bluuebunny/crossref_metadata_2025 huggingface dataset:
Fast Detection Of MYD88-L265P Mutation By PCR-RFLP In Chronic Lymphoproliferative Disorders
Authors: Argentou Nikoletta , Vassilopoulos George , Ioannou Maria , Germenis Anastasios , Speletas Matthaios
Published: October 2019

Introduction

Waldenstrom’s macroglobulinemia (WΜ) is an incurable disorder characterized by bone marrow infiltration from neoplastic lymphoplasmacytic B-lymphocytes and monoclonal IgM production. Recent data suggest a possible causal relationship of MYD88-L265P mutation with the pathogenesis of the disease (Treon Sp et al. N Eng J Med 2012;367:826-33). Additional studies have conformed that MYD88-L265P is characteristic of WΜ, but it is also rarely present in other chronic lymphoproliferative disorders (LPDs) (Jiménez C et al. Leukemia 2013,doi: 10.1038/leu.2013.62; Varettoni M et al. Blood 2013;121:2522-8). The purpose of this study was to analyse the prevalence of the aforementioned mutation in patients with WM and other LPDs, using a fast and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol.

Methods

Genomic DNA was extracted from the bone marrow of 11 patients with WΜ, 12 patients with B-cell chronic lymphocytic leukemia (CLL), 8 with splenic lymphoma with villous lymphocytes (SLVL). Furthermore, consecutive samples of peripheral blood and bone marrow isolated CD19+ cells, derived from a patient with monoclonal IgM gammopathy of undetermined significance (MGUS-IgM), were also retrospectively analysed for the presence of MYD88-L265P defect. The detection of the mutation was performed by PCR amplification of the MYD88 exon 5 region, followed by RFLP analysis, since the presence of the mutation results in the generation of BsiEI restriction enzyme site. PCR-RFLP results were also confirmed by direct sequencing of purified CD19+cells.

Results

The MYD88-L265P mutation was detected in 10 patients with WΜ (90.9%), in 1 patient with SLVL with markers of lymphoplasmatocytoid differentiation (12.5%) and was absent in all CLL patients. Interestingly, our PCR-RFLP protocol exhibited a greater sensitivity than direct sequencing, when applied to total bone marrow cells (12.5% vs 25%). Moreover, the patient with MGUS-IgM displayed the MYD88-L265P mutation in isolated CD19+cells of both bone marrow and peripheral blood in all consecutive samples and remains in a stable condition for the last 7 years.

Conclusion

PCR-RFLP is a rapid, sensitive and reliable technique for the detection of MYD88-L265P mutation in patients with WM and lymphomas with lymphoplasmatocytoid differentiation. Additionally, the presence of MYD88-L265P mutation in MGUS-IgM in the absence of disease progression for many years, suggests that this genetic defect alone is not sufficient to lead to overt neoplastic disease.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Role Of Monoclonal and Polyclonal Gammopathy Measured By Serum Free Light Chain and Immunofixation In Diffuse Large B-Cell Lymphoma
Authors: Kim Yu Ri , Kim Soo Jeong , Kim Yundeok , Hwang Doh Yu , Jang Ji Eun , Hyun Shin Young , Cheong June-Won , Min Yoo Hong , Kim Jin Seok
Published: October 2019

Background

Serum free light chain (FLC) assay has been used to evaluate the prognosis of several hematologic malignancies. We evaluated the prognostic role of monoclonal gammopathy (MG) and polyclonal gammopathy (PG) measured by serum FLC and immunofixation (IF) in DLBCL.

Patients and methods

We retrospectively reviewed 115 patients with DLBCL who treated with rituximab containing chemotherapy. MG was defined as elevated kappa (κ) or lambda (λ) FLC with abnormal κ to λ ratio or positive IF and PG was defined as elevated κ and/or λ FLC with normal κ to λ ratio and negative IF.

Results

Fifty-six (48.7%) patients had an elevated FLC. Twenty (17.4%) patients had MG and 39 (33.9%) patients accompanied by a PG. Two-year overall survival (OS) was 89.2%, and 2-year event-free survival (EFS) was 88.4%. Elevated FLC was associated with inferior OS and EFS (p=0.013, p=0.012). Patients with MG had an inferior OS and EFS compared to patients with normal FLC (p=0.006, p=0.011). In multivariate analysis, both elevated FLC and MG showed the significance for OS (HR 4.57, 95% CI 1.43-14.60, p=0.001 and HR 5.63, 95% CI 1.50-21.09, p=0.010). Elevated FLC did not show the significantly shorter OS or EFS in non-germinal center B-cell (GCB) type according to the Han’s criteria. However, MG was a significant prognostic factor for OS and EFS in non-GCB type (HR 6.28, 95% CI 1.21-32.62, p=0.029, HR 6.38, 95% CI 1.58-25.74, p=0.009).

Conclusion

It important to evaluate the associated MG and PG using serum FLC and IF for predicting the prognosis of DLBCL, especially in non-GCB subtype.

Disclosures:

No relevant conflicts of interest to declare.
Demographic Analysis Of 90 Polycythemia Vera Egyptian Cases : An Egyptian Reference Center Experience
Authors: Mattar Mervat , Tawfik Nihad , Elhosseiny Noha , Morad Mohamed
Published: October 2019

Objective

We report 90 cases of Polycythemia Vera ( PV )during the period between 2003 and 2013 followed up in an adult Cairo  hematology reference center

Results

Among 90 PV cases registered between 2003 and 2013, sixty eight cases (75.6%) of  whompresented in the last three years alone.  The age range was between 19 and 72 years with a mean of 53.5 years and 25 cases ( 27.78%) presented in the age group between 50-55 years.   Male : female ratio was 1.3:1.Fifty per cent of patients were urban while 50% lived in the country.

Symptoms included headache (24 cases) (26.67%), tinnitus (7 cases) (7.78%), fatigue (14 cases) (15.56%), weight loss (5 cases)(5.56%), fever and sweating( 7 cases)( 7.78%) , bone or joint pains (22 cases)(24.44%), parasthesias (13 cases)(14.44%), pruritis(7 cases) (7.78%)  or abdominal discomfort (16 cases)(17.78% (Fig 1)

Figure (1) : Incidence Of Symptoms Among 90 Egyptian PV Cases

Severity of symptoms was assessed by a simple score of zero (none), +1 (mild),+ 2 (moderate) and +3 (severe) (table1)

Nine cases (10%) presented by venous thrombosis , 4 of whom had portal vein thrombosis (4.44%) Cerebro-vascular strokes were noted in 5 cases (5.56%) and acute myocardial infarction in 2 cases (2.22%).

Splenomegaly was noted in 29 cases (32.22%) of cases and hepatomegaly in 13 cases (14.44%).

Associated conditions included hypertension in 24 cases (26.67%), gastrointestinal symptoms in 16 cases(17.78%) Diabetes mellitus in 15 cases (16.67%),Hepatitis C virus infection in 6 cases ( 6.67%), Hepatitis B in 2 cases ( 2.26%), coronory heart disease in 5 cases (5 .56%) and renal problems in 3 cases ( 3.33%). Bleeding episodes included 6 (6.67%) GI and 3(3.33%) gum bleeds.

Familial MPN, Cancer prostate, intestinal Schistosomiasis   and post-liver transplant done post- hepatitis C virus cirrhosis were reported in one case each.

Twenty two cases(24.44%) were controlled on Hydroxyuria only,16 cases 17.78%) on venesection plus hydroxyuria,16 cases(17.78%) on venesection only. Progression  was noted in 13 cases (14.44%) with marrow fibrosis in five cases (5.56%) and 14 cases (15.56%) were lost to follow-up. Mortality was seen in 10% (nine cases).

Conclusion

PV among Egyptians may have distinct presentations regarding incidence, age and  presentation.Simpler methods of symptom severity assessments may be more suitable for Egyptian patients.

Disclosures:

Mattar: Novartis, BMS, Amgen: Honoraria.
Protein Methyltransferase Inhibitors as Personalized Cancer Therapeutics
Authors: Copeland Robert A.
Published: October 2019

The protein methyltransferases (PMTs) constitute a class of enzymes that catalyze the methylation of lysine or arginine residues on histones and other proteins. A number of PMTs have been shown to be genetically altered in cancers through, for example: gene amplification, chromosomal translocations, point mutations, and synthetic lethal relationships [Copeland et al. (2013) Oncogene 32: 939-946]. The enzymes DOT1L and EZH2 provide two representative examples of altered PMTs that act as genetic drivers of specific human cancers. The enzymatic activity of DOT1L is associated with a chromosomal translocation that is universally found in patients with MLL-rearranged leukemia [Daigle et al. (2011) Cancer Cell 20: 53-65; Daigle et al. Blood, prepublished June 25, 2013; doi:10.1182/blood-2013-04-497644]. Point mutations at Y641 of EZH2 are found in a subset of non-Hodgkin lymphoma patients; the enzymatic activity of both wild-type and mutant EZH2 are required for pathogenesis in these patients [Sneeringer et al. (2010) Proc. Natl. Acad. Sci. USA 107: 20980-20985; Knutson et al. (2012) Nature Chem. Biol. 8: 890-896]. Also, deletion of the SMARCB1 subunit of the SWI/SNF chromatin-remodeling complex occurs in nearly all malignant rhabdoid tumors (MRTs), a childhood cancer with a particularly poor prognosis. An antagonistic relationship has been demonstrated between the biochemical action on chromatin of the SWI/SNF complex and EZH2 (in the context of the polycomb repressive complex 2), that is relieved in MRTs due to the SMARCB1 deletion. MRTs therefore demonstrate increased reliance on the enzymatic activity of EZH2 for proliferation and we have shown that MRT cells deficient in SMARCB1 are selectively killed by inhibition of EZH2 in cell culture and in mouse xenograft models [Knutson et al. (2013) Proc. Natl. Acad. Sci. USA 110: 7922-7927]. Drug discovery efforts have yielded potent, selective inhibitors of each of these targets that have now transitioned into phase I clinical trials. These inhibitors affect the appropriate histone methyl marks in cells, lead to selective cell killing that is dependent on genetic alteration of the target enzyme, and effect tumor growth inhibition in xenograft models. These data portend the effective use of selective PMT inhibitors as a novel modality for future personalized cancer therapeutics.

Disclosures:

Copeland: Epizyme: Employment, Equity Ownership.
Matters of Life and Death: Give and Take in the Bcl-2 Family
Authors: Green Douglas R.
Published: October 2019

The mitochondrial pathway of apoptosis, a major mode of cell death in vertebrate animals, is characterized by the process of mitochondrial outer membrane permeabilization (MOMP), in which proteins of the mitochondrial inter-membrane space gain access to the cytosol, thereby triggering caspases and apoptosis. This life-or-death decision is controlled by the proteins of the Bcl-2 family. These include the pro-apoptotic Bcl-2 effector proteins, Bax and Bak, that cause the permeabilization event, and the anti-apoptotic proteins, e.g., Bcl-2, Bcl-xL, Mcl-1, which prevent it. A third sub-family, the BH3-only proteins, which include Bid, Bim, Bad, Puma, and Noxa (among others), regulate the other two sub-families, promoting the activation of the effectors or blocking the activity of the anti-apoptotic proteins (or both). Structural studies have led to a greater understanding of these complex interactions and have provided the basis for the development of BH3-mimetics that show promise for cancer therapies. New structural studies have begun to elucidate the mechanisms whereby Bax and Bak become activated, opening new avenues towards promoting or inhibiting the mitochondrial pathway of apoptosis. A proposed “hit and run” model of effector activation, whereby a BH3-only protein engages Bak or Bax and then must be displaced as the effector proteins associate, was validated in these structural studies. Additional, emerging insights will be discussed in the context of a unified model of Bcl-2 protein interactions.

Disclosures:

No relevant conflicts of interest to declare.
Inherited and Acquired RUNX1 Mutations in Hematologic Disorders
Authors: Speck Nancy A. , Cai Xiongwei , Ge Jingping , Mason Philip J.
Published: October 2019

RUNX1, a DNA binding subunit of core binding factors, is frequently mutated or rearranged in hematopoietic malignancies, including acute myelogenous leukemia (AML), chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome (MDS). Mutations in RUNX1 can be early events in leukemia, and generate a long-lived pre-leukemic stem cell (pre-LSC). Additionally, it has been reported that loss of function RUNX1 mutations are particularly frequent in radiation-associated MDS and AML, suggesting that pre-existing RUNX1 mutations in a pre-LSC may predispose patients to MDS/AML following DNA damage. Discussion will focus on the phenotypic properties of Runx1-deficient pre-LSCs, and the mechanisms by which Runx1 deficiency contributes to these phenotypes. Pan-hematopoietic Runx1 loss in mice causes a G1 to S-cell cycle delay and decreases apoptosis of pre-LSCs. Runx1-deficient pre-LSCs are radiation- and chemotherapy-resistant, and this correlates with decreased p53 protein levels and an attenuated p53 pathway response. Both p53 protein levels and apoptosis are increased following treatment with Nutlin-3. Runx1-deficient pre-LSCs are smaller, consume less glucose, and produce less ATP than normal hematopoietic stem cells (HSCs). Runx1-deficient stem and progenitor cells have lower overall ribosomal content and skewing in the relative amounts of rRNA and mRNA encoding ribosomal proteins. Analysis of AKT pathway components suggests that the decreased ribosome biogenesis is unlikely to be primarily caused by lower AKT signaling. We hypothesize that one or more of the above-mentioned properties (low p53 levels, decreased metabolism) render Runx1-deficient pre-LSCs less sensitive to genotoxic stress than normal HSCs, allowing a Runx1-deficient pre-LSC population to both perdure and expand in the bone marrow.

Disclosures:

No relevant conflicts of interest to declare.
Oral Administration of Ibrutinib Is Ineffective at Preventing Scleroderma in Chronic Gvhd in Two Preclinical Mouse Models
Authors: Xu Yajing , Johnston Heather F , Forman Stephen J. , Zeng Defu
Published: October 2019

We have established two mouse models of chronic graft versus host disease (cGVHD): one is an MHC-matched DBA/2 donor to BALB/c recipient, and this model amplifies the role of CD4+ T and B cells in transplants; the other model is an MHC-mismatched C57BL/6 donor to BALB/c recipient, and this model amplifies the role of de novo-developed CD4+ T and B cells. BALB/c recipients in both models started to develop scleroderma ~35 days after transplantation of donor spleen and bone marrow cells and this peaked ~50 days after transplantation. BALB/c recipients given DBA/2 transplants also developed proteinuria due to IgG anti-dsDNA autoantibody deposition in the glomeruli. We have reported that donor CD4+ T and B cells play critical roles in the pathogenesis of chronic GVHD (J. Immunol 2012 and 2013). Administration by I.V. injection of depleting anti-CD20 prevented induction of chronic GVHD in both models (Biol. Blood and Marrow Transplant 2014). In the current studies, using these two models, we tested whether oral administration of Ibrutinib could prevent induction of cGVHD. Ibrutinib was gavaged at 12.5 mg/kg/day from day 0 to day 30 or given in drinking water at 35 mg/kg/day (0.16 mg/ml) from day 0 to day 50. We found that administration of Ibrutinib significantly delayed the onset of proteinuria in the model of DBA/2 donor to BALB/c recipient, but accelerated the onset and increased the severity of scleroderma in 20/20 recipients given DBA/2 transplants and 9/9 recipients given C57BL/6 transplants as measured by a clinical score system. Administration of Ibrutinib significantly reduced CD138+B220lo plasma cells but not CD138-B220+ B cells in the spleen and peripheral lymph nodes of both recipients. Administration of Ibrutinib slightly reduced CD4+ T cell numbers in the spleen but significantly increased CD4+ T cell numbers in the peripheral lymph nodes, although there was no significant impact on CD4+ T cell expression of IFN-g and TNF-α in both recipients. These results indicate that, although oral administration of Ibrutinib is able to reduce B cell activation and differentiation into plasma cells, it is ineffective at inhibiting the CD4+ T cell expansion that mediates scleroderma in the models of DBA/2 donor to MHC-matched BALB/c recipient and C57BL/6 donor to MHC-mismatched BALB/c recipient. Our result is in contrast to a previous report showing that oral administration of Ibrutinib prevented induction of chronic GVHD with bronchiolitis obliterans in a mouse model and reversed scleroderma in another mouse model (Poster 2591, AACR 2014, San Diego). Further discussion about the differences is warranted. (This work was supported by Nesvig Lymphoma Foundation).

Disclosures

No relevant conflicts of interest to declare.
Stimulation of Nitric Oxide Synthase Activity By Plasma Apolipoproteins: a Biomarker of Endothelial Function in Adults with Sickle Cell Disease
Authors: Wang Xunde , Mendelsohn Laurel , Freeman Lita , Vaisman Boris , Remaley Alan , Kato Gregory J.
Published: October 2019

Nitric oxide (NO) plays a critical role in maintaining basal vascular tone and regulating blood flow. Many factors, including shear stress and endogenous ligands such as vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF1), stimulate the activity of endothelial nitric oxide synthase (eNOS). We have developed a bioassay that measures stimulation of NOS activity by soluble factors in unfractionated human plasma in cultured human endothelial cells using a sensitive NOS activity assay with radiolabeled substrate.

The addition of 1% human plasma to the culture medium stimulates NOS activity 1.5-fold over background, with a linear response up to 10% plasma, which activates NOS 5.5-fold. We have concluded from several lines of evidence that this NOS-inducing activity in human plasma comes from high-density lipoprotein (HDL): The activity is heat-labile and sensitive to reducing agents; it is precipitable by ammonium sulfate and elutes as a broad peak on molecular exclusion and anion exchange columns; by immunoblot, the active fractions have large amounts of apolipoproteins apoA-I, apoE and paroxonase-1. We find that plasma from an apoA-I null mouse shows less stimulation of NOS activity in our bioassay than plasma from a wild type mouse and apoA-I transgenic mouse (p=0.06, Brown-Forsythe test; p=0.04, post-test for linear trend; see figure). Purified HDL fractions stimulate NOS activity four-fold, equivalent to unfractionated plasma, but further subfractionation of HDL components extinguishes its ability to activate NOS.

Remarkably, higher than median NOS-stimulating activity in our bioassay was associated with endothelial-dependent blood flow, detected by venous occlusion strain gauge plethysmography measurement of forearm blood flow induced by graded infusions of acetylcholine into the brachial artery in adults with sickle cell anemia (p=0.0013, two-way ANOVA, see figure). The NOS-stimulating activity in patient plasma also correlated with the plasma apoA-I level (Spearman r=0.64, p=0.0012).

In summary, our results suggest that circulating functional apoA-I and possibly other apolipoproteins, regulate NO production and endothelial function in adults with sickle cell disease, which is consistent with previous lines of evidence published by other investigators in subjects without sickle cell disease. Most strikingly, our plasma NOS stimulation assay appears to provide a useful research biomarker for endothelial function, applicable to frozen archived plasma biospecimens.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Risk Factors for Cerebral Vasculopathy in a Sickle Cell Disease Neonatal Cohort: A Prospective Study
Authors: Sommet Julie , Alberti Corinne , Couque Nathalie , Haouari Zinedine , Missud Florence , Holvoet Laurent , Ithier Ghislaine , Baruchel André , Benkerrou Malika
Published: October 2019

Children with sickle cell disease have a significant vascular morbidity, specifically cerebral vasculopathy (CV) that can be detected by transcranial Doppler. The aim of this study was to identify CV risk factors using longitudinal biological and clinical data including upper and lower respiratory tract evaluation in the newborn cohort of Robert Debre Pediatric Sickle Cell Referral Center. 375 patients with sickle cell anemia with a total of 2677 patient-years and a 7 years median follow-up were evaluated. Among the 59 children presenting CV, seven had a stroke. Overall, the incidence rate of CV was 2.20/100 patient-years (95% CI 1.64-2.76) and the incidence rate of stroke was 0.26/100 patient-years (95% CI 0.07-0.46). The cumulative risk of CV by the age of 11.3 years was 26.0% (95% CI 20.0%-33.3%). CV risk factors were assessed by a Cox model encompassing linear multivariable modelling of longitudinal quantitative variables. The final model retained four variables. Years with upper airways obstruction (HR=1.47, 95%CI 1.05-2.06), bronchial obstruction (HR=1.76, 95%CI 1.49-2.08), fetal hemoglobin level (HR=0.68 per 5% increase 95%CI 0.48-0.96) and reticulocyte count (HR=1.82 per 50000/mm3 increase 95%CI 1.10-3.01) were independent risk factors. An ancillary analysis performed for the 209 patients with α-thalassemia data (29 patients with CV and 180 without) found that α-thalassemia was protective in bivariate analysis but was not retained in multivariable analysis. The final Cox multivariable analysis for CV in this subgroup retained three variables. Years with upper airways obstruction and bronchial obstruction were significant risk factors for CV (with respectively HR=1.57 IC95% [1.05-2.32] and HR=1.73 IC95% [1.37-2.17]) whereas fetal hemoglobin was protective (HR=0.54 per 5% increase IC95% [0.31-0.94]). Reticulocyte count was no more retained as risk factor for CV after introducing α-thalassemia in the final multivariable model. Early evaluation and specific treatment for symptoms of asthma or nocturnal respiratory abnormalities could potentially reduce the risk of CV and stroke. They are currently under evaluation in a multicenter clinical trial with systematic nocturnal polysomnography.

Disclosures

No relevant conflicts of interest to declare.
Clinical Significance of CSF3R, SRSF2 and SETBP1 mutation in Chronic Neutrophilic Leukemia and Chronic Myelomonocytic Leukemia
Authors: Qiao Chun , Ouyang Yuan , Zhang Sujiang
Published: October 2019

Objective: To investigate the gene mutation and the clinical features of CSF3R, SETBP1 and SRSF2 in chronic neutrophilic leukemia (CNL) and chronic myelomonocytic leukemia (CMML) patients.

Method: Sequence analysis of CSF3R, SETBP1 and SRSF2 were performed in 10 CNL and 56 CMML patients whose clinical features were also studied.

Result: Among 10 CNL patients, 8(8/10, 80%) patients had CSF3R mutations and 7(7/8, 87.5%) of them were with CSF3R T618I. In 56 cases of patients with CMML, SRSF2 mutations were found in 14(14/56,25%), CSF3R in 4(4/56,7.1%) and SETBP1 in 3(3/56, 5.3%) patients. In CMML, compared to wild-type(wt) SRSF2 patients, SRSF2 mutated patients appeared to be more possible with SETBP1 mutations [1/14(7.1%) vs. 2/42(4.8%), P>0.05], less possible with CSF3R mutation [0/14(0%) vs. 4/42(9.5%), P<0.001]. The clinical characteristics such as age, gender, WHO category, FAB category, karyotype and blood cell counts did not reveal any difference between SRSF2 mutated and wtSRSF2 patients. Either SRSF2 mutated patients or SETBP1 mutated patients both had shorter overall survival (OS) and progression-free survival(PFS) when compared with those with wtSRSF2 (P<0.001 both) and wtSETBP1 (P<0.001 and P=0.02, respectively). No significant difference of OS and PFS between CSF3R mutated and wtCSF3R patients were observed. In multivariate analysis, SRSF2 mutation was an independent negative predictor for OS (HR, 3.307; 95% CI, 1.137 to 9.614; P=0.028) and PFS(HR, 15.431; 95% CI, 3.041 to 78.312; P = 0.001). What's more, SETBP1 mutation was also an independent negative predictor for OS(HR, 9.492; 95% CI, 1.183 to 76.128; P = 0.034).

Conclusion: The majority of patients with WHO-defined CNL have oncogenic mutations in CSF3R and the T618I mutation type is a highly sensitive and specific molecular marker of the disease. While mutations of SRSF2 are common in CMML and may be of prognostic significance. As a non-specific molecular marker, SETBP1 was found in CNL, CMML and other blood cancers, which have poor prognosis.

Disclosures

No relevant conflicts of interest to declare.
EZH2-Mediated Activation of Serine Biosynthetic Pathway Is Critical for Resistance of Clonal Cells to Stress-Related Apoptosis in Low-Grade Myelodysplastic Syndrome
Authors: Xu Feng , Chang Chunkang , Shi Wenhui , Li Xiao
Published: October 2019

Background

Increased cell apoptosis mediated by P53 or activated autoimmunity was frequent in low-grade MDS especially in 5q-syndrome and those with trisomy 8. However, MDS clonal cells can gradually acquired apoptosis resistance under different stressed conditions. The mechanism of apoptosis resistance is still not fully understood. The polycomb group protein EZH2 as an anti-apoptosis protein drives tumorigenesis. The association of EZH2 with apoptosis resistance of MDS clonal cells is worth further investigating.

Objective

The objective of this study is to investigate the role of EZH2 in the mechanism of apoptosis resistance of MDS.

Methods

The expression of EZH2 protein was analyzed by flow cytometry in 71 MDS patients. EZH2-targeted genes were identified through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. EZH2-targeted genes were confirmed through chromatin immunoprecipitation and luciferase reporter gene assay. Cell functional and metabolic experiments were also performed.

Results

We observed upregulation of the EZH2 expression in the patients with low-grade MDS especially in the individuals with trisomy 8 although downregulation in the patients with high-grade MDS. Based on the distinct EZH2 expression pattern, we identified an EZH2-targeted gene PSAT1, a serine biosynthetic critical enzyme through an integrated analysis of gene expression microarray analysis in MDS patients and cell line. Functionally, EZH2 can directly activate PSAT1 transcription by binding to its promoter and this effect is independent of its H3K27me3 enzymatic activity in vitro. Expanded sample analysis revealed the overexpression of PSAT1 in MDS patients especially in low-grade MDS. High expression of PSAT1 was associated with severe cytopenias, iron overload and activation of T lymphocytes-mediated autoimmunity. In vitro, overexpression of PSAT1 led to a significant proliferation advantage in the MDS and leukemia cell lines. Overexpression of PSAT1 could also downregulate apoptosis sensitivity to inflammatory cytokines and cytotoxic agents in vitro. However, knockdown of PSAT1 resulted in reduced cell growth capacity and increased the apoptosis sensitivity to cytotoxic agents. Further cancer metabolic experiments showed that overexpression of PSAT1 inhibited the ROS generation and induced an increase in glucose uptake, lactate and ATP production, which may make MDS clonal cells adaptive in the stress-related apoptosis environment.

Conclusion

EZH2/PSAT1 axis-mediated serine biosynthetic pathway is activated in low-grade MDS, which may confer MDS clonal cells growth advantages and resistance to stress-related apoptosis.

Disclosures

No relevant conflicts of interest to declare.
Phosphoproteomic Profiling of the Signaling Output of FLT3-ITD and Its AC220-Resistant Mutants Reveals Profound Signaling Differences and Differential Responsiveness to Inhibition of Downstream Kinases
Authors: Doebele Carmen , Kovar Johannes , Yepes Diego , Münch Silvia , Schnütgen Frank , Bohnenberger Hanibal , Köhler Anne , Urlaub Henning , Serve Hubert , Oellerich Thomas
Published: October 2019

Acute myeloid leukemia (AML) is a genetically heterogeneous disease where multiple mutations coincide in hematopoietic stem and progenitor cells leading to malignant transformation. One important class of mutations alters the function of signaling intermediates such as Fms-like tyrosine kinase 3 (FLT3), thereby helping AML cells to overcome the physiological communication with their microenvironment. Activating mutations in FLT3 are found in approximately 30% of adult AML cases. Particularly common are internal tandem duplications (ITD) in the juxtamembrane domain of FLT3, which are associated with poor clinical outcome. Recently, a phase II study of the second-generation FLT3 inhibitor AC220 (quizartinib) showed a complete remission rate of 44% to 54% in relapsed/chemotherapy refractory AML. However, secondary point mutations in the FLT3 tyrosine kinase domain have been reported as common causes of acquired clinical resistance to the FLT3 inhibitor AC220.

We used quantitative mass-spectrometry-based phosphoproteomics to elucidate and compare the signaling out-put of FLT3-ITD and its AC220-resistant mutants harbouring either the F691L 'gatekeeper' substitution or the D835V activation loop mutant in AML cell models. Our comprehensive signaling analyses profiled thousands of phosphorylation events in a site-specific manner and revealed marked differences in the signaling profiles of the FLT3 mutant variants. In general, we found differential activation of signal transducer and activator of transcription 5 (STAT5) and mitogen-activated protein (MAP) kinase signaling when comparing FLT3-ITD and the AC220-resistant mutants. Interestingly, some cytosolic tyrosine kinases showed differential activation patterns. For instance, spleen tyrosine kinase (SYK) signaling was significantly enhanced downstream of FLT3-ITD-F691L and cells harbouring this mutant showed increased responsiveness to compounds targeting SYK. Our resource study shows, how point mutations conferring resistance to AC220 impact the signaling output of FLT3-ITD and uncovers pathways whose inhibition might be useful to disrupt oncogenic signaling elicited by FLT3-ITD-F691L and -D835V mutants.

Disclosures

No relevant conflicts of interest to declare.
The Tyrosine Kinase-Binding and Proline-Rich Domains of Mutant CBL Are Essential for Leukemogenesis
Authors: Nadeau Scott , An Wei , Ahmad Gulzar , Mohapatra Bhopal , Zutshi Neha , Storck Matthew , Ogawa Seishi , Band Vimla , Band Hamid
Published: October 2019

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL are associated with myeloid malignancies, yet the molecular mechanisms by which this tumor suppressor becomes a dominant oncogene to initiate the leukemogenic process are unclear. In this study, we used systematic mutagenesis to delineate the importance of the various protein-protein interaction domains/motifs of a CBL mutant that is most frequently found in human leukemias, Y371H, in inducing leukemogenesis. We tested the impact of these secondary mutations on the ability of CBL-Y371H to impart hypersensitivity to cytokines and to upregulate the associated signaling pathways in the TF1 leukemia cell line model, and in mouse hematopoietic stem/progenitor cells from Cbl-null mice to mimic the lack of wildtype CBL expression in most of the mutant CBL bearing leukemia patients. The secondary mutations included: Cbl G306E, to abrogate the ability of the TKB domain of mutant Cbl to bind to activated tyrosine kinases; internal deletion of the proline-rich domain (AA 477-688) to abrogate interactions with SH3 domain containing partners; Cbl-Y700/731/774F triple phosphorylation site mutant predicted not to interact with the SH2 domain-containing partners; and Cbl-1-436 deletion construct lacking all C-terminal motifs. Analyses of stably expressed mutants in TF-1 cells for hypersensitivity to SCF1 demonstrated an essential role of an intact TKB domain and a particularly important role of the proline-rich domain. Transient retroviral expression in Cbl-null primary murine hematopoietic stem/progenitor cells confirmed these results. Deletion of the proline-rich domain led to substantially less tyrosine phosphorylation of the oncogenic mutant Cbl as well as of the c-Kit receptor upon SCF stimulation, and cells expressing this mutant lacked the sustained activation of Erk1/2 and Akt typically seen after SCF stimulation of the Cbl-Y371H-expressing cells. Together, our data provide conclusive evidence that interaction of leukemogenic mutant Cbl proteins with the upstream tyrosine kinase (via the TKB domain) and with partner proteins (via the proline-rich domain) provides a basic mechanism for gain of function phenotype of mutant Cbl proteins. Our studies suggest that identification of the leukemogenesis-critical partners of the proline-rich domain and targeting of the TKB-tyrosine kinase interface provide new therapeutic approaches against mutant Cbl-driven leukemias.

Disclosures

No relevant conflicts of interest to declare.
Immune Response in Acute Myeloid Leukemia with Mutated Nucleophosmin
Authors: Kuzelova Katerina , Brodská Barbora , Marková Jana , Helbig Grzegorz , Kyrcz-Krzemien Slawomira , Cetkovsky Petr
Published: October 2019

In our recent work, we described changes in HLA class I frequencies in AML patients with nucleophosmin (NPM) mutations. Several allelic groups were found to be depleted in patients with C-terminal NPM mutations (NPMc+), but not in patients with wild-type NPM, and these differences corresponded well to in silico prediction of NPM-derived peptides binding to HLA class I. Moreover, patients with "favorable" HLA type (i.e., expressing HLA alleles suitable for presentation of NPM-derived peptides) were found to have significantly better overall survival than those without suitable HLA alleles. Altogether, our results suggested that spontaneous anti-NPM immune response may prevent AML development and helps achieve durable cure.

In this contribution, an extended patient cohort (N = 71 patients with all available data) was divided into four groups according to HLA type (favorable/unfavorable) and Flt3-ITD status. Patients expressing suitable HLA alleles and not having internal tandem duplications in Flt3 gene (HLA+/Flt3-) had the best overall survival (Figure 1). The impact of unfavorable HLA type alone was comparable to that of Flt3-ITD positivity while the combination of both adverse factors was associated with particularly poor outcome (p = 0.0066 for trend).

On the basis of these results, we believe that NPMc+ AML patients with favorable HLA type (about 65% of all NPMc+ AML patients) could benefit from an immunomodulatory therapy. Besides inhibitory receptors (such as PD-1, CTLA-4, TIM-3), regulatory T-cells or decreased HLA expression, reduced antigen presentation by leukemic cells belongs to possible mechanisms of immune escape. Insufficiency of molecular complexes such as the proteasome or the transport-associated protein (TAP) can result in increased presentation of Class II-associated invariant chain peptide (CLIP). Indeed, increased CLIP expression on residual leukemic blasts has recently been reported to correlate with increased relapse risk in AML. Interestingly, valproic acid used in combination with standard chemotherapy has been described to significantly decrease the relapse rate in elderly AML patients with mutated NPM (but not in other AML subgroups). This could be associated with restored antigen presentation following treatment with histonedeacetylase inhibitors. In our experiments, we observed marked reduction of CLIP expression on primary NPMc+ AML blasts following in vitro treatment with suberoylanilide hydroxamic acid (SAHA, Figure 2). Both the fraction of CLIP-positive cells (open circles) and CLIP surface density on positive cells (closed circles) were reduced. HLA class I expression was normal and was not affected by SAHA treatment. Importantly, the effect on CLIP expression occurred even at subtoxic (0.5 µM) drug concentration.

Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Molecular Evidence of Ongoing Antigen Driven Somatic Hypermutation in the Natural History of Classic and Variant Hairy Cell Leukemia
Authors: Arons Evgeny , Davies Sarah , Still Katherine , Chai Nathan , Potocka Katherine , Zhou Hong , Kreitman Robert J.
Published: October 2019

Classic hairy cell leukemia (HCL) is a clonal chronic B-cell malignancy representing 2% of all leukemias, with neoplastic B lymphocytes in the blood, bone marrow and organs of the classic reticuloendothelial system. HCL variant (HCLv) is less common, typically has inferior prognosis, and usually lacks several HCL-related features including BRAF V600E, CD25, and Annexin A1. The malignant cells in HCL and HCLv patients have one or in unusual cases two different functional immunoglobulin heavy chain rearrangements. Significant variability can be observed in the 3rd complementarity determining region (CDR3), comprising the variable heavy (IGHV), diversity (IGHD), and joining (IGHJ) domains. A role for continued antigen selection in the pathogenesis and evolution of HCL and HCLv has been suggested but limited evidence has been found.

We examined HCL and HCLv-derived IGHV genes by massive parallel deep sequencing in 15 HCL and 7 HCLv cases. In all studied samples, a productive monoclonal tumor-derived IGH sequence was detected. Analysis of all reads sharing clonal CDR3 motifs revealed the existence of dominant subclone expansion and multiple subclonal variants. Analysis of phylogenetic trees indicated multiple branching and therefore exposure to multiple rounds of somatic hypermutation (SHM) in the evolution of clonally-related malignant cells. To investigate a possible cause of the high SHM activity and the large number of subclones in HCL and HCLv, we studied the expression of activation induced cytidine deaminase (AID), an enzyme essential for somatic hypermutations, expressed by germinal center (GC) B-cells where SHM occurs. In 5 HCL and 3 HCLv cases we detected a high expression level of AID mRNA, including wild-type and 2 splice variants; in 10 HCL, 4 HCLv, and 3 normal peripheral blood samples, AID expression was not detected using standard end-point PCR conditions. However, more sensitive real-time quantitative PCR detected AID transcripts in virtually all tested HCL and HCLv cases, although the range of transcript levels was large between different cases and varied with individual cases over time.

The data obtained for both HCL and HCLv fit a model of tumorigenesis in which the BRAF V600E mutation (or another event in HCLv) initiates neoplastic transformation in a GC B-cell committed to terminal differentiation, but still thereafter still targeted by ongoing SHM

Disclosures

No relevant conflicts of interest to declare.
A Randomized, Phase II Study with Biomarker Analysis of Panobinostat with or without Rituximab in Relapsed Diffuse Large B Cell Lymphoma
Authors: Assouline Sarit , Crump Michael , Holm Nielsen Torsten , Alcaide Miguel , Macdonald David , Tosikyan Axel , Haliotis Tina , Greenwood Celia , Klein Oros Kathleen , Morin Ryan , Johnson Nathalie , Mann Koren Kathleen
Published: October 2019

INTRODUCTION: Novel therapies which consider the molecular diversity of diffuse large B cell lymphoma (DLBCL) are needed to salvage patients who fail to respond to standard chemoimmunotherapy. The majority of DLBCL contain mutations in histone modifying enzymes (HME) which suggests that histone deacetylase inhibitors (HDI) should be active. Preclinical data suggest that they also augment the effect of rituximab.

METHODS: In this randomized phase II study, we evaluated the response rate and toxicity of panobinostat, a pan-HDI, 30 mg orally 3 times a week, with or without rituximab, in 40 patients with relapsed and refractory DLBCL (median 3 prior regimens, range1-9). Tumors were sequenced for mutations known to be associated with DLBCL, and serial biopsies were analyzed for changes in biomarkers.

RESULTS: 21 patients were treated with panobinostat alone and 19 with panobinostat and rituximab. Overall, 12/40 patients (30%) responded to panobinostat (95% confidence interval 16.6%-46.5%) the addition of rituximab did not increase responses. The median duration of response was 14.5 months (95% confidence interval 9.4 to not reached) (Figure). At a median follow up of 17.5 months for responders, 6 of 12 patients have not progressed. Patients with dual expression of MYC and BCL2 (6/27) and those with FAS mutations (5/33) did not respond although responses were seen in 5/10 patients with TP53 mutations. Responses were seen in 8/24 patients with mutations in HME genes. Patients with MEF2B mutations had the greatest odds of response (P<0.05) and a higher number of HME mutations tended to increase the odds of response. Changes in histone H3 acetylation and MYC expression were observed but did not correlate with response.

CONCLUSION: Panobinostat induces durable responses in some patients with relapsed DLBCL, and accompanying biomarker analyses identified DLBCL subgroups likely to respond.

Figure 1. Progression free survival overall and for responders, N=40 patients. Figure 1. Progression free survival overall and for responders, N=40 patients.

Disclosures

Assouline: Novartis: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Celgene: Consultancy. Off Label Use: panobinostat for the treatment of diffuse large B cell lymhpoma. Crump:Seattle Genetics: Consultancy; Celgene: Consultancy; Roche Canada: Consultancy. Tosikyan:Novartis: Consultancy.
CXCR4 Gene Dosage Is Critical for HSC Engraftment
Authors: McDermott David H. , Jacobs Paejonette , Liu Qian , Gao Jiliang , Murphy Philip M.
Published: October 2019

Introduction: Warts, Hypogammaglobulinemia, Infections and Myelokathexis Syndrome (WHIMS) is an autosomal dominant immunodeficiency resulting from gain-of-function mutations in the chemokine receptor CXCR4. We recently described a unique WHIMS patient who underwent spontaneous genetic and phenotypic reversion at approximately age 30 after being severely affected as a child. Her reversion was due to a single catastrophic genetic event known as chromothripsis (chromosome shattering) resulting in the deletion of one copy of 163 genes in addition to her mutant copy of CXCR4 on chromosome 2. This event was traced to a hematopoietic stem cell (HSC) that had spontaneously repopulated her bone marrow; however, which of the genes was responsible and the mechanism required further investigation.

Methods: Mouse models of CXCR4 haploinsufficiency (Cxcr4+/o) and WHIMS (Cxcr4+/S338X) were used in competitive bone marrow repopulation experiments transplanting whole bone marrow cells or purified HSC. Recipient mice were treated with / without lethal irradiation prior to transplant. Genome editing with TALENs and CRISPR-Cas9 technology was used to target CXCR4 for deletion in human cell lines.

Results: Cxcr4 haploinsufficiency markedly enhanced HSC engraftment potential in recipient WHIM mice whether the donor HSC were purified from whole bone marrow cells or not, and whether the recipient was conditioned by lethal irradiation or not. Enhanced engraftment by Cxcr4 haploinsufficient donor HSC also occurred in wild-type mouse recipients, but to a lesser extent, and was also HSC intrinsic. Genome editing experiments have been successful at deleting one or both copies of CXCR4 in human cell lines in up to 40% of treated cells, and in reducing CXCR4 surface expression.

Conclusion: While CXCR4 was already understood to be important in HSC biology, this patient and subsequent murine experiments have proven that the gene dosage of CXCR4 is a critical factor affecting HSC engraftment. Genome editing is a promising technology for deleting one copy of CXCR4, ideally the WHIM allele,in autologous HSC as a strategy to cure WHIM syndrome.

Disclosures

McDermott: US National Institutes of Health: Employment, Patents & Royalties: pending. Jacobs:US National Institutes of Health: Employment, Patents & Royalties: pending. Liu:US National Institutes of Health: Employment, Patents & Royalties: pending. Gao:US National Institutes of Health: Employment, Patents & Royalties: pending. Murphy:US National Institutes of Health: Employment, Patents & Royalties: pending.
Inflammatory Plasma Proteins Predict Disease Severity and Response to Therapy in Patients with LCH
Authors: Zinn Daniel , Lin Howard , Shih Albert , Scull Brooks , Cantu Miguel Dario , Abhyankar Harshal , Chakraborty Rikhia , Lim Karen Phaik Har , Peters Tricia L. , Lira Sergio , Tsz-Kwong Chris , McClain Kenneth , Merad Miriam , Allen Carl
Published: October 2019

Purpose: Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder with clinical manifestations ranging from single lesions to lethal multi-organ disease, and front-line therapy fails in the majority of patients. No histologic features of LCH lesions have yet been demonstrated to correlate with clinical outcomes.  We hypothesize that function of differentiated CD207+, including interactions with recruited activated lymphocytes, results in plasma protein profiles that reflect disease burden and tissue-specific DC-lymphocyte interactions. Plasma proteins may therefore inform mechanisms of pathogenesis and serve to predict risk and follow disease burden in LCH patients.

Methods: We evaluated inflammatory proteins in the plasma of LCH patients using the Luminex platform. We compared plasma concentrations of 121 analytes in 178 patients with LCH to 90 controls with discovery and validation sets. Comparisons were made based on disease status, age, and clinical risk categories. 152 samples collected from 78 patients at serial time points were also analyzed. Comparisons were made between diagnosis and cure, as well as multiple time points in therapy.

Results: Thirty-one analytes were significantly different between all LCH and controls, including proteins mediating chemotaxis and differentiation of lymphocytes and/or dendritic cells: osteopontin, SDF1A, 6CKine, CCL19, CCL20, sIL4R, IL20, and IL23. Twenty-three analytes were significantly different when comparing adult vs pediatric LCH. When comparing LCH vs control by age groups, 5 were significantly different in adults and 23 were different in children. Eighteen were different in pediatric low risk compared to high risk patients, with the most significant differences in inflammatory proteins including sTNFRI, sTNFRII, TNFa, sIL2Ra, and IL-8. Classifier analysis was able to predict risk organ involvement with a sensitivity of 91.5% and a specificity of 87.5%. Seventeen analytes changed significantly after curative treatment.

Conclusions: Distinct Plasma protein profiles exist in LCH, suggesting pathologic myeloid cells drive the inflammatory pathology of disease. Profiles are vastly different among children and adults with LCH, and differ based on burden of disease, suggesting separate inflammatory pathology. Seven analytes reliably predicted high-risk disease, and analysis of serial samples identified normalization of inflammatory proteins after cure.

Disclosures

Allen: Roche: Consultancy, Other: unpaid; NovImmune: Consultancy, Other: unpaid.