ALBERT

Description: Dyskerin gene is mutated in patients with X-linked dyskeratosis congenita (X-DC), which results in greatly reduced levels of telomerase activity. A genetic suppressor element (GSE) termed GSE24-2 has been isolated in a screening for cisplatin resistance. GSE24-2–expressing cells presented impaired telomerase inhibition following in vitro exposure to chemotherapies, such as cisplatin, or telomerase inhibitors. The promoter of the telomerase component hTERT was constitutively activated in GSE24-2 cells in a c-myc expression–dependent manner. Deletion analyses and mutagenesis of the human c-myc promoter demonstrated that the target sequence for activation was the nuclease hypersensitive element-III (NHEIII) site located upstream to the P1 region of the promoter. Further, expression of GSE24-2 in cell lines derived from patients with X-DC and in VA13 cells induced increased hTERT RNA and hTR levels and recovery of telomerase activity. Finally, expression of GSE24-2 was able to rescue X-DC fibroblasts from premature senescence. These data demonstrate that this domain of dyskerin plays an important role in telomerase maintenance following cell insults such as cisplatin treatment, and in telomerase-defective cells in patients with X-DC. The expression of this dyskerin fragment has a dominant function in X-DC cells and could provide the basis for a therapeutic approach to this disease.

Description: Key Points The mechanism of bone marrow failure (BMF) in PNH is not known. Novel CD1d-restricted, GPI-specific T cells are present in PNH patients and might be responsible for BMF.

Description: INTRODUCTION: A sizable proportion of elderly acute myeloid leukemia (AML) patients receive frontline hypomethylating agents (HMAs), namely azacitidine (AZA) and decitabine (DAC), as they are deemed unfit for intensive chemotherapy (ICT) by their treating physicians. A foreseeable high early death (ED) rate and lack of overall survival (OS) benefit under ICT are the main drivers for this decision. Several groups have published different predictive tools for ED or OS in elderly patients receiving ICT but, since ED in patients treated by HMAs is lower, the research activity has been restricted to OS in this population. METHODS: 415 elderly AML patients (264 M, 152 F) aged 61-90 receiving frontline HMAs (AZA 297, DAC 118), either in daily practice or within clinical trials (AZA 27, DAC 17), with complete relevant clinical information (see Table I) were available from the PETHEMA epidemiologic AML registry (NCT02006004). We analyzed the predictive value for ED (8wk) of the prognostic factors for OS/ED in AML included in the Walter, MRC/LRF, ALFA and ALMA scoring systems, namely age, WBC count, performance status (PS), MRC 2010 cytogenetics, platelet count and secondary disease, as well as the type of HMA. The potential predictors were categorized following previous published models (Walter, MRC/LRF, ALFA, ALMA). Cumulative early death rate at 8 weeks was calculated by the life-time method and the relevant strata were tested for univariate significance by the Wilcoxon test. All significant covariates were included in a Cox multivariate regression model and those significant for death at 8wk were included in a new predictive tool (HMA-EDS). Patients were assigned randomly in a 1:1 ratio to a training cohort (TC) and a validation cohort (VC). The different scoring systems (Walter, MRC/LRF, ALFA, ALMA, HMA-EDS) were checked for their prognostic impact on ED. Finally the 95% CI for the expected death rate at 8wk for the different strata of the new model was calculated for the full patient series. RESULTS: 51 patients out of 415 died and 13 were lost to follow-up before day 56 (cumulative ED rate at 8wk 13%, 95%CI 9-17%). Age, cytogenetics, secondary AML, platelet count and type of HMA were not significantly associated to ED. PS and WBC count strata confirmed their prognostic utility both in univariate and multivariate analysis (Table II). We developed the HMA-EDS by adding WBC (cutoffs 10 and 50, scores 1/2/3) and PS (0-1/2-4, scores 0/1) that classified patients in low-risk (score 1-2/ 84.6% of patients) and high risk (3-4/ 15.4% of patients) strata. When the prognostic utility for ED in the TC and the VC for the different scoring AML systems were checked, only HMA-EDS predicted ED in both cohorts (see Table III). The new EDS discriminates 2 different strata for ED at 8wk in unfit AML patients treated by HMA (see Figure 1 & Table III), namely a lower-risk group (ED rate 10%, 95% CI 6-14) and a high-risk group (ED rate 26%, 95% CI 14-38). CONCLUSIONS: WBC count and PS are the main predictors for ED in unfit AML patients treated by HMAs. A new tool (HMA-EDS) discriminates two different risk groups and supersedes other previously published prognostic systems (Walter's, Wheatley's MRC/LRF, ALFA and ALMA) for this purpose. This score could be useful to select patients for front-line HMA or even HMAs-based combination therapies, given that several cycles are usually needed to achieve a clinical response. We suggest that other patient-related covariates such as geriatric assessment be checked in future studies. Disclosures Ramos: Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria; Abbvie: Honoraria. Fernandez:Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Umbilical cord is an extra-embryonic-annex rich of both hematopoietic stem and progenitor cells (HSPC) and mesenchymal stem cells (MSC) and it is easily accessible. The HSPC derived from umbilical cord blood (UCB) are promising as graft for allogeneic bone marrow (BM) transplantation and as source of target cells for autologous HSPC gene correction. UCB-HSPC have several advantages compared to adult ones: a less risk of graft-versus-host disease, a higher frequency of progenitors with a greater clonogenic potential and more susceptibility to be transduced by lentiviral vectors. Nonetheless, the HSPC yield from single cord blood unit is not sufficient for these clinical approaches in adults. Therefore, ex-vivo expansion of HSPC in media supplemented by cytokines and/or in vitro culture systems with feeder layers, is a valid approach to exceed this limit. MSC are a component of BM-microenvironment that play a key role in supporting of hematopoiesis by ability to secrete soluble factors and probably by the direct cell-cell interaction too. In this work, we investigated the ability of umbilical cord extracellular matrix-MSC (Wharton's Jelly-MSC) to support the ex-vivo expansion of UBC- purified CD34+ cells. In particular, we evaluated the fold increase, and the frequency of CD34+ cell and CD34+subtypes during expansion at the following culture conditions: by direct contact with WJ-MSC layer, by exposure to the soluble factors secreted by WJ-MSC layer in transwell system. The fold expansion was compared with the CD34+ cells expanded in a customized serum-free medium. CD34+ cells were isolated by immuneselection from 8 fresh UCB. The WJ-MSC were isolated from UC cut-pieces by non-enzymatic procedure but thanks to their capacity to migrate to plastic substrate. At the confluence of 60-70% the WJ-MSC were treated with mytomicin-C to arrest the cell cycle. After 48h, the immune-selected CD34+ cells were seeded in WJ-MSC at the density of 5-10 x104 in 12 well plates by direct or indirect contact (by transwell system). CD34+cells were grown in absence of feeder layers at the same conditions. Early hematopoietic cytokines (Flt-3, TPO, SCF) were supplemented in all three conditions and freshly replaced every two days of culture. Numbers and frequency of CD34+cells were evaluated according to ISHAGE method and CD34+ subtyping was performed by four color method to investigated the co-expression of the primitive surface antigens (CD38, CD133, CD90). The frequency of CD34+ cells at day 5 of culture decreased only 10% and was about 50% after 8 days of culture in conditions. The expansion of CD34 + cells at direct contact with WJ-MSC was superior (5.5 fold increase) compared to that of the other two conditions (3 fold on average). At day 8of culture, the CD34+ cells expanded 12 fold at direct contact with feeder layer, about 7 fold in a transwell system and 6 fold in basic medium. No substantial differences in the grade of expansion was revealed in heterologous vs homologous co-cultures of HSPC/WJ-MSC. Noteworthy is that in the contact system in addition to the fluctuating CD34+ cells harvested from the medium (floating CD34+ cells), we found approximately 50% of the total CD34+ cells be adherent to WJ-MSC layer, these cells were released only after enzymatic proteolytic treatment. Subtyping the CD34+cell population growing in contact to the WJ-MSC or in the conditioned medium we found that the CD34+/CD133+cell population was maintained high (72% ±12 over the total CD34+ cells) as in unmanipulated CB-HSC. The CD34+CD38- cells decreased by 2,5 fold in both systems, as early as day 5 of culture. However, in the contact system this population was 3 times more represented in the attached CD34+ cell fraction. The CD34+/CD90+ subtype was also expanded (more than 8 fold) particularly in the attached fraction, as early as 5 days of culture and was maintained to the end WJ-MSC supported ex-vivo HSPC expansion with superior effect in a cell contact system. Two phenotypically different populations of HSPC developed in this system with an increased frequency of CD34+ cells that co-expressed markers typical of more early progenitors in the attached CD34+ cell fraction. We are assessing the significance of these differences by performing molecular and functional studies of WJ-MSC-supported HSPC. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

Description: Key Points This study strengthens the previous observation of elevated mitochondrial DNA copy number and future risk of chronic lymphocytic leukemia.

Description: Introduction: Studies have shown that a large monoclonal protein (MP) and immunoparesis in MGUS/SMM have predictive value of progression since they may be indirectly related to the degree of clonal expansion of bone marrow plasma cells (BMPC). A recently available assay, the Hevylite® (HLC), has allowed a more precise determination of MP and the quantification of isotype-matched immunosuppression (i.e. suppression of the monoclonal isotype but of the alternative light chain) which has shown prognostic value in some studies. In this study, we aim to evaluate the association between the alteration of the HLC parameters in MGUS patients with already known prognostic factors, but also, with less studied biomarkers such as circulating clonal plasma cells (cCPC) by Next Generation Flow (NGF) (Flores-Montero et al., Leukemia, 2017). Methods: A total of 175 MGUS patients diagnosed between October 2008 and September 2015 were included in the study. The median follow-up was 64 months (range: 1-100 months). MGUS and MGUS progression were defined according to the International Myeloma Working Group (IMWG) criteria. Clinical records were retrieved for all patients. HLC determinations were carried on a SPA+ turbidimeter analyzer and using specific reagents (Binding Site®, UK). The 6 HLCs pairs (IgGk, IgGl, IgAk, IgAl, IgMk and IgMl) were analyzed in all samples. HLC normal ranges were defined by the laboratory based on normal sera. Statistical analysis was done using IBM-SPSS-22. Results: HLC ratio was altered in 56.3% of the 111 patients with available sera (47.5% for IgG MGUS and 73.9% and 87.5% for IgA and IgM), respectively (p = 0.026). Patients with altered HLC ratio presented an MP significantly higher (Table 1) than those with normal ratio (0.62 g/dL vs 0.33 g/dL, p1.5 g/dL (p = 0.044)). Interestingly, it also correlated with a greater frequency and a greater amount of cCPC by NGF (68.29%, p=0.001; 0.2864 /mL vs 0.0139 /mL p=0.0001). Regarding BMPC infiltration, there was a correlation between altered HLC ratios and greater percentages of infiltration of BMPC (3.72% vs. 2.43%, p = 0.001) and higher percentage of BMPC measured by multiparameter flow cytometry (MFC) (54.42% vs 24.42%, p 1.5 mg/dL (100%, p = 0.044), non-IgG (78,12%, p = 0.003) and ratio of altered CLL (77,77%, p = 0.011). Patients with normal HLC ratios correlated with other low risk of progression to MM parameters, such as a MP

Description: Background We conducted an investigator-driven, multicenter, open label, randomized study to establish whether the source of factor VIII (FVIII) replacement (plasma-derived, pd; or recombinant, r) affects the rate of inhibitory alloantibodies in previously untreated patients (PUPs) with severe hemophilia A. Methods Between 2010 and 2014, 303 PUPs who provided consent through their tutors were screened at 42 participating sites in 14 countries from Africa, the Americas, Asia and Europe. The original aim was to screen 300 patients, randomize 270 (10% screening failure) and follow them for 50 exposure days (ED) or 3 years. Once the intended numbers were included, follow-up was terminated due to logistic and budgetary reasons. Screening criteria were age 5 treatments with blood components and 10 were not infused after randomization. The remaining 251 patients were analysed and 35 had truncated follow-up (25 dropout, 10 study termination). Patients were aged 0-81 months at randomization (median 14 months) and received between 1 and 50 infusions of FVIII concentrates (median 22). Of those who did not develop an inhibitor, over 70% had 〉20 ED. 76 patients developed an inhibitor, of which 50 were high-titred. The cumulative inhibitor incidence was 35.4% (95% confidence interval (CI95) 28.9-41.9%). 90% of inhibitors developed within 20 EDs, both for all and high-titre inhibitors. After randomization 125 patients received pdFVIII and 126 rFVIII. The putative confounders were equally divided between the two product class arms. There were 29 inhibitors (20 high-titred) in the group treated with the class of pdFVIII and 47 (30 high-titred) in those treated with rFVIII. The cumulative inhibitor incidence was 26.7% (CI95 18.3-35.1%) for pdFVIII and 44.5% (CI95 34.7-54.3%) for rFVIII (Figure). For high-titre inhibitors the cumulative incidence was 18.5% (CI95 12.1-26.9%) for pdFVIII and 28.4% (CI95 19.6-37.2%) for rFVIII. By univariate Cox regression analysis rFVIII was associated with an 87% higher incidence of inhibitors than pdFVIII (hazard ratio (HR) 1.87, CI95 1.18-2.97). For high-titre inhibitors the rate was 70% increased (HR 1.70, CI95 0.96-2.99). The associations did not materially change after adjustment for putative confounders: in adjusted models the rate remained 70-90% elevated for rFVIII vs pdFVIII. When analysis was restricted to sites that had not randomized patients to a second generation full length rFVIII or pdFVIII (n=131 patients, 25 inhibitors), the risk of other rFVIII concentrates vs pdFVIII was still twofold increased (HR 1.99, CI95 1.00-3.99). Conclusions The rFVIII product class was associated with a 1.87-fold higher incidence of inhibitors than the pdFVIII class. This difference remained even when second generation full length rFVIII concentrate was excluded from the analyses. The results of this randomized study have implications in the choice of product for management of PUPs, as inhibitor development remains a major challenge in the management of haemophilia A. (Funded by the Angelo Bianchi Bonomi Foundation, Italian Ministry of Health, Grifols, Kedrion and LFB - Registed at EudraCT 2009-001186-88). Figure 1. Figure 1. Disclosures Peyvandi: Octapharma: Other: Investigator; LFB, Kedrion, Novonordisk, Bayer, Roche, CSL Behring.: Consultancy, Honoraria, Research Funding. Mannucci:Novonordisk, Grifols, Kedrion, Bayer, Biotest, Baxalta: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Karimi:Octapharma: Other: Investigator. Young:Baxter, Grifols: Consultancy, Honoraria. Santagostino:Roche: Speakers Bureau; Bayer: Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Octapharma: Speakers Bureau; Biotest: Speakers Bureau; Novo Nordisk: Speakers Bureau; Kedrion: Speakers Bureau; Biogen/Sobi: Speakers Bureau; CSL Behring: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau. Mancuso:Baxter, Pfizer, CSL Behring, Baxter, Sobi/Biotest: Consultancy; Novo Nordisk, Bayer: Speakers Bureau. Mahlangu:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Research Funding; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bonanad:Baxalta: Research Funding. Ewing:Baxter, Novo Nordisk, Grifols, Bayer, Kedrion: Honoraria. Owaidah:King abdulaziz city for science, Novo Nordisk, Bayer: Honoraria, Research Funding. Kobrinsky:Octapharma: Speakers Bureau; CSL Behring: Speakers Bureau; Sanofi: Speakers Bureau; Kedrion Biopharma: Membership on an entity's Board of Directors or advisory committees. Kavakli:Baxter: Other: advisory board member and received educational and investigational support; Bayer: Other: advisory board member and received educational and investigational support; Novo Nordisk: Other: advisory board member and received educational and investigational support; Pfizer: Other: advisory board member and received educational and investigational support; Bio Products Laboratory: Other: received educational and investigational support; CSL Behring: Other: received educational and investigational support; Octapharma: Other: received educational and investigational support. Manco-Johnson:Baxter, bayer, biogen, CSL Behring, NovoNordish: Honoraria. Neme:Novo Nordisk and Pfizer: Other: fees for speaking. Wicklund:NovoNordisk, Bayer, Baxter (now Baxalta), Biogen-Idec, CSL-Behring, National Hemophilia Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees. Zulfikar:Eczacýbaþý-Baxter, Pfizer, Novo Nordisk: Consultancy, Honoraria, Research Funding.

Description: Transfusions (Tx) of Ultraviolet B (UVB)-irradiated peripheral blood mononuclear leukocytes (MNL) have been shown to induce humoral immune tolerance to major histocompatability complex (MHC) antigens (Blood 88:4375, 1996). To determine whether cellular immune tolerance to MHC antigens can be induced by the same approach, transplantation of bone marrow and spleen cells from tolerant donors across the H-2 barrier was conducted to study its effect on prevention of graft-versus-host disease (GVHD). After immune tolerance induction by four weekly Tx of UVB-irradiated BALB/c (H-2d) peripheral blood MNL into CBA/HT6 (H-2k) mice, bone marrow cells (BMC) and spleen MNL from tolerant or naive CBA mice were transplanted into lethally irradiated BALB/c mice. The transplanted mice were followed by measuring body weight, peripheral leukocyte counts, GVHD, survival, and cytokine response. All BALB/c recipient mice were fully engrafted with H-2k CBA donor cells after transplantation. The severity of GVHD was significantly attenuated in BALB/c mice transplanted with BMC and spleen MNL from tolerant CBA donor mice. The recovery of peripheral leukocyte and lymphocyte counts were faster and more complete in mice transplanted with cells from the tolerant donors. The serum cytokine profile after transplantation with tolerant donor cells showed increased interleukin-4 and reduced gamma interferon that are consistent with a polarized Th2 response. The results pooled from three separate experiments showed that BALB/c mice transplanted with 5 × 106 BMC and 4 × 105spleen MNL from tolerant CBA donors had better overall survival than the control group (72% v 17%, P = .018). The findings show that transplantation with bone marrow and spleen cells from tolerant H-2 disparate donor mice is associated with significant attenuation of GVHD and better outcomes. The results also support that transfusions of UVB-irradiated leukocytes may induce cellular immune tolerance.