB-approved, retrospective chart review to assess the indications, complications and risk factors associated with port placement in children and adolescents with SCD at the University of Oklahoma Health Sciences Center (OUHSC). The study period analyzed was 17.5 years from January 1st, 2000 to July 30th, 2018 and included patients from birth to 21 years of age, diagnosed with SCD who had homozygous sickle cell disease (HbSS) or compound heterozygous SCD - either sickle cell-β0-thalassemia, sickle cell-β+-thalassemia or sickle cell-hemoglobin C (HbSC) disease, and had ports in place for more than 7 days. Patients were identified systematically by querying the OUHSC Clinical Data Warehouse using diagnostic codes followed by chart review. Results: Thirty-two patients with SCD and ports were identified during the study period, out of which 31 patients had HbSS and one patient had HbSC disease. The median age at first port insertion was 8 years (range 1-20 years). A total of 63 ports were placed for a total of 99,272 port days with a median port life span of 1340 days. The two main indications for port placement were - either chronic transfusions for which 48 ports were placed for a total of 80,238 port days, or poor venous access (PVA) for which 15 ports were placed for a total of 19,034 port days. Out of the 48 ports placed for chronic transfusions, 6 ports were for transfusions for primary stroke prophylaxis, 22 ports were for transfusions for secondary stroke prophylaxis, 17 ports were for transfusions for recurrent vaso-occlusive episodes (VOE) and 3 ports were for transfusions after multi-organ failure. A total of 54 port complications occurred (malfunction=29, infection=20, thrombosis=3, difficult port access=1, and intractable pain over port site=1). From the data available, more ports were placed in the left subclavian vein (LSV=20) than right subclavian vein (RSV=4) and right internal jugular (RIJ=2), however rate of overall complications were similar between LSV and RSV - 0.57 complications/1,000 catheter days in LSV and 0.58 complications/1,000 catheter days in RSV. The rate of port associated infection, defined as a positive blood culture drawn from the port, was 0.2 per 1,000 port days. A total of 20 infections identified mostly gram-positive organisms (n=15) predominantly Staphylococcus, compared to gram-negatives (n=3), fungus with Candida albicans (n=1) and a rare acid-fast bacilli infection with Mycobacterium mucogenicum (n=1). The rate of thrombosis, identified radiologically using vascular doppler ultrasound, was 0.03 per 1,000 port days. The rate of premature port removal arising from complications was 0.36 per 1,000 port days. Ports placed for chronic transfusions had a lower rate of removal (0.31 per 1,000 port days) compared to ports placed for poor venous access (0.58 per 1,000 port days) with a ratio of 0.54 which approached statistical significance (p=0.09; CI 0.26-1.21). Conclusion: Ports in pediatric patients with SCD are associated with low rates of thrombosis, infection and malfunction. Ports may be a reasonable alternative for vascular access in patients with SCD - especially in patients who require chronic simple or exchange transfusions and have difficult access. Larger prospective studies will be needed to further assess the scope of use of ports in this population. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Despite advancements in the diagnosis and treatment of acute lymphoblastic leukemia (ALL), a need for improved strategies to decrease morbidity and improve cure rates in relapsed/refractory ALL still exists. Such approaches include the identification and implementation of novel targeted combination regimens, and more precise upfront patient risk stratification to guide therapy. New curative strategies rely on an understanding of the pathobiology that derives from systematically dissecting each cancer’s genetic and molecular landscape. Zebrafish models provide a powerful system to simulate human diseases, including leukemias and ALL specifically. They are also an invaluable tool for genetic manipulation, in vivo studies, and drug discovery. Here, we highlight and summarize contributions made by several zebrafish T-ALL models and newer zebrafish B-ALL models in translating the underlying genetic and molecular mechanisms operative in ALL, and also highlight their potential utility for drug discovery. These models have laid the groundwork for increasing our understanding of the molecular basis of ALL to further translational and clinical research endeavors that seek to improve outcomes in this important cancer.

Beschreibung: Myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) are clonal hematopoietic disorders resulting from genetic alterations in hematopoietic stem cells. These myeloid disorders are clinically heterogeneous and biologically complex. Despite major advances in understanding the genetic and molecular landscape of MDS/AML, along with the introduction of newer and targeted therapies, the cure rates in AML are still only about 60% in children, and much lower in adults. Exploiting the genetic tractability of the zebrafish (Danio rerio) vertebrate model, we are investigating the role of a novel epigenetic regulator, Chromodomain helicase DNA binding protein-1 (CHD1) in hematopoiesis and its misregulation leading to MDS and AML. CHD1 is located at chromosome 5q21, which lies within the most frequent breakpoints seen with the deletion of the long arm of chromosome 5 [del (5q)] in patients with MDS and AML. In addition to del (5q), we found that CHD1 levels are significantly lower in bone marrow cells of patients with other forms of MDS, relative to normal controls. Using CRISPR/Cas9-mediated targeted mutagenesis in zebrafish, we created chd1 homozygous mutant fish. We confirmed a marked decrease in chd1 gene expression in these mutant fish. Chd1 homozygous mutants are viable and fertile as adults, with no significant developmental or hematopoietic phenotypes observed during embryogenesis. As CHD1 can act as a tumor suppressor and is linked to the DNA damage response, we hypothesized that chd1 mutant zebrafish would be more sensitive to DNA damaging agents. Indeed, we found that chd1 mutants have increased sensitivity to ionizing radiation as evidenced by elevated brain cell death measured by whole mount imaging of live embryos and immunofluorescence for activated Caspase 3, a marker of apoptosis. We also generated chd1het; tp53het zebrafish to test whether chd1 haploinsufficiency could accelerate tumor rates in tp53 mutant fish. Single heterozygotes chd1het or tp53het usually do not form tumors at one year of age, but chd1het; tp53het double heterozygous zebrafish showed substantial tumor growth by one year of age. Taken together, our data suggest that CHD1 may play a key role in protecting genomic integrity, explaining why diminished CHD1 levels could contribute to the pathogenesis of MDS and AML. This genetic interaction may be especially crucial in patients with combined del (5q) and TP53 alterations, and could contribute to the increased severity seen in this group. Our findings suggest these and other CHD1-deficient patients may be resistant to standard therapies due to attenuated DNA damage responses, allowing their AML to survive DNA damage caused by conventional anti-cancer treatments. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer; B-lineage ALL (B-ALL) represents ~85% of cases. Transgenic humanMYC(hMYC), an oncogenic transcription factor in many blood cancers, is known to induce T-ALL in zebrafish. Until recently robust zebrafish B-ALL models did not exist, but we discovered fish of this transgenic line develop B-ALL also. To distinguish T-ALL from B-ALL, we use a transgenic marker,lck:GFP, where T-ALL fluoresce brightly while B-ALL fluoresce dimly. To define B- and T-ALL incidence, we monitored 〉600 fish by serial fluorescent microscopy and found 100% of fish developed ALL (64% T-ALL, 23% B-ALL, both 13%) by 10 months. To find transcriptional programs active in both ALL types, we performed RNA-seq on 10 T- and 14 B-ALL and compared these to T- and B-ALL induced by murineMyc(mMyc). Unexpectedly, we found B-ALL occur in distinct lineages in these related lines, withhMYCB-ALL expressingighzheavy chains, butmMycB-ALLighm. To investigate glucocorticoid responses, a key agent in ALL, we treated fishin vivowith dexamethasone (DXM).hMYCB-ALL were highly DXM-sensitive, but many recurred rapidly after treatment ceased. Studies lymphocytes or ALL cells from zebrafish typically require fish euthanization; it would be useful to obtain cells without euthanasia. Thus, we developed biopsy techniques in living fish to obtain lymphocytes from the thymus, kidney-marrow, and even scales. We found B and T cells can be obtained from thymus, with B cells abundant in scales. We can also recover ALL cells from scales of fish with B- or T-ALL. Analyses of normal lymphocytes and ALL cells from these anatomic regions prove that GFPhi cells express T cell genes, and GFPlo cells express B cell genes. These novel biopsy methods will permit studies ofex vivosamples from live fish. Overall, these new results and methods expand the utility of zebrafishhMYC-driven ALL, a model of the most important pediatric cancer. Disclosures No relevant conflicts of interest to declare.

Beschreibung: MYCis a key oncogene overexpressed by many cancers, however, its oncogenic mechanisms are poorly understood. MYC is also central to acute lymphoblastic leukemia (ALL), the most common and second most lethal pediatric malignancy. Much of MYC's oncogenicity has been attributed to its transcription factor function, but data suggest MYC also deregulates replication in transcription-independent fashion. As a known master regulator of cancer transcriptomes and epigenomes, we hypothesize that MYC dramatically alters both gene expression and replication timing (non-random spatiotemporal process where some part of the genome replicates early, and other late) in both types of ALL - B-ALL and T-ALL. Conceivably, MYC exerts oncogenic effects upon the ALL transcription and replication programs, with some changes shared by B- and T-ALL, and others unique to only one. We aim to address two novel questions not been investigated before. First, in ALL, do the same genetic loci show aberrant RNA transcriptionandDNA replication? Second, how similar are the affected loci in two closely-related, yet distinct, ALL types driven by the same oncogene? The basis of our project is a unique double-transgenicrag2:hMYC,lck:GFPzebrafish pre-clinical model we established, which is the only animal model proven to develop both B-ALL and T-ALL. We previously showed that gene expression profiles (GEP) differentiating zebrafish B- and T-ALL also distinguish human B- and T-ALL, making this an ideal model system to study human ALL. In this model, B-ALL and T-ALL are induced by human MYC(hMYC) regulated by aD.rerio(zebrafish)rag2promoter.Since B and T lymphoblasts both expressrag2, both lineages over-express MYC, causing highly-penetrant B- and T-ALL. Differential activity of aD. rerio lckpromoter causes B cells to fluoresce dimly and T cells to fluoresce brightly, allowing us to identify and purify B-ALL and T-ALL by fluorescent microscopy and fluorescence-based flow cytometry, respectively. This unique model enables comparing B- and T-ALL in one genetic background. We have purified 〉20 zebrafish ALL (both T-ALL and B-ALL) and isolated their RNA and DNA. We are now analyzing RNA-seq gene expression profiles (GEP) and replication timing (RT) profiles via next generation sequencing (NGS). We will compare both ALL types to identify mRNA signatures that are unique to, or shared by, both types. We seek loci that shift DNA replication from early-to-late, or late-to-early, to define the regions that replicate at the same time in both ALL types, versus loci that vary by ALL type. We will also interrogate these data to determine whether GEP and RT profiles correlate with each other, and with known MYC target genes. In conclusion, GEP and RT have never been analyzed in the same cancer sample, or in related cancers driven by the same oncogene. Exploiting our expertise with thehMYCzebrafish model, we are delineating how MYC alters transcription and replication, to ascertain if these affect the same loci and define which loci are unique to one ALL type or shared by both. MYC hyper-activity is seen ~70% of human cancers - making MYC a crucial oncogene in human cancer biology, so our findings are likely to inform not only mechanisms operative in ALL, but also other MYC-driven cancers. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, representing 〉25% of all cancers in children 0-14 years. Despite major advancements in pediatric ALL treatment, it remains the second most lethal childhood cancer, accounting for ~25% of deaths. The two types of ALL are precursor-B, or B-ALL, and precursor-T, or T-ALL, which have distinct molecular landscapes. Of these types, T-ALL comprises about 15% and 25% of pediatric and adult cases, respectively, and is historically considered more aggressive and treatment-resistant, with an inferior prognosis. In the precision medicine era, it is imperative to identify genetic alterations and aberrant gene expression patterns, to better understand tumor biology and improve treatment outcomes by identifying new therapeutic targets. Our study investigates a novel transcription factor, odd-skipped related transcription factor 2 (OSR2), which we hypothesize is a putative T-ALL tumor suppressor. We are using a zebrafish T-ALL model expressing transgenic human MYC (hMYC) regulated by a lymphoblast-specific promoter, rag2. Prior work in zebrafish and human T-ALL found low OSR2 levels in ~95% of T-ALL. Based on this, we then used RNA-seq to analyze 10 hMYC zebrafish T-ALL, confirming low-to-absent osr2 in all 10 T-ALL relative to wild-type (WT) T cells. We further confirmed decreased osr2 expression by qRT-PCR of additional T-ALL and WT thymocytes. We hypothesized that if OSR2 suppresses T-ALL, impaired zebrafish Osr2 function might increase T-ALL incidence and shorten latency. To test this, we bred osr2-mutant fish to rag2:hMYC transgenic animals to create three genotypes: heterozygous osr2-mutant (osr2het) fish, heterozygous hMYC (hMYChet) fish, and compound-heterozygote (osr2het;hMYChet) fish. We screened these genotypes for T-ALL incidence by serial fluorescence microscopy, with T-ALL subsequently confirmed by fluorescence-based flow cytometry. By 7 months of age, we found 9/18 (50%) of double-heterozygous fish developed T-ALL, compared to 0/7 hMYChet fish (p = 0.026); osr2het fish also did not develop T-ALL. Together, our findings suggest osr2 allelic loss accelerates MYC-driven T-ALL, supporting our hypothesis that osr2 is a T-ALL tumor suppressor. Disclosures No relevant conflicts of interest to declare.