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Abstract Introduction eIF5A is the only known protein to be modified by hypusination and is highly conserved across species. Hypusinated eIF5A, the predominant form in normal and cancer cells, is involved in cell survival and inflammatory pathway activation. siRNAs targeting eIF5A inhibit NF-kB activation and reduce pro-inflammatory cytokine production. Accumulation of the unhypusinated lysine form of eIF5A is associated with apoptosis. Mutants of eIF5A that cannot be hypusinated (e.g. eIF5AK50R) are pro-apoptotic in vitro and have anti-tumoral activity in vivo in multiple cancer types including melanoma and lung cancer. SNS01-T is a novel therapeutic with a dual mechanism of eIF5A modulation: inducing cell death via siRNA-mediated inhibition of hypusinated eIF5A while simultaneously causing over-expression of pro-apoptotic eIF5AK50R via a DNA plasmid with a B-cell promoter to induce tumor cell death. SNS01-T significantly inhibited tumor growth and increased survival in mouse models of myeloma (MM), mantle cell and diffuse large B-cell lymphoma. The phase 1-2 study of SNS01-T has completed 4 planned dosing cohorts 0.0125, 0.05, 0.2 and 0.375 mg/kg twice weekly IV for 6 weeks in pts with refractory B-cell cancers. Methods PK and PD secondary endpoints included characterization of PK by measuring pExp5A plasmid DNA and eIF5A siRNA in blood and bone marrow (BM), assessing potential immunogenicity of SNS01-T by measuring serum concentrations of antibodies against SNS01-T nanoparticles, and measuring serum concentrations of select proinflammatory cytokines by enzyme-linked immunosorbent assay in serum and plasma samples. Blood PK timepoints were 30 minutes before the first infusion and at 30 minutes, 2, 6, and 24 hours after the first infusions on Week 1, Week 3, and Week 6 and at the 4, 8, and 12 week visits after the last infusion; BM samples were collected 1 day after the final infusion. Serum and plasma PD sampling timepoints were 30 minutes before and at 2, 6, and 24 hours after the first infusion on Week 1, Week 3, and Week 6, and at 4 weeks after the final infusion. Cytokines assayed included TNF-α, IFN-α, IFN-ß, IFN-g, CXCL1, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12. Results Table 1 shows data available for interpretation as of August 2014. The remaining samples are under analysis and will be presented. Abstract 2121. Table 1:Data available for interpretation (number of patients)SNS01-T DoseOverall(n=18)0.0125 mg/kg(n=6)0.05 mg/kg(n=4)0.2 mg/kg(n=4)0.375 mg/kg(n=4)Blood PK DNA and RNA156441Bone marrow PK82321Serum antibodies to SNS01-T nanoparticles186444Serum and plasma cytokines156441 Plasmid and siRNA blood levels generally peaked 30 minutes post-dosing at weeks 1, 3 and 6 of dosing. Both plasmid and siRNA exhibited rapid clearance from the blood, with levels dropping to near pre-dosing levels within 24 hours of administration. pExp5A plasmid DNA was detectable in the bone marrow of 2 pts at cohort 1, 2 at cohort 2, 1 at cohort 3 and 1 at cohort 4. eIF5A siRNA was not detectable in bone marrow. No antibodies to SNS01-T nanoparticles were detected at any timepoint at any dose level. Cytokines remained within the expected range of inter-patient variability, similar to baseline across all timepoints at the first 2 dose levels. At dose level 3, levels of IL-6, IL-8 and TNF-α in particular increased at the 2 and 6 hour timepoints but had recovered to baseline levels 24-hours post dosing. This effect was more pronounced at the first infusion. Conclusions PCR analysis demonstrated the presence of both plasmid DNA and siRNA components of SNS01-T in blood at all dose levels, with a dose-dependent increase in plasmid copy number. Plasmid DNA was also detected in bone marrow collected 24 hours after the final infusion of SNS01-T. Pro-inflammatory cytokines did increase within hours of infusion but returned to baseline within 24 hours, synchronous with the clinical infusion reactions (see Abstract 70148). No evidence of an anti-SNS01-T antibody response was observed in any subject. Phase 2 trials are planned. Disclosures Bexon: Senesco: Consultancy. Craig:Senesco: PI Other. Siegel:Senesco: PI Other. Bensinger:Senesco: PI Other. Novitzky:Senesco: PI Other. McDonald:Senesco: PI Other. Gutierrez:Senesco: PI Other. Libby:Senesco: PI Other. van Rhee:Senesco: PI Other. Heidel:Senesco: Consultancy. Thompson:Senesco: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Barranco:Senesco: Consultancy. Taylor:Senesco: Research Funding. Browne:Senesco: Employment. Kurman:Senesco: Consultancy. Lust:Senesco: PI Other. Dondero:Senesco: Employment.

Abstract Abstract 92 BACKGROUND: All somatic cell types, including all B lineage cells, constitutively express a number of DNA repair proteins to maintain genomic stability and thwart tumorigenesis in the face of ongoing constitutive levels of DNA damage. Compromises in expression of these essential DNA repair factors have been shown to readily induce the development of various cancers including B cell lymphomas, suggesting expression levels are tightly regulated and limited in quantity. In germinal center (GC) B cells, however, additional DNA repair capacity is likely required to counterbalance the heightened level of mutagenic activity owing to the induced expression of activation-induced cytidine deaminase (AID) during somatic hypermutation (SHM). AID is a DNA editing enzyme which introduces somatic mutations into immunoglobulin (Ig) V regions at an estimated rate which is almost a million-fold higher than the spontaneous rate in somatic cells. HYPOTHESIS: We hypothesize that to maintain the genomic wellness of GC B cells which are undergoing SHM, there is a need for induction of an accompanying robust DNA repair system. We further hypothesize that inefficient induction of these repair genes may predispose to malignant transformation. METHODS: Using tonsillar tissue sections and purified tonsillar B cell subpopulations, we compared expression levels of various DNA repair genes and related these levels to AID expression across the subsets using immunohistochemistry, real-time RT-PCR, and Western blot analysis. To characterize the nature of signals capable of inducing expression levels of AID and/or DNA repair proteins, peripheral blood B cells were activated in vitro using a panel of stimuli, including coculture with activated CD4 T cells. As a surrogate measure of mismatch repair (MMR) activity in the relative absence of T cell help, we quantitated the number of somatic hypermutations at A/T sites in the Ig heavy chain variable (IGHV) region genes in a collection of IGHV sequences obtained from normal B cells and HIV-related lymphoma cells. RESULTS: Using immunohistochemistry, we observed that, similar to the expression of AID, DNA MMR genes are significantly induced in tonsillar GC B cells. These results were further validated using a more sensitive real-time RT-PCR assay and analysis by Western blotting. By expanding our DNA repair gene panel, we observed that proteins of homologous recombination, base excision repair and DNA single strand break signalling pathway are also similarly induced in GC B cells at RNA, protein, and functional levels compared to their expression in naïve and memory B cells. By contrast, expression of non-homologous end joining and DNA double strand break signalling molecules are unchanged. We have termed this selective induction of repair mechanisms in GC B cells as somatic hyperrepair (SHR). To identify pathways that lead to the activation of AID and SHR, we used an in vitro system and a variety of stimuli and we discovered that multiple B cell stimuli including CpG, CD40L, and anti-BCR could each independently induce the expression of AID while SHR induction strictly required the engagement of CD4+ T cells. This provocative observation suggests a novel role for CD4+ T cells in mitigating tumorigenesis of post-GC B cells through their ability to induce the SHR pathway in cells that have been induced to undergo SHM. To demonstrate the possible role of SHR in lymphomagenesis, we analysed the mutation pattern of IGHV genes from a panel of B cell lymphomas obtained from HIV infected (CD4+ T cell suppressed) patients. We found that HIV-related lymphoma cells displayed a significantly lower frequency of SHM at A/T positions relative to normal memory B cells, indicative of compromised MMR of their precursor cells during GC transit. Our findings resolve the lingering paradox that B cell malignancies are overwhelmingly prevalent under T cell suppression conditions such as HIV infection, post-organ transplant, and aging. Finally, our results also suggest for the first time that mounting efficient tumor suppression for some cells may depend on signals transmitted by neighboring cells and the specific microenvironment. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 669 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world but its pathogenesis remains largely unknown. More recently, a widely prevalent premalignant condition termed monoclonal B cell lymphocytosis (MBL) has been defined and similarly involves expansion of a CD19+CD5+ population of B cells. MBL has been the focus of a wide number of recent studies in hopes it can provide insight into the early pathological events that lead to clonal expansion of a pre-leukemic CLL-like clone. Previously, we and others identified the transcription factor lymphocyte enhancer-binding factor-1 (LEF-1) as one of several genes significantly over expressed in CLL B cells relative to blood CD19+ B cells from healthy adults. LEF-1 is crucial for the proliferation and survival of pro-B cells during B cell development and deregulated LEF-1 activation has been directly linked with leukemogenesis in a transgenic murine model. In the present study, we addressed three critical questions concerning LEF-1 and CLL: 1) do CD5+ normal B cells express LEF-1; 2) at what stage of transformation does LEF-1 expression first appear; and 3) what mechanism(s) underlies LEF-1 expression in leukemic B cells. Methodology: The goals of our study were to: 1) determine the LEF-1 expression status of normal CD 19+/CD5+ human B cells; 2) determine the expression status of LEF-1 in clonal B cells present in patients with MBL; and 3) identify regulatory mechanisms that control aberrant expression of LEF-1 in CLL B cells. In order to achieve the first two goals, a 3-color flow cytometry assay for CD19, CD5, and intracellular LEF-1 was developed. To achieve the third goal, we performed in silico analysis of the LEF-1 locus and correlated this with publicly available genome wide methylated CpG island recovery assay (MIRA) data describing the methylome of normal human B cells (Rauch, T. A. Proc Natl Acad Sci U S A. 2009; 106(3): 671-8). Results: Analysis of human umbilical cord blood B cells, a rich source of the CD19+/CD5+ B cell subset, revealed that all normal CD 19+/CD5+ human B cells are negative for LEF-1 protein expression. These data demonstrate LEF-1 expression by CLL B cells is truly aberrant and does not simply reflect the phenotype of CD5+ lineage B cells. We next tested if the CD19+/CD5+ cells obtained from patients with MBL express LEF-1 protein. In these analyses, we restricted our study to MBL patients with absolute B cell counts of less than 2.5 ×10 9 cells/(L) (range: 0.756 - 2.44 × 10 9 cells/(L)), which is significantly below the upper limit that defines the MBL to CLL transition. Of interest, each MBL sample analyzed (n=6) revealed the presence of two populations of cells: 1) CD19+/CD5+ B cells expressing LEF-1; and 2) CD19+/CD5- B cells lacking expression of LEF-1. To confirm the clonal nature of the CD19+/CD5+ cells from MBL patients we demonstrated that the cells were light chain restricted. These data clearly indicated that LEF-1 expression becomes deregulated in the premalignant state of MBL and may therefore represent an early event in CLL leukemogenesis. In order to identify differential pathways of LEF-1 regulation that may be altered in CLL and MBL B cells, in silico promoter analysis of LEF-1 was performed. We identified a number of potential transcription factor binding sites as well as a putative CpG island in the 5' promoter region of LEF-1. Using data from a human B cell genome wide methylation array, we were able to determine that this same putative LEF-1 promoter CpG island was highly methylated in normal human B cells. Methylation of promoter region CpG islands is known to play an important role in developmental regulation of gene expression and may be the operative mechanism underlying silencing of LEF-1 expression in normal B cells. Conclusions: LEF-1 expression is deregulated in MBL and appears to be an early and possibly key event in the transition of normal B cells into a premalignant/malignant state. Our data suggest that loss of epigenetic regulation of this developmentally important locus may play a role in aberrant LEF-1 expression in MBL and CLL B cells. Ongoing studies are aimed at determining the methylation status of the LEF-1 promoter in CLL B cells and the functional role of this protein in transcriptional regulation and survival of CLL and MBL cells. Disclosures: No relevant conflicts of interest to declare.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) has become the most common form of chronic liver disease. Determining the severity of NAFLD using the presence of nonalcoholic steatohepatitis (NASH) and stage of liver fibrosis is important, yet liver biopsy remains the gold standard despite limitations to its use. This study surveyed pediatric gastroenterologists across the United States to determine which guidelines are used to determine the severity of NAFLD in children, which patients undergo liver biopsy, and how these guidelines compare to each other and to clinical practice. METHODS: 140 pediatric gastroenterologists were surveyed electronically regarding current diagnostic practices for NAFLD. Responses were reviewed in Survey Monkey and compared to current North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN), American Association for the Study of Liver Diseases (AASLD) and European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines regarding liver biopsy for evaluation of NAFLD. RESULTS: 140 surveys were distributed with 54 responses, a 38.6% response rate. Participants included pediatric gastroenterologists, hepatologists and transplant hepatologists. 96.2% reported familiarity with NASPGHAN guidelines, 22.6% with ESPGHAN and 39.6% with AASLD. 88.7% of responders desired a generalized consensus regarding proper diagnosis. Pediatric gastroenterologists referred obese patients with abnormal liver enzymes suspected to have NAFLD 22% of the time, pediatric hepatologists 27.2%, and transplant hepatologists 39.9% (one-way ANOVA with P -value of 0.324). 61.5% of responders reported using Fibroscan and 59.6% used MR elastography to assess severity of NAFLD and presence of liver fibrosis when not pursuing liver biopsy. 92.5% of responders believed the presence of autoimmune antibodies was important in deciding when to proceed to liver biopsy, as well as 88.7% using abnormal screening labs and 75.5% using imaging suggestive of liver fibrosis. CONCLUSION: Comparison between the criteria that providers believed warranted a liver biopsy versus those included in society guidelines differed. The presence of autoimmune antibodies, abnormal screening labs, and liver fibrosis suggested on imaging all appeared to be important factors regardless of current guidelines. Providers also seem to rely on Fibroscan and MR elastography over liver ultrasound despite the lack of validated cutoff values in children.

BackgroundLymphatic dysfunction is hypothesized to be an underlying factor in the pathophysiology of yellow nail syndrome (YNS) (yellow nails, lymphedema, pleural effusions, and frequently chronic rhinosinusitis [CRS]). It is unclear why some YNS patients develop CRS. We quantified lymphatic and total vasculature of sinonasal mucosa in YNS patients and compared it to controls from CRS patients with (CRSwNP) or without polyps (CRSsNP).MethodsImmunohistochemistry was performed on archival sinonasal mucosal samples from 5 patients with YNS and 14 controls with antibodies against podoplanin and CD31, markers of lymphatics. Morphometric assessment was performed on digital images using ImageJ software.ResultsIn YNS, the number of lymphatics/mm2 ranged from 7 to 18/mm2 (controls: 6 to 43/mm2, p = 0.343), with a mean perimeter between 92 and 201 µm (controls: 42 to 280 µm, p = 0.482). Total vasculature density was higher than lymphatics, ranging between 189 and 1159 vessels/mm2, average 669 (controls: 139 to 1467/mm2, average 503, p = 0.257) with smaller average perimeter, 40 to 117 µm, mean 64.8 µm (controls: 42 to 92 µm, mean 65.3 µm, p = 0.965). Lymphatics constituted only a small fragment of the total vasculature, ranging from 1.15% to 4.76%, average 2.34% (controls: 0.81% to 10.58%, average 4.88%, p = 0.156). CRSwNP patients had significantly higher lymphatic density (p = 0.011) and ratio of lymphatics to total vasculature (p = 0.045) than patients with YNS or CRSsNP.ConclusionThis is the first histological analysis of sinus mucosa in patients with YNS. Vascular type, density, size, and distribution in the sinonasal mucosa of YNS patients are not statistically significantly different from those of the CRSsNP group. Lymphatic density and ratio to total vasculature is higher in CRSwNP patients.

Objectives To improve the trustworthiness of evidence, studies should be prospectively registered and research reports should adhere to existing standards. We aimed to systematically assess the degree to which endocrinology and internal medicine journals endorse study registration and reporting standards for randomised controlled trials (RCTs), systematic reviews (SRs) and observational studies (ObS). Additionally, we evaluated characteristics that predict endorsement of reporting or registration mechanism by these journals. Design Meta-epidemiological study. Setting Journals included in the ‘Endocrinology and Metabolism’ and ‘General and Internal Medicine’ 2017 Journal Citation Reports. Participants Journals with an impact factor of ≥1.0, focused on clinical medicine, and those who publish RCTs, SRs and ObS were included. Primary outcomes Requirement of adherence to reporting guideline and study registration as determined from the journals’ author instructions. Results Of the 170 (82 endocrinology and 88 internal medicine) eligible journals, endorsing of reporting standards was the highest for RCTs, with 35 (43%) of endocrine journals and 55 (63%) of internal medicine journals followed by SRs, with 21 (26%) and 48 (55%), respectively, and lastly, by ObS with 41 (50%) of endocrine journals and 21 (24%) of internal medicine journals. In 78 (46%) journals RCTs were required to be registered and published in adherence to the Consolidated Standards of Reporting Trials statement. Only 11 (6%) journals required registration of SRs. Internal medicine journals were more likely to endorse reporting guidelines than endocrine journals except for Strengthening the Reporting of Observational Studies in Epidemiology. No other journal characteristic proved to be an independent predictor of reporting standard endorsement for RCTs besides trial registration. Conclusion Our results highlight that study registration requirement and reporting guideline endorsement are suboptimal in internal medicine and endocrine journals. This malpractice may be further enhanced since endorsement does not imply enforcement, impairing the practice of evidence-based medicine.

Abstract Evidence regarding the effects of subclinical hypothyroidism (SCH) on adverse pregnancy outcomes and the ability of levothyroxine (LT4) treatment to prevent them is unclear. Available recommendations for the management of SCH during pregnancy are inconsistent. We conducted a nationwide survey among physicians assessing their knowledge of and current practices in the care of SCH in pregnancy and compared these with the most recent American Thyroid Association (ATA) recommendations. In this cross-sectional study, an online survey was sent to active US members of the Endocrine Society. This survey included questions about current practices and clinical scenarios aimed at assessing diagnostic evaluation, initiation of therapy, and follow-up in pregnant women with SCH. In total, 162 physicians completed the survey. ATA guidelines were reviewed by 76%, of whom 53% indicated that these guidelines actually changed their practice. Universal screening was the preferred screening approach (54%), followed by targeted screening (30%). For SCH diagnosis, most respondents (52%) endorsed a TSH level >2.5 mIU/L as a cutoff, whereas 5% endorsed a population-based cutoff as recommended by the ATA. The decision to initiate treatment varied depending on the specific clinical scenario; however, when LT4 was initiated, respondents expected a small/very small reduction in maternofetal complications. In conclusion, despite recently updated guidelines, there is still wide variation in clinical practices regarding the care of women with SCH in pregnancy. Highly reliable randomized trials are required to evaluate the effectiveness of the most uncertain treatment practices on the care of pregnant women with SCH.

e14132 Background: The use of PD-1 inhibitors (PD1) has been limited due to the lack of prognostic markers of response. Previous studies have suggested that indirect inflammatory markers such as neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR) may correlate with response to treatment and improved survival. We sought to evaluate the impact NLR and PLR in a real world cohort of patients (pts) with advanced solid tumors treated with PD1. Methods: Records of all pts treated with PD1 between 2011-2017 at the John Theurer Cancer Center were reviewed. NLR and PLR were registered at baseline and longitudinally. We dichotomized as low (lNLR/lPLR), and high (hNLR ≥5/hPLR ≥160). Univariate logistic regression and Cox proportional hazards were used for progression free survival (PFS), and overall survival (OS). Landmark analyses were performed at cycles 2 (C2), and 5 (C5). Results: 178 pts received 1131 cycles of PD1. Median age was 65 (range 24-93); 43% were female. ECOG was ≥1 for 85%. 142 pts (80%), and 89 (50%) received ≥2C and ≥5C respectively. In univariate analyses, baseline hNLR and hPLR were independently associated with inferior OS (median 7.8 vs 18.3 months; HR 1.77, 95% CI 1.21-2.43; p = 0.004), ) and (median 6.4 vs 15.6 months; HR 1.42, 95% CI 1.09-2; p = 0.04) respectively. On landmark analyses, lNLR and lPLR were associated with longer PFS and OS at 2C, and 5C. Longitudinally, lNLR and lPLR correlated with response rate; NLR decreased by 0.08 (95% CI: -0.19 to -0.04; p = 0.03) and PLR by 17 (95% CI: -29 to -14; p = 0.07) per month in responders compared with non-responders. hNLR or hPLR did not correlate with increase in autoimmune toxicities. Conclusions: hNLR, hPLR are adversely prognostic markers in pts receiving PD1 inhibitors in a “real world cohort”. These markers correlate with a longitudinal response and may help predict response. Further prospective studies are needed to determine their utility in decision making during treatment.