bluuebunny/crossref_metadata_2025 huggingface dataset:
High Musashi-2 Expression Is an Unfavorable Prognostic Factor in Adult B-Cell Acute Lymphoblastic Leukemia.
Authors: Mu Qitian , Wang Yungui , Chen Bing , Qian Wenbin , Meng Haitao , Tong Hongyan , Chen Feifei , Ma Qiuling , Ni Wangmao , Chen Sai-Juan , Jin Jie
Published: October 2019

Abstract 2508

Background:

Musashi-2 (MSI2) can inhibit translation of the mRNA encoding NUMB, the NOTCH signaling inhibitor, and play a vital role in the maintenance of hematopoietic stem cells. Musashi-1 (MSI1), another isoform of Musashi family, positively regulates NOTCH1 expression. Recent studies have demonstrated that MSI2 expression was up-regulated during chronic myelogenous leukemia (CML) progression and its high expression was associated with poor outcome of CML and acute myeloid leukemia (AML). However, the prognostic significance of MSI2 expression and the relation between MSI2 and NOTCH1 expressions in acute lymphoblastic leukemia (ALL) remain unknown.

Methods:

In this study, real-time PCR was used to measured expression levels of MSI2 and NOTCH1 signaling related genes in 116 B-ALL and 24 T-ALL patients, in whom more than 70% leukemic cells were morphologically detected in bone morrow (BM) at diagnosis. Clinical and Molecular data in relation with MSI2 expression level were analyzed.

Results:

In our B-ALL cohort, although MSI2 expression was not associated with gender, age, white blood cell (WBC), t(9;22)/BCR-ABL and IK6 variant of IKZF1, patients with high MSI2 expression had inferior overall survival (OS) (P=0.007) and event free survival (EFS) (P=0.002) than patients with low MSI2 expression. Multivariate analysis showed that high MSI2 expression was an independently prognosic factor in OS (P=0.001, HR=2.641, 95%CI, 1.494–4.668), EFS (P<0.001, HR=2.562, 95%CI, 1.513–4.218) and RFS (P=0.043, HR=2.057, 95%CI, 1.023–4.137). Moreover, MSI2 expression level had a positive correlation with that of NOTCH1 (P<0.001), but not c-MYC (P=0.432) or HES1 (P=0.509). Similarly, NOTCH1 expression level in patients with high MSI2 expression (0.98, range 0.02 to 13.58) was significantly higher than that in patients with low MSI2 expression (0.42, range 0.01 to 3.55, P=0.001), but c-MYC and HES1 expression levels between patients with high MSI2 expression and low MSI2 expression showed no significant differences (both P>0.05).

Conclusion:

Our data suggests MSI2 high expression can indicate poor prognosis in adult B-ALL accompanying with elevated NOTCH1 expression but not activating NOTCH1 downstream pathway.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide Genotype-Based Risk Model for Survival in Acute Myeloid Leukemia Patients with Normal Karyotype.
Authors: Kim Hyeoung-Joon , Choi Hangseok , Kim Yeo-Kyeoung , Sohn Sang Kyun , Moon Joon Ho , Kim TaeHyung , Zhang Zhaolei , Kim Dennis Dong Hwan
Published: October 2019

Abstract 2526

Introduction:

Single nucleotide polymorphism (SNP) is an inter-individual genetic variation which could explain inter-individual differences of response/survival to chemotherapy. The present study was attempted to build up risk model of survival for acute myeloid leukemia (AML) patients with normal karyotype (AML-NK).

Methods and materials:

A total of 247 patients with AML-NK was included into the study. Genome-wide SNP array (Affymetrix SNP-array 6.0) was performed in the discovery set (n=118), and genotypes were analyzed for overall survival (OS). After identifying significant SNPs for OS in single SNP analyses, risk model was constructed. Replication was performed in an independent validation cohort (n=129).

Results:

Out of 632,957 autosomal SNPs meeting genotype data filtration criteria, a total of 82 SNPs were selected and passed into the next step of validation in an independent cohort. In the risk model generation step, finally 4 SNPs (rs2826063, rs12791420, rs11623492 and rs2575369) were meeting stringent criteria for SNP selection as follows: 1) p-value < 0.10 from Cox proportional hazards regression model in adjustment with age and WBC counts at diagnosis; 2) minor allele frequency > 0.05; 3) call rate > 95.0%; 4) high linkage disequilibrium r2 < 0.8. These 4 SNPs were introduced into the risk model, and patients was grouped into 2 groups according to the number of deleterious variables including 4 SNPs and 2 clinical variables (i.e. age and WBC counts at presentation): risk score 0–2 as a low risk (n=80) and 3–6 as a high risk (n=38). The risk model could stratify the patients according to their OS in discovery (p=1.053656•10−4) and in validation set (p=5.38206•10−3). The risk model showed a higher AUC than those being incorporated only clinical or only 4 SNPs, suggesting improved prognostic stratification power of combined model.

Conclusion:

Genome-wide SNP based risk model obtained from 247 patients with AML-NK can identify high risk group of patients with poor survival using genome wide SNP data. (Clinicaltrials.govIdentifier:NCT01066338)

Disclosures:

No relevant conflicts of interest to declare.
Induction of NK Cell Reactivity in Breast Cancer by Fc-Engineered NKG2D-Ig Fusion Proteins
Authors: Raab Stefanie , Steinbacher Julia , Grosse-Hovest Ludger , Schmiedel Benjamin J , Steinle Alexander , Kanz Lothar , Salih Helmut R
Published: October 2019

Abstract 253

NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. Their capability to mediate Fc-receptor dependent effector functions like antibody dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of anti-tumor antibodies like Herceptin (Trastuzumab®), which is approved for treatment of breast cancer displaying HER2/neu-overexpression. Notably, only about 20% of breast cancer patients show overexpression of HER2/neu. Moreover, this antigen is also expressed on healthy cells, and application of Herceptin is associated with side effects. In contrast, ligands of the activating immunoreceptor NKG2D (NKG2DL) are widely expressed on malignant cells, but generally absent on healthy tissues. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as tumor-antigens for Fc-optimized NKG2D-Ig fusion proteins targeting breast cancer cells for NK cell ADCC and IFN-γ production. NKG2D-Ig fusion proteins with distinct modifications in their Fc portion were generated by amino acid exchange as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT) or Herceptin, our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcγRIIIa receptor (CD16), respectively. This resulted in lacking (NKG2D-Fc-KO) or highly enhanced (NKG2D-Fc-ADCC) NK cell activation. In cultures of NK cells and breast cancer cells, NKG2D-Fc-KO significantly reduced NK cell reactivity due to blockade of NKG2DL-mediated activating signals, while NKG2D-Fc-WT substantially enhanced NK reactivity by induction of ADCC and cytokine production. Notably, the effect of our NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT and, in case of HER2/neu low targets, also that of Herceptin, resulting in potently enhanced NK anti-tumor reactivity. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing cancer cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16 NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may thus constitute an attractive means for immunotherapy, especially of HER2/neu-low or -negative breast cancer.

Disclosures:

No relevant conflicts of interest to declare.
Human Natural Killer (NK) Cells: Differential Expression of Phosphatase and Tensin Homologue Deleted On Chromosome Ten (PTEN) During NK Cell Development Regulates Its Cytolytic Activity Against Leukemic Target Cells
Authors: Briercheck Edward L. , Trotta Rossana , Cole Jordan P. , Cole Tyler D. , Hartlage Alex S. , Martin Chelsea K. , Leone Gustavo , Caligiuri Michael A.
Published: October 2019

Abstract 254

Human NK cells are CD56(+)CD3(-) large granular lymphocytes characterized by the ability to directly kill virally infected or malignantly transformed cells. Five stages of human NK cell development can be identified in secondary lymphoid tissue. Stage 4 NK cells are immature CD56bright and have poor cytolytic activity against sensitive leukemic target cells at rest while stage 5 NK cells are mature CD56dim and have potent cytolytic activity against the same leukemic cells at rest. Both stage 4 CD56bright and stage 5 CD56dim NK cells can be found circulating in blood. We sought to determine the mechanism responsible for this different cytolytic activity by exploring changes in gene expression between CD56bright NK cells and CD56dim NK cells. We first observed that CD56bright NK cells expressed ∼5 fold greater amounts of PTEN protein over CD56dim NK cells by western blot (n=5, p < 0.04). Given that PTEN is a lipid phosphatase that opposes the PI3K/AKT pathway, we hypothesized that it may negatively regulate NK cell cytolytic activity. In order to test this, we used lentiviral infection to overexpress PTEN in the human NK cell line NK-92. Relative to cells infected with an empty lentiviral vector, NK-92 cells with overexpression of PTEN showed decreased cytotoxicity against sensitive leukemic target cells by at least 25% at all effecter:target ratios (n=4, p < 0.02). Next, we infected primary human NK cells with the same vector and showed an approximately 30% reduction in degranulation of cytolytic mediators as determined by CD107a mean fluorescent intensity (M.F.I.) when cultured with sensitive leukemic target cells (n=4, p<.08). Consistent with this, murine NK cells were isolated from FVB mice with a heterozygous germline deficiency in Pten (Pten+/−) and showed at least a 25% increase in cytotoxicity against sensitive lymphoma target cells (n=4, p<.0001). At the molecular level, when NK-92 cells overexpressing PTEN were bound to their sensitive leukemic target cells, they showed decreased activation along the AKT and ERK1/2 signaling pathways, which are known as positive regulators of NK cell cytotoxicity. Collectively, we identify a functional role for the tumor suppressor PTEN in normal human NK cell development. We demonstrate differential expression of PTEN in immature and mature human NK cells, show that it is a negative regulator of NK cell cytolytic activity, and suggest that this differential expression may in fact account for the difference in baseline cytolytic activity found in the CD56bright and CD56dim human NK cell subsets, possibly via the AKT and ERK1/2 pathways.

Disclosures:

No relevant conflicts of interest to declare.
Testing in Vitro Sensitivity of Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia.
Authors: Groth-Pedersen Line , Valentin Rebecca , Schmiegelow Kjeld
Published: October 2019

Abstract 2543

Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in children and adolescents. Although improved risk grouping, anti-cancer treatment, and supportive care have resulted in survival rates above 80 %, 15–20 % of patients experience a relapse, which is associated with an inferior prognosis. Relapse is caused by persistence of minimal residual disease (MRD) primarily in the bone marrow during chemotherapy, and methods for identification of drugs that are capable of eliminating the MRD cell population is necessary, but not currently available. In vitro sensitivity testing has been carried out at the time of diagnosis and shows predictive value of treatment outcome and correlation to MRD levels. However, the in vitro sensitivity profiles have not been applicable for treatment stratification, since the vast majority of leukemic cells present at diagnosis are rather chemosensitive, as opposed to the MRD population. Thus, the more sensitive clones mask the more resistant ones. We hypothesized that in vitro sensitivity testing of the more resistant MRD cells remaining in bone marrow after the induction therapy could help to stratify patients to individualized chemotherapy.

The greatest challenge in carrying out in vitro sensitivity testing on MRD cell populations is the low number of leukemic cells available, i.e. 0.1–5%. Traditionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flourometric microculture cytotoxicity assays have been used for in vitro sensitivity testing. They, however, need several million cells for in vitro sensitivity testing of 3 drugs. We aimed at developing a cell death assay applicable on small ALL cell populations. We show that a flow cytometry based assay using annexin V (reflecting exposure of phophatidylserine on the outer cell membrane during early apoptosis) and 7-Aminoactinomycin D (reflecting cell membrane rupture during late apoptosis) staining is a reliable method for evaluating early and late stages of cell death in small ALL cell populations. Both in ALL cell lines REH (B-cell precursor (BCP) ALL, t(12;21), Jurkat (t-ALL), RS4;11 (BCP ALL, t(4;11)) and Nalm-6 (BCP ALL, t(5;12)) and in primary ALL cell samples the results are highly reproducible and show the same relative sensitivity profile as when a large number of cells are used. This cell death assay only demands a total of 20.000 cells to determine the in vitro sensitivity for 3 drugs run in triplicates at 4 concentrations. This assay is thus applicable for sorted MRD populations accounting for 0.1% of the mononuclear cells in the bone marrow.

Disclosures:

No relevant conflicts of interest to declare.
Induction of NK Cell Reactivity Against AML Cells by Fc-Engineered RANK-Ig Fusion Proteins.
Authors: Nuebling Tina , Schmiedel Benjamin J , Azuma Miyuki , Schneider Pascal , Grosse-Hovest Ludger , Kanz Lothar , Salih Helmut R
Published: October 2019

Abstract 2625

NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. A clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies. In acute myeloid leukemia (AML) NK cells largely contribute to the therapeutic efficacy allogenic stem cell transplantation (SCT). Recently we demonstrated that AML cells functionally express the TNF family member RANK ligand (RANKL) which impairs NK cell anti-leukemia reactivity (Schmiedel et al., ASH annual meeting 2011). Here we developed a strategy to combine blocking of the NK inhibitory effects of RANKL with targeting of AML cells for NK cell ADCC. To this end we generated fusion proteins consisting of the extracellular domain of RANK and a human IgG1 Fc part that was modified by amino acid exchange. Compared to wild type RANK-Fc fusion protein (RANK-Fc-WT), our mutant RANK-Fc-ADCC (S239D/I332E) displayed highly enhanced affinity to FcγRIIIa (CD16) on NK cells. Primary AML cells expressed substantial levels of RANKL in 53 of 78 (68%) investigated patient cases, and our RANK-Ig fusion proteins bound to AML cells in a target antigen-specific manner. Treatment with both RANK-Fc-WT and RANK-Fc-ADCC clearly reduced the release of RANKL-induced immunomodulatory factors like TNF, IL-6, IL-8 and IL-10 by AML cells. When the effects of the fusion proteins on NK cell ADCC were studied we found that treatment with RANK-Fc-WT only slightly enhanced NK cell reactivity against RANKL-positive patient AML cells. However, RANK-Fc-ADCC potently induced NK cell ADCC and cytokine production in response to AML targets in a target antigen-dependent manner due to the functional properties of its engineered Fc moiety. Taken together, our Fc-engineered RANK-Fc-ADCC fusion protein may serve to modulate the cytokine milieu involved in AML pathophysiology and target RANKL-expressing leukemia cells for NK anti-tumor reactivity. Thus, RANK-Fc-ADCC constitutes an attractive immunotherapeutic means for the treatment of AML, e.g. for elimination of minimal residual disease after conventional therapy including SCT.

Disclosures:

No relevant conflicts of interest to declare.
PTEN Expression Is Decreased in Pediatric Burkitt Lymphoma Tissues but Does Not Correlate with AKT Activation.
Authors: Vladimir Rodic , Sheryl Tripp , Bayerl Michael G , Barnette Phillip , Schiffman Joshua D. , Perkins Sherrie L. , Miles Rodney R.
Published: October 2019

Abstract 2664

Background:

We have recently shown that a subset of pediatric Burkitt lymphoma (pBL) show gain of 13q31, containing the microRNA (miRNA) MIR-17–92 locus, as well as associated increased expression of miRNA from that locus. PTEN, a tumor suppressor phosphatase that negatively regulates the PI3K/AKT/mTOR pro-survival pathway, is negatively regulated by MIR-17–92. Therefore, we hypothesized that PTEN protein and mRNA expression would be decreased in pBL cases with 13q31 locus gain and high expression of MIR-17–92. Because PTEN negatively regulates the PI3K/AKT/mTOR pathway, we also assessed AKT activation in pBL tissues.

Methods:

We assessed PTEN protein expression by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) pBL tissues from a cohort of patients (n=25) where MIR-17–92 expression levels and status of the 13q31 locus have been previously determined (Schiffman/Miles, Brit J Haematol 155:477, 2011). From FFPE tissues, we assayed PTEN mRNA levels by quantitative RT-PCR. The results of protein and mRNA expression were then correlated with presence or absence of a 13q31 gain and the expression level of MIR17. To evaluate AKT activation in pBL, we assayed for phospho-(p)-AKT by IHC. In addition, PTEN, AKT, and p-AKT protein levels were evaluated by western blotting in Raji and Ramos BL cell lines.

Results:

PTEN protein was weakly expressed in 5/25 cases (20%) of pBL. PTEN protein showed moderate nuclear and cytoplasmic staining in a subset of paracortical and germinal center lymphocytes in reactive lymph node control tissues, and vascular endothelial cells provided internal positive control in tumor tissue. pBL tissues showed decreased relative PTEN mRNA expression compared to reactive lymph node controls (1.56 vs. 2.61, p<3E-07). There was no significant difference in PTEN mRNA levels between PTEN protein positive and negative cases (1.27 vs. 1.61, p=0.22); cases with or without 13q31gain (1.49 vs. 1.57, p=0.60); or cases with high or low expression of MIR17. p-AKT was detected by IHC in 1/25 cases (4%). The BL cell lines Raji and Ramos showed very low levels of PTEN protein but no detectable p-AKT; total AKT was readily detectable.

Discussion:

pBL shows decreased PTEN mRNA, and most cases are negative for PTEN protein expression. Unexpectedly, higher MIR-17–92 expression level or 13q31 gain did not show an inverse relationship with PTEN expression at the protein or mRNA level. Despite decreased PTEN expression, p-AKT was detected in only 1/25 pBL cases and was not detected in BL cell lines. These findings suggest that decreased PTEN expression does not lead to AKT activation in pBL. Ongoing studies will investigate the potential of alternate PTEN targets and/or alternate targets for MIR-17–92 in pBL.

Disclosures:

No relevant conflicts of interest to declare.
Adverse Prognostic Impact of Vascular Endothelial Growth Factor Gene Polymorphism in Patients with Non-Hodgkin Lymphoma.
Authors: Kim Min Kyeong , Eom Hyeon Seok , Lee Hyewon , Han Mi Hwa , Kim Hyun-Ju , Park Weon Seo , Yun Tak , Kong Sun-Young
Published: October 2019

Abstract 2674

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and progression of many types of cancer, and VEGF expression has been known to be associated with polymorphism of gene. However, the impact of polymorphisms of the VEGF gene on non-Hodgkin lymphoma (NHL) prognosis has not been fully elucidated. Here we investigated the association between VEGF polymorphisms and prognosis of NHL. The study involved 96 NHL patients treated at National Cancer Center, Korea. The median patient age was 57 years, and 60 patients (62.5%) were men. A total of 5 polymorphisms with heterozygous allele were analyzed. Clinical characteristics (e.g., cell lineage) and international prognostic index (IPI), including age, performance, LDH, stage and extra-nodal involvement, were evaluated and related to VEGF polymorphism. Hazard ratios (HRs) were determined in terms of risk for overall survival using Cox proportional hazard regression analysis. Diffuse large B cell lymphoma (n=73) was most common histologic type, and others were as follows: T-cell lymphoma (n=14), mantle cell lymphoma (n=6), and Burkitt lymphoma (n=3). IPI and stage were predictors for prognosis (p <0.01), and both index did not have correlation with VEGF polymorphisms. The genotype frequency of rs1570360 was GG (70%), GA (26%), and AA (4%), and it has significantly association with poor prognosis with HRs of 2.52 [95% confidence interval (CI), 1.36 to 4.67] in NHL patients and 2.41 [95% confidence interval (CI), 1.15 to 5.05] in DLBCL treated by rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), respectively. In IPI subgroup analysis, rs1570360 had association in higher IPI group (IPI ≥ 3) (p=0.02). These findings suggest that VEGF polymorphisms may be significant prognostic indicators for patients with NHL and DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
Cerebral Small Vessel Disease In Patients With Sickle Cell Disease: Initial Findings With Ultra-High Field 7T MRI
Authors: der Land Veronica van , Zwanenburg Jaco J.M. , Biemond Bart J , Hendrikse Jeroen , Mutsaerts Henk-Jan , Engelen Marc , Nederveen Aart J. , Majoie Charles B.L.M. , Luijten Peter R. , Fijnvandraat Karin , Nederkoorn Paul J.
Published: October 2019

Introduction

Almost 40% of patients with a severe form of sickle cell disease (SCD) develop small silent cerebral infarctions (SCIs), often located in the deep white matter, that may be associated with cognitive impairment. In patients with cardiovascular disease or risk factors, occurrence of these SCIs is a well known phenomenon, and considered as part of ‘cerebral small vessel disease’. Currently, it is unknown whether small vessel disease is the underlying cause of SCIs in sickle cell disease. If we can identify imaging features supporting the hypothesis that cerebral small vessel disease occurs in patients with SCD, this may provide new targets for therapeutic interventions. The aim of the present study is to investigate the brain imaging features of neurologically asymptomatic young SCD patients with ultra-high field 7T MRI.

Methods

Ten consecutive, neurological asymptomatic patients with SCD (homozygous hemoglobin S) without other cardiovascular risk factors were included (mean age 23 years, range 19-25). Brain lesions were scored using a 3D magnetization prepared fluid attenuated inversion recovery scan (MP-FLAIR) and a dual-echo T2*-weighted scan for the visualization of small arteries and veins.

Results

With the exception of two patients, all displayed round and circumscript infarcts in the deep white matter of the frontal or parietal lobe (Table). Most lesions (82%) were <5 mm. In one patient (A) we identified 164 white matter infarcts (Figure 1). Strikingly, one patient (patient B) had several cortical micro-infarcts (Figure 2) although she had no neurological symptoms. These were associated with an occluded A1 and A2 segment of the anterior cerebral artery. In addition, this patient had unique pattern of white matter lesions, consisting of irregular shaped white matter infarcts, some located periventricular, and others close to the cortical infarcts. In two other patients (patients C and D) we identified an isolated cortical micro-infarct despite the absence of large vessel vasculopathy. In patient C, we found numerous hypo-intense areas symmetrically distributed in the deep white matter of the frontal and parietal lobe (Figure 3). These areas were associated with prominent transmedullary veins.

Conclusion

In our series of young and neurological asymptomatic SCD patients, we found the typical imaging features of SCIs in 8 out of 10 patients. Our findings suggest that patients with SCD may have manifestations of cerebral small vessel disease before the occurrence of symptoms, even at a young age and without other cardiovascular risk factors.

Disclosures:

No relevant conflicts of interest to declare.
CMR Survey In a Large Cohort Of TI Patients
Authors: Meloni Antonella , Lai Maria-Eliana , Vacquer Stefania , Mattei Roberto , Argento Crocetta , Maddaloni Domenico , Maggio Aurelio , Salvatori Cristina , Restaino Gennaro , Positano Vincenzo , Lombardi Massimo , Pepe Alessia
Published: October 2019

Introduction

Little is known about cardiac involvement in thalassemia intermedia (TI) using cardiovascular magnetic resonance (CMR). We investigated myocardial iron overload (MIO), biventricular parameters, and myocardial fibrosis in a large cohort of TI patients, underlying the differences between transfusion-dependent and non-transfusion-dependent patients.

Methods

We studied 252 adult TI patients (119 females, 39.5±10.4 years) enrolled in the MIOT Network. MIO was assessed using a multislice multiecho T2* approach. Biventricular function parameters were quantified by cine sequences. Myocardial fibrosis was evaluated by late gadolinium enhancement acquisitions.

Results

One-hundred and eighty-eight (74.6%) patients showed no MIO in any segment, 56 (22%) had an heterogeneous distribution (52 with global heart T2*≥20 ms), and 8 (0.3%) showed an homogeneous MIO.

Left ventricular (LV) and right ventricular (RV) dilatations were present in 113 (45%) and in 49 (19%) patients, respectively. LV dysfunction was present in the 18.0% of the cases while RV dysfunction in the 3.63%. High LV mass indexes were present in 22 (8.7%) patients.

Fifty-two/227 (22.9%) patients showed myocardial fibrosis. Myocardial fibrosis was associated to LV dysfunction (P=0.001) and high mass indexes (P=0.038).

One-hundred and fourteen patients were non-transfusion dependent (transfusion requirements absent or sporadic) while 138 patients were transfusion-dependent (regular transfusions). The mean age at start of chronic transfusions was 11.8 ± 12.3 years. Table 1 shows the comparison between the two groups. Non-transfusion-dependent patients showed significantly higher global heart T2* values and MIO with a global heart T2* < 20 ms was detected in two of them (one requiring occasional blood transfusions and one non transfused). Biventricular end-diastolic volume index, stroke volume index, left ventricular (LV) mass index, and LV cardiac index were significantly higher in the non-transfusion dependent group.

Conclusions

CMR plays a key role in the management of TI patients. Heart iron (global heart T2* < 20 ms) was not common, but a quarter of the patients had some pathological segments. A consistent number of patients had the stigmata of the high cardiac output state cardiomyopathy. Myocardial fibrosis was related to the high cardiac output state. The signs of the high output state were controlled in the transfusion-dependent-patients.

Disclosures:

No relevant conflicts of interest to declare.
Modulation Of T Cell Function Through L-Arginine Metabolism: A New Therapy From An Old Enemy
Authors: Fletcher Matthew B. , Ramirez Maria Elena , Sierra Rosa , Raber Patrick , Rodriguez Paulo
Published: October 2019

Recent studies have suggested the relevance of different energy metabolic pathways in the balance between protective T cell immunity and T cell anergy in tumors. We and others have suggested the role of the depletion of the non-essential amino acid L-arginine as a mechanism for the induction of T cell suppression in tumors. Therefore, we hypothesize that it is possible to metabolically regulate T cell responses simply through the modulation of L-arginine. In this study, we aimed to determine the effect of a pegylated form of the human L-arginine-metabolizing enzyme arginase I (peg-Arg I) in T cell responses. Activation of naïve murine T cells and antigen-specific CD4+ or CD8+ T cells in the presence of peg-Arg I prevented cell proliferation and production of IFNγ in vitro and in vivo. Similarly, peg-Arg I impaired proliferation and IFNg production in T cells activated with PMA/Ionomycin, suggesting that the effect of peg-Arg I was independent of T cell receptor (TCR) signaling. In fact, the anti-proliferative effect induced by peg-Arg I correlated with an arrest of T cells in the G0-G1 phase of the cell cycle, a decreased expression of cyclin D3 and cdk4, and a major inhibition of de novo translation. Interestingly, treatment of T cells with peg-Arg I did not impair the expression of the activation marker CD69, which correlated with an intact mitochondrial biogenesis. As a result, peg-Arg I did not have an effect in oxygen consumption by mitochondrial respiration (OCR), but significantly blocked glycolytic pathways in activated T cells. Furthermore, peg-Arg I treated T cells increased the expression of genes associated with integrated stress responses (IRS) and arrest in translation including GCN2, Chop, and Atf4. We then tested the effect of peg-Arg I in mouse models of graft versus host disease (GVHD) and inflammatory bowel disease (IBD), both mediated through activated T cells. Peg-Arg I significantly extended the survival of mice in these 2 disease models, which was associated with a decreased production of IFNg. Altogether, these results suggest the potential effect of the modulation of the metabolism of L-arginine as a means to regulate T cell responses. Continuation of this study will advance in the understanding of the metabolic effects of L-arginine in T cell function, which could enable the development of therapies to modulate T cell responses in transplantation or autoimmunity.

Disclosures:

No relevant conflicts of interest to declare.
Age-Associated DNA Methylation Signature Reveals Premature Aging In Patients With Aplastic Anemia and Dyskeratosis Congenita Which Correlates With Telomere Shortening
Authors: Beier Fabian , Weidner Carola I. , Lin Qiong , Ziegler Patrick , Zenke Martin , Brümmendorf Tim H. , Wagner Wolfgang
Published: October 2019

Introduction

Telomere length (TL) reflects the replicative potential of a cell and is a useful parameter to assess chronological age of an organism. Aplastic anemia (AA) and dyskeratosis congenita (DKC) are bone marrow failure syndromes (BMFS) characterized by shortened telomeres compared to age-matched healthy individuals in peripheral blood leukocytes. Human aging is associated with DNA methylation (DNAm) changes at specific sites in the genome. The aim of this study was to analyze whether aging specific epigenetic modifications can be used to track donor age and reflect premature aging in patients with AA and DKC.

Methods

A large set of publically available DNAm profiles were used to identify an Epigenetic-Aging-Signature. The DNAm level of three corresponding CpG sites was then analyzed by pyrosequencing after bisulfite conversion. The results were used for linear regression models for age predictions. This method was applied with peripheral blood leukocytes of a training as well as a validation set. Furthermore, TL analysis by flow-FISH of peripheral blood leukocytes of 106 healthy donors was used to generate age-adapted reference values. Pyrosequencing and telomere length analysis were carried out in 16 AA and 6 DKC patients

Results

Our analysis revealed that DNAm levels at three AR-CpGs – located in the genes ITGA2B, ASPA and PDE4C – were best suited for age predictions. The mean absolute deviation from chronological age in the validation set of 69 donors was less than 5 years. Age-adapted telomere length analysis revealed significant telomere attrition in AA (- 1.1 kb) and DKC patients (-3.7 kb, all below the 1% percentile). In analogy, the epigenetic changes revealed premature aging in AA patients – particularly in those which revealed extensive telomere attrition.

Conclusion

Our results indicate that patients with AA and DKC undergo massive premature aging on the epigenetic level comparable to the observed telomere shortening in these diseases. Determination of this newly defined Epigenetic-Aging-Signature in combination with telomere length measurement may be used to further refine diagnosis of AA and DKC in the clinical setting.

Disclosures:

No relevant conflicts of interest to declare.
Aberrant 5-Hydroxymethylcytosine Levels Correlate With Poor Overall Survival In Acute Myeloid Leukemia
Authors: Kroeze Leonie I , Aslanyan Mariam G , van Rooij Arno , Koorenhof-Scheele Theresia N , Massop Marion , Carell Thomas , Boezeman Jan B , Marie Jean-Pierre , Halkes Constantijn J.M. , de Witte Theo M. , Huls Gerwin , Suciu Stefan , Wevers Ron , van der Reijden Bert A. , Jansen Joop H.
Published: October 2019

Background

Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In addition, changes in DNA methylation have been implicated in the pathogenesis of AML. Recently it was discovered that TET proteins are able to convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), which is an important intermediate in the demethylation pathway. In this study, we measured 5-hydroxymethylcytosine levels in AML patients, and correlated these with mutational status and overall survival (OS).

Patients and methods

Samples from 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years), included in the EORTC/GIMEMA AML-12 06991 clinical trial, were analyzed for mutations in DNMT3A, TET2, IDH1 and IDH2. 5-hydroxymethylcytosine levels were measured using HPLC-MS/MS.

Results

In healthy control cells, 5hmC levels were confined to a narrow range (1.5 fold difference), whereas in AML cells, a much wider range was detected (15 fold difference). In remission, 5hmC values were normalized to levels comparable to healthy bone marrow and peripheral blood, indicating that the aberrant 5hmC levels at diagnosis are intrinsic to the leukemic cells. Patients with mutations in TET2 and patients with mutations in IDH1/2 had significantly lower levels of 5hmC compared to patients without mutated TET2 and IDH1/2 (both P<.001), whereas mutations in DNMT3A did not influence 5hmC levels. Patients with bi-allelic TET2 inactivation displayed lower 5hmC levels than patients with one affected allele (P=.003). Among the patients that did not harbor TET2 or IDH mutations, still a wide variation in 5hmC levels was observed, and importantly, both low and very high 5hmC levels correlated with inferior OS (P=.02, HR=1.80 and P=.04, HR=1.97, respectively). Multivariate analysis revealed that abnormal levels of 5hmC were independent prognostic indicators for OS. The difference in OS could not be explained by an initial inferior response to therapy, however, the relapse rate was considerably higher in patients with low and very high 5hmC levels compared to patients with intermediate 5hmC.

Conclusion

Both low and very high levels of 5hmC are markers of poor prognosis in AML, lending further support for testing therapies targeting the DNA hydroxymethylation pathway.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Gene Regulation By JAK1 In Diffuse Large B-Cell Lymphoma
Authors: Rui Lixin
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) grows rapidly and represents 30% to 40% of newly diagnosed lymphomas. The most aggressive subtype, termed activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL), is dependent on the autocrine cytokines IL-6 or IL-10 for survival. Our recent work has demonstrated that secretion of the cytokines in these lymphoma cells is mediated by aberrant NF-κB activation, including gain-of-function mutations in the MYD88 gene. In this study, we will dissect the molecular mechanisms of the cytokine signaling in lymphomagenesis.

We have evidence that the Janus kinase downstream of the cytokine signaling is JAK1. I obtained a surprising result demonstrating that JAK1 localization is largely nuclear, and that JAK1 can modify the nuclear histone protein H3 through phosphorylating tyrosine 41 (H3Y41). We and others have discovered that this chromatin modification can be initiated by the homologue JAK2 in leukemia cells as well as in primary mediastinal and Hodgkin lymphoma cells. H3Y41 phosphorylation displaces the inhibitory protein HP1 from chromatin and activates gene transcription. Genome-wide mapping by Chip-Seq using antibodies specific for H3Y41 phosphorylation identified 2,582 candidate genes modified by activated JAK1 in ABC DLBCL cells. These include important oncogenes such as MYC, key NF-kB pathway genes, and AP1 genes whose function is not appreciated in this lymphoma. Given that inhibition of some of these genes by RNA interference or by pharmaceutical inhibitors is lethal to cultured ABC DLBCL cells, they will be considered potential molecular targets for therapeutic development.

Disclosures:

No relevant conflicts of interest to declare.
EZH2 Mediates Resistance To Apoptosis In Nktl By Activating Nfkb Signaling Through Repression Of TNFAIP3/A20 By H3K27 Trimethylation
Authors: Loong Koh Tze
Published: October 2019

Extranodal nasal-type natural killer/T-cell lymphoma (NKTL) is a rare and aggressive form of lymphoma with poor outcome. A better understanding of the molecular pathophysiology of the disease is needed to development better treatment and improve patient’s outcome. We previously showed that EZH2, a histone methyltransferase, is abnormally over-expressed in NK cell lines as compared to normal NK cells, and may therefore be important for the pathogenesis and treatment of NKTL. 3- Deazaneplanocin A (DZNep) is a drug that can deplete EZH2 levels. Downregulation of EZH2 by DZNep induces apoptosis in a panel of NKTL cell lines but not normal NK cells. We perform gene expression studies to identify possible mechanism by which DZNep induce apoptosis in NKTL. There is significant enrichment of NFKB target genes amongst the genes downregulated after DZNep treatment. In addition, 2 inhibitors of the NFKB pathways, TNFAIP3/A20 and NFKBIA were upregulated. The downregulation of these genes were validated by PCR. We further found that NFKB2 was downregulated after DZNep treatment on western blot suggesting that NFKB signaling was downregulated. Similar results were obtained when we silence EZH2 using siRNA suggesting that much of the effect of DZNep is mediated through downregulation of EZH2. When TNFAIP3/A20 was expressed in untreated NKTL cells, apoptosis was induced. Conversed with TNFAIP3/A20 was knocked down in cells treated by DZNep, DZNep induced apoptosis was reduced. These data suggest in NKTL, EZH2 downregulate TNFAIP3/A20 and activate NFKB leading to resistance to apoptosis. We next investigated if EZH2 directly regulate TNFAIP3/A20 expression at its promoter. Using chromatin immunoprecipitation, we found that there is high occupancy of EZH2 and H3K27me3 at the TNFAIP3/A20 promoter in NKTL. Upon EZH2 knockdown, EZH2 and H3K27me3 is markedly reduced at the promoter and there is concurrent increase in TNFAIP3 expression. Consistent with cell lines data, in clinical samples, there is a strong association between H3K27me3 and nuclear p50 expression on a tissue microarray of NKTL (Chi-square p-value 0.01). We have therefore uncovered a novel mechanism by which EZH2 activates the NFKB pathway through its histone methyltransferase activity on the promoter of TNFAIP3/A20 resulting in resistance of NKTL to apoptosis.

Disclosures:

No relevant conflicts of interest to declare.
Selection For Brentuximab Vedotin Resistant Lymphoma Cell Lines Leads To Downregulation Of Surface CD30 Expression
Authors: Chen Robert , Hou Jessie , Reddy Megan , Newman Edward , Forman Stephen J. , Kane Susan
Published: October 2019

Background

Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), specifically to cells expressing surface CD30. In pivotal phase II trials BV demonstrated a complete response rate (CR) of 34% in patients with relapsed/refractory Hodgkin lymphoma (HL) and CR of 50% in patients with relapsed/refractory anaplastic large cell lymphoma (ALCL). However, patients who do not attain CR will eventually develop progressive disease despite active treatment with BV. We aim to select for BV resistant lymphoma cell lines and examine for alterations in surface CD30 expression in cell lines.

Methods

In vitro experiments used two HL cell lines (L428, KM-H2) and one ALCL cell line (Karpas 299). The selection of BV resistant cell lines was done in two different approaches (pulsatile and constant exposure). Confirmation of BV resistance was done by MTS assays and cell counting assays. Measurement of surface CD30 was performed by flow cytometry using monoclonal mouse anti-human CD 30, clone Ber-H2.

Results

MTS assay showed the IC50 of parental cell lines to be: L428 (24 ug/ml), KM-H2 (9 ug/ml), and Karpas (16 ng/ml). The cell lines were incubated at supra-IC50 dosage until no proliferation was seen and then rescued with BV free media in the pulsatile approach. Only L428 was able to be rescued and kept in BV free media until consistent proliferation was seen (defined as incremental growth for 3 consecutive weeks). L428 was then incubated again in 50 mg/ml of BV until consistent proliferation was seen. For the constant exposure approach, the cell lines were incubated at sub-IC50 dosages and dosages of BV were adjusted up depending on cell proliferation rates. Only KM-H2 and Karpas 299 showed consistent proliferation in the constant exposure approach. We were able to select for 1 resistant line per cell type. Cell counting assay showed that all three resistant cell lines were able to proliferate in the presence of BV at dosages twice their respective IC50s (Figure 1, only L428 is shown). MTS assays showed that all resistant cell lines had increased IC50s (Table 1). Flow cytometry showed downregulation of surface CD30 expression in Karpas 299 and KM-H2 but not L428 (Table1). For Karpas 299, both the percentage of CD30+ cells and the median CD30 intensity decreased in the resistant line. For KM-H2, only the median CD30 intensity decreased in the resistant line.

Conclusion

Prolonged exposure of HL and ALCL cell lines to BV led to resistant cell lines. When compared to their parental cell lines, two out of three resistant lines showed downregulation in surface CD30 expression.

Disclosures:

Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau, Trave expenses Other.
CRM1 As a New Therapeutic Target For Non-Hodgkin Lymphoma
Authors: Shi Yuankai , Wang Jianfei , Han Xiaohong , Zhang Ningning , Wang Shuai , Yao Jiarui
Published: October 2019

Background

The nuclear export protein chromosomal region maintenance 1 (CRM1) may play a role in human neoplasia and serve as a novel target for cancer treatment. Investigators recently developed orally bioavailable selective inhibitors of nuclear export (SINEs) that irreversibly bind to CRM1 and block its function. Our objective was to evaluate the therapeutic efficacy of the novel SINEs KPT-185 and KPT-276 against NHL in vitro and in vivo and elucidate the mechanism of CRM1 inhibitor-mediated antitumor activity.

Methods

Cell viability, apoptosis and cell cycle were evaluated in 8 B and T cell lymphoma cell lines (Jeko-1, Mino, Granta519, Sp53, RL, Hut102, Hut78 and Jurkat) which were treated with KPT-185; Primary tumor cells from 5 patients and normal human lymphocytes from 2 healthy volunteers were treated directly with KPT-185. Tumor suppressor proteins were detected by western blot to explore the possible mechanisms of KPT-185 inducing lymphoma cells growth inhibition and apoptosis. BALB/c nude mice bearing Jeko-1 tumors were treated orally with KPT-276 (similar structure to KPT-185, but improved animal pharmacokinetics) to examine the efficacy and side-effects of KPT-276.

Results

KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (half-maximal inhibitory concentrations, 60-120 nM) and induced cell-cycle arrest and apoptosis in NHL cell lines and normal lymphocytes. The antitumor activity mainly consisted of regulating cell growth and apoptosis mechanisms by inducing caspase cleavage and downregulating the expression of antiapoptotic proteins such as CRM1, nuclear factor-kB, and survivin. Furthermore, oral administration of KPT-276 significantly suppressed tumor growth and prolonged survival in mice with NHL xenografts without any major toxic effects (P < 0.001). Analysis of tumor remnants in the mice demonstrated that KPT-276 trapped the antiapoptoic protein survivin within the nuclei of NHL cells.

Conclusions

We observed the biologic and pharmacologic activity of CRM1-inhibiting SINEs in NHL cells, primary NHL tumor samples, and a murine NHL xenograft model. SINE CRM1 inhibitors inhibited growth of lymphoma cells both in vitro and in vivo. The antitumor activity of the SINEs resulted primarily from induction of caspase activity and downregulation of expression of antiapoptoic proteins such as survivin and NF-kB. The preclinical in vitro and in vivo results reported herein support further study of CRM1-inhibiting SINEs as novel therapeutics for NHL.

Disclosures:

No relevant conflicts of interest to declare.
A Combined Molecular and Clinical Prognostic Index For Relapse and Survival In Cytogenetically Normal AML (PINA)
Authors: Pastore Friederike , Dufour Annika , Benthaus Tobias , Metzeler Klaus H. , Maharry Kati , Schneider Stephanie , Ksienzyk Bianka , Mellert Gudrun , Zellmeier Evelyn , Kakadia Purvi M. , Unterhalt Michael , Feuring-Buske Michaela , Buske Christian , Braess Jan , Sauerland Cristina , Heinecke Achim , Krug Utz , Berdel Wolfgang E. , Büchner Thomas , Woermann Bernhard J. , Hiddemann Wolfgang , Bohlander Stefan K. , Marcucci Guido , Spiekermann Karsten , Bloomfield Clara D. , Hoster Eva
Published: October 2019

Background

Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician it is difficult to know how to use the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pre-treatment patient and molecular characteristics.

Patients and methods

Two prognostic indices for CN-AML, one with regard to overall survival (PINAOS) and the other regarding relapse-free survival (PINARFS) were derived based on a cohort of 669 CN-AML patients treated within the AML Cooperative Group 99 (AMLCG99) study.

Results

Based on age (median: 60 years [range: 17-85 years]), performance status, white blood count, and presence or absence of NPM1 mutation, biallelic CEBPA mutation, and FLT3-ITD, patients were classified into three risk groups according to PINAOS and PINARFS: 29% of all and 32% of responding patients had low risk (5-year OS 72%; 5-year RFS 55%), 56% and 39% intermediate risk (5-year OS 28%; 5-year RFS 27%), and 15% and 29% high risk disease (5-year OS 3%; 5-year RFS 8%) (Figure 1). PINAOS and PINARFS further subdivided the European LeukemiaNet (ELN) favorable-genetic group as well as the ELN intermediate-I-genetic group. Both, PINAOS and PINARFS were confirmed in a large, independent, and comparable CN-AML cohort of 529 patients from the Cancer and Leukemia Group B (CALGB/Alliance) trials (Figure 2).

Conclusions

We have developed and validated the first prognostic indices specifically designed for CN-AML patients of all ages combining well-established molecular and clinical variables easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care by risk-adapted therapy of CN-AML.

Disclosures:

No relevant conflicts of interest to declare.