bluuebunny/crossref_metadata_2025 huggingface dataset:
Dose-Dense Rituximab Improves Outcome of Elderly Patients with Poor-Prognosis Diffuse Large B-Cell Lymphoma (DLBCL): Results of the DENSE-R-CHOP-14 Trial of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL).
Authors: Pfreundschuh Michael , Zeynalova Samira , Poeschel Viola , Haenel Mathias , Schmitz Norbert , Hensel Manfred , Reiser Marcel , Loeffler Markus , Schubert Joerg
Published: November 2019

Background: While 6 to 8 cycles of CHOP in combination with rituximab are widely accepted as standard regimen of care for aggressive lymphomas, the optimal dose and number of rituximab application have not been determined to date. In a previous pharmacokinetic study we had shown that the concomitant application of CHOP and rituximab does not achieve a plateau of rituximab trough levels until cycle 5 (Reiser, Blood 108, 778a, 2006). In order to achieve high rituximab levels early, we increased the number of rituximab applications.

Methods: 100 elderly patients with aggressive CD20+ B-cell lymphoma received 6 cycles of biweekly CHOP-14 combined with 12x rituximab (375 mg/m2) on days 0, 1, 4, 8, 15, 22, 29, 43, 57, 71, 85, and 99. Radiotherapy was planned to sites of initial bulk and/or extranodal involvement. The primary endpoint was event-free survival (EFS). 306 patients treated within the RICOVER-60 trial (Pfreundschuh et al., Blood 64a, 2006) with 6xCHOP-14 and 8 applications of rituximab served as control.

Results: 97/100 patients are evaluable for response. Dose-dense rituximab resulted in plateau trough serum levels of rituximab as early as day 1 of the first chemotherapy cycle and higher rituximab levels were maintained throughout the treatment compared to 8 bi-weekly applications. Because 3 therapy-associated deaths were observed among the first 20 patients treated, prophylaxis with levofloxacin, acyclovir and cotrimoxazol became mandatory for the following patients. Despite a less favorable study population DENSE-R-CHOP-14 resulted in a somewhat higher complete remission (83% vs. 78%) and lower progression under therapy rate (5% vs. 7%) rate, but event free and overall survival were not different compared to 8 biweekly applications of rituximab. However, a subgroup analysis of patients according to IPI risk group showed that DENSER-R-CHOP-14 resulted in a higher complete response rate of patients with poor-prognosis (IPI:3–5) disease (81% vs. 68%). This advantage translated into a better 1-year event-free survival rate (74% vs. 65%) of these patients.

Conclusion: In combination with 6 cycles of CHOP-14 densification of rituximab achieves higher rituximab serum levels and results in higher complete remission and event-free survival rates in elderly patients with poor-prognosis DLBCL. These observations from a phase-II trial must be confirmed in a randomized study.
Development of Osteonecrosis Is Associated with Difference in Upregulation of Antithrombin, Protein C and Protein S after Four Weeks of Dexamethasone during Induction Treatment in Childhood Acute Lymphoblastic Leukemia.
Authors: te Winkel Mariel L. , Pieters Rob , Appel Inge M. , Van Den Heuvel Marry M.
Published: November 2019

Osteonecrosis (ON) is a serious complication of chemotherapy for pediatric acute lymphoblastic leukemia (ALL). Impairment of microcirculation seems to be involved in the pathogenesis of ON. We hypothesized that variation in hypercoagulability plays a role in the etiology of ON during treatment of ALL. Dexamethasone (dexa) and asparaginase (asp) are important drugs in the induction treatment of pediatric ALL that may influence the coagulation system. In the current study differences in coagulation parameters between pediatric ALL patients with and without ON were evaluated. Blood samples were taken at diagnosis and at several time-points during induction of the DCOG-ALL9 treatment schedule. Procoagulant factors (fibrinogen, factor II,V,VII,IX,X), anticoagulant factors (antithrombin (AT), protein C and protein S), parameters of thrombin generation (prothrombin fragment 1+2 (F1+2), thrombin antithrombin (TAT) complex) and of fibrinolysis (α2-antiplasmin (a2AP), plasminogen, plasmin-α2AP (PAP) complex, D-dimers) were determined. In total 137 patients without ON and 24 patients with ON were evaluated. In addition plasminogen activator inhibitor type 1 (PAI-1) activity and tissue plasminogen activator antigen (tPA-ag) concentration were studied in 25 patients, 15 without ON and 10 with ON. At diagnosis no significant differences in any of the coagulation parameters were found between patients with and without ON. After 4 weeks dexa treatment (day 29), AT, protein-C activity and protein-S total (anticoagulants) were significantly less upregulated in ON positive patients as compared to ON negative patients (respectively p=0.043, p=0.029, p=0.023). During tapering of dexa and administration of 4 doses asp at day 29, 33, 36 and 40 these parameters significantly decreased in both ON positive and negative patients. However, the nadir of these anticoagulant proteins reached lower levels in the ON positive patients as compared to ON negative patients and in ON positive patients levels even reached below lower limits of normal reference ranges. No other dexa or asp induced differences in any of the other coagulation parameters were found between ON negative and positive patients. These results indicate that development of ON in pediatric ALL patients seems to be associated with less upregulation of AT, protein C and protein S after 4 weeks treatment with dexa (day 29). ON positive patients subsequently declined below the lower limit of normal reference range during tapering of dexa and administration of asp in contrast to ON negative patients. This may suggest a therapy induced hypercoagulable state contributing to the development of ON.
The Role of Autologous Stem Cell Transplantation (ASCT) in Patients with Advanced Waldenström’s Macroglobulinemia.
Authors: Kyriakou Charalampia , Canals C. , Sureda A. , Taghipour G. , Gisselbrecht C. , Mazza P. , Montserrat E. , Niederwieser N. , Indrak K. , Kolbe A. , Biron P. , Levis A. , Goldstone A. , Schmitz N.
Published: November 2019

Waldenström’s macroglobulinaemia (WM) is a relatively rare disorder that primarily affects elderly patients. Conventional therapies for symptomatic WM result in response rates of up to 70%. However, complete responses are rare and the disease remains incurable. Due to the indolent nature of the disease and the older age of patients the role of autologous stem cell transplantation (ASCT) in the treatment of patients with WM has not been analyzed in large series. In this retrospective multicenter study we report the outcome of 201 WM patients (132 male, 69 female), who underwent ASCT between 1992 and 2005. The median age at transplant was 53 years (22–73) and the median time from diagnosis to transplant was 18 months (3–239). Forty patients (20%) were in 1st maximum response (MR1), 24 (12%) in ≥MR2, 83 (41%) in PR1, 27 (13%) in ≥PR2 and 27(14%) were primary refractory to treatment. Conditioning regimens were BEAM (44%), TBI/Cyclophosphamide or Melphalan (28%), Melphalan (14%), BuCy (2%) and others (12%). The source of stem cells was PB in 188, BM in 10, and both in 3 patients. All patients but 3 had successful engraftment. With a median follow-up of 26 months (5–163), 112 (56%) patients are alive and free of disease, 73 (36%) patients have relapsed after a median of 14 months (1–110) post ASCT. Fifty-two patients died, 36(18%) from disease progression and 16(8%) from treatment toxicity. Non- relapse mortality was 6% at 1 year. The actuarial OS was 86% at 1 year, 75% at 3 years, and 61% at 5 years. The probability of relapse was 20% at 1 year, 38% at 3 years and 55% at 5 years with an estimated PFS of 74%, 54% and 33% at 1, 3, and 5 years respectively. Multivariate analysis revealed that, chemosensitive disease at the time of ASCT was the most important factor for NRM (p<0.001), relapse rate (p<0.01), PFS (p<0.001) and OS (p<0.001). In conclusion, this study suggests that ASCT is a safe procedure in patients with WM and that a significant proportion of heavily pre-treated patients with this disorder can respond to the procedure and achieve prolonged PFS.
Effect of the Percent Decrease in Monoclonal Protein with Induction Therapy on the Outcome after Autologous Stem Cell Transplantation in Patients with IgG and IgA Multiple Myeloma.
Authors: Reece Donna E. , Trieu Young , Pokoradi Alida , Xu Wei , Fung Sharon , Chen Christine , Kukreti Vishal , Mikhael Joseph , Trudel Suzanne , Stewart A. Keith
Published: November 2019

One potential approach to improve the results of autologous stem cell transplantation (ASCT) in multiple myeloma (MM) involves the use of more potent induction regimens; the achievement of deeper remission pre-ASCT should translate into longer progression-free (PFS) and overall survival (OS). To evaluate this hypothesis, we assessed the influence of the percent reduction in serum monoclonal protein (M protein) pre-ASCT in patients (pts) treated with dexamethasone-based regimens (most often VAD) on the best response, PFS and OS achieved after ASCT. Between 2000–2006, 376 pts with IgG (267) or IgA (109) non-progressive MM underwent ASCT. Median age was 59 (21–73) yrs; 60% were male. The median hemoglobin at diagnosis was 103 g/L, creatinine 98 μmol/L, β2-microglobulin 241 nmol/L and albumin 35 g/L. Maintenance therapy was given to 84 (22%) after ASCT and consisted of corticosteroids in 29, thalidomide in 10, both in 41 and other regimens in 4 pts. Patients were divided into 4 groups based on the per cent reduction in M protein after induction therapy:

group A, ≥ 99%; group B, 90–98%; group C, 50–89%; group D, < 50%.

Post-ASCT responses included complete remission (CR), very good partial remission (VGPR) (≥ 90% reduction in M protein), PR (≥ 50% reduction) and stable disease (SD). Median follow-up from diagnosis is 37.4 mos and from ASCT 24.1 mos. For all pts, the median OS from diagnosis is 90.8 mos (95% CI 73.9–129.1 mos) and from ASCT 63.9 mos (95% CI 50.8–69.4 mos), while the median PFS from ASCT is 21.3 mos (95% CI 19.1–23.3 mos). Maintenance therapy had no significant effect on PFS (p=0.49) or OS (p=0.59). The post-ASCT results in evaluable patients according to the percent reduction in M protein after induction therapy are summarized below. We conclude:

High-grade remissions after dexamethasone-based induction therapy are uncommon, with only 2% achieving ≥ 99% and 13% achieving 90–98% reduction in serum M protein; post-ASCT CR and VGPR rates were higher in these 2 groups; there was no significant difference in PFS or OS-based on protein response prior to transplant; whether newer induction regimens, which incorporate novel agents and which produce more CRs and VGPRs before ASCT, will confer better PFS and OS post-ASCT will be of great interest.

Table 1 Group N CR (%)1 VGPR (%)1 PR (%)1 SD (%)1 Median PFS (mo)2 Median OS (mo)3 1p<0.001;2p=0.77;3p=0.79 A 7 50 50 Not reached Not reached B 47 19 64 17 23.3 Not reached C 232 14 17 68 1 20.7 59.1 D 87 11 7 54 28 21.1 69.4 Figure Figure Figure Figure
Cloning of AML1-LAF4 Fusion Gene in Childhood T-Cell Acute Lymphoblastic Leukemia with t(2;21)(q11;q22) by Bubble PCR Method for cDNA.
Authors: Chinen Yoshiaki , Taki Tomohiko , Nishida Kazuhiro , Shimizu Daisuke , Okuda Takashi , Yoshida Naohisa , Kobayashi Chie , Koike Kazutoshi , Tsuchida Masahiro , Hayashi Yasuhide , Taniwaki Masafumi
Published: November 2019

The AML1/RUNX1 gene is frequently rearranged by chromosomal translocations in acute leukemia. To date, more than 10 fusion genes involving AML1 have been cloned, such as AML1-MTG8 in acute myeloid leukemia (AML) with t(8;21), AML1-EVI1/MDS1 in therapy-related AML/myelodysplastic syndrome with t(3;21), and TEL/ETV6-AML1 in B precursor ALL with t(12;21). We analyzed a pediatric patient having T-cell acute lymphoblastic leukemia (T-ALL) with t(2;21)(q11;q22), and identified that the LAF4 gene on 2q11.2–12 was fused to the AML1 gene on 21q22 using the bubble PCR method for cDNA. The genomic breakpoints were within intron 7 of AML1 and intron 7 of LAF4 resulting in the in-frame fusion of exon 7 of AML1 and exon 8 of LAF4. LAF4 gene is a member of the AF4/FMR2 family, and was previously identified as a fusion partner of MLL in B-precursor ALL with t(2;11)(q11;q23), although AML1-LAF4 was in T-ALL. LAF4 is the first gene fused with both AML1 and MLL in acute leukemia. These findings provide new insights into the common mechanism of AML1 and MLL fusion proteins in the pathogenesis of ALL. In this study, we first applied the panhandle PCR method that is usually used for cloning the fusion partners of MLL or NUP98; however, no fusion transcripts could be obtained. Therefore, we searched for another method for cloning the fusion transcripts and successfully adapted the bubble PCR method for cloning the novel AML1-LAF4 fusion transcript. To date, bubble PCR has been performed for cloning unknown genomic fusion points but not fusion cDNAs. Using double-strand cDNA, we could apply the bubble PCR method for cloning fusion cDNA with rare non-specific products. Bubble PCR for cDNA could amplify in both 5′ to 3′ and 3′ to 5′ directions of the gene or transcript and handle any exons fused to unknown partners for amplification easily. This method will contribute to identifying numerous novel translocation partners more easily, and these findings may help to clarify the role of the fusion proteins in leukemogenesis.
High Frequency of AML1/RUNX1 Mutations in Specific Cytogenetic Subgroups in De Novo Acute Myeloid Leukemia.
Authors: Dicker Frank , Haferlach Claudia , Kern Wolfgang , Haferlach Torsten , Schnittger Susanne
Published: November 2019

Somatic mutations in the DNA-binding domain, the socalled Runt homology domain, of the AML1/RUNX1 gene have been identified to occur in acute myeloid leukaemia (AML) with the highest incidence in AML M0, in therapy-related myelodysplastic syndrome (t-MDS), in therapy-related AML (t-AML) and AML after MDS (s-AML). Cytogenetic aberrations that are associated with RUNX1 mutations (RUNX1mut) have been reported to be trisomy 13 in AML and trisomy 21 in myeloid malignancies, but also loss of chromosome 7q, mainly in t-MDS but rarely in t-AML. So far the majority of RUNX1mut have been described in secondary or therapy-related cases. Thus, we characterized a cohort of 119 patients (pts) with de novo AML and compared these results to 19 MDS and s-AML, 2 t-MDS (n=2) and 8 t-AML. The cohort was selected for specific cytogenetics with high reported frequencies of RUNX1mut: trisomy 13 (n=17), trisomy 21 (n=9), −7/7q- (n=34). In addition pts with normal karyotype (NK) (n=42), inv(3)/t(3;3) (n=12), trisomy 8 (n=11), complex karyotype (n=13) and 10 pts with various other cytogenetic aberrations (other) were analyzed. The incidence of RUNX1mut in the different cytogenetic subgroups was: 94% (16/17) in +13, 56% (5/9) in +21, 29% (10/34) in −7/7q-, 10% (4/42) in NK, 17% (2/12) in inv(3)/t(3;3), 18% (2/11) in +8, 0% (0/13) in complex karyotype and 20% (2/10) in other, respectively. Based on clinical history we observed RUNX1 mutations in: 6/19 (32%) in MDS/s-AML, 1/10 (10%) in t-MDS/t-AML and 34/119 (29%) in de novo AML. Of the 6 RUNXmut cases with MDS/s-AML the karyotypes were heterogeneous NK (n=1), −7 (n=2) +13 (n=1), +21 (n=1), and inv(3) (n=1). The only recurrent cytogenetic aberration in MDS/s-AML was −7, thus the frequency of RUNXmut in the MDS/s-AML group with −7 was 2/8 (25%). Also the only RUNX1mut case with t-AML revealed a −7. These data correspond to those reported in the literature. We further focussed on the analyses of RUNX1 in de novo AML which is rarely reported so far. In the de novo AML group only we detected RUNX1mut with the highest frequency in +13 (16/16; 100%) followed by +21 (4/8; 50%) −7 (7/21; 33%), + 8 (2/10, 20%), inv(3) (1/8; 12.5%), and NK (3/33; 9.1%). In addition, in the group with “other” aberration 2/8 were mutated. Interestingly, these 2 mutated cases displayed a high number of trisomies including +8 and +13. No RUNX1mut were detected in AML with complex karyotype (n=10). These data for the first time show that RUNX1mut are not strongly correlated to MDS, s-AML or t-AML. With almost the same frequency they can be observed in de novo AML if specific cytogenetic groups are considered. Thus the RUNXmut seem to be more related to these cytogenetic subgroups than to the MDS, s-AML or t-AML.
Detection of Genomic Lesions in Childhood Precursor-B Cell ALL in Diagnosis and Relapse Samples Using High Resolution Genomic Profiling.
Authors: Kuiper Roland P. , van Leeuwen Frank N. , Keijzers-Vloet Suzanne T.M. , van Reijmersdal Simon V. , Hehir-Kwa Jayne Y. , van Wering Elisabeth R. , van der Velden Vincent H.J. , van Dongen Jacques J.M. , Schoenmakers Eric F.P.M. , van Kessel Ad Geurts , Hoogerbrugge Peter M.
Published: November 2019

Due to advances in therapeutic regimens developed during the last two decades, the majority of children with acute lymphoblastic leukemia (ALL) respond well to therapy. However, in approximately 25% of the patients relapses occur. Chomosome aneuploidies and recurrent chromosomal translocations are of considerable prognostic importance, and are routinely used in the course of clinical decision making. Current technological developments in molecular cytogenetic techniques have revealed that genetic lesions driving tumorigenesis frequently occur at the submicroscopic level and, consequently, escape standard cytogenetic observations. Therefore, we have previously performed high resolution genomic profiling of precursor-B-cell ALL samples obtained at diagnosis, using 250k NspI SNP-based oligoarrays from Affymetrix (Kuiper et al., 2007). By doing so, we detected multiple de novo genetic lesions, some of which were subtle and affected single genes. Many of these lesions involved recurrent (partially) overlapping deletions and duplications, encompassing various established leukemia-associated genes, such as ETV6, RUNX1, and MLL. Importantly, the most frequently affected genes were those controlling G1/S cell cycle progression (e.g. CDKN2A, CDKN1B, and RB1), followed by genes associated with B-cell development. The latter group included the B-lineage transcription factors PAX5, EBF, E2-2, and IKZF1 (Ikaros), as well as genes with other established roles in B-cell development, i.e., RAG1 and RAG2, FYN, PBEF1, or CBP/PAG. Here we have selected 34 additional precursor-B cell ALL cases that suffered from relapses 6 months to 9 years after diagnosis. Lesions affecting genes involved in G1/S cell cycle progression and B-cell development were observed with similar frequencies in the diagnosis and relapse samples as compared to our previous cohort of patients with unknown therapy response. However, additional (secondary) lesions were observed in the relapse samples in nearly all patients analyzed, indicating that these relapse samples are genomically distinct. In addition, several cases were encountered in which the diagnosis and relapse samples carried alternative lesions affecting the same gene(s), including CDKN2A and PAX5, suggesting that inactivation of these genes were secondary but essential events required to develop a full blown leukemia.
Evaluation of Platelet Behavior in Response to Varied Conditions in a Microfluidic Flow Cells.
Authors: Conant Carolyn G , Schwartz Michael A , Ionescu-Zanetti Cristian
Published: November 2019

Abstract 5075

Platelet function under shear flow can be tested using a variety of instruments ranging from cone and plate viscometers to microfluidic flow cells. While these methods offer relevant data, they generally suffer from high blood volume requirements, low throughput and complex operating protocols. We have developed a flow-based well plate microfluidic device that operates at very wide shear ranges (from 0-200dyn/cm2) and use minimal blood volumes ('1ml per experiment). Here we present data using the microfluidic device to examine platelet response over time on coated surfaces. Flow channels were coated with fibronectin, von Willebrand factor (vWF), and collagen I. Whole blood was introduced at increasing shear stress ranging from 0-200dyn/cm2. Devices were used to study dose responses to abciximab (ReoPro) on vWF and collagen I at 200 and 10dyn/cm2 respectively by examining percent platelet coverage following application of flow. Within the concentration range tested, 1.25-20 μg/ml (26.5nM-470nM), for both collagen and vWF, the IC50 fell slightly below 26.5nM. We compared the data generated in the microfluidic channels to data generated in traditional parallel plate flow chambers. While response to coated surfaces and ReoPro were predictably comparable, the time to set up the microfluidic device and the volume of blood used were significantly decreased, 6X and 10X respectively, leading to higher throughput in one experimental session and more data generated from one donor. Therefore, the use of such microfluidic devices can greatly streamline studies with multiple variables enabling many data points to be generated from single donors within a single device.

Disclosures

Conant: Fluxion : Employment. Schwartz:Fluxion : Employment, Equity Ownership. Ionescu-Zanetti:Fluxion: Employment, Equity Ownership.
STAT3-Activated GM-CSFRα Shuttles To The Nucleus and Protects CLL Cells From Apoptosis
Authors: Li Ping , Rozovski Uri , Harris David , Zhiming Lui , Ferrajoli Alessandra , Bueso-Ramos Carlos E. , Hazan-Halevy Inbal , Grugurevic Srdana , Wierda William G. , Burger Jan A. , O'Brien Susan , Faderl Stefan H. , Keating Michael J. , Estrov Zeev
Published: November 2019

Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates the survival, proliferation, differentiation, and activation of hematopoietic cells, dendritic cells, and T cells. Upon GM-CSF binding, the α and β subunits of the GM-CSF receptor (GM-CSFR) dimerize and activate signaling. While exploring a possible role for GM-CSF in treating chronic lymphocytic leukemia (CLL), we found that GM-CSF did not enhance the phosphorylation of STAT3, Akt, or ERK, suggesting that GM-CSF does not directly affect CLL cells. Indeed, as in normal B cells, flow cytometry analysis of CLL cells did not detect GM-CSFRβ. However, unlike in normal B cells, GM-CSFRα (CD116) was present in CLL cells. Using confocal microscopy, cell fractionation studies, and GM-CSFRα antibody epitope mapping, we detected GM-CSFRα not only on the surfaces but also in the cytosol and nuclei of CLL cells, suggesting that GM-CSFRα has functions that are unrelated to receptor-ligand interaction. Because STAT3 is constitutively activated in CLL cells and because the promoter of the GM-CSFRα gene harbors putative STAT3 binding sites, we hypothesized that STAT3 activates GM-CSFRα in CLL cells. Both chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) analyses revealed that STAT3 binds to the promoter of GM-CSFRα. We therefore cloned the GM-CSFRα promoter in multiple myeloma cell line MM1 and used luciferase assay we identified STAT3 binding activity. To investigate the function of GM-CSFRα, we induced the overexpression of GM-CSFRα in 293FT cells and analyzed the RNA and protein that co-immunoprecipitated with GM-CSFRα. We identified 7200 RNA transcripts that co-immunoprecipitated with GM-CSFRα, including SKI and MAFA oncogenes, two serine/threonine kinase genes, and DEFT1P2 and DEFP1P, which encode proteins that belong to members of the death effector domains family, known to have a role in modulating apoptosis. Overall, the transcripts that co-immunoprecipitated with GM-CSFRα belong to survival pathways, the JAK/STAT pathway, and hematopoietic lineage pathways. Mass spectrometry analysis revealed that housekeeping proteins, chaperon proteins, KAP1, and ISG-15 co-immunoprecipitated with GM-CSFRα. We found that GM-CSFRα–bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFRα–bound ISG-15 inhibited the nuclear factor-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα-siRNA induced the apoptosis of CLL cells. Taken together, our data suggest that STAT3 induces the transcription of GM-CSFRα, which has distinct, ligand-independent, anti-apoptotic activities.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide Mapping and Subtype Classification of Long Non-Coding RNA in Acute Myeloid Leukemia
Authors: Chen Rong , Malouf Gabriel G , Zhang Jianping , Huang Xuelin , Weinstein John N , Spano Jean-Philippe , Molldrem Jeffrey J. , Plunkett William , Su Xiaoping
Published: November 2019

Background: Most efforts to characterize acute myeloid leukemia (AML) have focused so far on genetic and epigenetic aberrations, which can ultimately lead to altered protein-coding gene function. The roles of long non-coding RNAs (lncRNAs), which orchestrate cell physiology and act as key regulators of the AML oncogenic state, remain uncharacterized globally.

Material and Methods: We performed a genomic analysis of GENCODE lncRNAs in 179 clinically annotated cases of de novo AML, using The Cancer Genome Atlas (TCGA) RNA-Seq data. In addition, we described global correlations between lncRNAs and the expression of cis- and trans-acting genes. We also established lncRNA-based subtype classification based on distinct signatures and then correlated that classification with fusion transcripts and genetic alterations.

Results: Using stringent criteria (RPKM ≥1 in at least 10% of AML), we identified 2,913 expressed lncRNAs and used an integrative analysis to predict those that are potential regulators of AML oncogenic state. The expression of 1,935 (66.4%) lncRNAs showed positive correlations with the mRNA expression of their neighboring genes, while only 14 (0.4%) of the lncRNAs showed negative correlations. Gene ontology analysis using GREAT revealed enrichment of cis-neighboring genes in the PML body gene set (p=8.2x10-7). Unsupervised clustering of lncRNA-based expression showed five robust molecular clusters (C1 to C5), which were highly correlated with the mRNA-based classification. Of those, three clusters (C1, C2 and C5) were tightly associated with recurrent fusion transcripts; cluster C1 (n=16) was composed exclusively of promyelocytic leukemias, while cluster C5 (n=45) was enriched for MLL-rearranged cases (24.4%), and cluster C2 (n=31) was enriched for MYH11-CBFB or RUNX1-RUNX1T1 (55.2%) rearranged cases. Importantly, cluster C4 (n=30), which includes cytogenetically normal leukemias, was highly enriched for NPM1 (p=2.3x10-11) and FLT3 (p=1.6x10-4) mutations; conversely, cluster C3 (n=53) was highly enriched for recurrent copy-number alterations as well as RUNX1 (p=0.001) and TP53 somatic mutations (p=0.004). We further discovered a core of 37 lncRNAs significantly associated with a MLL-signature and 16 lncRNAs with a NPM1-mutated signature.

Conclusion: This study describes the first genome-wide mapping and characterization of lncRNAs in AML and proposes a robust lncRNA-based classification. This classification may serve in defining core lncRNAs that orchestrate key oncogenic states in the different clinical subtypes.

Disclosures

No relevant conflicts of interest to declare.
Mutation in Retinoic X Receptor-γ Is a Possible Mechanism of All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia(APL): Identifying Genetic Changes Related to Drug Resistance in APL Using Whole Exome Sequencing
Authors: Youk Jeonghwan , Koh Youngil , Park Hyunkyung , Kim Dae-Yoon , Lee Chan-Soo , Lee Junglim , Kim Hyo Jung , Yoon Hwi-Joong , Ahn Kwang-Sung , Jung Jong-Sun , Kim Inho , Park Seonyang , Yoon Sung-Soo
Published: November 2019

Background

Acute promyelocytic leukemia (APL) has the best prognosis among acute myeloid leukemia (AML). However, a subset of APL patients is not cured with all-trans retinoic acid (ATRA) combined with anthracycline-based cytotoxic chemotherapy. Some mechanisms such as increased ATRA metabolism have been suggested to acquired resistance to ATRA. However genetic mechanism of ATRA resistance has not been characterized at all. Hence, in this study, we tried to reveal genetic alterations attributable to ATRA resistance.

Methods

First, we performed whole exome sequencing (WES) using DNA of three APL patients who showed resistance to ATRA based treatment. These included two patients who failed to achieve complete remission (CR) after induction chemotherapy, and one patient who experienced relapse after CR despite of consolidation treatment. DNA extracted from bone marrow at the time of diagnosis was used for analysis, while DNA extracted from saliva at the time of CR was used as germline control. Calling of single nucleotide variants (SNV) was performed using internal pipeline called Adiscan (http://www.syntekabio.com). Annotation was performed using Polyphen-2. SNV’s found by WES were validated by direct Sanger sequencing. The frequency of those validated SNV’s was defined in a separate APL cohort.

Results

We identified 34 somatic non-synonymous SNV’s in three patients. Polyphen-2 algorithm predicted 9 among 34 SNV’s to damage protein function. Sanger sequencing revealed 8 over 9 SNV’s to be validated. These validated SNV’s include RXRG M77R, N6AMT2 A78S, TXNDC15 D198E, B3GALTL A444T, RBBP8NL E182G, TNPO3 L173W, BHMT M185I and ADAMTS5 G85D. When these 8 SNV’s were genotyped in a separate cohort, none of these SNV’s was found in the APL cohort composed of 30 ATRA sensitive patients, suggesting these SNV’s would be truly related to ATRA resistance in APL. Especially, when a simulation using amber molecular dynamics was performed, we observed 1) Increase in hydrogen bonding, 2) Decreased helix folding structure, 3) Decreased energy state in RXRG mutant case.

Conclusions

WES identified eight SNV’s which were unique in ATRA resistant cases. Among those mutations, RXRG could be a promising nonsynonymous mutation that explains the genetic mechanism of ATRA resistance.

Disclosures

No relevant conflicts of interest to declare.
Somatic Mutational Landscape of AML with Inv(16) and t(8;21) Identifies Two Distinct Patterns in Relapse Tumors
Authors: Sood Raman B. , Hansen Nancy F , Donovan Frank X , Carrington Blake , Maskeri Baishali , Young Alice , Bucci Donna , Chandrasekharaappa Settara , Mullikin James C , Bloomfield Clara D. , Marcucci Guido , Liu Paul
Published: November 2019

Acute myeloid leukemia (AML) is a heterogeneous disease with a wide prognostic spectrum ranging from poor to good depending upon the underlying mutations and/or cytogenetic abnormalities. Although AMLs with inv(16)/t(16:16) or t(8,21), collectively referred to as core binding factor leukemias (CBF-AMLs), are classified as prognostically favorable, such patients often succumb to their disease following relapse after an initial response to cytarabine/anthracyclin-based treatment regimens. Thus, to develop successful treatment strategies, it is critical to understand the mechanisms leading to disease relapse and target them with novel therapeutic approaches. To pursue this goal, we applied genomic approaches (whole exome sequencing and single nucleotide polymorphism arrays) on DNA from samples collected at sequential time points (i.e., diagnosis, complete remission and relapse) in seven patients with inv(16) and six patients with t(8;21). We identified mutations in several previously identified AML driver genes, such as KIT, FLT3, DNMT3A, EZH2, SMC1A, SMC3, WT1 and NRAS. Three relapse samples showed mosaicism for monosomy/disomy of the region of chromosome 3 containing GATA2. Overall, our data revealed two distinct profiles that support different mechanisms of relapse: 1) diagnosis and relapse blasts harbor the same driver gene mutations, indicating the intrinsic resistance of the major clones present at diagnosis to treatment regimen used; 2) diagnosis and relapse tumors have different driver gene mutations, indicating disease clonal evolution possibly through treatment selective pressure. Furthermore, our data has identified previously unreported putative driver genes for AML. Among these, we identified same somatic variant (R222G) in DHX15, an RNA helicase involved in splicing, in two patients at diagnosis. The variant was also detected at relapse in one of these patients. Functional validation of the mechanistic roles of wild type and mutated DHX15 in hematopoiesis and leukemogenesis, respectively, is ongoing in in vitro and in vivo models.

Disclosures

No relevant conflicts of interest to declare.
Differences of Somatic Mutations and Gene Expression in Blasts of Transient Leukemia and Acute Myeloid Leukemia of Down Syndrome
Authors: Chen Jian , Yousif Fouad , Beck Timothy , McPherson John D , Hitzler Johann K.
Published: November 2019

Background: Transient leukemia (TL) occurs in 30% of newborns with Down syndrome (DS) and typically resolves spontaneously. Approximately 20% of infants with TL go on to develop acute myeloid leukemia of DS (DS-AML) within the first four years of life. The blasts of both TL and DS-AML harbor somatic mutations of GATA1 . The objective of this study was to identify additional genetic events, which associated with the progression of TL to DS-AML.

Methods: Leukemic blasts of TL, DS-AML and normal T lymphocytes were sorted from blood and bone marrow samples of five patients who successively developed both disorders. In addition, blasts of one patient with subsequent relapse of DS-AML were analyzed. Mutational spectrum and gene expression and were determined by exome sequencing and RNASeq (Illumina HiSeq2000). The presence of mutations, which were identified with this approach in DS-AML blasts, was examined by droplet digital PCR in TL blasts (BioRad QX200).

Results: Blasts of TL overall harbored fewer mutations than those of DS-AML. Mutations of cohesin and RAS pathway genes were identified in a subset of DS-AML but not TL. In the patient who developed a relapse, different cohesin gene mutations were detected at initial diagnosis of AML and relapse; a minor clone present at initial diagnosis of AML emerged as the predominant clone at relapse. Concordant somatic GATA1 mutations were present in both TL and DS-AML blasts derived from the same patient. In contrast, other genetic events, which were detected in DS-AML blasts by exome sequencing, were confirmed to be absent in TL (by droplet digital PCR). The majority of differentially expressed genes showed higher expression levels in blasts of TL compared to DS-AML. They included genes encoding chemokines and related to IL1 and TGFb signaling.

Conclusions: The pathogenic sequence starting with TL and culminating in AML is uniquely initiated in children with DS by somatic mutation of GATA1. In contrast, the events associated with the transformation of TL to DS-AML resemble progression factors also found in non-DS AML. These progression events were not detectable even in minor subclones of TL suggesting they are acquired after the onset of TL.

This research was supported by funding from the Canadian Cancer Society Research Institute and Ontario Institute for Cancer Research.

Disclosures

No relevant conflicts of interest to declare.
FASN Is Critical for Maintenance of NUP98 Fusion-Induced Leukemias
Authors: Shima Yutaka , Kitabayashi Issay
Published: November 2019

NUP98 is a component of the nuclear pore complex (NPC), which plays an important role in molecular traffic between the cytoplasm and the nucleus. The NUP98 gene is rearranged and fused to several partner genes, such as HOXA9 and DDX10, in acute myeloid leukemia and myelodysplastic syndromes and the leukemia with NUP98 rearrangement has a poor prognosis. The role of the NUP98 moiety of NUP98 fusion proteins is not well investigated. NUP98 has two Phe-Gly (FG) repeat domains, which are characteristic of the NPC proteins. To investigate the role of the NUP98 moiety of NUP98 fusion proteins, we purified the protein complexes formed with wild-type NUP98-HOXA9 and its mutants that lack the each FG repeat domain and looked for the proteins that interact with the NUP98 moiety. Mass spectrometry analysis identified that fatty acid synthase (FASN) interacted with the FG repeat domain of NUP98-HOXA9. These data suggest two possibilities. One is that NUP98-HOXA9 may affect the activity of FASN. The other is that FASN may affect activity of NUP98-HOXA9. To test the former possibility, we tested the effect of NUP98-HOXA9 on the activity of FASN in vitro, and we found that NUP98-HOXA9 reduced the FASN activity. FASN is known to be essential for cell growth. In fact, the colony formation of the NUP98-HOXA9-immortalized mouse myeloid stem/progenitor cells was inhibited by shRNA for FASN.　Because NUP98-HOXA9 reduced FASN activity, we hypothesized that FASN activity is low in NUP98 fusion gene-transformed cells and FASN inhibition selectively inhibits the colony formation of these cells. To test this hypothesis, we examined the effect of orlistat, which is the inhibitor of FASN on the colony formation. Orlistat strongly inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells, but it did not severely affect, the colony-forming activities of other fusion genes-transformed cells and normal progenitor cells. Other FASN inhibitors inhibited the colony formation of NUP98-HOXA9- and NUP98-DDX10-transformed cells. NUP98-HOXA9- and NUP98-DDX10 did not affect the expression level of FASN. The inhibition of FASN did not affect the expression of Hoxa genes and Meis1 gene, which are upregulated by NUP98 fusions. These results suggest that FASN activity is low but is essential for NUP98 fusion-mediated leukemia cells, and that FASN can be a therapeutic target for NUP98 fusion gene-mediated leukemia.

Disclosures

No relevant conflicts of interest to declare.
The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome Positive Acute Lymphatic Leukemia
Authors: Mian Afsar , Rafiei Anahita , Oancea Claudia , Ottmann Oliver G. , Ruthardt Martin
Published: November 2019

The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL.

Disclosures

No relevant conflicts of interest to declare.
KLF4 Acts As a Tumor Suppressor in T-Acute Lymphoblastic Leukemia and Negatively Regulates Human T Cell Development and Homeostasis
Authors: Xu Bing , Li Peng
Published: November 2019

The transcription factor Kruppel-like factor 4 (KLF4) may induce tumorigenesis or suppress tumor growth in a tissue-dependent manner. We found that overexpression of KLF4 induced not only human acute T-acute lymphoblastic leukemia (T-ALL) cell lines but also primary samples from T-ALL patients to undergo apoptosis through the BCL2/BCLXL pathway in vitro. T cell-associated genes including BCL11B, GATA3, and TCF7 were negatively regulated by KLF4 overexpression. Especially, KLF4 induced SUMOylation and degradation of BCL11B. However, the KLF4-induced apoptosis in T-ALL was rescued by the in vivo microenvironment. Furthermore, the invasion capacity of T-ALL to hosts was compromised when KLF4 was overexpressed. In normal human T cells, the overexpression of KLF4 severely impaired T cell development at early stages, but the blockage of T cell development was resumed by restoration of GATA3 or ICN1. In summary, our data demonstrate that KLF4 acts as a tumor suppressor in malignant T cells and that downregulation of KLF4 may be a prerequisite for early human T cell development and homeostasis.

Disclosures

No relevant conflicts of interest to declare.
Loss of PTEN in Pediatric T-Cell Acute Lymphoblastic Leukemia Patients: Proteomic and Molecular Characterization
Authors: Bonaccorso Paola , Bugarin Cristina , Buracchi Chiara , Fazio Grazia , Paganin Maddalena , Basso Giuseppe , Biondi Andrea , Cazzaniga Giovanni , Lo Nigro Luca , Gaipa Giuseppe
Published: November 2019

Signaling networks such as the PI3K/Akt/PTEN/mTOR pathway play a role in the modulation of the aggressiveness of T-Cell Acute Lymphoblastic Leukemia (T-ALL), but the significance of an aberrant activation of this pathway is poorly investigated. The master regulator of PI3K-AKT signaling is the lipid phosphatase and tensin homolog (PTEN); decreased or absent PTEN expression or activity could be associated with a constitutive activation of PI3K/Akt/PTEN/mTOR signaling pathway in T-ALLs. We investigated PTEN Exon7 mutations in diagnostic DNA samples from 67 pediatric T-ALL enrolled in AIEOP ALL2000 and R2006 protocols according to methods by Bandapalli et al. (Haematologica, 2012). We also investigated the PI3K/Akt/PTEN/mTOR pathway using Western Blotting (WB) and Flow Cytometry (FC) in parallel. WB analysis was performed using standard RIPA buffer for protein extraction. FC analysis of protein expression was applied as previously described (Gaipa G et al, Leukemia 2008), and protein levels were measured as % of positive cells compared to isotype control. Positivity or negativity by WB was established by presence or absence of a protein band, while for FC a threshold for positivity was set at ≥ 1% of positive cells. PTEN Exon 7 mutations were identified in 11 out of 67 (16.4%) of patients. According to samples availability, PI3K/Akt/PTEN/mTOR pathway was studied in 9 out of 67 patients (3 PTEN Exon 7 mutated and 6 wild type). PTEN protein resulted completely absent in all three PTEN Exon 7 mutated patients, by contrast PTEN was expressed in all 6 PTEN exon 7 wild type patients (mean by FC 46.98% ± 28.58%). This finding was fully confirmed when WB was applied to the same samples. We did not observe any statistically significant differences in p4EBP1 or mTOR levels in Exon 7 mutated patients as compared to wild type. By contrast, PTEN Exon 7 mutated blasts showed lower phospho-S6 levels compared to PTEN Exon7 wild type patients (mean 3.0% of positive cells vs 32.0%, p=ns). In conclusion, our data show a frequency of PTEN Exon 7 mutations of 16.4%, in agreement with the report by Bandapalli et al. (17.3%). Interestingly, we observed a strong association between the PTEN Exon 7 mutation and the total absence of PTEN protein in the pediatric T-ALL patients studied (data not reported so far to our knowledge). Moreover, although the differences are not statistically significant, p-S6 expression resulted consistently lower in mutated patients as compared to wild type patients. If confirmed in a larger cohort of pediatric T-ALLs, our data could open new insights in the significance of PTEN Exon 7 mutation in pediatric T-ALL and its associated functional profile.

Disclosures

No relevant conflicts of interest to declare.
Identification of Minor Histocompatibility Antigens Based on the 1000 Genomes Project for Application in Therapeutic Dendritic Cell Vaccination
Authors: Oostvogels Rimke , Lokhorst Henk M. , van Elk Maureen , van den Oudenalder Kelly , Spierings Eric , Minnema Monique C. , Mutis Tuna , Spaapen Robbert
Published: November 2019

Minor histocompatibility antigens (mHags) are highly immunogenic, HLA-bound polymorphic peptides playing prominent roles in the development of the graft vs host disease (GvHD) as well as the therapeutic graft vs tumor (GvT) effect of allogeneic stem cell transplantation (allo-SCT). To date, a small subset of mHags has been identified as genuinely hematopoietic system-specific. This small number of conceptually GvT-associated mHags are currently being tested in various clinical trials for the feasibility and efficacy of inducing an exclusive GvT effect without GvHD in patients with relapsing haematological malignancies after allo-SCT. Nonetheless, a broader and timely application of mHags as therapeutic tools in allo-SCT, as well as the development of appropriate GvHD prevention strategies still requires more insights that can be acquired through the identification of a much larger set of mHags presented by various HLA molecules in different ethnic populations. Recently, the introduction of genome-wide association (GWAS)-based strategies, despite a number of drawbacks, significantly accelerated the identification of mHags. To develop a strategy that can overcome the drawbacks of these earlier developed strategies, we now integrated our previously developed GWAS methodology [1,2] with the comprehensive genomic databases from the 1000 Genomes Project [3]. We show that the data set of the 1000 Genomes Project is suitable to identify all theoretical possible mHags, hereby enabling us to delineate important yet unknown characteristics of mHags. Furthermore, we demonstrate the actual power of this novel approach by the rapid and unambiguous identification of the HLA-DP4 restricted mHag UTDP4-1, which -despite extensive efforts in the past 15 years- could not be identified using any of the previously developed biochemical, molecular biological or genetic strategies. The 1000 Genomes Project-based identification of mHags thus represents a very rapid, convenient and robust method for the identification of new mHags, that will not only improve the understanding of the yet unknown facets of mHags but may also significantly enlarge the arsenal of targets for immunotherapy in haematological cancers.

Spaapen et al., J.Exp.Med. 2008 Nov 24;205(12):2863-72. Oostvogels et al., Leukemia 2013 Mar;27(3):642-9. Abecasis et al., Nature 2012 Nov 1;491(7422):56-65.

Disclosures

Lokhorst: Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Mutis:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding.