Phosphoinositide lipids play a key role in cellular physiology, participating in a wide array of cellular processes. Consequently, mutation of phosphoinositide-metabolizing enzymes is responsible for a growing number of diseases in humans. Two related disorders, oculocerebrorenal syndrome of Lowe (OCRL) and Dent-2 disease, are caused by mutation of the inositol 5-phosphatase OCRL1. Here, we review recent advances in our understanding of OCRL1 function. OCRL1 appears to regulate many processes within the cell, most of which depend upon coordination of membrane dynamics with remodeling of the actin cytoskeleton. Recently developed animal models have managed to recapitulate features of Lowe syndrome and Dent-2 disease, and revealed new insights into the underlying mechanisms of these disorders. The continued use of both cell-based approaches and animal models will be key to fully unraveling OCRL1 function, how its loss leads to disease and, importantly, the development of therapeutics to treat patients.

Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.

Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.

Objective: Clinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind. Methods: To address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of lowfrequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood. Results: Analytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89e0.95]) and reproducible (>0.87 [95% CI: 0.87 e0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples. Conclusions: This is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours.

Data reported until today suggested a pivotal role of nuclear DNA mutations in the process of carcinogenesis. Recently more and more authors claim that disruption of mitochondrial DNA should not be excluded from this analysis. mtDNA have been reported in many cancers of head and neck region. Mitochondrial D-loop has been proven to be mutation hot - spot with majority of mutations in the positions 303 to 315 of poly-C tract. Data show that 37% of patients with premalignant lesions and 62% with carcinoma in situ are positive for mtDNA mutations. Moreover mutations in genes encoding ND2, ND5, COIII, CYTB, and ATP6 were observed in 17% of patients. Mutations in mitochondrial rRNA genes occured in similar number of cases. Neoplastic cells undifferentiation and disease progression is accompanied by multiplication of mtDNA number and increased mtDNA content. mtDNA content corellates with the stage of the disease. mtDNA mutations faciliate cell proliferation and inhibit apoptosis by increasing the production of ractive oxygen species (ROS). Cells harbouring mutated mtDNA have increased proliferation rate, as increased ROS concentration may act as an endogenous growth factor.

Introduction: Patients with leukemic indolent B-cell Non-Hodgkin Lymphomas (LI B-NHL) that do not easily fall into a distinct pathological category with standard diagnostics can pose a challenge as their optimal management is uncertain. Lack of a clear diagnostic label can deny patients access to novel therapies as regulatory approval of drugs is largely granted within histological categories. Moreover, these patients may be excluded from participation in clinical trials. We report findings from a prospective study evaluating targeted next generation sequencing (NGS) of circulating malignant B-cells in this setting. Methods: Over a period of 4 years, 108 patients with LI B-NHL, deemed unclassifiable on standard peripheral blood (PB) diagnostics, were prospectively enrolled from 14 centres around the UK including ours. Patients with non-clonal lymphocytosis, confirmed CLL-type MBL, CLL (Matutes score 4 or 5), MCL, HCL, high grade lymphoma on standard PB diagnostics were excluded. Clinical and morphological characteristics were analysed. PB immunophenotyping was used for characterisation including ROR1 expression. CD15+ cells were positively selected with magnetic beads for germline DNA. Tumor-germline pairs were analysed with theEuroclonality-NGS DNA capture panel, and ARResT/Interrogate and complementary pipelines, to characterise clonal immunoglobulin rearrangements, translocations and somatic mutations (Stewart et al. ASH annual meeting 2019). Germline variants were subtracted to improve somatic mutation detection. Results: Median age was 72yrs with M:F ratio of 2:1. Incidental lymphocytosis on a routine blood count was the commonest mode of presentation with only a third of patients symptomatic at presentation. Median lymphocyte count was 12.4 x 109/L at the time of sampling. Just under 50% of cases had partial or complete CD5 expression, CD19 and 20 expression was moderate to strong in most cases while ROR1 was negative in most cases. NGS was able to assign a diagnostic category in 11/90 (12%) cases based on detection of IGH;CCND1 (6), IGH;BCL2 (2), IGH;BCL3 (2) translocations and in one case a BRAF mutation with confirmation of hairy cell leukaemia on immuno-morphology. These tests were not triggered by standard diagnostic pathways at initial presentation. Two novel translocations of uncertain significance were detected - RAD51B:BIRC3 and IGHMswitch-MICALL2/INTS1. Clonal IGHV-IGHD-IGHJ rearrangements were detected by NGS and/or Sanger sequencing with predominantly IGHV3 and IGHV4 gene usage, IGHV4-34 being the most common with 15 cases. The majority of rearrangements showed somatic hypermutations above 2% and none could be assigned to the 19 major CLL-associated stereotyped subsets (using ARResT/AssignSubsets). Somatic mutations were detected in 74/108 cases. The most frequently mutated genes were TP53 in 16/108 (15%) and the hotspot MYD88 L265P mutation in 14/108 (13%) with VAF of 20-46%. Other variants detected were distributed among genes associated with NFkB signalling and chromatin remodelling (Figure 1). The MYD88 L265P mutation was present in a fifth of cases with mutations detected (14/74) and was the sole change seen in 7 cases. Accompanying mutations in the remaining cases included CD79B (2), POT1 (1), NFKBIE (1). No concomitant CXCR4 mutations were detected. Median age of the MYD88 L265P cohort was 68 years with male predominance (M:F=10:4). 9/14 presented with an incidental lymphocytosis and median count of 11.18 x 109/L. Lymphadenopathy was present in 3/14 (21%) while 7/14 (50%) had splenomegaly. A paraprotein was detected in 6/14 cases (4 IgM, 1 IgG, 1 IgA). CD5 was expressed in 6/14 cases by flow cytometry. CD19 and 20 were uniformly positive, CD79b was variable and 12/14 cases tested did not express ROR1. Conclusion: This prospective study outlines the value of a targeted NGS panel in enhancing the diagnosis of unclassifiable LI B-NHL thereby improving patient access to novel therapies and clinical trials. It highlights recurrent MYD88 mutations in a subset of patients that have splenomegaly as a frequent feature. 5 year clinical follow up data, currently being gathered in both the MYD88 mutated and overall cohort, will be valuable in further characterisation and risk stratification to inform management of these patients. Disclosures Furtado: Abbvie: Other: Conference Support. El-Sharkawi:Abbvie: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Innate: Consultancy. Iyengar:Abbvie: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Honoraria; Beigene: Consultancy.

Diamond-Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome that manifests as a profound macrocytic anemia and classically presents within the first year of life. Heterozygous mutations in, or genomic loss of one of several Ribosomal Protein (RP) genes have been identified in over 50% of DBA patients, most commonly RPS19, accounting for 25% of all cases. DBA shares a similar erythroid phenotype to the 5q- subtype of myelodysplastic syndrome in which anemia is thought to arise from heterozygous loss of RPS14. Anemia in these conditions is at least partially due to p53-mediated apoptosis and cell cycle arrest of erythroid progenitors. To further study the role of p53 in the pathogenesis of DBA and 5q- syndrome, we employed genome editing tools to generate stable Rps14 and Rps19 knockout zebrafish lines. We generated Transcription Activator-Like Effector Nucleases (TALENs) targeting exon 1 of rps19 and exon 1 of rps14 as well as Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) single guide RNAs (sgRNA) targeting exon 2 of rps19. TALENs or CRISPRs were injected into p53m214k/m214k zebrafish embryos at the single-cell stage. This zebrafish line carries a mutated p53 that is insensitive to DNA damage and hence prone to tumor formation. rps19 CRISPR sgRNAs were injected with mRNAs encoding Cas9, Cas9D10A nickase, and a ssDNA guide with a human DBA mutation. For each cohort of embryos injected, genomic DNA analysis from 20 phenotypically normal embryos from each clutch was screened to determine the efficacy of cleavage by TALEN and CRISPR using MiSeq. Mutations were identified in 30% (rps14 TALEN) 29% (rps19 TALEN), 27% (rps19 Crispr Cas9) and 12% (rps19 Crispr Cas9D10A) of reads. None of the rps19Crispr Cas9D10A carried the ssDNA guide mutation, rather single nucleotide variants and indels similar to those observed with Cas9. The remaining embryos from each F0 clutch were raised in order to generate stable mutant lines in the F1 generation; however, early, overt tumor growth was noted in all RP injected lines. Tumors were observed from 4 months post fertilization compared with 9 months for uninjected controls. F0 RP mosaic fish continued to develop tumors earlier than uninjected counterparts. At 10 months of age tumor development was statistically significantly higher in rps19 and rps14 TALEN and rps19 Cas9D10A and trended towards significance in rps19 Cas9 injected fish. Overall survival was significantly reduced in each of the cohorts compared to p53m214k/m214k uninjected controls (p<0.0001). Preliminary histology of grown tumors has shown melanomas and malignant peripheral nerve sheath tumors. Zebrafish injected with an unrelated TALEN targeting a zinc transporter (SLC30A10), into p53m214k mutant embryos do not show any increase in tumor formation compared to uninjected controls. Notably several RP’s have been shown to be haploinsufficient tumor suppressor genes in their own right in zebrafish and drosophila models. To determine if the early tumor development in p53m214k zebrafish was simply additive to a potential tumor suppressor effect of Rps14 or Rps19 alone, we injected WT embryos with the rps14 and rps19 TALENS. High mortality in rps14 and rps19 TALEN injected WT embryos impeded this analysis; however recent published reports on stable Rps19 mutant zebrafish do not report an increase in tumor incidence. Interestingly, embryo survival was not affected when TALENs were injected into p53m214/+. Analysis of these zebrafish is ongoing. Our results show that loss of Rps14 or Rps19 accelerates the development of tumors in the p53m214k/m214k mutant line. This effect is independent of the RP or the method of mutation (TALEN vs CRISPR), indicating that off target effects are unlikely to be responsible for this observation. As these are mosaic F0 fish, it is possible that tumors may arise from cells with homozygous RP mutations. Further molecular analysis will reveal this. We have now identified 2 stable Rps19 mutant lines, and tumor analysis of F1 fish from these lines is ongoing. In conclusion, we have shown that loss of Rps14 or Rps19 cooperates with a loss of function p53 mutation to accelerate tumor formation and death. Our results highlight the importance of caution in using p53 suppressors as a therapeutic option in RP deficient patients. Disclosures No relevant conflicts of interest to declare.