ALBERT

Description: Abstract 4559 Objective: Allogenetic hematopoietic stem cell transplantation (Allo-HSCT) can be used to cure some genetic leukodystrophy patients in current. This clinical study is to investigate efficacy and practicability of Allo-HSCT in treatment of genetic leukodystrophy in China. Methods: Three boy diagnosed genetic leukodystrophy (2 cases of metachromatic leukodystrophy, 1 case of X□|linked adrenoleukodystrophy) had undergone allogeneic Stem cell Transplantation. A 32 month old boy accepted HLA 8/10 high-resolution matched unrelated umbilical cord blood as graft, which contained nucleated cells 13.5×107/kg. A 2 year-old boy accepted HLA high-resolution 10/10 matched unrelated peripheral blood stem cells (PBSC) as graft which contained mononuclear cells 8 ×108/kg. The conditioning regimen consisted of cyclophosphamide (60–100 mg/kg), fludarabine (150–200mg/kg), TT (5mg/kg) and busulfan (12–13.2 mg/kg). A 12 year-old boy accepted sibling HLA low resolution 6/6 (A, B, DRB1) matched bone marrow as graft which contained mononuclear cells 1.23 ×108/kg. The conditioning regimen consisted of busulfan (16mg/kg) and cyclophosphamide (200mg/kg). Cyclosporine A and mycophenolate mofetil were used to prevent graft versus host disease (GVHD). ATG-Fresenius (15 mg/kg) and low dose of methotrexate were added for prophylaxis of GVHD in unrelated PBSC transplantation. XY chromosome by the FISH method or quantitative PCR for short tandem repeat (STR) were used to identify the chimerism. ASA level in peripheral blood leukocytes and C26:0 plasma level were monitored after transplantation respectively. Results: Conclusion: Allogenetic hematopoetic stem cell transplantation is effective and feasible in the treatment of some childhood genetic leukodystrophy patients. These patients should accept allo-HSCT in the early stage of disease in order to get good survival. Disclosures: No relevant conflicts of interest to declare.

Description: Current study analyses the effect of splenectomy on outcomes of hematopoietic stem cell transplantation (HSCT) for patients with β-thalassemia major (TM). Twenty-two class II and III (according to Nanfang’s criteria, see Blood, 2012;120 (19): 3875-3881) patients with TM had a pre-transplant splenectomy. The outcomes of the 22 transplants were compared with 193 transplants in class II and III patients between Aug. 2008 and Dec. 2013. Patients in the splenectomy group were older (8.9±5.0 vs. 6.0±3.5 year old; p=0.001) and class III patients were more (8/22 vs. 10/193, p=0.001) than non-splenectomy. In splenectomy group 10 patients received graft from HLA matched sibling, 9 from unrelated donors and 3 from haploidentical parents plus unrelated cord blood, respectively. In non-splenectomy group 82 patients received graft from matched sibling and 121 from unrelated donors, respectively. 213 cases engrafted in 4 weeks, Splenectomized patients had a significantly shorter time to absolute neutrophil count 〉500/mm3 (14.5±5.2 vs 19±4.2 days p=0.001). There were no significantly different at time to platelet 〉2000/mm3 (16.2±5.8 vs 16.1±5.3 days, p=0.415) and hemoglobin 〉80g/L (15.3±4.8 vs 13.1±5.3 days p=0.135). Secondary rejection occurred in 2 patients (at 3rd month and 4th month post transplantation, respectively) in splenectomy group, but rescued successfully by second transplants from unrelated donor one year later and from haploidentical parent two years later, respectively. The patients received graft from father, one died of complications of VOD; other died of lung infection on forty-five days post transplantation. Two patients rejected their grafts in non-splenectomy group. The mean RBC transfusion was 4u (0u-12u). The mean platelet transfusion in splenectomy group had reduced (4.4±4.5 vs 7.1±4.8 units p=0.016), with 17 patients transfused less than 6u Platelet. The incidence of acute and chronic GVHD, late infections were rare and not significantly different between the two groups. No patients died of infection due to splenectomy. Patients underwent HSCT after splenectomy had a high rate of OS (95.5% vs 92.8% p=0.001). Although TFS was lower in splenectomy group than non-splenectomy group (88.5% vs 92.3% p=0.001), this may be due to more older, class III patients and higher rate of rejection (2/22 vs 2/193). Conclusion, our study showed pre-transplant splenectomy patients with β-thalassemia major was associated with faster engraftment and decreased require of platelet and bloodtransfusion support. Disclosures No relevant conflicts of interest to declare.

Description: Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell (HSC) disorder that manifests as hemolytic anemia, venous thrombosis, and deficient hematopoiesis. PNH concomitant with inherited hemolytic anemia has been rarely reported. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the only curative therapeutic measure. Here, We present our first allo-HSCT form unrelated donor for a 23-year-old patient with PNH and heterozygous α-thalassemia. Case report: Flow cytometry of the patient's peripheral blood demonstrated that 7.9% and 11.9% of the erythrocytes were CD59 and CD55 deficient, respectively. Thalassemia gene analysis revealed heterozygous α-thalassemia. The patient was diagnosed with PNH and heterozygous α-thalassemia, and became transfusion dependent 1 year later and was admitted to our hospital in March 2011. Conditioning was consisited of Cyclophosphamide (d-10 to d-9), Fludarabine(d-8 to d-4), Busulfan (d-7 to d-5), Thiotepa (d-4) and ATG-F (d-3 to d-1). Stem cell source was peripheral blood stem cells (PBSC) harvested on the 16th October 2011 from a HLA-matched (8/8) unrelated donor. The infused mononuclear cell (MNC) and CD34+cell does were 8×08/kg and 1.38×106/kg body weight of the recipient, respecitively.The time to platelets engraftment and leukocyte engraftment was 21 and 25 days,respecitively.The patient did not accoured acute graft versus host disease (aGVHD). Out-patient were followed up for 2 years, the patient's liver and kidney function, urine which showed normal, as well as reviewed the implant remains for independent plantentry. Conclusions: The present case suggests that allo-HSCT can be used to cure patient with PNH and heterozygous α-thalassemia. Allo-HCT from unrelated donors is a new and potentially curative option for patients with PNH and inherited hemolytic anemia. Disclosures No relevant conflicts of interest to declare.

Description: Hepatic veno-occlusive disease (VOD) is a common complication of hematopoietic cell transplantation (HCT), especially patients with β-thalassemia major (TM). To estimate whether incidence of VOD recently decreased after using NF-08-TM protocol (NP), 311 TM-HCTs performed from February 2003 to June 2013 were analyzed. 241 patients received NP in or after 2009 and 70 received non-NP before 2009. VOD was diagnosed by Seattle criteria (SC) or Baltimore Criteria (BC). Patients were stratified by Nanfang (NF) criteria. A total of 31(10.0%) and 14 (4.5%) HCTs developed VOD (6 and 5 developed severe VOD) in SC and BC cohorts, respectively. The incidence of VOD was significantly lower in NP versus non-NP groups and in Class 2 versus Class 3 groups. Overall survival was significantly higher in NP versus non-NP cohorts. Rate of VOD in alternative donor transplant (ADT) was similar to that in matched sibling transplant (MST). Requirement of platelet and value of D-dimer significantly increased in the VOD patients. Our study showed the incidence of VOD significantly decreased in our center after using NP. ADT was similar to MST on rate of VOD. NF criteria of stratification can indicate occurrence of VOD. The SC can be more suitable criteria for early diagnosis. Disclosures No relevant conflicts of interest to declare.

Description: Background: Lymphoepithelioma-like carcinoma (LELC) is a rare high-grade carcinoma that can occur throughout the body. It is usually associated with Epstein-Barr virus (EBV) infection in patients from Asian countries. Pulmonary LELC occurs mostly in Asian women,, in their fifth or sixth decade, with no previous history of smoking. Optimal treatment has not been clearly established. Treatment options are based on surgery for early stage and on multimodal therapy for advanced stages.There is no report on children treated with programmed cell death 1 (PD1) inhibitor. Case Description: Case 1, a 12-year-old female who was admitted for cough and serious neck swelling. PET-CT showed multiple lymph nodes enlargement in the whole body ;among them, the anterior mediastinal lesions were fused with each other and the boundary between adjacent large vessels was unclear; multiple lung and pleural metastasis . EB virus in blood was 7.48×104copies/mL.Bone marrow morphology: Naive lymphocytes account for 2.5%, some lymphocytes are irregular; Mature plasma cells easy to see.Bone marrow immunotypes: 0.56% CD19-CD5-CD10-abnormally mature B lymphocytes. Biopsy: (left neck lump) LELC, EBERs(+).Partial remission was achieved after 2 courses of paclitaxel / carboplatin/ apatinib protocal. Due to delay with varicella, the tumor came back. After the third course of treatment,she showed chest tightness and pleural effusion, EB virus in blood increased, PDL1 protein was TPS 80% positive. After one PD1 antibody combined with chemotherapy,lymph nodes and thoracic lesions significantly reduced (Image1-3), blood EB virus turned to negative. She is now continuing with PD1 inhibitor. Case 2 , 10-year-old boy with huge right mouth bottom and right neck mass was diagnosed as LELC with EBER(+), TP53 (3%, +), CMYC (80% +). After chemotherapy, surgery and radiotherapy,he achieved complete remission. However, multiple metastases in mediastinum lymph nodes, pleura and lung happened and surgery, chemotherapy followed by automatic stem cell transplantation and pulmonary radiotherapy were given. CT-led puncture biopsy of suspected pulmonary residual lesion showed chronic inflammation with the mild growth of the alveoli, no malignant tumor.The child took oral Tegafur as maintenance regimen. 2 months later, recurrence appeared in lymph nodes, lung ,pleural and acetabular with positive EB virus in pleural effusion. Gene detection with NGS, which includes 312 gene-wide exon region, 208 gene hotspots and 16 fusion genes, did not show mutations such as gene mutations, amplification, and fusion with a clear clinical correlation. Tumor mutation load (TMB) was low, however,the boy had attained good response after PD1 antibody followed by rescue regimen and negative blood EB virus .The 2 patients didn't develop side effects correlating with PD1 inhibitor. Conclusion: To our knowledge, this is the first report of the use of PD1 inhibitor in children with metastatic LELC. These 2 refractory cases were all Epstein-Barr virus (EBV)-driven LELC,are more likely to respond to PD-1 blockade regardless of PD1 expresssion or TMB. EB virus burden disappeared after PD1 blockade,which perhaps can explain the anti-tumor effect.More cases are needed to verify the potential benefits of PD1 inhibitors to treat refractory LELC in children. Figure Disclosures No relevant conflicts of interest to declare.

Description: Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM. We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013). To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (〉1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (〉1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p

Description: High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.

Description: 14-3-3 proteins are chaperone and scaffold proteins that exert a widespread influence on cellular processes through binding to serine/threonine-phosphorylated residues on target proteins, forcing conformational changes or influencing their interactions with other molecules. Altered 14-3-3 expression is associated with development and progression of cancer. We therefore evaluated the status of all 14-3-3 isoforms in plasma cells disorders in publically available gene expression profiling (GEP) data. Using independent patient datasets, we observed a consistent higher expression of YWHAE (coding gene for the isoform 14-3-3ε) in MM and plasma cell leukemia (PCL) patients, while no consistent differences were observed with the other isoforms. Moreover, we also confirmed higher expression of YWHAE in our RNA-seq data from 420 newly-diagnosed MM patients, with relatively low expression in normal plasma cells. Finally, 14-3-3ε was also found to be constitutively expressed at protein level in primary patient MM cells and in a large panel of MM cell lines, with significantly lower expression in healthy donor B cells. To evaluate if 14-3-3ε represents a functional dependency in MM, we performed genetic perturbation of YWHAE in a panel of MM cell lines. Depletion of YWHAE using 3 different shRNA inhibited cell proliferation and induced cell apoptosis across 5 different cell lines, independently of their genetic background. We next performed CRISPR-Cas9-mediated YWHAE knock out (KO) in H929 and JJN3 cells and observed a significant decrease in cell viability and a robust apoptotic response. H929 YWHAE KO cells infected with FLAG-YWHAE addback lentiviral construct completely rescued this phenotype, confirming that loss of YWHAE is responsible for the defective cell viability and apoptotic phenotype. These observations were corroborated by ectopic overexpression of YWHAE in H929 WT cells that significantly promoted MM cell viability. To elucidate the underlying molecular mechanisms, proteins immunocomplexed co-precipitated with FLAG in H929 KO cells with 14-3-3ε-FLAG addback were analyzed by mass spectrometry. Protein analysis revealed interaction of 14-3-3ε with a large number of proteins, enriched in mTORC1, PI3K-AKT-mTOR and unfolded protein response (UPR) pathway-related genes. Among these, TSC2 and mTORC1 proteins were further studied. WB analysis confirmed interaction of 14-3-3ε with p-mTOR (S2448) and its upstream negative regulator p-TSC2 (S939), while mTORC1 downstream targets, p-p70 S6k and p-4E-BP1, did not interact with 14-3-3ε. WB analysis also revealed activation of TSC2 and consequent inhibition of mTORC1 (via decrease of p-mTOR S2448 levels) in YWHAE KD cells. YWHAE-FLAG addback reversed these effects. Additionally, GEP data in KD cells confirmed a significant impact on mTORC1 pathways. Importantly, YWHAE expression highly correlated (R〉 0.8) with genes involved in the mTORC1 pathway, including PSMC4, COPS5, EIF2S2, in our RNA-seq dataset, demonstrating a clinical significance of 14-3-3ε and mTORC1 cooperation in the context of myeloma. One of the most conserved functions of mTORC1 is to promote translation. We therefore assessed the impact of YWHAE on global translational efficiencies in MM cells, and observed significant impact on nascent protein synthesis by YWHAE modulation. 14-3-3ε KD induced 4EBP1 de-phosphorylation through inhibited mTORC1, and concomitantly induced EIF2α phosphorylation. Both effects inhibited translation initiation complex formation, mechanistically supporting a strong protein synthesis arrest. These data show the modulation of several hubs of the signaling apparatus controlling translation initiation in response to YWHAE modulation, ultimately producing a marked protein synthesis inhibition. Deregulated translational control is a central feature of MM. Our findings highlight a unique function for YWHAE as promoter of MM cell survival through regulation of mTOR-dependent protein synthesis and apoptosis. Pharmacological inhibition of YWHAE/14-3-3ε is therefore a possibility to specifically target malignancies with deregulated translational control such as MM. Disclosures Anderson: C4 Therapeutics: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

Description: We used a novel NF-08-TM transplant protocol based on intravenous busulfan, cyclophosphamide, fludarabine, and thiotepa in 82 consecutive patients with β-thalassemia major (TM), including 52 with allogeneic peripheral blood stem cell transplantation (PBSCT) from unrelated donors (UDs) with well-matched human leukocyte antigens and 30 with hematopoietic stem cell transplantation (HSCT) from matched sibling donors (MSDs). The median age at transplantation was 6.0 years (range, 0.6-15.0 years), and the ratio of male-to-female patients was 56:26. The median follow-up time was 24 months (range, 12-39 months). The estimated 3-year overall survival and TM-free survival were 92.3% and 90.4% in the UD-PBSCT group and 90.0% and 83.3% in the MSD-HSCT group. The cumulative incidences of graft rejection and grades III-IV acute graft-versus-host disease were 1.9% and 9.6%, respectively, in the UD-PBSCT group and 6.9% and 3.6%, respectively, in the MSD-HSCT group. The cumulative incidence of transplant-related mortality was 7.7% in the UD-PBSCT group and 10.0% in the MSD-HSCT group. In conclusion, UD-PBSCTs using the well-tolerated NF-08-TM protocol show similar results to MSD-HSCTs and can be used to treat β-thalassemia patients in the absence of MSDs.

Description: Abstract 4592 Objective Chronic lymphocytic leukemia (CLL) is a heterogeneous malignant hematologic disease; the response to chemotherapy partly determines the prognosis of patients. Fludarabine, which is frequently used in the treatment of CLL, induces CLL cell apoptosis by activating p53 and then down stream BH3-only family members, consequent to the induction of DNA damage. To identify which is the key factor in this apoptosis pathway, we incubated CLL cells with an active metabolite of fludarabine, 9-β-D-arabinosyl-2-fluoroadenine (F-ara-A), in vitro and then analyzed the role of Puma, Noxa and Bim in p53-mediated apoptosis of CLL cells. Methods CLL cells from 15 CLL patients were incubated in medium with 3.5 mM fludarabine. Isometric cells were incubated with medium only, as blank control. CLL cells were harvested after 48-hour incubation. The apoptosis of cells was measured by staining with annexin V-FITC/propidium iodide (PI). The mRNA levels of Puma, Noxa, Bim and Bcl-2 genes were quantified using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with SYBR Green by ABI LightCycler. Fluorescence in situ hybridization (FISH) analysis was used to detect ATM and p53 gene deletions. CLL cells were evaluated for immunoglobulin heavy chain variable region (IgVH) gene and p53 gene mutational status by PCR and sequencing. The function of p53 gene was detected by flow cytometry. Results 1. The percentage of apoptosis at the beginning of incubation was 7.41% (4.11%-10.71%), and the percentages of spontaneous and fludarabine-induced apoptosis after 48 h incubation were 31.40% (18.14%-44.66%) and 59.6% (43.72%-75.48%), respectively. When CLL cells were cultured in vitro, the apoptosis was raised obviously (P=0.0002). Mean fludarabine-induced apoptosis was higher than the mean spontaneous apoptosis of CLL cells (P=0.0019). 2. After 48 h incubation without fludarabine, the expression level of Puma was significantly higher in CLL cells (P=0.0317), but the expression levels Noxa (P=0.5404), Bim (P=0.3067) and Bcl-2 (P=0.1747) were not changed. Treatment of CLL cells with fludarabine further enhanced Puma expression in all samples compared to cells in medium only (P=0.0359), but the expression levels of Noxa (P=0.8063), Bim (P=0.5703) and Bcl-2 (P=0.5322) were not changed. 3. In this analysis, 4 patients carried p53 abnormality and 3 patients carried heterozygous ATM gene deletion. The level of p21 in cytoplasm was not raised in CLL cells from patients with p53 abnormality after incubated with fludarabine. Elevation of p21 was detected in CLL cells carried heterozygous ATM gene deletion, after fludarabine induction. 4. It was appeared that apoptosis level (P=0.1704) and Puma expression level (P=0.5334) of CLL cells cultured in medium only were indepented of p53 status, but apoptosis level (P=0.0109) and Puma expression level (P=0.0430) were up-regulated higher by fludarabine in CLL cells with functional p53 than in ones with dysfunctional p53. Fludarabine-induced apoptosis level (P=0.228) and Puma expression level (P=0.164) in CLL cells with heterozygous ATM gene deletion were similar to p53 wild type ones. 5. Fludarabine-induced apoptosis and the expression level of Puma seemed to be higher in CLL cells with mutated IgVH than those with ummutated IgVH, but the difference was not significant. Conclusion Functional p53 was necessary in fludarabine-inducd apoptosis. CLL cells with p53 gene deletion and/or p53 mutation showed dysfunction of p53. CLL cells with heterozygous ATM gene deletion carried functional p53. Fludarabine induced CLL cells apoptosis via activating p53 and then up-regulating its downstream effector Puma. Only Puma was the essential factor for p53-mediated apoptosis, and Noxa and Bim seemed to play a more restricted role. Disclosures: No relevant conflicts of interest to declare.