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Summary The nucleoside analogue decitabine can deplete the epigenetic regulator DNA methyltransferase 1 (DNMT1), an effect that occurs, and is saturated at, low concentrations/doses. A reason to pursue this molecular‐targeted effect instead of the DNA damage/cytotoxicity produced with high concentrations/doses, is that non‐cytotoxic DNMT1‐depletion can cytoreduce even p53‐null myeloid malignancies while sparing normal haematopoiesis. We thus identified minimum doses of decitabine (0·1–0·2 mg/kg) that deplete DNMT1 without off‐target anti‐metabolite effects/cytotoxicity, and then administered these well‐tolerated doses frequently 1–2X/week to increase S‐phase dependent DNMT1‐depletion, and used a Myeloid Malignancy Registry to evaluate long‐term outcomes in 69 patients treated this way. Consistent with the scientific rationale, treatment was well‐tolerated and durable responses were produced (~40%) in genetically heterogeneous disease and the very elderly.

The DNA methyltransferase inhibitor azacytidine (aza) may reactivate pathways associated with plasma cell differentiation, cell cycle control, apoptosis, and immune recognition and thereby restore sensitivity to lenalidomide (len) and dexamethasone (dex) in relapsed and/or refractory multiple myeloma (RRMM). We aimed to develop an aza regimen that reaches epigenetically active levels 8 times in 28 days with less bone marrow toxicity than the myeloid malignancy standard of 7 consecutive doses to enable safe combination with len. Aza was escalated from 30 mg/m2 once a week up to a predefined maximum of 50 mg/m2 twice a week in combination with GFR-adjusted len (≥ 60 mL/min: 25 mg, 3059 mL/min: 10 mg) day 1 to 21 every 28 days and dex 40 mg once a week followed by a limited expansion study to a total N of 23 at the highest tolerated dose. Fifty-one patients (pts) with RRMM were screened, 42 were treated and 41 were evaluable for response based on at least 1 response assessment or progression after treatment start. The median number of prior lines of therapy was 5 (1-11) and 81% (34) were refractory to len and/or pomalidomide (pom). Two DLTs occurred in different cohorts, 1 neutropenic fever in 1/6 pts on the aza 40 mg/m2 twice a week GFR ≥ 60 mL/min cohort and 1 GGT elevation in 1/6 pts on the aza 50 mg/m2 GFR 30-59 mL/min cohort. An MTD was not reached and aza 50 mg/m2 SC twice a week was chosen for the expansion study. At least possibly related Grade 3/4 AEs occurred in 28 pts (67%) with the following in > 1 pt: neutropenia (N = 16, 38%), anemia (N = 6, 14%), lymphopenia (N = 5, 12%), thrombocytopenia (N = 4, 10%), leukopenia (N = 4, 10%), febrile neutropenia (N = 4, 10%), fatigue (N = 3, 7%), fever (N = 2, 5%), and infection (N = 2, 5%). At a median follow up time for alive pts of 60.2 months (range: 36.1-82.5 months), the overall response rate (≥ partial response) and clinical benefit response rate (≥ minor response) was 22 and 32%, respectively, with 4 very good partial responses (10%), 5 partial responses (12%), and 4 minor responses (10%). The median PFS was 3.1 months (95% confidence interval [CI]: 2.1-5.1 months), median TTP 2.7 months (95% CI: 2.1-7.5 months), and median OS 18.6 months (95% CI: 12.9-33.0 months). Achieving at least minor response and reaching TTP > 6 months was associated with approximately 35% lower median plasma levels of the enzyme that inactivates aza, plasma cytidine deaminase (CDA, P 0.5% in 6 and by > 5% in 3 of the 8 pts, most pronounced in 2 pts with clinically convincing aza contribution who achieved a reduction in overall differentially methylated CpGs by 23 and 68%, respectively, associated with increased expression of immunoglobulin genes. The study demonstrated tolerability of twice a week SC aza at 50 mg/m2 with len and dex in RRMM and suggested aza may help overcome the len/pom refractory state, possibly by activating differentiation pathways. Relatively low response rates and association of clinical benefit with low plasma levels of the aza inactivating enzyme CDA suggest the aza regimen will need to be optimized further and pt selection may be required to maximize benefit.

BackgroundMean platelet volume (MPV), may help to determine atherosclerosis threat. Cardiovascular disease is one of the common causes of morbidity and mortality in patients having psoriasis.ObjectiveTo examine MPV as a biomarker for subclinical atherosclerosis in psoriasis patients, compared with i

Most superficial mycotic infections of human skin are due to dermatophytes. Children are frequently affected due to different predisposing factors, particularly overcrowding in classrooms. This study aimed to estimate the prevalence of dermatophytes infections and their related risk factors among school children in Menoufia Governorate, Egypt. Six public primary and preparatory schools were randomly selected and their pupils (n = 3464) were asked to complete a predesigned questionnaire covering both personal data and suspected risk factors for superficial dermatophyte infections. The children were also examined for dermatological diseases. Any suspected lesions were biopsied for mycological examination. The prevalence of clinically suspected dermatophytes infections was 1.41%, whereas the prevalence of culture confirmed cases was 0.98%. The most common clinical type was tinea capitis with a prevalence of 1.01%. Microsporum canis was the only isolated organism from the suspicious lesions with a 69.4% positivity rate. A higher prevalence was observed among boys, low socio-economic pupils and those with a family history of dermatophyte infections. Pet contact and sharing towels and caps among pupils were significant risk factors. Dermatophyte infection is still prevalent among basic school pupils. Fortunately, it is related to preventable risk factors. We recommend regular screening and use of educational health programmes for kids to control it.

To the editor: The course of aplastic anemia (AA) is often complicated by the development of clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and secondary myelodysplastic syndromes (sMDS).[1][1][⇓][2][⇓][3][⇓][4]-[5][5] Identification of patients at risk for development of

Background Postoperative pain is a common, distressing symptom following arthroscopic knee surgery. The aim of this study was to compare the potential analgesic effect of dexmedetomidine after intrathecal versus intra-articular administration following arthroscopic knee surgery. Methods Ninety patients undergoing unilateral elective arthroscopic knee surgery were randomly assigned into three groups in a double-blind placebo controlled study. The intrathecal dexmedetomidine group (IT) received an intrathecal block with intrathecal dexmedetomidine, the intra-articular group (IA) received an intrathecal block and intra-articular dexmedetomidine, and the control group received an intrathecal block and intra-articular saline. The primary outcome of our study was postoperative pain as assessed by the visual analogue scale of pain (VAS). Secondary outcomes included the effect of dexmedetomidine on total postoperative analgesic use and time to the first analgesic request, hemodynamics, sedation, postoperative nausea and vomiting, patient satisfaction, and postoperative C-reactive protein (CRP) levels. Results Dexmedetomidine administration decreased pain scores for 4 h in both the intrathecal and intra-articular groups, compared to only 2 h in the control patient group. Furthermore, there was a significant reduction in pain scores for 6 h in the intra-articular group. The time to the first postoperative analgesia request was longer in the intra-articular group compared to the intrathecal and control groups. The total meperidine requirement was significantly lower in the intra-articular and intrathecal groups than in the control group. Conclusions Both intrathecal and intra-articular dexmedetomidine enhanced postoperative analgesia after arthroscopic knee surgery. Less total meperidine was required with intra-articular administration to extend postoperative analgesia to 6 h with hemodynamic stability.

T-cell large granular lymphocyte leukaemia (T-LGLL) is a chronic clonal lymphoproliferative disorder of cytotoxic T lymphocytes (CTL), which commonly occurs in older patients and is often associated with autoimmune diseases. Among 246 patients with T-LGLL seen at our institution over the last 10 years, we encountered 15 cases following solid organ or haematopoietic stem cell transplantation. Here, we studied the clinical characterization of these cases and compared them to de novo T-LGLL. This experience, represented a clear picture of the intricate nature of the disease manifestation and the complexities of several immune mechanisms triggering the clonal expansion.

Compound heterozygous germline mutations in CTC1 gene have been found in patients with atypical dyskeratosis congenita (DC), whereas heterozygous carriers are unaffected. Through screening of a large cohort of adult patients with acquired bone marrow failure syndromes, in addition to a DC case, we have also found extremely rare or novel heterozygous deleterious germline variants of CTC1 in patients with aplastic anaemia (AA; n = 5), paroxysmal nocturnal haemoglobinuria (PNH; n = 3) and myelodysplastic syndrome (MDS; n = 2). A compound heterozygous case of AA showed clonal evolution. Our results suggest that some of the inherited CTC1 variants may represent predisposition factors for acquired bone marrow failure.

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2′-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy.

Acute promyelocytic leukemia (APL) is characterized by PML-RARA fusion caused by the t(15;17)(q24;q21) translocation. Although PML-RARA fusion explains the dedifferentiation in most of APL patients, it still does not entirely represent the unique cause of all the clinical manifestations of the disease failing to determine the full leukemic phenotype. Up to 40 % of APL patients have an additional chromosomal abnormality other than PML-RARA. Murine studies have reported that additional cytogenetic abnormalities and secondary somatic mutations (for instance FLT3-ITD) might contribute to leukemia progression. Indeed, mice expressing mutant PML-RARA develop definitive leukemia after one year, suggesting that additional hits are required for transformation. Moreover, no distinct genetic signature has been characterized by next generation sequencing (NGS). This confirms that no gene has been reproducibly identified. APL respond to all-trans retinoic acid (ATRA) in the great majority of patients. However, one quarter of APL develop ATRA resistance suggesting that additional secondary chromosomal abnormalities might be evolving resistance. Combination of low dose arsenic, modify certain epigenetics, with ATRA decreased resistance potential and improved response. In the line with other possible factors involved in ATRA resistance, is the broad nature of the targets of ATRA. A molecular core network of ATRA's targets has been clustered in differentiation, growth factors and nuclear receptors possibly cooperating with PML-RARA and additional chromosomal abnormalities. Herein, we aimed to characterize the gene mutations and chromosomal abnormalities playing key roles in cellular differentiation and epigenetic regulation and to correlate the occurrence of these alterations with treatment response and survival outcomes in APL. We took advantage of a large cohort of APL patients (n=145). Median age of the cohort was 50 yrs (19-85); equal gender distribution; median blood counts were: [WBC 6.2 x 109/L (0.4-155); 37% had leukopenia], hemoglobin [9.8 g/dL (2.7-16.2); 32% had anemia] and platelets [29 x 109/L (range of 0-228); 93% had thrombocytopenia]. In terms of karyotype, 15% of the patients carried +8, 7% had complex karyotyping (≥3 cytogenetic abnormalities), 2% had -7/del (7q) or del (12p), 1% had -17/del(17p), and 1 patient had -5. Mutational analysis of 30 genes panel, identified 141 mutations carried by 65% (94/145) of APL patients. The most frequent mutations were observed in FLT3-ITD (61/143; 43%), WT1 (26/139; 23%), and ASXL1 (7/136; 5%) genes. Less frequent mutations were found in 3.7% of CEBPA, KRAS, and NRAS genes as well as in CBL, EZH2, TET2 (3% each) genes. Additionally, we noted that all mutations were recurrent in specific functional pathways and patients carried mutations in more than 1 gene of the same pathway. Of note, cell signaling and proliferation genes (CBL, NRAS, KRAS, KIT, FLT3) were the most frequently mutated (77/141, 55%) and impacted OS (HR: 1.7, P=0.02). Moreover, transcriptional factors which are often mutated in AML (e.g. CEBPA, TP53, NPM1, RUNX1, WT1) as well as major determinants of cell's fate were markedly mutated (38/141, 27%) suggesting that genetic impairment of signaling and transcription might contribute to the lack of differentiation observed in APL phenotypes. Mutations in epigenetic genes and histone methyltransferases (ASXL1, BCORs, DNMT3A, EZH2, IDH1/2, TET2) were also found in 18/141 (13%) while genes regulating cell proliferation and RAS family (CBL, NRAS, KRAS, NPM1) were enriched in 16/132 (12%) of APL cohort. We then analyzed the genetic picture of remission (APLRm, n=131, 90%) and relapsed (APLR, 1sr relapsed to ATRA, n=14, 10%) patients. Acknowledging the low number of APLR, we observed that molecular mutations did not make a key difference in APLRvs. APLRm [except for a complete lack of mutations in epigenetic pathways (0% vs. 13%)]. Contrarily, specific cytogenetic abnormalities were more common in APLR compared to APLRm as the case of +8 (36% vs. 11%; P= .02) and -17/del(17p) (2/14 vs. 0/131; P= .008). In sum, our study demonstrates that PML-RARA might be accompanied by additional acquired chromosomal change with a variety of genetic mutations in key pathways driving cellular differentiation. These molecular/ cytogenetic associations could determine resistance to ATRA and overall APL patients' survival. Disclosures No relevant conflicts of interest to declare.

Patients presented with pancytopenia in the presence of bone marrow (BM) hypoplasia, aplastic anemia (AA), may get cured by specific medical care, if patients are under 40 years, mainly in the form of allogeneic hematopoietic stem cell transplantation (allo-HSCT) which is the treatment of choice for severe AA (SAA) in young age. In Egypt, SAA may get allo-HSCT only with availability of matched sibling (MSD). Success rate of allo HSCT-MSD is still suboptimal although it is a life challenging and expensive procedure with an outcome difficult to predict. Reports suggest that measurement of serum ferritin may help in predicting favorable MSD-alloHSCT outcome. Evidences showed that total infused CD34 cells and/ or measurements of hematogones (HGs) levels at the time of engraftment by flow cytometry may better predict the outcome of MSD-alloHSCT. HGs, normal B lymphocyte precursors, levels may be affected by the status of BM and humoral immunity. Elevated HGs have been noticed in healthy children, called reactive HGs. Regenerative HGs exist in non-malignant disorders such as autoimmune diseases, congenital cytopenia, acquired immunodeficiency and neoplastic disorders such as lymphoma. In addition they have been found in response to regeneration of humoral immunity post chemotherapy and post allo-HSCT. We aimed to examine predictive value of HGs in comparison to pre-transplant ferritin and/ or infused CD34⁺ counts for a favorable outcome of SAA patients treated with allo-HSCT from MSD. This is a prospective cross-sectional study, following institutional approval. In total 21 patients with SAA were consented and enrolled. All patients were treated with MSD allo-HSCT, between December 2013 and January 2016. Patients > 40years age, clinically unfit, with abnormal cytogenetic, diagnosed with inherited BM failure or paroxysmal nocturnal hemoglobinuria (PNH) were excluded. HGs were measured in BM samples at engraftment using flow cytometry where cells were stained with an anti-CD34, anti-CD38, anti- CD10, anti-CD19, and lineage (Lin) mixture (anti-CD3, -CD4, -CD8 -CD20, -CD14, and -CD56). Early HGs referred to both CD38ˉCD10⁺ CD19ˉCD34⁺ and CD38-CD10⁺CD19ˉCD34ˉ. Late HGs referred to CD38⁺CD10⁺CD19⁺CD34ˉ, and CD38⁺CD10ˉCD19⁺CD34ˉ cellss. The mean ± SD of early HG was 0.8 ± 1.6 and median with range was 0.05 (0-7.8) while mean ± SD of late HG was 0.6 ± 0.5 (p=0.4) and median with range 0.4 (0-1.4). Total HG mean ± SD was 0.7±1.2 and median with range 0.2 (0 - 7.8).Interestingly the mean ± SD of HG levels were higher in survivors vs non-survivors (2.3 ± 3.4 vs. 0.2 ± 0.2; P=0.01). Infused CD34 count (10^6) were in survivors vs non-survivors (6.7 ± 3 vs 7.8 ± 4) while pre-transplant serum ferritin levels (pm/mL) were in survivors vs non-survivors (1493 ± 1025 vs 1213 ± 371) without showing significant differences. Using ROC curve analysis, it showed the following for engraftment HGs%, best cut-off value was 0.37%, with AUC of 0.88, likelihood ratio (LR) of 3.85, it was the most sensitive 76.9% (95% CI; 35.4-84.8) and specific 80% (95% CI; 28.4 - 99.5) predicting favorable MSD-alloHSCT outcome. Infused CD34⁺ count showed best cut-off level of 6.8 x 10⁶ cells/kg, 0.63 for AUC, 3.13 for LR; 62% sensitivity (95% CI; 46.2-95) and 80% specificity (95% CI; 28.4-99.5). Pre-transplant serum ferritin showed best cut-off level of 1337, 0.59 for AUC; 1.6 LR; 64% for sensitivity (95%CI; 35.14 - 87.24) and only 60% specific (95% CI, 14.66 - 94.73). Moreover, patients with high HG (>0.37%; 57% vs. In sum HGs% FCM assay may be a highly sensitive and specific biomarker predicting HSCT outcome in SAA patients.. Estimating HGs% at engraftment time seem to be a promising predictor for the outcome of MSD-alloHSCT in SAA patients. SAA patients with HGs 0.37% or higher had better OS. Disclosures No relevant conflicts of interest to declare.

T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (>500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Rationale: Dose-reduction of a nucleoside analog reduces cytotoxicity and hence can increase safety, but with less cytotoxicity-driven efficacy as a trade-off. Such an efficacy trade-off, however, may not apply to the nucleoside analog decitabine, because it has a molecular-targeted action of depleting DNA methyltransferase 1 (DNMT1) separable from cytotoxicity, and that occurs and is saturated at low concentrations. In fact, higher decitabine doses/concentrations, by causing cytotoxicity, limit feasible exposure times for this molecular effect that can cytoreduce p53-null, chemorefractory myeloid malignancies via terminal-differentiation instead of apoptosis, simultaneously sparing normal hematopoiesis. Hence, we designed decitabine application to avoid cytotoxicity and increase DNMT1-targeting and describe here results in 69 patients (25 of whom were previously described with shorter follow-up (NCT01165996)(J Clin Invest 2015; 125(3):1043-55]). Methods: IRB approved Myeloid Malignancy Registry (16-020) and Sample Repository protocols (5024) curated demographic, laboratory, intervention, outcome, and mutational data in myeloid malignancy patients after written informed consent; 69 of 1694 patients in the Registry received the alternative decitabine regimen: (i) Dose: 0.1-0.2 mg/kg (~3.5-5 mg/m2), verified to deplete DNMT1 without cytotoxicity in primate and human studies; (ii) Schedule: 1-2X/week, frequent and distributed, to increase S-phase dependent DNMT1-depletion; (iii) Route: subcutaneous, to avoid high peak concentrations.A standard starting dose of 0.2 mg/kg was reduced to 0.15 mg/kg if infection risk was high (e.g., ANC10% or for lack of response to 1X/week. Neutropenia from treatment was managed by interruption then resumption upon neutrophil recovery (same dose or lower by 0.05 mg/kg). Routine anti-emetic prophylaxis was not required. Results: Non-cytotoxic DNMT1-depletion was confirmed by bone marrow gH2AX and DNMT1 measurements (published for 1st25 patients). The 69 patients had MDS 54%, MDS/MPN 14%, MPN 17% and AML 15%. Their median age was 69 years (range 45-89). Prior therapies (77%) were 5-azacytidine 29%, lenalidomide 19%, erythropoietin 26%, hydroxyurea 7%, ruxolitinib 7%, cytarabine 4%. The side-effect was neutropenia (non-cytotoxic DNMT1-depletion skews myeloid differentiation away from GMP and to EMK), complicated by fever/infection in 31 patients (45%), with 1 septic death. Eight of these 31 patients (25%) had fever/infection before treatment. Blood count improvements meeting IWG criteria for response (hematologic improvement [HI]/complete remission [CR]) occurred in 30 patients (43%; CR 14%) (Fig 1A) and were durable(median treatment duration in HI/CR 82 weeks [range 14-347]). Treatment decreased bone marrow myeloblasts, even in cases without HI/CR (Fig 1B). HI/CR was achieved in MDS/AML containing monosomy 7, trisomy 8, complex cytogenetic abnormalities and/or multiple mutations (Fig 1C). Cytogenetics were normalized in 10/36 (28%) (Fig 1C). Patients with HI/CR had better overall survival (median 31 vs18 months) (p=0.036) (Fig 1D). Predictors of HI/CR were higher baseline neutrophils (median 3.04 vs 1.23 x109/L; p=0.002)and higher marrow cellularity (median 70 vs48%; p=0.044)(Fig 1E, F). Conclusion: Myeloid malignancies containing diverse genetic abnormalities responded sustainably (up to 6.5 years follow-up) to a decitabine regimen designed and demonstrated to be non-cytotoxic yet DNMT1-targeting, consistent with scientific data validating DNMT1 as a mutation-agnostic target that operates in a final common pathway of myeloid transformation. Although this single-agent regimen is non-curative, we postulate that avoidance of cytotoxicity, combined with target-validity, enabled sustained disease control/transfusion-freedom in several patients. Reported links between some mutations, e.g., in TET2, and decitabine response could be via higher neutrophils that in turn enable frequent decitabine exposures, a basic requirement for response. Also fundamental, HI/CR requires not just suppression of malignant clones but marrow capacity to support and recover functional hematopoiesis, suggesting why low baseline marrow cellularity predicted non-response. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy. Saunthararajah:Novo Nordisk: Consultancy; EpiDestiny: Consultancy, Equity Ownership, Patents & Royalties. OffLabel Disclosure: Alternative dose and scheduling of Decitabine for myeloid neoplasms

Large granular lymphocytic leukemia (LGLL) is an indolent disease and often associated with autoimmune disorders such as rheumatoid arthritis. The association of humoral immune disorders resulting from abnormal B cell activity, may affect original LGLL pathogenesis, clinical presentation and management through modifying disease presentation, progression and/ or resistance to standard care. Coexistence of T cell LGL leukemia with B cell abnormalities has previously been identified in the literature although described in sporadic case reports. However, no large case series or cohorts have been collected so far to study the frequency of the B-cell dyscrasia (BCD) associated with LGLL and describe clinical/ hematological findings in patients with this co-association. Here, we conducted a retrospective review of patients diagnosed with LGL leukemia at The Cleveland Clinic Foundation to search for any associated BCDs. We then classified our population into 2 groups: LGLL with BCD vs. LGLL without BCD, and comprehensively compared them for baseline, clinical and molecular characteristics. A total of 244 T-LGL patients were collected and studied. All cases were uniformly diagnosed with LGLL if 3 out of 4 following criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) presence of abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. Molecular studies including targeted deep sequencing for STAT3mutations were performed. Bone marrow biopsy results were reviewed to exclude other conditions. Endpoints of the study were death or lost to follow up. In our cohort, we found a frequent manifestation of humoral immune system abnormalities. We identified coexisting BCD in 45% (109/ 244) of LGLL patients, of whom 28 (11.2%) had monoclonal gammopathy of unknown significance (MGUS), and 13 (5.2%) had chronic lymphocytic leukemia (CLL/SLL). Six LGLL patients had multiple myeloma (2.4%). Moreover, polyclonal hypergammaglobulinemia (n=28, 11.2%) or hypogammaglobulinemia (n=14, 5.6%) was reported in 42 LGLL-patients (16.8%). The frequency of other disorders of B-cell origin was also examined. The total incidence of B-cell abnormalities in our LGLL cohort was 45%. Indeed an heterogeneous appearance of other B-cell disorders was observed including mantle cell lymphoma (n=2), DLBCL (n=6), marginal zone lymphoma (n=3), Waldenstrom's macroglobulinemia (n=1), Burkitt's lymphoma (n=1), indolent lymphoma (n=1), Hodgkin's lymphoma (n=1), non-Hodgkin's lymphoma (n=3), neck lymphoma (n=1), and smoldering myeloma (n=2). Patient with LGLL-BCD were older as compared to the ones without (median age: 62 vs. 63 years; ≥60 years: 57% vs. 69%, respectively), although the difference was not statistically significant (P=0.07). Gender was equally distributed (male: 54%, n=132; female: 46%, n=112) in patients who developed BCD. Conventional cytogenetics showed that patients without BCD were more often associated with abnormal cytogenetics (24%, n=9) as compared to LGLL-BCD (9%, n=5). Interestingly, BCD was found in 55 men and 54 women in whom only 6 patients had NK-LGLL while the remaining (n=103 patients) had T-LGLL suggesting a higher association with LGLL of T- rather than of NK-cell origin. Leukopenia was observed in 25/109 patients, with average absolute lymphocytes of 4.18 k/µL and LGL count of 2333 k/µL. Blood count showed: neutropenia in 44, anemia in 65, and thrombocytopenia in 29 out of 109 LGLL patients with BCD. TCR rearrangements were seen in 74 while somatic STAT3 mutations were observed in 37 LGLL patients while more enriched (44%, n=52) in LGLL without BCD. The association of other autoimmune conditions e.g., rheumatoid arthritis, was not different between the two groups (15% vs. 16% in LGLL with BCD vs. without; P=0.8). In sum, our investigation shows that BCD were frequent in LGLL and coexisted in 45% of the patients, commonly in the form of MGUS, and/ or hypergamaglobulinemia. Perhaps, the co-association of B-cell pathology with LGLL suggests that the two diseases either share pathogenetic driving mechanisms to enhance both B cells and T cells clones or that immunological dysfunction in setting of B cell dyscrasia could trigger/potentiate LGL expansion and/or transformation in this context. Disclosures No relevant conflicts of interest to declare.

Tramadol, one of the most commonly abused drugs in Middle East, impacts spermatogenesis and disturbs reproductive hormones in animal studies. We aimed to investigate tramadol impact on sperm quality and on levels of testosterone, prolactin and gonadotropins, in tramadol abusers (n = 30) to age-matched control (n = 30). Abusers had significantly low percentages of sperm motility, normal forms and vitality compared with control (95% CI -40.7 to -19.3, -13.5 to -9.3 and -31.9 to -9.7 respectively). Hypoandrogenism (95% CI -4.5 to -2.8), hyperprolactinaemia (CI (95%) 4.9 to 9.4) and hypergonadotropinaemia (95% CI 2.9 to 7.2 for FSH and 2.0 to 7.8 for LH) were observed in tramadol abusers vs controls. Smokers (26 of 30), concurrently abusing other drugs (11 of 30) and asymptomatic leucocytospermic (15 of 30) patients subgroups significantly abused tramadol beyond 3 years (p = .02,.001, = .03 respectively) and in excess450 mg/day (p = .02, = .01, = .005 respectively). Progressive motility (a + b%) was significantly low in young men25 years old (p = .03) subgroup. Tramadol abuse is associated with poor sperm quality, hyperprolactinaemia and hypergonadotropic hypogonadism. We recommend semen analysis for tramadol long-intakes, question sperm donors and follow-up studies to prevent and reverse tramadol-induced testicular damage.

Introduction: Many theories are suggested to explain aetiology of vitiligo, such as autoimmune, genetic and biochemical. Interleukin-8 (IL-8) is a pro-inflammatory chemokine which is evaluated in the pathogenesis of some skin diseases, like psoriasis, however, in vitiligo, few studies were reported regarding this issue. Aim: The aim of the present study was to investigate serum level of IL-8 and IL-8 mRNA in patients with different types of vitiligo to validate its role in aetiopathogenesis of vitiligo. Materials and Methods: This case-control study was conducted on 39 vitiligo patients and 15 age and gender matched healthy controls. The participants were selected from Dermatology Outpatient Clinic, Menoufia University Hospitals from October 2014 to October 2015. They were subjected to clinical history, examination and full general examination. Detailed dermatological examination including VASI score was applied. IL-8 serum level was measured by ELISA and IL-8 mRNA was quantitated by Real Time-PCR. The statistical analysis was done using SPSS, version 20.0. Mann Whitney U test was used to compare two quantitative not normally distributed. Chi-square test (χ2 ) was used to study association between two qualitative variables. Spearman correlation test was applied to assess correlation between two continuous quantitative variables. A p≤0.05 was considered significant. Results: IL-8 serum level and mRNA concentration mean values were significantly elevated in vitiligo patients (26.25±43.28 pg/ mL and 8.48±11.92 ng/mL) than controls (0.57±0.50 pg/mL and 0.60±0.32 ng/mL) (p=0.002, for both), and they showed significant positive correlation with each other (r=0.622; p≤0.001). Moreover, both of them revealed significant high values in localised vitiligo (62.20±74.39 pg/mL and 12.42±13.85 ng/mL) than generalised (18.39±29.49 pg/mL and 7.61±11.52 ng/mL) (p≤0.000 and p=0.004), respectively. Conclusion: Serum IL-8 chemokine and its mRNA increased significantly in vitiligo patients indicating that it may have a dynamic role in vitiligo development and participate in its pathogenesis.

Perturbation in iron homeostasis is a hallmark of some hematologic diseases. Abnormal sideroblasts with accumulation of iron in the mitochondria are named ring sideroblasts (RS). RS is a cardinal feature of refractory anemia with RS (RARS) and RARS with marked thrombocytosis (RARS/-T). Mutations in SF3B1, a member of the RNA splicing machinery are frequent in RARS/-T and defects of this gene were linked to RS formation. Here we showcase the differences in iron architecture of SF3B1-mutant and wild-type (WT) RARS/-T and provide new mechanistic insights by which SF3B1 mutations lead to differences in iron. We found higher iron levels in SF3B1 mutant vs WT RARS/-T by transmission electron microscopy/spectroscopy/flow cytometry. SF3B1 mutations led to increased iron without changing the valence as shown by the presence of Fe(2+) in mutant and WT. Reactive oxygen species and DNA damage were not increased in SF3B1-mutant patients. RNA-sequencing and Reverse transcriptase PCR showed higher expression of a specific isoform of SLC25A37 in SF3B1-mutant patients, a crucial importer of Fe(2+) into the mitochondria. Our studies suggest that SF3B1 mutations contribute to cellular iron overload in RARS/-T by deregulating SLC25A37.

e19072 Background: T-cell large granular lymphocytic leukemia (LGLL) has been reported after solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT); yet its underlying pathophysiological evolution has been poorly explained. Methods: We retrospectively screened a cohort of 243 LGLL patients at Cleveland Clinic for disease development post-transplantation and postulated several possible responsible pathophysiological mechanisms. Results: Five percent (n = 12) of the patients had a history of transplant. Of these, 67% (n = 8) had SOT (kidney, liver or heart) while 33% (n = 4) had HSCT. Twenty-five percent were females while 75% were males. The median age was 56 years (range: 15-65). The median time from transplant to diagnosis was 7 years. Conventional cytogenetic showed normal karyotype in 89% of the patients. TCR gene re-arrangement by PCR was detected in all tested individuals. Targeted deep sequencing identified somatic STAT3 mutations in 17% (n = 2) of the cases. Fifty percent (n = 6) had EBV DNA, IgM and/or IgG post-transplant. Forty-two percent (n = 5) had CMV DNA, IgM and/or IgG; of which 80% (n = 4) were post-transplant and 20% (n = 1) pre-transplant. The median survival from disease diagnosis was 64.3 months (8.37-232.9). Indeed, T-LGL clonal expansion resulted from long-term stimulation by viral antigens including EBV and CMV causing continuous cytotoxic T cell immune response or by “oncomodulation” as explained in 83% of patients. The latter theory suggests that CMV infects tumor cells and modulates their malignant properties, without direct transformation. Immunosuppression in setting of pre-transplant exposure to CMV, caused reactivation of the latent oncogenic viral genome as seen in 8% of patients. In 17% of cases that were seronegative for viral genome, graft allo-antigenic stimulation has triggered long-term recipient LGLL expansion from antigenic mismatch. Somatic STAT3 mutations, which are highly expressed in leukemic LGL, has led to aberrant signaling and transformation (17%). Conclusions: Our study highlights the complex nature of LGLL evolution post SOT and HSCT and points out confounding mechanisms. Certainly, investigating pre- vs. post-transplantation baseline, clinical and molecular characteristics is warranted to deeply understand this phenomenon.

Germ line (GL) alterations of telomerase machinery genes may lead to inherited telomeropathies, but recent analysis of large control populations revealed that some of the previously assumed pathologic variants are present in comparable frequencies in healthy individuals. Pathogenic telomerase gene variants can be found, but are rare in idiopathic aplastic anemia (AA) and are associated with excessive telomere attrition. Previously, in a cohort of patients with MDS, pathogenic germ line variants of telomerase genes were very extremely rare. Recently a patient with bone marrow failure and liver cirrhosis presented with a biallelic CTC1 gene mutation. CTC1 is a member of the CTC complex, located on Chr 17p13.1, and critical for telomere replication. GL alterations of this gene were found to cause inherited disease, including Coats plus (CP), dyskeratosis congenital (DC) and cerebroretinal microangiopathy with calcifications and cysts (CRMCC) in a compound heterozygous manner. Our patient (age 28) had compound heterozygous CTC1 germline mutations (p.Lys242Leufs*41 and p.Cys985del) resulting in early childhood presentation of DC, subsequent severe AA at the age of 19, and a significant shortening of telomeres. Both parents as confirmed CTC1 variant carriers showed normal blood counts. Based on this index case and literature reports, manifest CTC1 diseases follow a recessive trait, however, but it is possible that heterozygous carriers may indeed have also an increased, albeit attenuated risk of BMF leading to a later manifestation or incomplete penetrance. To test this possible scenario we screened a cohort of acquired BMF (n=538) with AA/PNH (172), MDS/AML (n=366), with deep NGS of all coding regions of CTC1. In total, we identified 10 heterozygous CTC1 variants in 10 unrelated patients (8 AA/PNH and 2 MDS); 4/10 variants (1 stop gain R1202X and 3 frameshift deletions D405fs, P999fs, E454fs) were fulfilled the criteria of Tier-1 lesions and were found in AA/PNH patients (4/172, 2.3%), The remaining 6 missense variants were of uncertain significance or likely benign(Tier-2), 2 of which (H1092G and E1136K) were found in a compound heterozygous configuration in a AA/PNH patient. K438N, as a novel missense variant, was recurrently present in 2 MDS pts. Given the expected frequency of the CTC1 variants found in controls (104/33370, 0.3%), CTC1 variants appear to be enriched in AA/PNH subgroup (p Because of the curious co-incidence of heterozygous CTC1 variants and AA/PNH, we further analyzed this subgroup for the presence of other telomerase gene mutations, 11/172 Tier-1 variants, while 6 Tier-1 GLVs detected in MDS. Most frequent SNVs were found in POT1 with 5/172 (3%) in AA/PNH and 3/366 (0.8%) in MDS/AML. Notably, in one AA/PNH patient POT1 stop gain variant (Q364X) was co-concurrent with CTC1 heterozygous frame shift deletion (P999fs). The enrichment of variants in TELO-related genes in AA/PNH subgroup was found in comparison to MDS/AML (p In sum, our results indicate that heterozygous CTC1 variants were associated with otherwise typical AA with clonal outgrowth of PNH clone and the presence of this variant does not seem to preclude response to immunosuppression. Disclosures Maciejewski: Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n = 81) and non-male factor (NMF, n = 52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p 0.001). Sperm chromatin damage was correlated with percentage of CMA(3)- positive sperm (r = 0.64, p 0.001) and with sperm concentration (r = -0.36, p 0.001), motility (r = -0.21, p 0.05), and morphologically normal spermatozoa (r = -0.29, p 0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA(3)- positive sperm (p 0.001) in ungrouped patients. It was related to both percentage of CMA(3)- positive sperm and serum DHEA in the MF group (p 0.001 and p 0.05, respectively). Sperm chromatin maturity assessed by CMA(3) test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.

Infertility affects about 15-20% couples attempting pregnancy and in about half cases the problem lies in the male. Among the sperm parameters, linear progressive motility is one of the most important predictors of fertility potential. Though genetic and chromosomal abnormalities are important aetiological factors in the pathogenesis of male infertility, the mechanism involved in impaired sperm motility is poorly understood. Here we report mitochondrial DNA (mtDNA) mutations with increased seminal reactive oxygen species (ROS) levels and higher DNA fragmentation level in the sperm resulting in decreased ATP production which plays an important role in sperm motility defect. Thus it is important to understand the aetiology of asthenozoospermia and to distinguish if infertile men harbour nuclear or mtDNA mutation as they are very important prognostic markers. This case study also highlights that routine semen parameters are very modest predictors of fertility outcome but ROS estimation and DNA integrity analysis by Comet assay have better diagnostic and prognostic capabilities. Thus this study is a detailed and comprehensive workup of an infertile asthenozoospermic male.

Summary Male Wistar rats (n = 54) received daily supplementation of red palm oil (RPO: 0, 2, 4 ml). Subgroups were subsequently injected with saline, cumene hydroperoxide (cHP, 10 μm) or t-butyl hydroperoxide (tbHP, 20 μm) over a 60-day period after which animals were sacrificed. Epididymal sperm motility, concentration, reactive oxygen species (ROS), lipid peroxidation and enzymes were measured. Sperm concentration, motility, superoxide dismutase (SOD) concentration, glutathione (GSH) and catalase (CAT) activities were significantly lower, while dichlorofluorescein (DCF) and malondialdehyde (MDA) were higher in sperm of hydroperoxide-treated animals compared to controls (P 0.05). RPO supplementation can successfully attenuate the oxidative stress-induced sperm damage due to organic hydroperoxide exposure. We therefore propose that a daily intake of RPO supplement to the diet might be helpful in protecting males against the adverse effects of high ROS in sperm function and help preserve fertility.

The cytosine analogue decitabine alters hematopoietic differentiation. For example, decitabine treatment increases self-renewal of normal hematopoietic stem cells. The mechanisms underlying decitabine-induced shifts in differentiation are poorly understood, but likely relate to the ability of decitabine to deplete the chromatin-modifying enzyme DNA methyltransferase 1 (DNMT1), which plays a central role in transcription repression. HOXB4 is a transcription factor that promotes hematopoietic stem cell self-renewal. In hematopoietic precursors induced to differentiate by the lineage-specifying transcription factor Pu.1 or by the cytokine granulocyte-colony stimulating factor, there is rapid repression of HOXB4 and other stem cell genes. Depletion of DNMT1 using shRNA or decitabine prevents HOXB4 repression by Pu.1 or granulocyte-colony stimulating factor and maintains hematopoietic precursor self-renewal. In contrast, depletion of DNMT1 by decitabine 6 hours after the differentiation stimulus, that is, after repression of HOXB4 has occurred, augments differentiation. Therefore, DNMT1 is required for the early repression of stem cell genes, which occurs in response to a differentiation stimulus, providing a mechanistic explanation for the observation that decitabine can maintain or increase hematopoietic stem cell self-renewal in the presence of a differentiation stimulus. Using decitabine to deplete DNMT1 after this early repression phase does not impair progressive differentiation. ©2010 AACR. Fil: Hu, Zhenbo. Cleveland Clinic; Estados Unidos Fil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Cleveland Clinic; Estados Unidos Fil: Gu, Xiaorong. Cleveland Clinic; Estados Unidos Fil: Mahfouz, Reda. Cleveland Clinic; Estados Unidos Fil: Ng, Kwok Peng. Cleveland Clinic; Estados Unidos Fil: Ebrahem, Quteba. Cleveland Clinic; Estados Unidos Fil: Copelan, Edward. Cleveland Clinic; Estados Unidos Fil: Singh, Harinder. University of Chicago; Estados Unidos Fil: Maciejewski, Jaroslaw P.. Cleveland Clinic; Estados Unidos Fil: Saunthararajah, Yogen. Cleveland Clinic; Estados Unidos

Objective To investigate sperm viability, incidence of apoptosis, and intracellular basal and induced reactive oxygen species (ROS) in sperm fractions. Design Prospective controlled study. Setting Center for Reproductive Medicine at a tertiary care hospital. Method(s) Liquefied seminal ejaculates (n = 12) prepared by density gradient centrifugation were reconstituted to 2 mL with phosphate-buffered saline. Oxidative stress was induced by hydrogen peroxide (H 2 O 2 , 100 μM). Sperm viability, intracellular ROS, and incidence of apoptosis/necrosis in neat, immature, and mature sperm fractions were assessed. Result(s) Before H 2 O 2 exposure, mature spermatozoa fractions showed a significantly lower incidence of apoptotic sperm and intracellular O 2 −• levels but higher amounts of intracellular H 2 O 2 compared with neat semen. Higher levels of intracellular H 2 O 2 were demonstrated in immature sperm fractions compared with neat or mature fractions. In all sperm fractions, intracellular H 2 O 2 levels correlated with the intracellular concentration of O 2 −• . After H 2 O 2 exposure, neat semen showed a significantly higher percentage of apoptosis compared with the prepared mature spermatozoa. However, no differences were observed in the incidence of apoptosis between immature and mature sperm fractions. Conclusion(s) There is a differential shift of both intracellular H 2 O 2 and O 2 -• in each sperm fraction that may affect sperm quality. Sperm apoptosis is related to intracellular H 2 O 2 levels, which in turn are affected by intracellular O -• levels. Oxidative stress was not associated with an increased incidence of apoptosis in immature or mature sperm fractions.

NPM1 is the most frequently mutated gene in acute myeloid leukemia (AML). Unfortunately, there are no 'precision' or rational treatments for this subtype of AML.To elucidate molecular mechanisms of pathogenesis, we performed the first comprehensive, unbiased analysis of the endogenous NPM1 protein-interactome using mass-spectrometry (LC-MS/MS). This approach identified abundant amounts of the master transcription factor driver of monocyte lineage-differentiation PU.1 (SPI1). The NPM1/PU.1 interaction causes PU.1 functional deficiency when NPM1 is mutated, because mutant-NPM1 dislocates PU.1 into the cytoplasm with it.This was confirmed using six different methods: (i) Immunoprecipitation (IP)-LC-MS/MS from nuclear and cytoplasmic fractions of wildtype (wt) and NPM1 -mutated AML cell lines (n=2); (ii) IP-Western blot (WB) from nuclear/cytoplasmic fractions of NPM1 -mutated/wt AML cell lines (n=2); (iii) WB of nuclear/cytoplasmic fractions of NPM1 -mutated/wt AML cell lines (n=5); (iv) immunofluorescence microscopy (IF) of NPM1 -mutated/wt AML cell lines (n=5); (v) IF of NPM1 -mutated/wt primary AML cells (n=6); and (vi) cotransfection of HEK293 cells to express PU.1 + mutant vs wt-NPM1 followed by IF. Re-introduction of Pu.1 into the nucleus of Pu.1-null myeloid precursors which are differentiation-arrested and exponentially proliferating repressed key myeloid precursor genes (e.g., Hoxa9) and triggered terminal monocytic differentiation. Even though primary AML cells (n=900) express the PU.1/RUNX1/CEBPA monocyte differentiation-driving master transcription factor circuit at levels comparable to or exceeding that in normal monocytes, the expression of ~300 monocyte terminal-differentiation genes was suppressed. Importantly, the genes affected are strongly positively correlated (avg. rho 0.7) with PU.1 expression in normal hematopoiesis, consistent with functional disruption of PU.1 in AML. To translate these observations into a treatment option for NPM1 -mutated AMLs, we were guided by additional observations. First, the NPM1/PU.1 protein complex is exported by the nuclear export protein XPO1. XPO1-mediated nuclear export is inhibited by the small molecule selinexor. We found that sub-cytotoxic /low nanomolar concentrations of selinexor locked mutant-NPM1/PU.1 in the nucleus, releasing terminal monocytic differentiation of NPM1 -mutated AML cells both in vitro and in vivo . Briefly, NSG mice were xenotransplanted with NPM1 / FLT3 -mutated primary AML cells and observed until AML engraftment (≥20%) was confirmed. Selinexor was then administered by oral gavage at 2 mg/kg 4X/week (Fig1A), which is 10-fold lower than the usual in vivo cytotoxic dose (15-20 mg/kg) - low doses are well-tolerated and sufficient to promote non-cytotoxic differentiation of AML cells. After 50 days of treatment, bone marrow (Fig1B) and spleen (Fig1C) AML burden was significantly lower in selinexor vs vehicle treated mice. In addition, selinexor treated mice preserved murine hematopoiesis (Fig1D) and IF confirmed the partial nuclear restoration of PU.1 (Fig1E). Terminal monocytic differentiation of AML cells was evident by Giemsa-stained morphology (Fig1F) and flow cytometry (Fig1G). RUNX1 and CEBPA remain in NPM1 -mutated AML cell nuclei at high levels - PU.1 usually cooperates with these master transcription factor partners to exchange corepressors for coactivators and activate differentiation genes. Accordingly, IP-LC-MS/MS of endogenous nuclear CEBPA demonstrated enrichment for corepressors. Depletion of one of these corepressors, DNMT1, using non-cytotoxic concentrations of decitabine or 5-azacytidine (clinical DNMT1-depletors), also induced terminal-differentiation. Moreover, the granulocytic direction of differentiation naturally downregulated NPM1, an event inherent to CEBPA-driven granulocytic (but not monocytic) differentiation. This approach readily induced terminal-differentiation in NPM1 -mutated AML cells selected over 52 weeks of culture for resistance to selinexor, shown by exponential growth in selinexor 20 nM. The mechanisms by which mutant-NPM1 creates leukemic self-replication (proliferation uncoupled from differentiation) are thus reversed by non-cytotoxic molecular targeted clinical drugs. We are evaluating the combination of selinexor with non-cytotoxic DNMT1-depletion in vivo and clinical trials are planned. Disclosures Landesman: Karyopharm Therapeutics: Employment.

Objective To examine simultaneously the levels of hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 −• ) using chemiluminescence and flow cytometry. Design Prospective laboratory study. Setting Reproductive research lab in a tertiary hospital. Patient(s) Semen samples from 18 healthy male volunteers. Intervention(s) Sperm preparation and measurement of reactive oxygen species (ROS) by chemiluminescence using luminol and lucigenin before and after H 2 O 2 exposure and by flow cytometry using dichlorofluorescin diacetate (DCFH-DA) for H 2 O 2 and dihydroethidium (DHE) for O 2 −• . Main Outcome Measure(s) Sperm count, motility, viability, and ROS levels. Result(s) Immature sperm fractions showed significantly higher levels of ROS measured by either luminol or lucigenin compared with the neat and mature fraction. ROS levels were detectable by flow cytometry in chemiluminescence-negative samples. Both mature and immature sperm fractions had a significantly higher percentage of cells positive for H 2 O 2 compared with neat semen. On the other hand, the percentage of O 2 −• -positive cells in neat semen was significantly higher compared with the percentage found in mature fractions but significantly lower than that in the immature sperm fractions. Conclusion(s) We recommend ROS measurement by flow cytometry on the basis that it requires a lower sperm count, is comparable to chemiluminescence, and has higher specificity for intracellular ROS in viable spermatozoa. Samples tested negative by chemiluminescence still may have high intracellular H 2 O 2 generation that can be detected by flow cytometry.

Objective: To examine the relationship among sperm apoptosis, sperm chromatin status, and DNA ploidy indifferent sperm fractions.Design: Prospective study.Setting: Reproductive research center in a tertiary care hospital.Intervention(s): Sperm prepared by density gradient were evaluated for sperm count, motility, apoptosis, andsperm chromatin assessment.Main Outcome Measure(s): Sperm count, sperm motility, toluidine blue (TB) results, DNA fragmentation index(%DFI), high DNA stainability, DNA cytometry, and early and late apoptosis.Result(s): Sperm motility was related to late apoptotic and subhaploid apoptotic sperm (r ¼ 0.56 and 0.53,respectively). The sperm %DFI showed significant correlation with late apoptotic and subhaploid sperm (r ¼0.62 and 0.68). TB-stained sperm were significantly correlated with late apoptotic sperm (r ¼ 0.51). SignificantlyhigherproportionsofhaploidspermandlightblueTB-stainedspermwereseeninmaturecomparedwithimmaturefractions.Conclusion(s): Even in semen samples with low %DFI, semen processing results in a lower incidence of nuclearimmaturity and subhaploidy, but the incidence of late apoptotic sperm remains unchanged. Therefore, simulta-neous evaluation of apoptosis and sperm chromatin status is important for processing sperm in assisted reproduc-tive procedures. (Fertil Steril 2009;91:1110–8. 2009 by American Society for Reproductive Medicine.)Key Words: Sperm DNA integrity, sperm apoptosis, sperm fractions, sperm DNA cytometry

Objective To establish cutoff value, sensitivity, specificity and intra- and interobserver variability of total antioxidant capacity (TAC) in seminal plasma from healthy donors (controls) and infertile patients. Design Seminal plasma from proven fertile donors (n = 55), nonproven fertile donors (n = 45), and infertile patients (n = 42) were examined for TAC level. Setting Reproductive research center in a tertiary care hospital. Patient(s) Infertile patients from male infertility clinic of various diagnoses. Intervention(s) Seminal plasma TAC measurement by a colorimetric assay using the TAC assay kit, receiver operating characteristics curve. Main Outcome Measure(s) Seminal plasma TAC levels, cutoff value, sensitivity, and specificity. Result(s) Proven fertile donors showed higher TAC values (median and range): 1700 (1440–2290 μM); compared with the infertile patients: 1310 (1040–1600 μM). The best cutoff to distinguish between fertile controls and infertile men was 1420 μM. At this threshold, specificity was 64% and sensitivity 76%. Conclusion(s) Total antioxidant capacity of the seminal plasma as measured by the colorimetric assay is a reliable and simple test for the diagnosis and management of male infertility.

Objective To optimize the L-carnitine (LC) concentration as a supplement in embryo culture medium and to investigate the effect of LC on developing embryos. Design Experimental study. Setting Reproductive research center at a tertiary hospital. Intervention(s) To optimize the LC concentration, 420 mouse embryos were divided into seven groups and incubated with different LC concentrations (0, 0.3, 0.6, 1.2, 2.5, 5.0, and 10 mg/mL). To investigate the effect of LC on the developing embryos, 500 mouse embryos were divided into three groups and incubated with either actinomycin-D (AD; 0.005 μg/mL), hydrogen peroxide (H 2 O 2 ; 500 μmol/L), or tumor necrosis factor α (TNF-α; 500ng) with and without LC 0.3 or 0.6 mg/mL. Blastocyst development rate (%BDR) and DNA damage were examined for all groups. Main Outcome Measure(s) Effect of LC on embryogenesis. Result(s) Significant improvement in %BDR was seen at LC 0.3 mg/mL compared with the control (p = 0.006). L-Carnitine at 0.3 and 0.6 mg/mL significantly reduced the blocking effect of AD, H 2 O 2 , and TNF-α and significantly decreased the level of DNA damage. Conclusion(s) Embryo culture medium supplementation with LC may offer a novel and a cost-effective technique to improve the embryogenesis of cultured embryos. This may be beneficial in improving IVF outcomes.

BACKGROUND. Mutational inactivation in cancer of key apoptotic pathway components, such as TP53/p53, undermines cytotoxic therapies that aim to increase apoptosis. Accordingly, TP53 mutations are reproducibly associated with poor treatment outcomes. Moreover, cytotoxic treatments destroy normal stem cells with intact p53 systems, a problem especially for myeloid neoplasms, as these cells reverse the low blood counts that cause morbidity and death. Preclinical studies suggest that noncytotoxic concentrations of the DNA methyltransferase 1 (DNMT1) inhibitor decitabine produce p53-independent cell-cycle exits by reversing aberrant epigenetic repression of proliferation-terminating (MYC-antagonizing) differentiation genes in cancer cells. METHODS. In this clinical trial, patients with myelodysplastic syndrome (n = 25) received reduced decitabine dosages (0.1–0.2 mg/kg/day compared with the FDA-approved 20–45 mg/m2/day dosage, a 75%–90% reduction) to avoid cytotoxicity. These well-tolerated doses were frequently administered 1–3 days per week, instead of pulse cycled for 3 to 5 days over a 4- to 6-week period, to increase the probability that cancer S-phase entries would coincide with drug exposure, which is required for S-phase–dependent DNMT1 depletion. RESULTS. The median subject age was 73 years (range, 46–85 years), 9 subjects had relapsed disease or were refractory to 5-azacytidine and/or lenalidomide, and 3 had received intensive chemoradiation to treat other cancers. Adverse events were related to neutropenia present at baseline: neutropenic fever (13 of 25 subjects) and septic death (1 of 25 subjects). Blood count improvements meeting the International Working Group criteria for response occurred in 11 of 25 (44%) subjects and were highly durable. Treatment-induced freedom from transfusion lasted a median of 1,025 days (range, 186–1,152 days; 3 ongoing), and 20% of subjects were treated for more than 3 years. Mutations and/or deletions of key apoptosis genes were frequent (present in 55% of responders and in 36% of nonresponders). Noncytotoxic DNMT1 depletion was confirmed by serial BM γ-H2AX (DNA repair/damage marker) and DNMT1 analyses. MYC master oncoprotein levels were markedly decreased. CONCLUSION. Decitabine regimens can be redesigned to minimize cytotoxicity and increase exposure time for DNMT1 depletion, to safely and effectively circumvent mutational apoptotic defects. TRIAL REGISTRATION. Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01165996","term_id":"NCT01165996"}}NCT01165996. FUNDING. NIH (R01CA138858, {"type":"entrez-nucleotide","attrs":{"text":"CA043703","term_id":"24344623","term_text":"CA043703"}}CA043703); Department of Defense (PR081404); Clinical and Translational Science Award (CTSA) (UL1RR024989); and the Leukemia and Lymphoma Society (Translational Research Program).

Apoptosis plays an important role in the pathogenesis of male infertility. Failure of fertilization in assisted reproduction (ART) despite the use of morphologically normal and motile spermatozoa may be due to an ongoing process of apoptosis. Therefore, the use of apoptotic spermatozoa may be one of the reasons behind the suboptimal success rates in ART. Sperm selection methods currently used routinely in conjunction with ART procedures lack the ability to specifically target apoptotic spermatozoa. To address this deficiency, a protocol that includes the selection of non-apoptotic spermatozoa using magnetic activated cell sorting (MACS) has been proposed.

Cancer chemotherapy is typically toxic. This problem could be addressed by using differences between cancer and normal cells for controlled delivery of drugs to cancer cells. One such difference is the ubiquitously elevated glutathione expression in cancer cells. We report a simple and versatile synthesis of water-soluble gold nanoparticles passivated with amine-containing molecules, which allow for controlled drug release via ligand exchange with bio-available glutathione. Taking methotrexate-passivated gold nanoparticles (Au:MTX) as an example, drug delivery and controlled release via glutathione-mediated ligand exchange was evaluated. Furthermore, the possibility of using Au:MTX to improve therapeutic index in acute myeloid leukemia (AML) models was examined in vitro and in vivo. Au:MTX exhibited cancer selectivity in vitro. Au:MTX had an elevated potency toward an AML cell line THP-1 in a dosage range of 1–5 nM, and therefore an enhanced delivery of drug, whereas normal hematopoietic stem/progenitor cell (HSPC) growth was minimally affected by Au:MTX and MTX treatments within the same range of dosage. In vivo efficacy and safety of Au:MTX was evaluated in a murine xenotransplant model of primary human AML. Au:MTX treatment, compared to control groups including MTX-only and Au nanoparticle-only treatments, produced better leukemia suppression without added toxicity, indicating an enhanced therapeutic index.

Somatic mutations constitute clonal markers now amenable to monitoring by deep NGS. While transient and low frequency clones have been described in AA, their pathophysiologic link to the overtly clonal complication of AA, secondary MDS following AA (sMDS), has not been established. Clarification of this relationship may provide clues as to the genesis of sMDS. Identification of predictive markers for AA patients at risk for this complication is necessary. The etiology of sMDS within AA may include either expansion of a preexisting clone or truly late clonogenic events. In both instances, progression may result in clonal escape. To address these questions we studied 258 AA and 60 PNH patients, identifying 35 patients (11%) who evolved to sMDS. Cytogenetic analysis showed abnormal karyotype in 76% cases; 5% had complex karyotype and -7/del(7q) was present in 67% of cases. The presence of a PNH clone was detected in a similar proportion of cases that transformed to sMDS vs. those that did not (P=.76). For comparison, we have also analyzed primary de novo cases of MDS (pMDS) with (N=19) and without (N=161) -7/del(7q). In contrast to sMDS, -7/del(7q) was present in 12% of cases of pMDS. Using WES on 8 cases and a 60 gene targeted panel on 15 cases, confirmed mutational events and chromosomal aberrations were found in 21/23 patients with sMDS; 18/23 cases of sMDS had at least 1 confirmed somatic mutation. By comparison of mutational profiles ASXL1, RUNX1, PIGA, SETBP1, and CBL were most common in sMDS (26.1%, 21.7%, 18%, 13% and 13%, respectively). Because sMDS included a high proportion of patients with -7/del(7q), we compared sMDS with -7/del(7q) to pMDS with -7/del(7q) for coexisting mutational events. Mutations in RUNX1 appeared to be more frequent in sMDS vs. pMDS (27% vs. 0%, P=.03). In contrast, TP53 was more common in pMDS (7% vs. 32%, P=.1). Similarly, there were several other distinctive differences between all sMDS and pMDS irrespective of cytogenetics: mutations in U2AF1 were common in pMDS, mutations in RUNX1 appeared to be more frequent in sMDS vs. pMDS (22% vs. 5.5%, P=.02). Mutations in PIGA gene constituted a marker for sMDS derivation from AA. To discern a possible biological relationship we have also compared mutational profiles of hypocellular pMDS and sMDS, but no significant differences aside from PIGA prevalence were found. If sMDS is derived from mutations present at the AA stage, one would expect overlaps in the mutational spectrum of AA before and after evolution to MDS. DNMT3A, BCOR/BCORL1, and PDGFR family mutations were found at higher frequency in AA while ASXL1, RUNX1, SETBP1, PIGA, and CBL were higher in sMDS. Thus, cross-sectional analysis suggests that most of the clonal events occurring during the course of AA do not initiate sMDS. To further examine these findings we performed serial sequencing analyses: in 7 patients with sMDS WES was performed and clonal architecture was analyzed. We then queried whether mutations present in MDS were detected in archival samples at presentation using deep targeted NGS (depth 5-10x104 rds. In 4/7 cases the alterations appeared to be ancestral events for sMDS evolution. When an additional 68 AA cases were studied by deep NGS, somatic mutations were present in 31% of AA patients at presentation. Patients with clonal events at presentation tended toward worse progression free survival compared to patients without mutations (P=.1). Mutations found at both initial presentation and upon evolution were suggestive of a slow expansion of previously cryptic clones (ASXL1, CUX1, TET2, CBL, RUNX1, and SETBP1). Patients with these gene mutations (n=21) before immunosuppressive therapies (IST) had worseoverall survival compared to patients without these mutations (n=47; P=.009). To assess the potential impact of IST, we also investigated a subset of 37 patients (25 responders/ 12 refractory) following IST. Clonal somatic events were identified in 42 of them, but there was no association between the response to IST and somatic mutations at presentation (P=.7). Our results demonstrate that while subclonal mutations indicative of oligoclonal hematopoiesis are frequent in AA, the presence of specific permissive ancestral somatic events at the outset of AA predisposes patients to sMDS, a feature that has diagnostic and prognostic implications. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

Acute myeloid leukemia (AML) is self-renewal by immature myeloid precursors that fail to differentiate. An influential 'leukemia stem cell' model thus proposes that leukemogenic proteins augment or introduce a stem cell-like self-renewal program into cells, e.g., by upregulating signaling or transcription factors (TF) emblematic of stem cells (e.g., HOX). We investigated how the most recurrently mutated protein in AML, mutant nucleophosmin (mNPM1), causes leukemic cell expansion. The results challenge this model, but most importantly, open the door to rational targeted therapy for mNPM1 AML. One way of examining for stem cell programs in AML cells is to look at expression patterns of master TF that regulate expression of hundreds of genes and dictate cell fates. Of these select TF, the master TF that create hematopoietic stem cells (HLF etc.) are minimally or not expressed. Instead, there are very high levels of the master TF that drive monocyte and granulocyte lineage fates, PU.1 (SPI1) and CEBPA. Clearly, however, the lineage-programs intended by PU.1/CEBPA are inefficiently executed if at all - mNPM1 AML patient bone marrows had 85-97% cells with a granulocyte-monocyte progenitor phenotype, accumulated at the expense of downstream mature cells (Quek et al, JEM 2016). This aggregation at a lineage-committed, intermediate, naturally proliferative level of the hematopoietic hierarchy suggests an alternative model - instead of introducing a poorly-defined stem cell self-renewal program, mutant proteins disable differentiation programs which usually quench MYC-driven proliferation intrinsic to lineage-progenitors. To better understand how mNPM1 interacts with cellular machinery, we used mass-spectrometry to comprehensively document the protein interactions of endogenous NPM1 in AML cell nuclear and cytoplasmic fractions, the first analysis of this kind. Notably, the NPM1 protein interactome was enriched for PU.1. Critically, wild-type (wt) NPM1/PU.1 was in the nucleus of wtNPM1 AML cells, but mNPM1/PU.1 was in the cytoplasm of mNPM1 AML cells. This was evident clearly also by Western blot of cell fractions and by IF microscopy of primary AML cells and cell lines. Is cytoplasmic dis-location of PU.1 sufficient to explain persistent hematopoietic precursor proliferation? We used murine Pu.1 knock-out hematopoietic precursors transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER) to answer this question - Pu.1 relocation from the cytoplasm to the nucleus by tamoxifen triggered monocytic differentiation that terminated proliferation. Moreover, Pu.1-ER cells, like mNPM1 AML cells, highly express Hox genes, rapidly suppressed upon Pu.1 relocation to the nucleus. Thus, Pu.1 dominantly controls Hox and proliferation, as befitting of a master TF, and persistent HOX expression, like persistent progenitor proliferation, can be caused by Pu.1 loss-of-function. Protein macromolecules like NPM1 require transport factors to exit (exportins) the nucleus. A specific exportin, XPO1, was the major exportin found in the NPM1 interactome. XPO1 interactions with transported cargo can be inhibited by the small molecule drug KPT330. KPT330 10-20 nM rapidly re-located mNPM1 and PU.1 to the nucleus, downregulated MYC, upregulated p27/CDKN1B, upregulated monocyte but not granulocyte differentiation markers, induced morphologic changes of monocyte differentiation, and terminated proliferation of mNPM1 AML cells. The same low nanomolar treatment did not induce differentiation of wtNPM1 AML cells (THP1). Moreover, these KPT330 levels are not toxic to normal hematopoiesis (also shown by others). Thus, rather than gain-of-function of elusive stem cell-like self-renewal, the most frequently mutated protein in AML creates self-renewal by disabling a differentiation program that quenches intrinsic MYC-driven proliferation of lineage-progenitors. These observations are a mechanistic rationale to select refractory/relapsed mNPM1 AML patients for treatment with low well-tolerated doses of KPT330, with a defined molecular pharmacodynamic objective of returning PU.1 to the nucleus, to produce cell cycle exits by differentiation rather than p53-mediated apoptosis (to address chemotherapy resistance), to spare precious normal HSC (good therapeutic index), and directly reverse the basis for leukemic self-renewal (proliferation without differentiation). Figure. Figure. Disclosures Landesman: Karyopharm Therapeutics Inc: Employment, Other: stockholder. Saunthararajah:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine.

s / Biol Blood Marrow Transplant 21 (2015) S266eS321 S318 (higher CT value), whereas the presence of an increased number of symptoms ( 3) was associated with a higher viral load. Lower viral load at initial surveillance testing correlated with an increased likelihood of being asymptomatic initially and remaining asymptomatic throughout the viral shedding episode. These results provide initial support for the use of viral load as a clinical endpoint in interventional trials.

Purpose: The cytidine analogs 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase 1 (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Experimental design: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity, and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated patients with MDS (n = 90) and cytarabine-treated patients with acute myeloid leukemia (AML) (n = 76). Results: By high-performance liquid chromatography (HPLC), plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females than in males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared with high S-phase fraction disease (e.g., MDS vs. AML), because in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female patients with MDS treated with 5-azacytidine/decitabine. Conclusions: Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy. Clin Cancer Res; 19(4); 938–48. ©2012 AACR.

The most frequently mutated gene in de novo acute myeloid leukemia (AML) is nucleophosmin (NPM1). How mutated NPM1 (mNPM1) confers clonal advantage is unknown. We hypothesized that mNPM1 interferes with the function of one or more master transcription factors (TF) essential for myeloid differentiation. We used mass-spectrometry to comprehensively analyze the protein interactome of endogenous NPM1 and mNPM1 in AML cell nuclear and cytoplasmic fractions, and examined the cell fate implications of observed interactions. NPM1 was found in abundance in both nuclear and cytoplasmic fractions of wtNPM1 AML cells, but only in the cytoplasm of mNPM1 AML cells. In the nucleus of wtNPM1- and cytoplasm of mNPM1-AML cells, the NPM1/mNPM1 interactome was enriched for the master TF driver of monocytic differentiation PU.1 and the RARA co-factors STAT1 and CNOT1. Accordingly, by both Western blot (WB) of cell fractions and immunofluorescence (IF) microscopy, PU.1 was localized in the cytoplasm of mNPM1 AML cells together with mNPM1. Another master differentiation-driving TF highly expressed in AML cells, CEBPA, was not in the NPM1 interactome nor localized to cytoplasm. Nuclear export of NPM1 is dependent on the nuclear export protein XPO1 (CRM1). Selinexor (KPT-330) is an anti-cancer drug that inhibits protein trafficking to cytoplasm by covalently binding to the XPO1 cargo binding pocket. Selinexor 20-50nM relocated both mNPM1 and PU.1 into the nucleus in mNPM1 AML cells, demonstrated by IF and cell fraction WB. Importantly, this nuclear relocation prompted terminal monocytic differentiation of mNPM1 AML cells. The RARA ligand ATRA at 10nM transferred RARA and STAT1 but not mNPM1/PU.1 into the nucleus and triggered terminal granulocytic rather than monocytic differentiation, presumably enabled by high baseline intra-nuclear CEBPA. mNPM1 AML is distinguished by high HOX gene expression. To evaluate a potential cause-effect relationship, Hox expression was examined in Pu.1 knock-out hematopoietic precursors retrovirally transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER). Addition of estrogen to translocate Pu.1-ER into the nucleus substantially decreased Hox expression accompanied by terminal monocytic differentiation. Thus, nuclear export of PU.1 and RARA cofactors with mNPM1 causes the differentiation arrest that defines AML. Low nanomolar concentrations of selinexor transfer PU.1 to the nucleus and trigger terminal monocytic differentiation, while low nanomolar concentrations of ATRA transfer RARA/STAT1 to the nucleus and trigger terminal granulocytic differentiation. These are p53-independent pathways of cell cycle exit distinct from p53-dependent apoptosis that mediates chemotherapy effect. Thus selinexor and/or ATRA differentiation therapy may offer the ∼50% of mNPM1 AML patients with chemo-refractory disease a needed novel, mechanistically rational treatment option. Citation Format: Xiaorong Gu, Reda Mahfouz, Ji Zhang, Francis Enane, Zhenbo Hu, Tomas Radivoyevitch, David Wald, Maria Paola Martelli, Brunangelo Falini, Yosef Landesman, Jaroslaw Maciejewski, Yogen Saunthararajah. Cytoplasmic localization of PU.1 with mutated NPM1 causes myeloid differentiation arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2872.

In vivo, half-lives of cytidine analogues such as 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), are determined largely by cytidine deaminase (CDA), an enzyme that rapidly metabolizes these drugs into inactive uridine counterparts. Genetic factors influence CDA activity, and hence, could impact 5-azacytidine/decitabine levels and efficacy, a possibility requiring evaluation. Using an HPLC assay, plasma CDA activity was confirmed to be decreased in individuals with the CDA SNP A79C. More interestingly, there was an even larger decrease in females. Explaining the decrease in enzyme activity, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass-spectrometry, were significantly higher in females. In mathematical modeling, the detrimental effect of shortening half-life of S-phase specific therapy was amplified in low S-phase fraction disease (e.g., MDS). Accordingly, in multivariate analysis of MDS patients treated with 5-azacytidine/decitabine, overall survival was significantly worse in males.

Only 5-azacytidine (5Aza) and decitabine (Dec), both DNA methyltransferase (DNMT1) depleting drugs, are FDA-approved to treat all myelodysplastic syndrome (MDS) subtypes. Neither agent is curative. What are the mechanisms of resistance? MDS is genetically heterogeneous, thus, this question has been approached by attempting to correlate MDS genetics with response, or by examining genetics at relapse. Unfortunately, results have been contradictory/inconclusive. Another approach is to consider that achievement of the intended molecular pharmacodynamic (PD) effect of DNMT1-depletion is a minimum requirement for response, superceding other biological considerations including genetics/susceptibility to apoptosis or differentiation. As such, a logical first-step is to examine whether relapse is driven by sub-clones thriving despite DNMT1-depletion, or from failure to deplete DNMT1 in the first place. In both murine and clinical studies, relapse was from failure to deplete DNMT1 (Leukemia 2011;25(11):1739-50; Oncotarget 2012;3(10):1137-45; J Clin Invest 2015;125(3):1043-55). Thus, the question can be reframed as: Why is molecular PD not achieved, and can mechanisms be identified to extend response? Achievement of molecular PD by 5Aza/Dec depends on intra-cellular exposure/half-life (t½). Intra-cellular t½ depends in turn on extra-cellular exposure time (plasma t½) and pyrimidine metabolism enzymes that phosphorylate 5Aza/Dec, trapping these drugs in cells. There is controversy regarding the identify of the enzymes that phosphorylate and trap 5Aza: UCK1, UCK2 or both. To answer this question, we correlated UCK1, UCK2 and DCK expression with sensitivity of NCI60 cancer cell lines (n=60) to 5Aza or Dec. 5Aza sensitivity correlated strongly with UCK2 expression but not UCK1 or DCK (corr. coeff.: 5AzaGI50 vs UCK2-0.28, p=0.03; vs UCK1 0.22, p=0.1; vs DCK -0.07, p=0.5). Dec sensitivity correlated with DCK expression but not UCK2 or UCK1 (corr. coeff.: DecGI50 versus DCK-0.27, p=0.04; vs UCK1-0.01, p=0.9; vs UCK2-0.19, p=0.2). There was greater than 2-fold variation of UCK2/DCK expression in primary MDS CD34+ bone marrow cells, not linked with WHO-subtypes or genetics, suggesting flexible use of one or the other enzyme for pyrimidine salvage, although a trend for higher DCK expression was noted in MDS containing mutated TP53 (GSE58831). Does treatment select for sub-clones utilizing salvage enzymes that avoid drug? DCK and UCK2 expression was serially measured by QRT-PCR in bone marrow from MDS patients treated with a Dec regimen rationalized for non-cytotoxic DNMT1-depletion (0.1-0.2 mg/kg SC 1-3X/week, n=12) or with conventional 5Aza (n=6). UCK2 consistently increased (up to 100-fold) and DCK consistently decreased at relapse on Dec, with vice-versa for 5Aza (Fig. 1). Mechanism-based solutions to such resistance were evaluated in xenotransplant models of human MDS-AML that phenocopy human disease with bone marrow infiltration and death by cytopenia. Candidate solutions investigated were (i) Dec + thymidine, or HU, or methotrexate, to increase DCK (by inhibition of ribonucleotide reductase [RNR]); (ii) 5Aza or Dec at lower dose to decrease Cmax and avoid cytotoxicity + tetrahydrouridine (THU) to increase plasma t½/Tmax for DNMT1-depletion (THU inhibits the enzyme cytidine deaminase that rapidly inactivates 5Aza and Dec in vivo); (iii) 5Aza alternating or combined with Dec, since 5Aza-mediated increases in DCK are expected to increase sensitivity to Dec, while Dec-mediated increases in UCK2 are expected to increase sensitivity to 5Aza; Of these interventions, reduced dosage 5Aza or Dec + THU, and alternating THU-5Aza with THU-Dec, but not simultaneous THU-5Aza+THU-Dec, nor the RNR inhibitors, increased response and survival to striking extents (approaching 1 year)(Fig. 2). Bone marrow gH2AX and DNMT1-expression by flow-cytometry and preservation of murine hematopoiesis confirmed the non-cytotoxic, epigenetic mechanism of action (Fig. 2). We show that straightforward shifts in pyrimidine metabolism demonstrably account for much of the resistance to 5Aza or Dec. Fortunately, such resistance is amenable to logical, non-toxic, clinically applicable solutions, validated in pre-clinical in vivo models. Clinical trials of non-cytotoxic THU-Dec (IND#112914) and alternating THU-Dec/THU-5Aza, incorporating companion PD/metabolism biomarkers, are planned. Disclosures Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Saunthararajah:Not applicable: Other: patent applications around decitabine, 5-azacytidine and tetrahydrouridine. Off Label Use: decitabine used by route of administration and dosages other than FDA approved regimens.

Historically, the evolution rate of aplastic anemia (AA) to MDS approaches 15% in 10 yrs; thus, AA can be considered a major predisposition factor for secondary MDS (sMDS). The likely etiology includes expansion of a preexisting clone or truly late clonogenic events. In both instances, progression can be a result of a clonal escape, but confirmation of the presence of mutant cells at presentation would indicate that initial autoimmune processes may represent a tumor surveillance reaction. We studied 326 patients with AA and 47 patients with paroxysmal nocturnal hemoglobinuria (PNH) and identified 36 cases (progression rate: 11% in median follow up of 6 years) that evolved to MDS or AML (median time to progression: 3.2 yrs.; transplanted patients were not censored). Cytogenetic analysis upon progression showed abnormal karyotype in 83% of cases; 7% had complex karyotype and -7/del(7q) was present in 62% of cases. The presence of a PNH clone was detected in 17% of cases that transformed to sMDS vs. 35% in non-progressors (P=.1). For comparison, we have also analyzed primary de novo cases of MDS (pMDS) with (N=94) and without (N=557) -7/del(7q). In contrast to sMDS, -7/del(7q) was present in 14.4% of cases in pMDS. Because sMDS following AA or PNH included a high proportion of patients with -7/del(7q), we compared sMDS with -7/del(7q) to pMDS with -7/del(7q) for coexisting mutational events. Mutations in RUNX1, CBL, SETBP1 and ASXL1 appeared to be more frequent in sMDS vs. pMDS (28.6% vs. 2.1%, 21.4% vs. 2.1%, 21.4% vs. 5.3%, 21.4% vs. 10.6%, P=.003, P=.02, P=.07, P=.37, respectively). In contrast, TP53 and DMT3A were more common in pMDS (7.1% for sMDS vs. 17%, 0% for sMDS vs. 8.5%, P=.69, P=.59). Similarly, there were several other distinctive differences between all sMDS and pMDS irrespective of the cytogenetics: mutations in SF3B1, SRSF2, NPM1, DNMT3A were common in primary AML but entirely absent from cases after AA; mutations in RUNX1 and SETBP1 appeared to be more frequent in sMDS vs. pMDS (26.3% vs. 8.3%, 21.1% vs. 3.2%, 15.8% vs. 3.9%, P=.03, P=.005, respectively). Whole exome NGS was performed after progression, with confirmed somatic mutations subsequently tracked back by targeted deep NGS applied to serial samples starting at initial presentation. Confirmed mutational events and chromosomal aberrations were found in 19/36 patients with sMDS; 17/19 cases of sMDS had at least 1 confirmed somatic mutation. Remarkably, in retrospective analysis in 6/7 cases studied serially, at least one of the identified mutations was detectable at presentation when deep targeted sequencing (depth 5,000~20,000 reads) was performed. In 5 of these cases the alterations appeared to be ancestral events for sMDS evolution. When anadditional 77 AA or PNH cases were studied by deep sequencing, somatic mutations were present in 48% of AA patients at presentation. Detection of clonal events at presentation was associated with an increased risk of subsequent MDS evolution (14/37 mutant cases vs. 3/40 nonclonal cases evolved, P=.002). Mutations found at both initial presentation and upon evolution were suggestive of a slow expansion of previously cryptic clones (ASXL1, CUX1, TET2, CBL, RUNX1, and SETBP1). Patients with these genes (n=18) had worseoverall survival compared to patients without these mutations (P=.03). To assess the potential impact of immunosuppressive therapies (IST), we also investigated a subset (out of 77) of 53 patients (39 responders and 14 refractory cases) following IST. Clonal somatic events were identified in 27 of them, but there was no association between the response to IST and somatic mutations at presentation. Our results demonstrate that while subclonal mutations indicative of oligoclonal hematopoiesis are frequent in AA, the presence of permissive ancestral somatic events at the outset of AA predisposes patients to sMDS, a feature that had diagnostic and prognostic implications. Disclosures Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2μM administered 2x/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy. Fil: Lavelle, Donald. University of Illinois at Chicago; Estados Unidos. Jesse Brown VA Medical Center; Estados Unidos Fil: Vaitkus, Kestis. University of Illinois at Chicago; Estados Unidos. Jesse Brown VA Medical Center; Estados Unidos Fil: Ling, Yonghua. Ohio State University; Estados Unidos Fil: Ruiz, Maria A.. University of Illinois at Chicago; Estados Unidos Fil: Mahfouz, Reda. Cleveland Clinic; Estados Unidos Fil: Peng Ng, Kwok. Cleveland Clinic; Estados Unidos Fil: Negrotto, Soledad. Cleveland Clinic; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Smith, Nicola. National Cancer Institute; Estados Unidos Fil: Terse, Pramod. National Cancer Institute; Estados Unidos Fil: Engelke, Kory J.. Avanza Laboratories; Estados Unidos Fil: Covey, Joseph. National Cancer Institute; Estados Unidos Fil: Chan, Kenneth K.. Ohio State University; Estados Unidos Fil: DeSimone, Joseph. University of Illinois at Chicago; Estados Unidos. Jesse Brown VA Medical Center; Estados Unidos Fil: Saunthararajah, Yogen. University of Illinois at Chicago; Estados Unidos. Cleveland Clinic; Estados Unidos

Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the deoxycytidine analogue decitabine that deplete the chromatin-modifying enzyme DNA methyl-transferase 1 without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine upregulated the key late differentiation factors CCAAT enhancer-binding protein and p27/cyclin dependent kinase inhibitor 1B (CDKN1B), induced cellular differentiation and terminated AML cell cycle, even in cytarabine-resistant p53-and p16/CDKN2A-null AML cells. Leukemia initiation by xenotransplanted AML cells was abrogated but normal hematopoietic stem cell engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S phase specific decitabine therapy. In xenotransplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared with conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy. © 2011 Macmillan Publishers Limited All rights reserved. Fil: Ng, K. P.. Cleveland Clinic; Estados Unidos Fil: Ebrahem, Q.. Cleveland Clinic; Estados Unidos Fil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Cleveland Clinic; Estados Unidos Fil: Mahfouz, R. Z.. Cleveland Clinic; Estados Unidos Fil: Link, K. A.. Cincinnati Children’s Hospital; Estados Unidos Fil: Hu, Z.. Cleveland Clinic; Estados Unidos Fil: Gu, X.. Cleveland Clinic; Estados Unidos Fil: Advani, A.. Cleveland Clinic; Estados Unidos Fil: Kalaycio, M.. Cleveland Clinic; Estados Unidos Fil: Sobecks, R.. Cleveland Clinic; Estados Unidos Fil: Sekeres, M.. Cleveland Clinic; Estados Unidos Fil: Copelan, E.. Cleveland Clinic; Estados Unidos Fil: Radivoyevitch, T.. Case Western Reserve University; Estados Unidos Fil: MacIejewski, J.. Cleveland Clinic; Estados Unidos Fil: Mulloy, J. C.. Cincinnati Children’s Hospital; Estados Unidos Fil: Saunthararajah, Y.. Cleveland Clinic; Estados Unidos

Apoptosis was induced in immature and mature sperm in the presence or absence of poly (ADP-ribose) polymerase (PARP) inhibitor. The association of cleaved (cPARP) with sperm morphology was examined using sperm deformity index (SDI) score. The SDI scores are associated with PARP cleavage as an early marker of apoptosis.

To standardize the TUNEL assay by establishing inter- and intraobserver variability, interassay variability, cutoff values, sensitivity and specificity of the assay, and studying the distribution of the DNA damage in a population of infertile men referred to a clinical andrology laboratory.Seminal ejaculates from 25 healthy male volunteers (controls) and 194 infertile men (with male factor infertility) referred to an andrology laboratory were examined for DNA damage by TUNEL assay using flow cytometric analysis.Both the inter- and intraobserver variability and interassay variability was small (10%). DNA damage in the controls was 11.9 ± 6.8% vs. 29.5 ± 18.7% in patients (P.001). The cut-off value of 19.25% maximized the observed sensitivity (64.9%) and specificity (100%) of the assay. The distribution of DNA damage in the patients was as follows: 14.9% (29 of 194) with DNA damage between 0% and 10%; 22.7% (44 of 194) between 10% and 20%; 8.8% (17 of 194) between 20% and 30%; and 17.5% (34 of 194) between 30% and 40%. Finally, 27.3% (53 of 194) had TUNEL values40%.We report a detailed standardization of the TUNEL assay for clinical use, as well as reference ranges for DNA damage in normal healthy donors and infertile men. A cut-off of 19.25% with observed 100% specificity established in our program can differentiate infertile men with DNA damage from healthy men. This test can be offered to infertile patients who are idiopathic, have severe oxidative stress-related abnormal semen quality, and contribute to the infertility problem of the couple who are considering assisted reproductive techniques.

The objective of this study was to elucidate the missense Glu298Asp polymorphism within exon 7 of the endothelial nitric oxide synthase (eNOS) gene in infertile men with astheno- zoospermia and its potential role in sperm motility. In this prospective controlled study conducted in our andrology unit, we investigated the frequency of the 894G.T polymorphism (Glu298Asp variant) within exon 7 of the eNOS gene in 70 infertile men and 60 healthy men. Sperm motion kinetics were assessed with computer-assisted semen analysis. The presence of G.T, a single nucleotide polymorphism (SNP) in exon 7 of the eNOS gene (NCBI SNP cluster rs1799983; GenBank accession number NG_011992; protein accession number NP_000594) was determined by allele- specific polymerase chain reaction followed by restriction fragment length polymorphism analysis. Sequencing analysis was used to confirm the specific genotype. The 894G.T eNOS allele (T) was found at a higher frequency in the patients with asthenozoospermia (60% vs 22.5% in the control group; P 5 .02). The percentage of progressive motile sperm (grade a + b) was lower in the asthenozoospermic infertile men with the homozygous eNOS (TT) genotype than in the wild-type eNOS (GG) (P 5 .02) and heterozygous eNOS (GT) genotypes (P 5 .01). However, the percentage of progressive motile sperm (grade a + b) was higher in the wild-type vs mutant eNOS (TT) (P 5 .03) and heterozygous eNOS (GT) genotypes (P 5 .04). Our findings suggest that the T allele encoding for aspartic acid of the eNOS (Glu298Asp) gene may contribute to poor sperm motility.