bluuebunny/crossref_metadata_2025 huggingface dataset:
S136 REPROGRAMMING HEMATOPOIETIC STEM CELL FUNCTION VIA MODULATION OF MITOCHONDRIAL ACTIVITY
Authors: Girotra M. , Rincon‐Restrepo M. , Oggier A. , Coukos G. , Naveiras O. , Rezzi S. , Vannini N.
Published: June 2019

Background:

Hematopoietic stem cells (HSCs) are a small group of cells that ensure a steady production of all mature blood cells throughout an organism's lifetime. HSCs mediate this process by giving rise to progenitors that in turn produce terminally differentiated mature blood cell lineages via several rounds of cell division and well‐orchestrated differentiation steps. Cellular metabolism has emerged as a crucial regulator guiding this complex process. Especially, HSCs differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. This distinct metabolic state protects the HSCs from cellular damage inflicted by reactive oxygen species (ROS) in active mitochondria. However, whether this change in the metabolic program is the cause or a consequence of the unique function of HSCs remains unknown.

Aims:

Having previously shown that mitochondrial activity can be used as a reliable readout for HSC fate, here we asked if modulation of mitochondrial activity results in enhancement of HSC function.

Methods:

We used in vitro mitochondrial activity and in vivo long‐term blood reconstitution assays as readouts of murine and human HSC functionality.

Results:

We previously demonstrated that modulation of mitochondrial metabolism in murine and human HSCs, by chemical uncouplers of the electron transport chain or via NAD+ boosting agent Nicotinamide Riboside (NR), results in better long‐term blood production in serially transplanted mice (Vannini N∗, Girotra M∗. et al., Nature Communication 2016 and, Vannini et al., Cell Stem Cell, in press).

Here we proceeded to carry out a screen, using mitochondrial activity as readout, to identify metabolic modulators that enhance HSC function. We found two novel candidates, a natural compound and a vitamin precursor, that modulate mitochondrial activity in both mouse and human HSCs, and resulted in enhanced HSC function post bone‐marrow transplantation. Interestingly, we found that these candidates mediate their effects partially by inducing mitophagy, supporting recent studies highlighting the role of mitophagy as a key driver of HSC function. Moreover, our preliminary analysis reveals that they mediate similar effects in aged human HSCs, making them ideal candidates to revert age‐associated myeloid bias in human patients.

Summary/Conclusion:

Our data establishes a causal relationship between mitochondrial metabolism and HSC function via mitophagy. It also provides a valuable tool to identify optimal ex vivo conditions for HSC expansion, to improve the outcome for patients suffering from bone marrow insufficiency and to restore the functionality of the immune system in aged individuals.
PF193 NO BLASTS AT DAY 8 IS BETTER THAN MORE IN CORTICO‐SENSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA OF CHILDREN
Authors: Frikha I. , Turki I. , Medhaffer M. , Hdijji S. , Ben Amor I. , Charfi M. , Kassar O. , Ghorbel M. , Kallel F. , Elloumi M.
Published: June 2019

Background:

Acute lymphoblastic leukemia (ALL) had a good prognostic in children and young adults. However, some parameters were predictive of a worse outcome, particularly the poor response to prephase.

Aims:

We analysed in this study the impact of persistent blasts at day 8 of pre‐phase in cortico‐sensitives patients with ALL.

Methods:

Between january 2000 and December 2015, we retrospectively analysed the data of 264 children and young adults treated according the EORTC 58951 protocol. Cortico‐sensitivity (Cs) was considered if peripheral blast count is less than 1000/mm3 at day 8 of pre‐phase. The group of cortico‐sensitives ALL was subdivided on two subgroups : the first with no blasts at day 8 (Cs = 0) and the second with more (Csǂ0). We analysed the impact of cortico‐sensitivity on complete remission (CR), relapse and survival (overall survival (OS), event free survival (EFS) and disease free survival (DFS)).

Results:

One hundred ninety two children and 72 young adults were treated according to the EORTC 58951 protocol. Median age was 10 years (1 to 30 years). Sex ratio was 1,5. B and T phenotype were observed in respectively 64 and 36% of cases. The ALL were stratified in low risk (11 patients), average risk (182 patients), and high risk (71 patients). Two hundred and nine patients (79%) were cortico‐sensitive, among them 150 patients (72%) had no blast at day 8 of pre‐phase. The therapeutics results in our study and into the two groups of cortico‐sensitivity were shown in this table :

Summary/Conclusion:

Cortico‐sensitivity is one of several parameters that predict good outcome in ALL. Moreover, our study showed that no blasts in peripheral blood smears at day 8of prephase can be considered a good first marker of early blastic clearance and good outcome. In fact, this parameter does not discussed previously in the literature.

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PF199 TARGETING BRD4 IN MLL‐PTD AML
Authors: Pepe F. , McNeil B. , Ozer H.G. , Karunasiri M. , Bill M. , Garzon R. , Dorrance A.
Published: June 2019

Background:

Mixed lineage leukemia (MLL) gene encodes for a nuclear protein that regulates genes involved in development and hematopoiesis. De‐regulation of MLL by chromosome translocations causes acute myeloid leukemia (AML). In addition, about 5–11% of AML patients have MLL‐partial tandem duplication (PTD) mutations. MLL‐PTD is associated with poor prognosis and shorter remission duration in AML. Preliminary studies from our group indicate that when MLL‐PTD is knockdown in AML blasts we observe a mRNA expression profile that is very similar to that of bromodomain 4 (BRD4) inhibition in AML.

Aims:

We hypothesized that MLL‐PTD interacts with BRD4 forming a complex that regulate key genes in MLL‐PTD positive AML. Therefore treatment with BRD4 inhibitors, such as JQ1, could be a valid therapeutic strategy for MLL‐PTD AML patients.

Methods:

To identify proteins forming the transcriptional complex regulated by BRD4 and MLL, we conducted a Rapid Immunoprecipitation Mass spectrometry of Endogenous protein (RIME) study using BRD4 and MLL antibodies. Successively, we treated MLL‐PTD positive primary patient cells and CD34+ cord blood from healthy donors as controls with JQ1, and conducted RNA‐seq and ChIP‐seq studies. Finally, we conducted in vitro and in vivo studies on MLL‐PTD harboring samples to assess JQ1 treatment efficiency in inducing cell death in MLL‐PTD AML.

Results:

RIME analysis using BRD4 and MLL antibodies showed that the 75% of the proteins were in common between the two groups. PANTHER gene ontology analysis identified common pathways between them, suggesting that MLL and BRD4 could be part of similar protein complexes. Next, we treated MLL‐PTD+ primary patient cells, and CD34+ cord blood cells as controls with JQ1 or vehicle, and conducted RNA‐seq and ChIP‐seq studies. RNA expression and peak intensity on promoter region results were combined to identify genes aberrantly expressed in MLL‐PTD+ samples. We identified 98 up‐regulated and 32 down‐regulated genes that were differentially expressed d by JQ1 treatment only in AML samples. The top deregulated genes were validated by RT‐qPCR and ChIP in both human and murine MLL‐PTD+ samples. Next, we treated the MLL‐PTD+ AML cell line (EOL1) and primary murine AML cells from Mll‐PTD/Flt3‐ITD model with JQ1 and showed that JQ1 treatment induced more apoptosis and reduction of living cells in MLL‐PTD+ models respect the relative controls (fold change living cells at 48 hours WT: 0.81, Mll‐PTD+: 0.42, p‐value: 0.0247). Last, treatment of the Mll‐PTD/Flt3‐ITD xenotranplanted BoyJ mice with JQ1 significantly increased their survival compared with controls (median survival CTRL: 75 days, JQ1: 109 days, Mantel‐Cox p‐value: 0.0195). In addition, we observed a reduced engraftment in serial re‐transplantation assays.

Summary/Conclusion:

Our data support developing a therapeutic strategy to target BRD4 in MLL‐PTD AML.

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PF249 NEXT‐GENERATION‐SEQUENCE‐BASED GENE MUTATION DETECTION AND ITS CLINICAL SIGNIFICANCE IN PATIENTS WITH ACUTE MYELOID LEUKEMIA
Authors: Yu J. , Li T. , Li Y. , Li Y. , Xing H. , Sun H. , Wan D. , Liu Y. , Wang C. , Xie X. , Jiang Z.
Published: June 2019

Background:

Acute myeloid leukemia (AML) is a biologically heterogeneous disease with many different cytogenetic disorders and gene mutations. The advances of the next‐generation sequence (NGS) made it more practical for the clinical research to explore the cytogenetic analysis and mutation testing for the classification and risk assessment of AML patients.

Aims:

In this study, we sought to investigate the differences in molecular gene mutations between different type of AML patients and the response from those newly diagnosed AML patients to induction chemotherapy.

Methods:

We retrospectively evaluated 274 hospitalized non‐M3 AML patients. A next‐generation sequencing assay covering 43 genes was used to investigate recurrently mutated genes. The mutational status of fusion genes was determined by real time PCR. Clinical follow up was based on the patients recent clinic visits. Correlation analysis was done based different groups classified by different categories.

Results:

The median age at diagnosis was 45 year‐old (range 16–81 years). 231 patients were 59 years or younger with the median age of 41 years. Forty‐three patients were 60 years or older with a median age of 69 years (range 60–81 years). At least one non‐synonymous gene mutation was detected in 233 patients (85%). The median number of gene mutations was two (range 0–8). Comparing to the 59 years or younger patients, there was a significantly higher incidence of gene mutation in 60 years and older patients (p <0.01). The mean number of molecular gene mutations at diagnosis was higher in older patients than younger patients (2.92 vs 1.48, p < 0.05). Older patients had significantly higher frequency of ASXL1 (23.3% vs 13.9%, p <0.05), TP53 (11.6% vs 1.7%, p <0.05), IDH2 (18.6% vs 4.3%, p < 0.05) mutations, higher frequency of CEBPA (21.2% vs 14%, p < 0.05). Among 11 patients with no gene mutation received DA (daunorubicin 80 mg/m2 per day on days 1–3 and cytarabine 100 mg/m2 twice per day on days 1–7) as the induction therapy, 8 patients (73%) achieved CR after 1 course induction treatment. Meanwhile, among 21 patients with one or more gene mutations received DA induction therapy, only 12 patients (57%) achieved CR after 1 course induction treatment. More data need to be collected regarding the significance of each individual gene fusion correlating with induction chemotherapy.

Summary/Conclusion:

A higher incidence of cytogenetic disorders was found in older AML patients with higher burdens of molecular mutations, comparing to younger patients. Also, significantly higher frequency of ASXL1, TP53, IDH2 and CEBPA had been found with older patients. Patients with fewer gene fusion had a better chance of achieving CR when treated with cytarabine and daunorubicin induction chemotherapy.
PF270 IS ACUTE MYELOBLASTIC LEUKEMIA IN CHILDREN UNDER TWO YEARS OF AGE A SPECIFIC ENTITY? A REPORT FROM THE FRENCH ELAM02 STUDY GROUP
Authors: Blais S. , Boutroux H. , Pasquet M. , Leblanc T. , Fenneteau O. , Gandemer V. , Bertrand Y. , Ducassou S. , Michel G. , Nelken B. , Petit A. , Cuccuini W. , Gouache E. , Marceau A. , Baruchel A. , Lapillonne H. , Leverger G.
Published: June 2019

Background:

The clinical and biological characteristics of children under two years (infants) with acute myeloid leukemia (AML) are different from those of older children.

Aims:

We aimed to describe the specific characteristics of this population and the potential factors that influence the prognosis.

Methods:

We analyzed data concerning 438 children with newly‐diagnosed AML treated in the ELAM02 protocol between March 2005 and December 2011, of which 103 were under two years old at diagnosis. The evaluation criteria were overall survival (OS) and event‐free survival (EFS) of infants versus older children. The clinical and biological features were secondary criteria.

Results:

Infants presented more frequent extra‐medullary presentation than older children. They had a significantly higher proportion of skin lesions and central nervous system involvement (15% vs 3%, P < 0.0001 and 26% vs 12%, P = 0.0005, respectively). The global incidence of KMT2A rearrangements was nearly 55% for infants versus 11% for older children (P < 0.0001). Median five‐year OS was 70.4% for infants versus 71.4% for older children (P = 0.83). Five‐year EFS was 67% for infants versus 58% for older children (P = 0.27).

Summary/Conclusion:

Infants with AML represent a cohort of patients with specific clinical and biological features. These remarkable differences had no significant impact on their outcome in the ELAM02 protocol.
PF292 LOW‐DOSE CYTARABINE WITH OR WITHOUT GLASDEGIB IN NEWLY DIAGNOSED PATIENTS WITH ACUTE MYELOID LEUKEMIA: LONG‐TERM ANALYSIS OF A PHASE 2 RANDOMIZED TRIAL
Authors: Papayannidis C. , Smith B.D. , Heuser M. , Montesinos P. , Sekeres M.A. , Oriol A. , Schiller G. , Candoni A. , Jamieson C. , Hoang C. , Ma W.W. , Zeremski M. , O’Connell A. , Chan G. , Cortes J.
Published: June 2019

Background:

Glasdegib is a potent, selective, oral inhibitor of the Hedgehog signaling pathway, approved in the US – in combination with low‐dose cytarabine (LDAC) – for the treatment of newly diagnosed acute myeloid leukemia (AML) in patients unable to receive intensive chemotherapy due to comorbidities or age (75 years or older).

Aims:

To evaluate long‐term efficacy and safety of LDAC with or without glasdegib in patients newly diagnosed with AML unable to receive intensive chemotherapy due to comorbidities or age.

Methods:

In this follow‐up analysis from the phase 2 BRIGHT AML trial (NCT01546038), newly diagnosed patients with AML ineligible for intensive chemotherapy were randomized 2:1 to glasdegib + LDAC or LDAC alone (study design was reported previously: Cortes et al., Leukemia 2018). This long‐term analysis evaluated efficacy and safety after approximately 20 months of additional follow‐up.

Results:

As of October 11, 2018, 116 patients were assigned to treatment with glasdegib + LDAC (n = 78) or LDAC alone (n = 38). The median follow‐up time for glasdegib + LDAC and LDAC alone was 43.4 and 42.0 months, respectively. The median overall survival was higher with glasdegib + LDAC vs LDAC alone (Table). Improvement in overall survival was consistent across the prespecified subgroups. The main cause of death in both arms was disease progression (both during study and follow‐up). The incidence of adverse events (AEs) and serious AEs on glasdegib was generally lower long term (after 90 days) than short term (during the first 90 days) (83.7% and 51.2% vs 98.7% and 65.3%, respectively).

Summary/Conclusion:

Addition of glasdegib to LDAC vs LDAC alone continued to demonstrate improved overall survival in patients with AML in this analysis; improvement was consistent across groups stratified by cytogenetic risk. Long‐term follow‐up confirmed that treatment with glasdegib was associated with an acceptable safety profile.

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PF343 CRISPR/CAS9 MEDIATED ELANE KNOCKOUT ENABLES NEUTROPHILIC MATURATION OF HSPCS AND IPSCS OF SEVERE CONGENITAL NEUTROPENIA PATIENTS
Authors: Nasri M. , Ritter M. , Mir P. , Dannenmann B. , Amend D. , Makaryan V. , Xu Y. , Zeidler C. , Dale D.C. , Klimiankou M. , Welte K. , Skokowa J.
Published: June 2019

Background:

Autosomal‐dominant ELANE mutations are the most common cause of severe congenital neutropenia (CN). Although the majority of CN patients respond to daily granulocyte colony stimulating factor (G‐CSF), approximately 15 % do not respond to this cytokine at doses up to 50 μg/kg/day and approximately 15% of G‐CSF treated patients will develop myelodysplasia (MDS) or acute myeloid leukemia (AML).

Aims:

“Maturation arrest,” the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated CN. Because mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and “maturation arrest” corrected by CRISPR/Cas9 mediated gene editing.

Methods:

We applied CRISPR/Cas9‐sgRNA ribonucleoprotein (RNP) mediated ELANE knockout (ELANE KO) in induced pluripotent stem cells (iPSC) and primary hematopoietic stem and progenitor cells (HSPC).

Results:

We used iPSC from 2 CN patients and HSPC from 4 CN patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE. We observed that granulocytic differentiation of ELANE KO iPSC and HSPCs was comparable to healthy individuals and phagocytic function of the ELANE KO neutrophils was also normal. Knock down of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils.

Summary/Conclusion:

These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients’ HSPCs followed by autologous transplantation may be an alternative therapy for CN.
PF417 PONATINIB IN CHILDHOOD PHILADELPHIA POSITIVE LEUKEMIAS: THE EXPERIENCE OF THE INTERNATIONAL REGISTRY OF CHILDHOOD CHRONIC MYLELOID LEUKEMIA (I‐CML‐PED‐STUDY)
Authors: Millot F. , Suttorp M. , de Bont E. , Kalwak K. , Nelken B. , Ducassou S. , Bertrand Y. , Baruchel A.
Published: June 2019

Background:

Ponatinib is effective in adults with Philadelphia positive (Ph+) leukemias but only few case reports are available regarding the use of this so far not‐licensed drug in children and adolescents.

Aims:

To describe the profile of tolerance and efficacy of compassionate use of ponatinib in children and adolescents with Ph+ leukemias.

Methods:

Data from 11 children with chronic myeloid leukemia (CML) registered to the I‐CML‐Ped‐Study and from 3 children with Ph+ acute lymphoblastic leukemia (Ph+ ALL) treated with ponatinib were retrospectively collected.

Results:

In 11 girls and 3 boys (median age 14 years, range: 4.5–17) ponatinib was used as a second (n = 2), third (n = 5), fourth (n = 2), fifth (n = 3), sixth (n = 1) or eighth (n = 1) treatment line. Ponatinib was administered as single therapy in 7 patients while the 7 remaining patients (advanced phases CML n = 5, Ph+ ALL n = 2) received additional chemotherapy. The status of the disease when ponatinib was started was: CML advanced phases (accelerated phase, n = 2; blast crisis, n = 6), CML without achievement of major molecular response (MMR, n = 2), T315I mutation (n = 1) and in Ph+ ALL patients molecular relapse (n = 2) or marrow relapse (n = 1). Six patients were previously transplanted. Ponatinib was used as a bridge to transplant in 5 cases. Dose administered ranged between 15–41 mg/m2 (median: 27 mg/m2) and median duration of ponatinib treatment was 2.5 months (range: 0.5–14 months). Treatment responses in the 8 patients in CML‐advanced phases were: no response (n = 2), complete hematologic response (n = 1), MMR (n = 3), MR4.5 (n = 1) or unknown (n = 1). In patients in CML chronic phase, achievement of MMR was obtained in 2 of the patients without prior achievement of MMR while no response was observed in the patient with mutation T315I. Two of the 3 patients with Ph+ ALL achieved MR4.5 and MR5 while the remaining patient did not respond. Grade 1 to 4 side effects were recorded in 5 of 7 patients treated with ponatinib alone; toxicity grade 3–4 was reported in 2 patients (hematologic toxicity). No vascular event related to ponatinib was observed.

Summary/Conclusion:

With the limitation of the retrospective nature of this study, ponatinib may be a reasonable additional treatment option for children with Ph+ leukemias who failed to several lines of therapy.
PF421 PLANNED PREGNANCY IN FEMALE PATIENTS WITH CHRONIC MYELOID LEUKEMIA RECEIVING TYROSINE KINASE INHIBITOR THERAPY
Authors: Dou X. , Qin Y. , Huang X. , Jiang Q.
Published: June 2019

Background:

Pregnancy has become an important issue in female patients with chronic myeloid leukemia (CML) on tyrosine kinase inhibitor (TKI). Female patients with CML who wish to conceive are currently advised to discontinue TKI before conception due to the recognized teratogenic effects of TKI. However, it is not known what the ideal degree and duration of response a female patient should achieve before stopping TKI‐therapy for pregnancy. Concrete infant outcomes for female patients who discontinued TKI‐therapy immediately once pregnancy was confirmed are also lacking.

Aims:

To explore disease and fetal outcomes of planned pregnancy in female patients with CML on tyrosine TKI therapy.

Methods:

Data of female patients with CML proceeding with a planned pregnancy were retrospectively reviewed.

Results:

A total of 17 female CML patients who achieved at least a major molecular response (MMR) during imatinib (n = 13) or nilotinib (n = 4) therapy prior to a planned pregnancy were enrolled. At the time of TKI interruption, patients had received TKI‐therapy for a median of 49 months (range, 6–102 months), 6 were in MMR, 2 in MR4, and 9 in MR4.5. TKI‐therapy was discontinued at a median of 6 weeks (range, 2–15 weeks) before conception in 4 patients and at a median gestational age of 4 weeks (range, 2–5 weeks) after determination of pregnancy in 13 patients. Apart from one patient who suffered a spontaneous abortion, 16 patients delivered uneventfully at full term. A total of 10 patients lost MMR at a median of 3 months (range, 1–9 months) after stopping TKI; 8 patients lost MR2 at 4 months (range, 2–7 months) and 3 lost complete hematological response (CHR) at 3 months (range, 3–4 months) after TKI cessation. None of the patients progressed to an advanced phase. Log‐rank analyses showed achievement of MR4 (p = 0.030), MR4.5 (p = 0.031), CCyR (p = 0.049) or MMR duration (p = 0.040) ≥3.5 years were significantly associated with longer MMR‐failure free survival. Each of the 10 patients losing MMR during pregnancy regained MMR at a median of 4 months (range, 2–7 months) after resuming TKI‐therapy, and 1 patient who maintained MR4.5 during pregnancy remained off treatment at 6 months after delivery. All 16 babies that were successfully delivered have continued normal growth and development during a median follow‐up of 19 months (range, 7–78 months).

Summary/Conclusion:

Planned pregnancy might be safe and practicable in female patients with CML on TKI‐therapy. Achieving deep molecular response and importantly MMR duration ≥3.5 years before conception were significantly associated with maintaining MMR during pregnancy.

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PF538 GENETIC EVOLUTION PATTERNS IN PATIENTS WITH CHRONIC MYELOMONOCYTIC LEUKEMIA TO SECONDARY ACUTE MYELOID LEUKEMIA: AN ANALYSIS OF 36 PAIRED SAMPLES
Authors: Su Y.‐J. , Kuo M.‐C. , Wang P.‐N. , Wu J.‐H. , Lin T.‐H. , Huang T.‐Y. , Lin T.‐L. , Chang H. , Hung Y.‐S. , Shih L.‐Y.
Published: June 2019

Background:

Chronic myelomonocytic leukemia (CMML) are associated with a tendency to secondary acute myeloid leukemia (sAML) progression.

Aims:

We sought to examine the patterns of genetic evolution by analyzing a cohort of matched paired samples collected at initial diagnosis and at sAML transformation.

Methods:

Mutational analyses of 33 genes related to myeloid neoplasms on paired bone marrow samples from 36 patients with CMML at both diagnosis and sAML phase were performed. The samples were analyzed by PCR‐based assays followed by direct sequencing and/or next generation sequencing (NGS). The allele frequency of mutated genes was determined by variant allele frequency with NGS and/or pyrosequencing for validation.

Results:

Mutational analyses on the 36 paired CMML/sAML samples showed that 92% of patients had at least one mutated gene at initial diagnosis. Gene mutations involving epigenetic regulators (65%), myeloid transcription factors (52%), spliceosome (50 %), and signaling pathway (47%) were frequently detected, followed by BCOR (11%), NPM1 (6%) and SETBP1(6%), mutations of cohesin complex were rare (3%), and none had TP53, WT‐1 or BCORL1 mutations. Co‐existed mutations with different gene classes were present in 78% of CMML patients at diagnosis. The median time from CMML to sAML was 7.2 months (ranged from 1 to 89.6 months). At sAML transformation, 97% of patients had at least one mutated gene, mutation frequencies of epigenetic regulators (62%) and spliceosome (56%) remained mostly unchanged at sAML phase. Acquisition of additional gene mutations was observed in 47% of patients during sAML progression, in which signaling pathway mutations emerged most frequently (RTK/RAS/JAK‐STAT: 5/3/1), followed by transcription factors (3 acquiring RUNX1 mutations and 3 CEBPa). Emergence of new mutant subtypes of the same genes with or without disappearance of pre‐existed mutants was found in 3 RUNX1, and one each for CBL, PTPN11 and TET2 mutated patients during sAML progression. In addition, 8 patients had apparently increase in allele burden (>20%) for 6 genes. Loss of mutations was found in 4 (11%) patients during sAML progression, one each for PTPN11, JAK2V617F, CSF3R and TET2. Eight patients had apparently decrease in allele burden (>20%) for 4 different genes (N‐RAS, PTPN11, CBL and RUNX1).

Summary/Conclusion:

The present study on a relative large cohort of matched paired CMML/sAML samples showed that genetic evolution from CMML to sAML exhibited different patterns, including stable mutations, acquisition of new mutations, subclone selection or expansion of pre‐existed clones. Loss of mutation might occur but infrequently.
PF571 IDENTIFICATION OF HLA‐E AS A KEY GENE IN MULTIPLE MYELOMA BASED ON BIOINFORMATIC ANALYSIS AND DESIGN HIGH‐AFFINITY PEPTIDES TARGETING HLA‐E BASED ON COMPUTATIONAL TECHNIQUES
Authors: Yang Y. , sun M. , yu Z. , Liu Z. , Wei M.
Published: June 2019

Background:

Multiple myeloma (MM) is a common plasma cell malignancy that is currently incurable. Finding potential drug targets and new drugs are crucial for the treatment of thisdisease. Microarray technology is widely used to investigate differential mRNA expression and identify biomarkers in disease, which provides important clues to identifying new treatment targets and then new drugs could be designed.

Aims:

To find new treatment target for MM and design targeting affinity peptide.

Methods:

Two datasets (GSE6691 and GSE39754) were obtained from the Gene Expression Omnibus (GEO) database, and a protein‐protein interaction (PPI) network of differentially expressed genes (DEGs) was constructed to identify hub genes.Key clusters in the PPI network were analyzed by Molecular Complex Detection (MCODE). The gene both in hub genes and key clusters could be considered as potential treatment target and will be verified in clinical patients’ bone marrow samples. As the treatment target identified, crystal structure is obtained from Protein Data Bank and affinity peptide drug designed by Molecular Operating Environment (MOE) software.

Results:

HLA‐E was identified as hub gene and also located in one of the key clusters which could be a potential therapeutic target for MM.The high expression of HLA‐E in myeloma cells was confirmed by measuring HLA‐E mRNA expression in bone marrow samples from MM patients and healthy volunteers from Shengjing Hospital.The crystal structure of HLA‐E from the Protein Data Bank(PDB ID: 3CDG) was used to find the key active area of HLA‐E. MOE softwarewas used to design high‐affinity targeting peptides using key interaction amino acids as model peptides. A peptide library was constructed and screened to identify peptides with the highest stability and affinity for HLA‐E. Three high‐affinity peptides were selected and verified by peptide‐protein docking. Thus, these peptides could serve as targeting heads to find myeloma cells that express high levels of HLA‐E.

Summary/Conclusion:

HLA‐E could be recognized as a new treatment target for MM. At the meanwhile, affinity peptide(NALDEYCEDKNR) could be considered as a new targeting drug.

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PF584 C‐MYC‐DRIVEN AND PRMT5‐DEPENDENT REGULATION OF MULTIPLE MYELOMA CELL PROLIFERATION THROUGH SMCHD1 GENE
Authors: Peipei X. , guo D.
Published: June 2019

Background:

The protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetrical bimethylation of arginine residues, which plays an important regulatory role in the life process. Transcription factor c‐Myc is widely proved involved in the alteration and development of tumors, while the regulatory mechanism of c‐Myc‐mediated gene expression has not been fully understood.

Aims:

In this article we try to investigate the functional role of PRMT5 and c‐Myc in regulating multiple myeloma cell proliferation

Methods:

The protein and mRNA expression levels of PRMT5 in myeloma cells and normal cells were detected by Western Blot and qPCR. Lentivirally transduced shRNAs targeting PRMT5 (sh‐PRMT5) was constructed using lentivirus mediated RNAi technology, and was packaged to infect MM cells to select positive colonies. The effect of PRMT5 on the proliferation of MM cells was detected by cck‐8 assay. Annexin V/7‐AAD double staining flow cytometry was used to detect cell survival and apoptosis rate. The expression of apoptotic suppressor gene c‐Myc in sh‐PRMT5 cell lines was detected by Western Blot and qPCR. Finally, double‐knockdown of PRMT5 and c‐myc was performed and gene expression differentiation were identified by RNA sequencing (RNA‐seq).

Results:

PRMT5 expression in MM cell lines (RPMI8226 and U266) was relatively high compared with peripheral blood mononuclear cells (PBMCs), which was correlated with progression‐free survival (PFS) and overall survival (OS) in MM patients. After sh‐PRMT5 infection to MM cell lines, puromycin selection was performed and efficient gene knockdown was evaluated by Western Blot and qPCR. After targeted silencing of PRMT5 expression, the proliferation of MM cells was decreased and apoptosis was significantly increased (P < 0.05). C‐Myc protein and mRNA levels were significantly downregulated after PRMT5 gene silencing. The results showed that PRMT5 promoted the proliferation of MM cells and inhibited the apoptosis of MM cells by regulating the expression of apoptotic suppressor gene c‐Myc. Subsequently, RNA‐seq confirmed SMCHD1 as the common candidate target gene of c‐myc and PRMT5. Therefore, we speculated that PRMT5 and c‐Myc may jointly regulate SMCHD1 gene to promote the proliferation of MM cells.

Summary/Conclusion:

Altogether, our study not only proposed the mechanism PRMT5 and c‐myc in regulating multiple myeloma related gene expression, but also provided a new strategy and theoretical basis for clinical treatment of multiple myeloma.
PF622 CARFILZOMIB‐LENALIDOMIDE‐DEXAMETHASONE IN THE MANAGEMENT OF LENALIDOMIDE‐REFRACTORY MULTIPLE MYELOMA
Authors: Cerchione C. , Martinelli G. , Nappi D. , Pareto A.E. , Di Perna M. , Ferrara K. , Ferrari A. , Simonetti G. , Pane F. , Catalano L.
Published: June 2019

Background:

Carfilzomib is an epoxyketone proteasome inhibitor of second generation, proved to be effective and safe in relapsed and refractory Multiple Myeloma (rrMM), in combination with dexamethasone or lenalidomide and dexamethasone.

Aims:

In this retrospective observational trial, it has been evaluated efficacy and safety of carfilzomib, in combination with lenalidomide‐dexamethasone (KRD) as salvage regimen in patients with rrMM, refractory to lenalidomide, whose prognosis is particularly severe.

Methods:

41 patients (23 M/18 F), with rrMM, median age at diagnosis 63.7 years (r. 43–82), median age at start of treatment 67 years (r. 48–84) previously treated with several lines of treatments (median 3, r. 2–11), underwent to KRD regimen (ASPIRE trial schedule) for a median treatment cycles of 8 (r 2–18). ISS was equally distributed, and all patients had previously been treated with bortezomib and IMIDs, and were refractory to this agents. 61% (19/31) of them had undergone at least to a single ASCT.

Results:

According to IMWG criteria, after a median follow‐up of 9 months (r. 2–18), ORR was 68,2% (28/41: 9 CR, 12 VGPR, 7 PR) with 5 progressive diseases (PD) and 8 patients in stable disease (SD): this can be considered as an impressive result in this subset of rrMM patients, refractory to lenalidomide. In particular, for 11 patients, KRD was, after having achieved at least a PR, a bridge to second/third autologous SCT. Median time to response was 1.3 months (r.1–4), median OS from diagnosis was 62 months (r. 9–170), median OS from start of Carfilzomib was 11 months (r. 2–18). Carfilzomib was well tolerated, with grade 2 anemia in 39% (16/41) of patients, successfully managed by ESAs, without necessity of blood transfusions; 29% (12/41) grade 3–4 neutropenia (pegfilgrastim in primary prophylaxis was given, no ospedalization was required, no septic shocks were observed); 34% (14/41) grade 2, 21% (9/41) grade 3 and 12% (5/41) grade 4 thrombocytopenia, without hemorrhagic events and transfusion‐dependency. Moreover, it was observed pneumonia in 39% (16/41) of patients, treated by common antibiotic drugs and always solved. A cardiac monitoring was performed for all patients: hypertension (grade 2–3) in 34% (14/41) of patients; fatigue in 39% (16/31) of patients.

Summary/Conclusion:

Carfilzomib‐Lenalidomide‐Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, also lenalidomide, and it could be considered as a bridge to a second autologous or allogenic SCT.
PF645 EXPOSURE‐RESPONSE ANALYSIS & DISEASE MODELING FOR SELECTION OF OPTIMAL DOSING REGIMEN OF ISATUXIMAB AS SINGLE AGENT IN PATIENTS WITH MULTIPLE MYELOMA
Authors: Thai H.‐T. , Liu L. , Koiwai K. , Brillac C. , Van de Velde H. , Nguyen L. , Veyrat‐Follet C. , Semiond D.
Published: June 2019

Background:

Isatuximab is an anti‐CD38 monoclonal antibody with multiple modes of action for killing tumor cells through direct tumor targeting and immune cell engagement [1].

Aims:

Exposure‐Response (E‐R) analysis and disease modelling of tumor burden were performed to evaluate the relationship between isatuximab exposure and efficacy outcome and to support dosing regimen selection for isatuximab as a single agent in relapsed/refractory multiple myeloma (RRMM) patients.

Methods:

The E‐R analyses were conducted in 194 RRMM patients evaluable for pharmacokinetics (PK) from two monotherapy trials, a phase 1/2 trial (NCT01084252)[2] and a phase 1 trial (NCT02514668). Isatuximab was administered intravenously at doses from 1 to 20 mg/kg once a week or every 2 weeks. Several isatuximab exposure parameters were tested and their association with the probability of achieving an objective response (CR, VGPR or PR) was examined by logistic regression modeling. Baseline covariates were also considered in the model to reduce their potential confounding effects. Disease progression was captured in a subset of 122 evaluable patients with the dynamics of the serum M‐protein and accounted for dropout using a joint model. Trial simulations were then performed to evaluate different dosing regimens of interest using both models.

Results:

The E‐R relationship was best described by Emax model, in which Ctrough at 4 weeks and percent of bone marrow plasma cells were significant predictors of overall response rate (ORR). Patients with bone marrow plasma cells lower than 50% were more likely to respond. For a given bone marrow plasma cell value, higher probability of response to treatment was obtained with higher Ctrough at 4 weeks. Longitudinal data of M‐protein was adequately described by a tumor growth inhibition model [3,4] and provided more insights into the response of patients over time. Therefore, a high loading dose of 20 mg/kg weekly over 4 weeks was chosen for maximizing the tumor response and a maintenance dose of 20 mg/kg every 2 weeks appeared sufficient to sustain efficacy. Clinical trial simulation demonstrated that this dosing regimen presented a probability of success of 76% to reach 30% ORR (5,000 trials with 100 patients) and would allow 52% reduction of serum M‐protein from baseline at 2 months of treatment. In addition, isatuximab appeared to be well tolerated at this dose level.

Summary/Conclusion:

Model‐based drug development has been successfully applied to support phase II isatuximab monotherapy dosing regimen selection in RRMM patients. This approach increases both the robustness of dose selection decision making and the chances of success for future clinical trials.
PF726 TOLERABILITY OF TREATMENTS FOR RELAPSED/REFRACTORY MULTIPLE MYELOMA: A SYSTEMATIC REVIEW
Authors: Nassim M. , Nilsson J. , Zavisic S. , Malmenäs M. , Fotheringham I. , Landfeldt E.
Published: June 2019

Background:

Multiple myeloma is a fatal cancer characterized by excessive proliferation and improper function of plasma cells in the bone marrow.

Aims:

To conduct a systematic literature review of the tolerability of treatments for relapsed/refractory multiple myeloma.

Methods:

We searched MEDLINE, Embase, and the Cochrane Library for English language full‐text articles published between 2000 and 2018 reporting results from randomized controlled trials of treatments for relapsed/refractory multiple myeloma. Two independent investigators screened article titles and abstracts for eligibility and subsequently reviewed full‐text versions of selected records. For all articles included in the review, the following data were extracted: Author, year of publication, setting, sample, treatments, and tolerability results.

Results:

The search resulted in the identification of 3488 unique publications. Of these, 3188 were excluded following title and abstract screening and 75 after full‐text review. A total of 22 articles were included for data synthesis. Identified randomized controlled trials involved an average of 477 patients (range: 80 to 768) and covered a total of 14 interventions for relapsed/refractory multiple myeloma. Of these, 14% were monotherapies and 86% combination therapies. Across included randomized controlled trials, the most frequently occurring adverse event during treatment was neuropathy, observed in 1%–67% of patients, followed by thrombocytopenia (1%–57%), pain (1%–24%), neutropenia (1%–52%), and diarrhea (1%–68%). Treatment discontinuation (all causes) was reported at between 31% and 95%, and discontinuation due to adverse events at between 7% and 34%.

Summary/Conclusion:

Current therapies for relapsed/refractory multiple myeloma are associated with several serious adverse events, resulting in treatment discontinuation. Yet, tolerability profiles vary markedly between interventions and patient populations. These data should be helpful to inform therapeutic decisions in relapsed/refractory multiple myeloma to enhance patient satisfaction and treatment compliance.
PF783 TREG DOWNREGULATION IS ASSOCIATED WITH INCREASE OF T CELL RESPONSES AGAINST IMMUNOGENIC ANTIGENS AND CLINICAL RESPONSES IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIES AFTER DONOR LYMPHOCYTE INFUSION
Authors: Greiner J. , Götz M. , Schmitt M. , Wiesneth M. , Bunjes D. , Hofmann S.
Published: June 2019

Background:

Cytotoxic T‐cell responses against malignant cells play a pivotal role in maintaining remission and prolonging overall‐survival after allogeneic stem cell transplantation (allo‐SCT) and donor lymphocyte infusion (DLI), however they are not well characterized so far.

Aims:

In this study, we focused on the detection of immune responses in patients before and after DLI. A broader epitope‐specific T cell activity is associated with clinical response of patients treated with DLI and additionally reduced regulatory T cell frequency may contribute to clinical response in patients after DLI.

Methods:

For a better characterization of the T cell responses, frequency and diversity of leukemia‐associated antigen (LAA)‐specific cytotoxic T cells was assessed using ELISpot and pMHC multimer assays. Furthermore, the frequency of regulatory T cells (Treg) before and after DLI was analyzed. Results were correlated to the clinical course of the patients.

Results:

Independently of their clinical response, over the course of DLI 7/11 patients (63.6%) showed an immunological response through an increase in the number of recognized epitopes. Comparing early screening and maximum response after DLI there was a significant increase (p = 0.02) in epitope recognition. A significant increase in the mean augmentation from 1 to 4 of the spotted epitopes in the course of DLI was detected in the cohort of clinical responders (R) compared to non‐responders (NR) who did not show any dynamics in epitope recognition with a median of 3 to 4 recognized LAA.

The proportion of the CD4+CD25highFoxP3+ Treg within the CD4+CD25high T cell fraction decreased significantly in R from a median of 72.9% to 54.6% when comparing the variation at the time points before and after DLI (p = 0.04). In NR there was no significant change in the Treg fraction in the course of DLI.

In general, significantly more LAA‐derived T cell epitopes (p = 0.02) were recognized in R when compared to NR. Moreover, the frequency of Treg in R decreased significantly (p = 0.008) while remaining stable in NR.

Summary/Conclusion:

Taken together, an increase of specific CTL responses against several LAA after DLI was detected. This study suggests that decreasing numbers of Treg as well as enhanced LAA diversity in T cell responses contribute to clinical outcome of patients treated with DLI.
PF794 DETECTION OF ANTIBODIES DIRECTED AGAINST DOMAIN 1 OF BETA 2 GLYCOPROTEIN I IN PATIENTS WITH ANTIPHOSPHOLIPID SYNDROME
Authors: Kerényi A. , Nagy G. , Antal‐Szalmás P. , Kappelmayer J.
Published: June 2019

Background:

Antiphospholipid syndrome (APS) is an acquired thrombophilia caused by antiphospholipid autoantibodies (aPL). Diagnosis of APS is based on laboratory and clinical criteria (thrombosis or pregnancy‐related complications). The detection of lupus anticoagulant (LA), IgG and/or IgM isotype anti‐cardiolipin (ACA) and anti‐β2 glycoprotein I antibodies (aβ2GPI) constitutes the laboratory tests of APS. The β2GPI protein consists of 5 domains and an association of thrombosis with the presence of antibodies directed against domain 1 (aD1β2GPI) was suggested in the literature.

Aims:

The aim of this study was to investigate the presence of IgG isotype aD1β2GPI and its association with thrombosis in our patient cohort.

Methods:

Using a chemiluminescence immunoassay (INOVA QUANTA Flash β2GPI‐Domain I, ACL Acustar) we have measured aD1β2GPI titer in 51 patients having IgG isotype aβ2GPI (APS with thrombosis: n = 21; APS with pregnancy loss: n = 7; other autoimmune disease: n = 18; aPL positive asymptomatic patients, n = 5).

Results:

In patient samples the concordance between the presence of IgG isotype aD1β2GPI and aβ2GPI was 67% and the correlation between these antibody titers was high (r = 0.71). We have found that the level of IgG isotype anti‐β2GPI was significantly higher in thrombotic APS patients compared to all other patient groups. The aD1β2GPI titer was the highest in thrombotic patients however, there was no significant difference in the antibody titer among the patient groups. The IgG isotype aβ2GPI and aD1β2GPI titers were significantly higher in triple positive patients (LA, ACA and aβ2GPI positive) than in double positive patients (ACA and aβ2GPI positive). In the aD1β2GPI positive group 47% of patients had thrombosis, while in the aD1β2GPI negative group this figure was only 29%, however, the association of aD1β2GPI positivity with thrombosis was non‐significant.

Summary/Conclusion:

This study provides further evidence that the large majority of IgG isotype aβ2GPI is directed against domain 1. Since the concordance between the two antibody positivity was not 100% and aD1β2GPI negative patients can present with thrombosis, we conclude that monitoring aD1β2GPI cannot replace detection of aβ2GPI in the laboratory diagnosis of APS.
S851 A FULLY HUMAN BISPECIFIC ANTIBODY FUNCTIONALLY RESCUES FACTOR VIII DEFICIENCY EX VIVO
Authors: Wang W. , Blackwood J. , Magliozzi R. , Moraes L. , Sinopoli A. , Chi B. , Sellick C. , Pearce C. , Theurl I. , Hollins J. , Dickson K. , Bradley A. , Lee E.‐C.
Published: June 2019

Background:

Haemophilia A is caused by deficiency of Factor VIII (F.VIII), an essential blood‐clotting cofactor. Prophylaxis or on‐demand treatment with recombinant FVIII forms the current standard of care to prevent bleeding in Haemophilia A patients in the developed world. However, approximately 30% of severe haemophilia A patients develop inhibitory antibodies against F.VIII. Moreover, FVIII treatment requires venous access which is burdensome. The recent successful clinical trials and subsequent regulatory approval of a F.VIII‐mimetic bispecific antibody, Hemlibra, in Haemophilia A patients with inhibitors have provided much needed hope for suffers and their family. However, whilst Hemilibra represents a major breakthrough in the field, there remain some concerns about balancing the risks of bleeding with those of thrombosis when using this agent.

Aims:

To develop a new fully human FVIII mimetic bispecific antibody that can support the assembly of a functional Factor IXa (F.IXa) and Factor X (F.X) complex, thereby replacing F.VIII function in vivo.

Methods:

Using Kymab's bispecific antibody platform, we developed KY1049, a fully human common light chain (CLC) bispecific antibody, to test as an effective functional mimetic of F.VIII. KY1049 bispecific heterodimer was purified using standard Protein A chromatography, followed by cation ion exchange chromatography (cIEX). The mass and heterodimer purity of the bispecific molecule was confirmed using Mass Spectrometry (MS). A combination of in vitro and ex vivo assays was used to characterize the cIEX purified bispecific antibody, including chromogenic FXase and Activated Partial Thromboplastin Time (aPTT). The affinities of the FIX arm and FX arm of KY1049 bispecific to FIX and FX, respectively, were determined using Surface Plasmon Resonance (SPR).

Results:

cIEX purified KY1049 molecule was confirmed as a single species by MS. After denaturing, the molecular weights of the two heavy chains and one light chain matches in silico predicted molecular weights. Purified KY1049 actively generated activated F.X (F.Xa) and functionally rescued the clotting defect in F.VIII‐depleted plasma in the aPTT assay. Importantly, ex vivo activities of KY1049 indicates its therapeutic benefit is likely to be similar in efficacy to Hemlibra. KY1049 binds F.IX and F.X simultaneously by SPR, but the affinities are different from those reported of Hemlibra.

Summary/Conclusion:

We have developed KY1049, a FVIII mimetic bispecific antibody, that demonstrates biological activities comparable to those reported for Hemlibra. Its therapeutic benefit is likely to be at least as effective as Hemlibra. Furthermore, initial analysis of the KY1049 molecule also showed some differences between the two molecules which could be harnessed to enhance its physical properties and activity.