ALBERT

Description: Background: Multiple myeloma (MM) is the second most common hematological malignancy. This disease remains incurable as nearly all patients will relapse and become refractory to established MM therapy. Thus, new treatment option for relapsed or refractory (R/R) MM is needed, particularly those with different mechanisms of action. One such approach is to inhibit histone deacetylase (HDAC) and produce synergistic anti-myeloma activity via mechanisms of epigenetic modulations. In 2015, panobinostat was approved by US FDA as the first HDACi to treat R/R MM in combination with bortezomib and dexamethasone. Bisthianostat is a novel bisthiazole-based HDACi evolved from the thiazole-thiazoline cap group in natural product Largazole (Nan et al., ACS Med Chem Lett. 2014). It is orally available and displayed inhibition against a series of MM cell lines. Here we presented preliminary in-human findings from CH-020PI study, an ongoing phase 1 study of bisthianostat. (Trial registered at ClinicalTrial.gov: NCT03618602) Methods: CH-020PI is a first-in-human study to investigate the safety, tolerability, pharmacokinetics, and efficacy of bisthianostat in R/R MM patients. It is a single center, open-label, single arm, dose escalating phase I study. A standard 3+3 cohort design with 100mg as the starting dose was used to determine the maximum tolerated dose of bisthianostat. This study comprised two phases: a pharmacokinetics phase and an expansion phase. In the pharmacokinetics phase, a single-dose of bisthianostat was administered on day 1, and then multiple-dose was administered on a twice-weekly schedule for 4 consecutive weeks. Patients in the expansion phase received continuous bisthianostat twice weekly until progressive disease or unacceptable toxicities. Results: Until 30 June 2019, 8 patients were enrolled at 3 dose levels from 100 to 400mg. The median age at enrollment was 62 years (range, 51-70 years). The median number of previous lines of therapy was 5 (range, 2-6). Per protocol, all of 8 patients were evaluable for pharmacokinetics, toxicities and efficacy. In the pharmacokinetic evaluation, for all the 8 patients tested at day 1, the peak concentration of bisthianostat was reached within 2.3 hours; half life time were around 4 hours; bisthianostat uptake represented by AUClast were in good proportion to the level of dose as 100, 200 and 400mg, respectively. Similar results were observed at day 28. Any grade hematological treatment-related adverse events (AEs) occurred in 4 of 8 patients (50%), while grade 3/4 hematological AEs occurred in 2 (25%) patients. Any grade non-hematological treatment-emergent AEs were observed in 3 (37.5%) patients; no grade 3/4 non-hematological AEs were reported. No patient discontinued the treatment of bisthianostat due to AEs. Except patient 007 (200mg cohort) experienced a grade 2 nausea, no patients experienced diarrhea, nausea, or vomiting. It is worthy to note that gastrointestinal toxicity is common with the use of panobinostat, a FDA-approved HDAC inhibitor. Overall single-agent efficacy was modest, and stable disease (SD) was observed in 4 (50%) patients. At the time of data cut-off for statistical analysis, no dose-limiting toxicity has been observed. Conclusions: Bisthianostat proved to be well absorbed and tolerated. It exhibited modest anti-tumor efficacy in our cohort of heavily pretreated patients with R/R MM. This phase I clinical trial is currently ongoing, and future trials should compare different doses and schedules of the combination in order to optimize the treatment tolerability and enhance its efficacy. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3633 Circadian rhythm is present in human and all eukaryotes with a 24-hour cycle. Circadian genes use transcriptional-translational feedback loops to control circadian rhythms. Therefore, the marked characteristic of circadian systems is the strong daily cycling of clock gene mRNA, clock protein, and clock-controlled gene RNA and protein. Recent studies have demonstrated that expression of some of the circadian genes display daily oscillation in peripheral tissues including liver, eye, lung, heart, spleen, kidney, peripheral blood and bone marrow. Circadian rhythms regulate various functions of human body and disruption of circadian rhythm has been associated with cancer development and tumor progression. In this study the expression profiles of the 9 circadian genes (hPER1, hPER2, hPER3, hCRY1, hCRY2, hCLOCK, hBMAL1, hCK1e and hTIM) in peripheral blood (PB) total leukocytes from 95 chronic myeloid leukemia (CML) and 54 healthy volunteers were investigated. For comparison of circadian gene expression between PB mononuclear cells (PBMCs) and polymorphonuclear cells (PMNLs), another group of 10 healthy volunteers were also investigated. Collection of PB was carried out at four time points: 20:00, 02:00, 08:00, and 14:00, respectively. In healthy individuals, the daily oscillation was shown for hPER1, hPER2, hPER3, and hCRY2 with peak expression levels at 8:00AM, and the expression of the nine genes displayed similar profile both in PBMCs and in PMNs. In contrast, oscillations of these four genes were abolished in newly diagnosed pre-imatinib mesylate treated and blast crisis-phase CML patients and partial recoveries of oscillations were observed in CML patients with complete cytogenetic response and major molecular response. In some serial monitored individual patients, the recoveries of oscillations of circadian gene expression accompanied with the disappearance of BCR-ABL transcripts were also noted. Expression of hCRY1, hCLOCK, hBMAL1, hCK1e and hTIM did not oscillate both in healthy individuals and CML patients. Updated results on more healthy volunteers and serial monitored CML patients will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Advances in hemophilia care have led to improved life expectancy and a cohort at risk for age-related comorbidities such as hypertension and cardiovascular diseases. Several studies have shown an increased prevalence of hypertension in patients with hemophilia compared to age-matched general population. However, causes of the increased prevalence of hypertension in patients with hemophilia are unclear. Hemophilia-specific risk factors such as renal bleeding or micro-bleeding may be implicated, but data are limited and conflicting regarding the association between hematuria, renal insufficiency and hypertension. In this two-centre prospective cohort study, we aim to assess the prevalence of gross or microscopic hematuria detected on routine surveillance urinalysis and microscopy, and determine the impact of hematuria on blood pressure and renal function. Methods: 135 adult males with mild-severe hemophilia A and B followed by the British Columbia Adult Bleeding Disorders Program (n=56) and the University of California, San Diego Hemophilia Treatment Center (n=79) were included. Screening urinalysis/microscopy were performed in all patients during routine clinic visits. Hematuria was defined as either a self-reported history of gross hematuria, or 〉 3 red blood cells per high-power field on urine microscopy in the absence of urinary tract infections. Hypertension was defined as systolic blood pressure (SBP) ≥140mmHg or diastolic blood pressure (DBP) ≥90mmHg on ≥2 occasions, or use of anti-hypertensive medications. Univariate and multivariate logistic regression analysis were used to examine the significance of hematuria and other potential hypertension risk factors. Results: The prevalence of hypertension was 44% in this population, 71% of whom were on anti-hypertensives, of whom 43% achieved blood pressure control (SBP

Description: Introduction: Recombinant Factors VIII Fc (rFVIIIFc) and IX Fc (rFIXFc) fusion proteins are extended half-life (EHL) factor products approved in Canada in 2014 and became available through the Canadian Blood Services in January 2016. Real-world product utilization and clinician practice patterns in EHL prescription are unknown. Objectives: This is a prospective Canadian multi-centre observational study to describe product utilization, clinical and patient-reported outcomes (PROs) in patients pre- and post-switch to rFVIIIFc/ rFIXFc products compared to those who remained on standard half-life (SHL) products. The primary outcome is intra-individual change in annualized factor consumption from 12-month period pre-switch to 24-month post-switch to rFVIIIFc/rFIXFc. This analysis describes the baseline clinical characteristics and intra-individual changes in PROs from the cohort who completed baseline, 3-month and 12-month assessment before June 2018 in this ongoing study. Methods: Males aged ≥12 years with moderate and severe hemophilia A and B (factor level

Description: Abstract 1843 Senescence is a specialized form of growth arrest that it is generally irreversible and can be induced by telomere attrition, oxidative stress, oncogene expression and DNA damage signaling. Senescent cells display a typical upregulated senescence-associated (SA)-b-galactosidase activity and novel changes in chromatin architecture, the formation of SA heterochromatic foci (SAHF). Changes in gene expression, such as upregulated p16, p53, and p21 expression and silencing of E2F target genes, have been characterized to promote the establishment of senescence. Besides, by the actions of HP1g, HMGA, and DNMT proteins to produce a repressive chromatin environment, the transcription of proliferation-associated genes can be suppressed. Therefore, senescence has been suggested to functions as a natural brake to tumor development. In this study, we first sought to establish an in vitro senescence model using doxorubicin (DOX) and paclitaxel to treat CML cell line K562. We found that 50 nM DOX induced senescence, but did not induce apoptosis. In contrast, 10 nM and 100 nM paclitaxel induced apoptosis but did not induce senescence, and lower doses of paclitaxel (1 nM and 5 nM) had no effect on the cells. p53 and p16-pRb are the two major senescence pathways. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells is supposed to be established by a pathway independent of p53 and p16-pRb pathways. Indeed, the expression of the typical SA-premalignant cell markers (CDC6, Ki67, p19, p38, PU1, DNMT1, HMGA1, HP1g) did not change in the DOX-induced senescent K562 cells although the typical SA-b-galactosidase staining and SAHF were apparent. MicroRNA profiling revealed that miR-375 was upregulated in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor could rescue the proliferation ability suppressed by DOX (p 〈 0.05). The identification of miR-375 targets should help us to elucidate the substitution pathway that is responsible for the DOX-induced senescence in the absence of both p16 and p53 genes. With the observation that DOX treatment induced cells entering senescence but eventually lead to cell death, we also investigated if the alternative mode of cell death, autophagy, was involved. By examining the expression patterns of the 26 human autophagy-related (ATG) genes, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. Both ATG9 and ATG18 are crucial for the formation of autophagosome. Hence, in addition to the upregulation of miR-375, our results also demonstrated that senescence induced by DOX in K562 cells is associated with the initiation of autophagy. Updated results on the identification of miR-375 targets and the regulation of senescence and autophagy pathway will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4613 Circadian rhythms regulate various functions of human body and disruption of circadian rhythm has been associated with cancer development and tumor progression. Circadian clock genes use transcriptional-translational feedback loops to control circadian rhythms. Many transcriptional regulators are histone acetyltransferases (HAT) or histone deacetylases (HDAC). As clock function and integration of inputs rely on transcriptional regulation, it is possible that chromatin is remodeled during circadian cycles and in response to signals that regulate the clock. SIRT1 (sirtuin 1) is a HDAC that has recently been identified as a crucial modulator of the circadian clock machinery. To date, at least 7 SIRT genes (SIRT1–7) have been identified. In our previous report we have demonstrated the daily expression patterns of PER1, PER2, PER3, CRY1, CRY2, and CKIe in peripheral blood (PB) of healthy individuals were abolished in chronic myeloid leukemia (CML) patients and partial recoveries of daily patterns were observed in CML patients with complete cytogenetic response (CCyR) and major molecular response (MMR) post-imatinib treatment [J Biol Rhythms 2011]. In this study we further investigated the expression profiles of the 7 SIRT genes (SIRT1–7) in PB total leukocytes from 49 CML and 22 healthy volunteers. Collection of PB was carried out at four time points: 2000 h, 0200 h, 0800 h, and 1400 h, respectively. In PB total leukocytes of healthy individuals, the daily pattern of SIRT1 (p 〈 0.01) and SIRT5 (p 〈 0.05) expression level peaked at 0200 h, and SIRT2 (p 〈 0.01) peaked at 0800 h. Daily pattern expression of these 3 genes was abolished in newly diagnosed pre-imatinib mesylate treated and blast crisis-phase CML patients. Partial daily patterns of gene expression recoveries were observed in CML patients with CCyR and MMR. In some serial monitored individual patients, the recoveries of oscillations of SIRT1, 2, and 5 genes expression accompanied with the disappearance of BCR-ABL transcripts were also noted. The expression of SIRT3, 6, and 7 did not show a time-dependent variation among the healthy and CML patients. SIRT4 expression was undetectable both in the healthy and CML patients. Updated in vitro study results of the regulation of SIRT1, 2, and 5 genes on circadian clock genes expression will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Anti-factor VIII antibodies are a serious complication of factor replacement therapy in hemophilia A patients. Neutralizing antibodies could previously only be detected via the functional and gold-standard Bethesda assay with or without the Nijmegen modification. The introduction of an enzyme-linked immunosorbent assay (ELISA) screening test provides a less laborious alternative to the Bethesda assay and also has a high sensitivity. Unlike the Bethesda assay, the ELISA can potentially detect non-neutralizing antibodies, which raise the possibility of false positive screens. As it is important that both clinicians and the coagulation laboratory understand the clinical performance of ELISA screening, this study evaluated the sensitivity and specificity of ELISA and Bethesda assays used in both a laboratory validation study and in subsequent clinical experience. Methods Results from all active adult and pediatric congenital hemophilia A patients who underwent both Bethesda assay and ELISA in British Columbia, Canada as of July 2013 were included in this study. The sensitivity and specificity were compared to the provincial coagulation laboratory validation study of the GTI Factor 8 Antibody ELISA kit against the classical Bethesda assay conducted in both hemophilia A and normal controls in 2010. Optical density (OD) readings were retrieved for eligible samples and from the laboratory validation study. Results 35 samples from 26 different patients were used in the validation study performed in 2010. Of 147 adult and 86 pediatric hemophilia A clinic patients active during the study period, 56 samples from 16 adults and 17 children underwent concurrent Bethesda assay and ELISA screen between November 2010 and June 2013. Since ELISA implementation, 389 ELISA screens and 187 Bethesda assays were performed. ELISA screens resulted in the avoidance of Bethesda assays in 85% (330/389) of samples submitted during that period. Specificities and positive predictive values were lower in the clinical sample due to a larger number of false positives (n=18; 32%) relative to the validation study (n=3; 9%), while sensitivity and negative predictive values remained at 100% (Table 1). Interestingly, 67% (2/3) of false positive ELISA screens in the laboratory setting had Bethesda positive histories; however, in the clinical sample, only 6% (1/18) had a distant history of inhibitors. ODs were available for all validation study samples and for 54 clinical samples. Redefinition of our OD cutoff to improve specificity was prevented by false positive patients with strongly positive ODs. Conclusions The ELISA screen used in this setting is highly sensitive for anti-factor VIII antibody detection in congenital hemophilia A. However, compared to pre-clinical data, our 2.5 years of clinical experience reveals a high incidence of false positives resulting in a significantly lower specificity. Our approximate cost savings in British Columbia as a result of avoided Bethesda assays due to ELISA screens (n=330) is $90 per test, and may exceed $200 per test in the United States. Additional testing and follow-up on positive ELISA results can be an inconvenience in both time and blood sampling, especially in the pediatric population where inhibitor status is more closely monitored. The cost ramifications of this follow up testing could not be quantified in this study. The potential implications for the detection of non-neutralizing anti-FVIII antibodies in hemophilia A need to be further explored and long-term study and monitoring of these discrepant patients is warranted. Disclosures: No relevant conflicts of interest to declare.

Description: Studies in large-scale genome sequencing have shown that only 2% of the mammalian genome encodes mRNAs, but the most part is transcribed as long and short non-coding RNAs (ncRNAs). The ncRNAs with gene regulatory functions are starting to be seen as a common feature of mammalian gene regulation. Genomic imprinting is a form of epigenetic regulation and imprinted genes are silenced in a parental-specific manner. Although the exact mechanism how imprinted ncRNA regulates gene expression remains largely unknown, it is general accepted that imprinted ncRNAs binds to chromatin modifying complexes, such as PRC2, TRX, and G9a, and generates specific silencing of genomic loci both in cis and trans. Imprinting is associated with many human diseases or syndromes (e.g. Prader-Willi, Angelman, Beckwith-Wiedemann, Retts, and Silver-Russell syndromes) and various cancers (e.g. breast, prostate, and colorectal cancers), but its role in leukemogenesis remain elusive. In this present study, the expression of a panel of 24 human imprinted ncRNA genes (AMPD3, C15orf2, COPG2, CPA4, GABRB3, H19, IGF2, IMPACT, INPP5F, L3MBTL, NR3251, NR3252, PEG3-AS, PPP1R9A, PRIM2, RASGRF1, RTL1, SFMBT2, SLC22A3, SNURF, TCEB3C, TSPAN32, ZNF215, ZNF264) and a panel of 66 human histone modifying enzymes (HME) genes was investigated in 68 newly-diagnosed acute myeloid leukemia patients with chromosome normal (AML-CN), 115 AML patients with chromosome abnormal (AML-CA), and 85 healthy individuals using real-time quantitative RT-PCR. Altered expression of 9 imprinted ncRNA genes (C15orf2, COPG2, H19, IGF2, IMPACT, PEG3-AS, PRIM2, SLC22A3, ZNF215) and 16 HME genes were observed. In AML-CN, patients’ survival days are correlated with the expression levels of H19 (p 〈 0.01), IMPACT (p 〈 0.05), DNMT3L (p 〈 0.05) and AURORA (p 〈 0.01). In AML-CA, patients’ survival days are correlated with the expression levels of PGE3-AS (p 〈 0.01), PRIM2 (p 〈 0.01), SLC22A3 (p 〈 0.05), and ZNF215 (p 〈 0.01). Multiple linear regression analysis further revealed the expression level of H19 and ZNF215 can be used as predictors for 2-year survival for AML-CN patients (p = 0.002) and AML-CA patients (p = 0.040), respectively. Cox proportional hazard model was used to analyze the hazard ratio (HR) for H19 (HR=0.868, 95.0% Confident Interval: 0.797-0.945, p = 0.001) and ZNF215 (HR=0.904, 95.0% Confident Interval: 0.821-0.995, p =0.040). In addition to survival, analysis has also been performed to correlate patients’ clinical parameters and expression levels of these altered genes and to correlate the expression levels between imprinted ncRNA genes and HME genes (results will be presented at the meeting). From our preliminary results, it is reasonable to hypothesize that loss imprinting of imprinted ncRNA is critical for the leukemogenesis of AML and under CN or CA conditions different ncRNAs are activated and affect different imprinted gene expression and thus leading to different clinical outcomes. Based on our findings, we will further perform in vitro functional analysis to elucidate the functions and mechanisms of these imprinted ncRNAs in AML tumorigenesis. Updated results of these analyzes will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Circadian rhythm is present in all eukaryotic and some prokaryotic life forms. This time-keeping system is organized in a hierarchical fashion and composed of a self-sustained central pacemaker in the suprachiasmatic nuclei of the anterior hypothalamus and peripheral oscillators in most body cells. A recent study demonstrated that mice deficient in the clock gene mPer2 are cancer prone and display a deregulated temporal expression genes involved in cell cycle regulation, such as c-Myc, Cyclin D1, Cyclin A, and Mdm-2. These mice display salivary gland hyperplasia, and a large portion of them develops lymphomas. The results revealed that Per2 is an essential circadian gene and it is associated with proliferation control in mammals. In a more recent study, it was demonstrated that an age-dependent decay of the circadian clock both at the behavioral and the molecular levels was observed in mPer1−/−mCry2−/− double-mutant mice. To gain further insights into the roles of circadian genes in chronic myeloid leukemia (CML), we analyzed peripheral blood from 21 healthy individuals and 35 CML patients (18 in blastic crisis and 17 in chronic phase) by real-time quantitative RT-PCR for the expression of circadian genes PER1, PER2, and PER3. In blastic crisis cases, the expression levels of all three PER genes were significantly impaired than in healthy individuals (PER1, 1:5.97, p 〈 0.005; PER2, 1:13.51, p 〈 0.0001; PER3, 1:2181.33; p 〈 0.0001); whereas, in chronic phase, only the expression levels of PER2 and PER3 were significantly impaired (PER1, 1:1.23, p 〉 0.5; PER2, 1:2.47, p 〈 0.05; PER3, 1:14.22; p 〈 0.0001). Mutational analysis of the whole gene and methylation analysis of CpGs sites at the promoter area were further performed to investigate the possible mechanisms. No mutation was found within the coding regions of the three PER genes in all CML cases. Methylation analysis using methylation-specific PCR and direct sequencing showed no methylation in the promoter areas of both PER1 and PER2 genes. In contrast, most of the CpG sites were methylated in the promoter area of PER3 gene in CML cases and none of these CpG sites were methylated in healthy individual cases. In addition, the methylated CpG frequencies of PER3 gene differed in patients at blastic crisis and at chronic phase (CpG, 8.24 ± 0.73 vs. 4.48 ± 0.48, p 〈 0.0001; T/CpG, 10.47 ± 0.67 vs. 14.67 ± 0.46, p 〈 0.0001; TG, 1.24 ± 0.32 vs. 0.81 ± 0.16, p 〉 0.05). Demethylation by treatment of hypermethylated K562 cells with 5′-aza-2′-deoxycytidine resulted in partial reactivation of PER3 expression. Our results suggest that the downregulated PER3 expression in CML was due to the inactivation of PER3 gene by methylation and the differential expression was correlated to the ratio of methylated CpG sites at the promoter region. We hope to explore the role of circadian genes in the tumorigenesis of leukemia and to establish circadian genes as novel and useful markers for diagnosis and references for therapies in leukemia.

Description: Abstract 1599 Infectious agents have been shown to contribute to the development of certain lymphoid malignancies. The different distribution of lymphoid malignancies in Asian and Western populations suggests possibly different etiologies in lymphomagenesis in Asian populations. Herpes zoster infection, commonly seen in immunocompromised persons, has been reported to be associated with lymphoid malignancies, but the results are controversial and large-scale studies from Asian populations are lacking. In this study we performed a population-based matched-controlled prospective study on Taiwanese patients using the National Health Insurance Research Database which provided 1,000,000 random subjects from 1996 to 2007. We defined herpes zoster by compatible ICD-9-CM (International Classification of Disease, 9th Revision, Clinical Modification) codes of herpes zoster (053.0–053.9) on at least one service claim for inpatient or outpatient care. The cases were identified by compatible ICD-9-CM codes including Hodgkin's disease (code 201.0–201.9), non-Hodgkin's lymphoma (code 200.0–200.8, 202.0–202.9), multiple myeloma (code 203.0–203.1), and lymphoid leukemia (code 204.0–204.9). Patients who had been diagnosed with any lymphoid malignancies or any cancers (code 140.0–199.1) before herpes zoster, and who had been diagnosed with other viral infections (code 045.0–052.9, 054.0–066.9, 071–079.9) and HIV infection (code 042) before the diagnosis of lymphoid malignancies were excluded. We randomly selected 169,983 control subjects (4 for every herpes zoster patient), matched with the study group in terms of age, sex and the year and month of index visit. Of 42,498 patients with herpes zoster prior to the diagnosis of any malignancies, the mean age was 48.92 years (± 20.67 years), with a mild female predominance (52.4%). Patients with herpes zoster infection had a lower monthly income (p 〈 0.001), and tended to live in urban areas (p 〈 0.001). Among the patients with herpes zoster, 2.42% subsequently developed cancer, and 0.11% lymphoid malignancy. Among the controls, 2.26% of the patients subsequently developed cancer, and 0.06% lymphoid malignancy. Patients with herpes zoster had a significantly increased risk of developing any cancers (excluding lymphoid malignancies, crude HR: 1.07, 95% CI: 1.01–1.15), and lymphoid malignancies (crude HR: 1.82, 95% CI: 1.29–2.55) compared with the control group. After adjusting for Charlson disease index, income category, and residence using Cox proportional hazard regressions, patients with herpes zoster had an increased risk of developing lymphoid malignancies (adjusted HR: 1.72, 95% CI: 1.22–2.42, p = 0.0019), but did not have an increased risk of developing non-lymphoid malignancies (adjusted HR: 1.00, 95% CI: 0.93–1.07, p = 0.895). These data suggest that preceding herpes zoster infection is an independent risk factor for the subsequent development of lymphoid malignancies in Taiwanese subjects. Further studies are warranted to explore the role of herpes zoster in the pathogenesis. Disclosures: No relevant conflicts of interest to declare.