ALBERT

Beschreibung: Introduction: Hematopoietic stem cell transplantation (HSCT) is a potentially curative or consolidative therapy for a large number of hematological diseases. Sexual dysfunction (SD) and abnormal level of the sexual hormone are common in patients after HSCT, which are usually caused by intensive myeloablative conditioning. The change of sexual hormone level and SD resulted in the poor quality of life in this population after transplantation. The current aims of this study were to determine: (i) the incidence rate of SD and the association with androgen post both autologous (auto) and allogeneic (allo) HSCT; (ii) multi-factors analysis between SD and clinical characteristics, primary diease, donor type, cGVHD, etc; (iii) the association of androgen with cGVHD and glucocorticoid (GC) therapy. Methods: From April 2010 to February 2019, a total of 126 (74 males and 52 females) patients with hematological diseases undergoing HSCT were enrolled in our study. The reason for the small sample of patients was that only 126 patients completed our Sexual Function Questionnaire. Controls were 108 healthy, age and gender matched persons came from Medical Examiniation Center of our hospital. Assessment indexes included clinical characteristics, donor type, GVHD incidence, sex hormone levels, and Sexual Functioning Questionnaire (SFQ). The SFQ was implemented by the team members of our research group through a telephone interview, email, paper letter, and WeChat. All of the information and privacy of each patient was strictly conserved. Results: 1. Clinical characteristics of the 126 patients who underwent HSCT were shown in Table 1. The median age of the patients was 38 years old (range 16-66) and the follow up after HSCT was from 6 months to 7 years. The predominant disease spectra were multiple myeloma (MM) and acute leukemia in auto- and allo-HSCT group, respectively. Our results showed a significant difference in gender (P

Beschreibung: Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

Beschreibung: Key Points Novel crosstalk between SMO and NF-κB representing additional level of NF-κB regulation independent of genetic constitutive activation. SMO activates NF-κB by recruiting Gαi and Gα12 to activate PKCβ/CARMA1 and assembling CARMA1/BCL10/MALT1/TRAF6 to SMO.

Beschreibung: Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log275% reduction in both cell lines, p

Beschreibung: Introduction: Although recent studies have refined the classification of B-progenitor and T-lineage acute lymphoblastic leukemia into gene-expression based subgroups, a comprehensive integration of significantly mutated genes and pathways for each subgroup is needed to understand disease etiology. Methods: We studied 2789 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,322 cases) or T-ALL (n=467) treated on Children's Oncology Group (n=1,872) and St. Jude Children's Research Hospital trials (n=917). The cohort comprised childhood NCI standard-risk (41.8%; age range 1-9.99 yrs, WBC ≤ 50,000/ml), childhood NCI high-risk (44.5%; age range ≥10 to 15.99 yrs) and AYA (9.9%; age range 16-30.7 yrs). Genomic analysis was performed on tumor and matched-remission samples using whole transcriptome sequencing (RNA-seq; tumor only; n=1,922), whole exome sequencing (n=1,659), whole genome sequencing (n=757), and single nucleotide polymorphism array (n=1,909). Results: For B-ALL, 2104 cases (90.6%) were classified into 26 subgroups based on RNA-seq gene expression data and aneuploidy or other gross chromosomal abnormalities (iAMP21, Down syndrome, dicentric), deregulation of known transcription factors by rearrangement or mutation (PAX5 P80R, IKZF1 N159Y), or activation of kinase alterations (Ph+, Ph-like). For T-ALL, cases were classified into 9 previously described subtypes based on dysregulation of transcription factor genes and gene expression. In 1,659 cases subject to exome sequencing (1259 B-ALL, 405 T-ALL) we identified 18,954 nonsynonymous single nucleotide variants (SNV) and 2,329 insertion-deletion mutations (indels) in 8,985 genes. Overall, 161 potential driver genes were identified by the mutation-significance detection tool MutSigCV or by presence of pathogenic variants in known cancer genes. Integration of sequence mutations and DNA copy number alteration data in B-ALL identified 7 recurrently mutated pathways: transcriptional regulation (40.6%), cell cycle and tumor suppression (38.0%), B-cell development (34.5%), epigenetic regulation (24.7%), Ras signaling (33.0%), JAK-STAT signaling (12.0%) and protein modification (ubiquitination or SUMOylation, 5.0%). The top 10 genes altered by deletion or mutation in B-ALL were CDKN2A/B (30.1%), ETV6 (27.0%), PAX5 (24.6%), CDKN1B (20.3%), IKZF1 (17.6%), KRAS (16.5%), NRAS (14.6%), BTG1 (7.5%) histone genes on chromosome 6 (6.9%) and FLT3 (6.1%), and for T-ALL, CDKN2A/B (74.7%), NOTCH1 (68.2%), FBXW7 (21.3%), PTEN (20.5%) and PHF6 (18.2%) (Figure 1A). We identified 17 putative novel driver genes involved in ubiquitination (UBE2D3, UBE2A, UHRF1, and USP1), SUMOylation (SAE1, UBE2I), transcriptional regulation (ZMYM2, HMGB1), immune function (B2M), migration (CXCR4), epigenetic regulation (DOT1L) and mitochondrial function (LETM1). We also observed variation in the frequency of genes and pathways altered across B-ALL subtypes (Figure 1B). Interestingly, alteration of SAE1 and UBA2, novel genes that form a heterodimeric complex important for SUMOylation, and UHRF1 were enriched in ETV6-RUNX1 cases. Deletions of LETM1, ZMYM2 and CHD4 were associated with near haploid and low hypodiploid cases. Deletion of histone genes on chromosome 6 and alterations of HDAC7 were enriched in Ph+ and Ph-like ALL. Mutations in the RNA-binding protein ZFP36L2 were observed in PAX5alt, DUX4 and MEF2D subgroups. Genomic subtypes were prognostic. ETV6-RUNX1, hyperdiploid, DUX4 and ZNF384 ALL were associated with good outcome (5-yr EFS 91.1%, 87.2%, 91.9% and 85.7%, respectively), ETV6-RUNX1-like, iAMP21, low hyperdiploid, PAX5 P80R and PAX5alt were associated with intermediate outcome (5-yr EFS 68.6%, 72.2%, 70.8%, 77.0% and 70.9%, respectively), whilst KMT2A, MEF2D, Ph-like CRLF2 and Ph-like other conferred a poor prognosis (55.5%, 67.1%, 51.5% and 62.1%, respectively). TCF3-HLF and near haploid had the worst outcome with 5-yr EFS rates of 27.3% and 47.2%, respectively. Conclusions: These findings provide a comprehensive landscape of genomic alterations in childhood ALL. The associations of mutations with ALL subtypes highlights the need for specific patterns of cooperating mutations in the development of leukemia, which may help identify vulnerabilities for therapy intervention. Disclosures Gastier-Foster: Bristol Myers Squibb (BMS): Other: Commercial Research; Incyte Corporation: Other: Commercial Research. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding. Raetz:Pfizer: Research Funding. Borowitz:Beckman Coulter: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Angiolillo:Servier Pharmaceuticals: Consultancy. Relling:Servier Pharmaceuticals: Research Funding. Hunger:Jazz: Honoraria; Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel.

Beschreibung: Key Points CYR61/CCN1 is a bone marrow microenvironmental biomarker for myeloma progression and for transformation of MGUS and asymptomatic disease to overt myeloma. CCN1 reduces myeloma bone disease and tumor growth and is a potential therapeutic target for myeloma.

Beschreibung: Background: Relapse remains the main cause of treatment failure post transplantation. Relapse prevention is an important strategy for acute myeloid leukemia (AML) patients. M ethods: We retrospectively analyzed the results of21 high risk AML patients who received a median number of 3 courses (range, 2 - 8) of decitabine (DAC) maintenance treatment (20mg/m2/d ×5d every 3 months for 1 year). Meanwhile, another 63 high risk AML patients without any prophylactic treatment after transplantation were included as a control group for 1:3 pair matched study. Results: With median follow-up of 23 months, 20 out of 21 (95.2%) patients maintained complete molecular remission (CMR) in DAC group, while 35 out of 63 (55.6%) patients maintained CMR in control group. Comparing with control group, the patients of DAC group had higher 3-year overall survival (OS) rates and 3-year leukemia free survival (LFS) rates (92.9% vs 51.8%, P =0.003; 94.1% vs 54.7%, P=0.002 respectively). Moreover, DAC maintenance was well tolerated in all patients and grade 3/4 or 4/4 hematological toxicities were observed in 11 of 21 (52.4%) patients. Conclusion: Our results suggested that DAC maintenance therapy was an effective and safe treatment option to prevent relapse after transplantation for high risk AML patients. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background MicroRNAs (miRNAs) are a class of small non-coding single stranded RNAs whose dysregulation of expression plays an important role in cancer development. Circulating miRNAs are novel biomarkers in several cancers including acute myeloblastic leukemia and diffuse large B cell lymphoma. Thus, we explored whether the miRNAs in plasma could be a useful clinical biomarker for multiple myeloma (MM) patients. Methods MiRNA microarray was conducted to determine deregulated miRNAs in plasma of MM patients. TaqMan real-time PCR was used to validate the results. Receiver operating characteristic (ROC) analysis was performed to reveal the diagnostic accuracy of the significantly deregulated miRNAs as potential biomarkers. The Kaplan-Meier and log-rank tests were used to assess the correlation of miRNAs with progression-free survival (PFS). Results The expression levels of five miRNAs in plasma were up-regulated while eight miRNAs were down-regulated in MM patients compared to normal controls according to microarray. Validation of the five miRNAs detected as promising biomarkers was carried out. MiR-483-5p was found to have potential as diagnostic biomarkers in myeloma. ROC analysis revealed that miR-483-5p had considerable diagnostic accuracy, yielding an ROC-AUC (the areas under the ROC curve) of 0.722 (sensitivity 61%, specificity 75%). The expression levels of miR-483-5p are associated with Durie-Salmon (DS) staging system and ISS staging system (p 〈 0.01). After a median follow up of 10 months (range: 2-60 months) from diagnosis, the median PFS of patients highly expressing miR-483-5p and of low expression patients was 11 and 21 months, respectively (p=0.04). Conclusions These data demonstrate that miR-483-5p highly expresses in the plasma of MM patients. MiR-483-5p may serve as a diagnostic and prognostic biomarker in MM. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1823 Poster Board I-849 The proteasome inhibitor bortezomib, a novel anti-myeloma (MM) agent, has recently drawn considerable attention to its anabolic actions on bone formation in patients with MM. Bortezomib was reported to enhance the activity of Runx2/cbfa1, an essential transcription factor for osteoblast (OB) induction, in mesenchymal stem cells to induce OB differentiation. However, because over-expression of Runx2 unexpectedly suppresses terminal OB differentiation or mineralization, there may be critical factors involved in OB differentiation in concert with Runx2 to achieve terminal OB differentiation in the treatment with bortezomib. Proteasome inhibition accumulates a variety of proteins and induces ER stress or unfolded protein response. Among proteins induced by ER stress, activating transcription factor-4 (ATF-4) plays a critical role in OB differentiation. ATF-4 is expressed in osteoprogenitors and preOBs following Runx2, and acts in concert with Runx2 to facilitate terminal maturation of OBs. However, it is unknown whether a change in ATF-4 protein levels plays any role in OB differentiation induced by proteasome inhibition. In the present study, we therefore explored the role of ATF-4 in OB differentiation by proteasome inhibition in Runx2-expressing immature OB lineage cells. Bortezomib dose-dependently increased ATF-4 protein levels in primary bone marrow stromal cells and ST-2 stromal and MC3T3-E1 preosteoblastic cell lines at concentrations higher than 10 nM as early as 3 hours. Because serum bortezomib levels reach around 100 nM (Cmax) with T1/2 of 3 hours after iv injection at therapeutic doses, bortezomib treatment in MM patients is expected to enhance ATF-4 protein levels in OB lineage cells. Interestingly, bortezomib treatment did not change mRNA levels of ATF-4 as well as βTrCP1, E3 ligase for ATF-4. Because translation of ATF-4 mRNA is triggered by ER stress response, it is plausible that the ATF-4 accumulation by bortezomib is mediated by the suppression of proteasomal degradation with subsequent induction of ER stress response. MM cell-derived factors and TGF-β released from bone by enhanced bone resorption suppress OB differentiation in MM bone lesions. Treatment with bortezomib was able to accumulate ATF-4 in the presence of MM conditioned media (CM) or TGF-β to the levels similar to those without MM CM nor TGF-β. Furthermore, bortezomib enhanced promotor activity of osteocalcin, a marker of mature OBs, as well as BMP-2-induced mineralized nodule formation in MC3T3-E1 cells, and these effects of bortezomib were suppressed by ATF-4 siRNA. These results demonstrate that bortezomib treatment accumulate ATF-4, and suggest that the effect of bortezomib on OB differentiation is mediated via an accumulation of ATF-4 protein in OB lineage cells. We have previously demonstrated that OB differentiation is suppressed in MM bone lesions, and that differentiated OBs suppress MM cell growth and survival. Thus, resumption of bone formation by bortezomib may further suppress MM cell growth in concert with its direct anti-MM actions. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 2856 Germline and somatic mutations (heterozygous) in Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling processes, have been identified in juvenile myelomonocytic leukemia (JMML), a childhood myeloproliferative disease (MPD), and pediatric acute leukemias. These mutations cause hyperactivation of Shp2 catalytic activity and enhance the binding of mutant Shp2 to signaling partners. Ptpn11 mutations are sufficient to drive the development of JMML-like MPD and acute leukemias in mice, suggesting that they play a causal role in the pathogenesis of hematological malignancies. However, the mechanisms by which Ptpn11 mutations induce these malignancies are not completely understood and the signaling partners that mediate the pathogenic effects of Ptpn11 mutations have not been explored. We previously generated a line of conditional knock-in mice with Ptpn11E76K mutation, the most common and most active Ptpn11 mutation found in JMML and acute leukemias. Induced knock-in of this mutation in hematopoietic cells resulted in MPD with full penetrance as a result of aberrant activation of hematopoietic stem cells (HSCs) and myeloid progenitors (J. Exp. Med., 2011). Recently, we discovered that the interaction between Shp2 E76K and Gab2, a prominent interacting protein of Shp2 and a scaffolding protein important for cytokine-induced PI3K/Akt/mTOR signaling, was greatly enhanced, and that mTOR was highly activated in Ptpn11E76K/+ MPD cells. To address the role of Gab2 and mTOR in the pathogenesis of Ptpn11E76K/+ mutation-induced MPD, Ptpn11E76K/+/Gab2-/- double mutant mice were generated and their phenotypes were compared with those of Ptpn11E76K/+ single mutant mice. MPD phenotypes were markedly attenuated in Ptpn11E76K/+/Gab2-/- double mutant mice. Overproduction of myeloid cells in the bone marrow was alleviated, and splenomegaly was diminished in the double mutants. Myeloid cell infiltration in the liver also decreased. Cytokine (IL-3 and GM-CSF) sensitivity of myeloid progenitors was significantly decreased in Ptpn11E76K/+/Gab2−/− mice as compared to that in Ptpn11E76K/+ mice. Hyperactivation of HSCs and excessive myeloid differentiation caused by Ptpn11E76K mutation were largely corrected by deletion of Gab2. Furthermore, we treated Ptpn11E76K/+ mice with Rapmycin, a specific and potent mTOR inhibitor, which substantially diminished MPD phenotypes. Collectively, this study reveals the essential role of the Gab2/PI3K/mTOR pathway in mediating the pathogenic effects of Ptpn11E76K/+ mutation and suggests that Gab2 and mTOR are potential therapeutic targets for the treatment of Ptpn11-associated hematological malignancies. Disclosures: No relevant conflicts of interest to declare.