Increased Soluble GPVI Levels in Cirrhosis: Evidence for Collagen Induced Platelet Activation In Vivo

Abstract Background Cirrhosis is a consequence of prolonged inflammation arising from chronic liver disease of different aetiologies. It is characterised by tissue fibrosis, the deposition of collagen-rich extracellular matrix tissue within the liver. Glycoprotein VI (GPVI) is platelet-specific collagen receptor that is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligation. The shed extracellular region of GPVI can be detected in plasma and used as a measure of GPVI-dependent platelet activation in vivo. Several lines of evidence suggest that GPVI-dependent platelet activation occurs in cirrhosis. Platelets have been shown to accumulate at sites of collagen-rich fibrotic tissue. Circulating levels of collagen are increased in cirrhosis. Collagen-induced platelet aggregation responses are reduced in vitro with cirrhosis. Based on these results, we hypothesised that soluble GPVI (sGPVI) levels are increased in patients with cirrhosis. As such, the aim of this study was to quantify sGPVI levels in patients with cirrhosis and compare to healthy controls. Methods Compensated cirrhotic patients were recruited at the Mater Misericordiae University Hospital, Dublin, Ireland. The diagnosis of cirrhosis was based on clinical examination, blood tests, and radiological examination (nodular surface, larger right lobe, coarse echotexture). Exclusion criteria were decompensated cirrhosis, recent thrombotic events, and antiplatelet and/or anticoagulant therapies. Healthy controls were recruited at the Royal College of Surgeons in Ireland and Beaumont Hospital, Dublin, Ireland. Blood samples were collected into vacutainers containing 3.2 % sodium citrate as anticoagulant. sGPVI levels in platelet poor plasma were measured using an in house custom ELISA. Results 57 patients with mixed aetiology cirrhosis and 55 healthy controls were recruited. In the patient group, 42% of patients had alcoholic liver disease (ALD), 30% had hepatitis C (HCV), 7% had non alcoholic fatty liver disease (NAFLD), 5% had Hepatitis B (HBV), 5% had autoimmune hepatitis (AIH), 5% had cryptogenic liver disease, 4% had hereditary haemochromatosis (HH), and 2% had primary biliary cholangitis (PBC). sGPVI levels were significantly increased in patients with cirrhosis (5.8 ± 0.6 ng/ml, n = 57) compared to healthy controls (3.2 ± 0.4 ng/ml, n = 55, p < 0.0001). There was no significant difference between sGPVI levels in AIH (4 ± 1 ng/ml, n = 3), ALD (5.6 ± 1 ng/ml, n = 24), cryptogenic (12 ± 5 ng/ml, n = 3), HBV (3.1 ± 1 ng/ml, n = 3), HCV (5 ± 0.6 ng/ml), or NAFLD (5.3 ± 1.1 ng/ml, n = 4). sGPVI levels did not correlate with platelet count (r = 0.12, p = 0.3) or parameters of liver cell function (albumin, bilirubin, prothrombin time, and liver stiffness measurements). Conclusion sGPVI levels are significantly increased in patients with mixed aetiology cirrhosis. This indicates collagen induced platelet activation is occurring in vivo and suggests the presence of an underlying coagulopathy in patients with cirrhosis. Disclosures Ní Áinle: Actelion Pharma: Research Funding; Leo Pharma: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

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Characterization of Platelet Activation in Patients with Eisenmenger Syndrome

Blood ◽

10.1182/blood-2018-99-116914 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4973-4973

Author(s):

Gordon Haire ◽

Karl Egan ◽

Barry Kevane ◽

Michael Fay ◽

Anne Fortune ◽

...

Keyword(s):

Congenital Heart Disease ◽

Board Of Directors ◽

Platelet Activation ◽

Research Funding ◽

Congenital Heart ◽

Plasma Concentrations ◽

Published Data ◽

Healthy Controls ◽

Eisenmenger Syndrome ◽

Advisory Committees

Abstract Background Eisenmenger Syndrome (ES) is an extremely rare and serious complication of congenital heart disease in which pulmonary vascular resistance overcomes that of the systemic circulation causing bidirectional or reversed flow across an intracardiac shunt. Patients with ES have an increased incidence of both thrombotic and bleeding complications which occur through poorly understood mechanisms. We have recently demonstrated a critical mechanistic role for platelets in supporting abnormal hypercoagulability in patients with ES using calibrated automated thrombography (Kevane & Ní Áinle et al., J Thromb Haemost 2018). Aims Based upon our published data, we further hypothesized that patients with ES would have higher plasma concentrations of platelet activation markers. Therefore, the aim of this study was to measure plamsa levels of soluble P-Selectin (sP-Selectin) and soluble glycoprotein VI (sGPVI) in patients with ES and in matched healthy controls. Methods Patients over the age of 18 with Eisenmenger Syndrome and healthy controls were recruited from the cardiology service at a large tertiary referral centre (Mater Misericordiae University Hospital) incorporating the Irish national congenital heart disease service. Platelet poor plasma (PPP) was generated from participants' blood by centrifugation at 2000xg for 10 minutes at room temperature. Commercially available ELISA assays employing quantitative sandwich immunoassay techniques were performed according to manufacturer protocol to measure plasma sP-Selectin and sGPVI levels. All experiments were performed in duplicate with results expressed as mean +/- standard error of mean. Comparisons between groups were made utilising the Student's t-test with a p-value < 0.05 deemed to represent statistical significance. Results During the study period, >14,000 patients attended the MMUH cardiology service. Of these, 14 consecutive patients with a diagnosis of ES and 10 matched healthy controls were recruited. Patients with ES had significantly elevated plasma concentrations of both soluble P-Selectin [38.5 +/- 8.3 vs. 11.3 +/- 4.2 ng/ml (p < 0.05)] and soluble GPVI [10.5 +/- 2.4 vs. 1.5 +/- 1.4 ng/ml (p < 0.01)] compared to healthy controls. Conclusion Building upon our recently published data in this population, a large cohort given the extreme rarity of this condition, increased concentrations of markers of platelet activation in individuals with ES further suggest a mechanistic role for platelets in ES-mediated hypercoagulability. This phenotype may be a consequence of vascular pathology associated with pulmonary hypertension in these patients. As such, therapies targeted at underlying vascular pathologies may act to ameliorate prothrombotic tendencies and we aim to characterize response to therapy during this ongoing study. Disclosures Ni Ainle: Leo Pharma: Research Funding; Actelion: Research Funding; Bayer: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer: Membership on an entity's Board of Directors or advisory committees.

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Dysfunctional HDAC8 Impacts Genomic Integrity and Is a Novel Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153382 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1610-1610

Author(s):

Zuzana Chyra ◽

Srikanth Talluri ◽

Rao Prabhala ◽

Mehmet K. Samur ◽

Anil Aktas-Samur ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Hdac Inhibitors ◽

Dependent Manner ◽

Dna Breaks ◽

Advisory Committees

Abstract The histone modifications and associated changes in chromatin structure and function have emerged as important epigenetic mechanisms impacting gene expression and have significant translational relevance in cancers, including multiple myeloma (MM). Epigenetic intervention with histone deacetylases (HDACs) inhibitors is emerging as a promising therapeutic strategy in combination with current anti-myeloma agents. Although pan-HDAC inhibitors have been shown to be effective both in preclinical and clinical setting, they seem to be associated with toxicity. It is, therefore, extremely important to understand the biological and molecular roles of individual HDACs to then selectively target them to limit toxicities observed with pan-HDAC inhibitors. Based on our observation that elevated HDAC8 expression correlates with poor overall survival in MM patients in three different datasets including one publicly available dataset (GSE39754), we evaluated its functional role in MM. HDAC8, a member of class I HDAC isoenzymes, is responsible for the deacetylation of lysine residues on the N-terminal part of the core histones as well as non-histone proteins. We performed genetic modulation of HDAC8 by loss-of-function studies, using shRNA as well as siRNAs targeting HDAC8. Downregulation of HDAC8 in 3 different MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with induction of cell apoptosis. Consistently, treatment with a selective and potent HDAC8 inhibitor (OJI-1) caused a significant inhibition of MM cell growth in a panel of 20 MM cell lines (IC50 = 80 nM) in a time- and dose-dependent manner, while having a minimal impact on six PBMC samples from healthy donors both in resting and activated state (IC50 = 150 nM). The mechanism of cell death was apoptosis as demonstrated by annexin-labeling. Importantly, both the HDAC8 knockdown and OJI-1 treatment inhibited DNA breaks as evidenced from γH2AX expression or a single cell gel electrophoresis method to visualize and quantitate DNA breaks. HDAC8 inhibition also caused inhibition of RAD51 foci and HR activity, as measured by strand-exchange assay. Interestingly, non-homologous end joining in MM cells was not impacted by these treatments. Consistent with these data, the overexpression of HDAC8 in MM as well as in normal cells increased DNA breaks and HR activity. Furthermore, the inhibition of HDAC8 (by knockdown and OJI-1) inhibited, whereas its overexpression increased genomic instability, as assessed by micronucleus assay, in surviving MM cells. We also demonstrate that HDAC8 interacts with RAD51 and impacts its acetylation. The treatment of MM cells with HDAC8 inhibitor (OJI-1) increased RAD51 acetylation. Next, we examined the in vivo efficacy of the HDAC8 conditional knockdown in a human xenograft mouse model, using H929 cells injected subcutaneously in SCID mice. HDAC8 knockdown not only caused a significant reduction in tumor growth but also increased survival (p=0.0016) compared to mice injected with control cells. Evaluation of tumors from these mice confirmed in vivo inhibition of DNA breaks and HR activity, and induction of apoptosis following HDAC8-knockdown. HDAC8 inhibitor OJI-1 also synergistically increased the cytotoxicity of existing MM drugs including dexamethasone, bortezomib and lenalidomide. In conclusion, our results demonstrate that elevated HDAC8 in MM cells is involved in inhibition of apoptosis but also contributes to increased DNA breaks and dysregulation of homologous recombination and genome stability. Therefore, HDAC8 is a novel target for therapeutic application in MM. Selective and potent HDAC8 inhibitor OJI-1 has shown a favorable therapeutic index with synergistic effect in combination with existing MM drugs. Disclosures Hajek: Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Munshi: Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy; Legend: Consultancy.

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Incidence and Prognostic Relevance of ASXL2 Mutations in Adult CBF-AML with t(8;21)(q22;q22): A Study of the German-Austrian AML Study Group (AMLSG)

Blood ◽

10.1182/blood.v126.23.3818.3818 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3818-3818 ◽

Cited By ~ 1

Author(s):

Nikolaus Jahn ◽

Mridul Agrawal ◽

Lars Bullinger ◽

Daniela Weber ◽

Andrea Corbacioglu ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Prognostic Impact ◽

Dependent Manner ◽

Trisomy 8 ◽

Advisory Committees ◽

Mutation Status ◽

Paired Samples ◽

Significant Difference ◽

High Incidence

Abstract Background: The ASXL2 (Additional Sex comb-like 2) gene on chromosome 2p23.3 encodes an epigenetic regulator that is thought to act through histone modification and thereby regulating gene transcription in a context-dependent manner. Recently, ASXL2 mutations (ASXL2mut) were found with a high incidence (~23%) in a cohort of 110 pediatric or adult AML patients (pts) with t(8;21)(q22;q22) (Micol et al., Blood 2014). Aim: We assessed the frequency and prognostic impact of ASXL2mut in the context of other clinical and genetic factors in a large clinically well-defined cohort of intensively treated adults with t(8;21) AML. In addition, the stability of ASXL2 mutation status was analysed in a subset of pts at the time of relapse. Methods: Diagnostic samples from 204 adults with t(8;21);RUNX1/RUNX1T1 -positive AML [median age: 49 years, range: 18-82] were analysed for ASXL2mut in exon 11 and 12 using a combination of PCR-based amplification and subsequent direct DNA-sequencing. Paired samples at diagnosis and relapse were evaluated for the ASXL2 mutation status in a subset of 22 pts. Additional mutation analyses were performed for KIT, FLT3 (ITD and TKD), NRAS, and ASXL1 genes. All pts received intensive treatment either within AMLSG trials (n=155) or according to standard chemotherapy regimens. Results: Thirty four ASXL2mut were identified in 33 (16.2%) of the 204 pts. All mutations (frame-shifts, n=32; non-sense, n=2) created stop codons leading to premature protein truncation with loss of the terminal PHD domain; 27% of the mutations affected codon T740 in exon 12. At diagnosis, there was no significant difference between pts with ASXL2mut and ASXL2 wildtype (ASXL2wt) with respect to sex, WBC, haemoglobin, platelets, LDH serum levels, and circulating or bone marrow blasts. Of note, ASXL2mut were not associated with increasing age, a finding which is commonly observed for ASXL1 mutations in AML. In terms of secondary chromosome aberrations ASXL2mut were frequently associated with del(9q) (P=.006), whereas they never co-occurred with trisomy 8. There was no significant association between ASXL2mut and all other gene mutations analysed. Analysis for ASXL2 mutation status in 22 paired samples obtained at diagnosis and relapse showed a high stability since the mutation was still present in two pts at relapse whereas none of the remaining 20 ASXL2 wildtype cases acquired ASXL2mut. There was no difference in complete remission (CR) rate after double induction between pts with ASXL2mut (88%) and those with ASXL2wt (92%); the same was true when comparing pts with ASXL1 or ASXL2 mutations (ASXL1/2mut) as one group (93%) versus those with ASXL1/2 wildtype. Neither ASXL2mut nor ASXL1/2mut did impact the endpoints event-free-survival, cumulative incidence of relapse, relapse-free and overall survival. Conclusions: Beside KIT and NRAS, ASXL2 is among the most frequently mutated genes in t(8;21) AML. ASXL2mut did not impact achievement of CR or any survival endpoint. Nevertheless, the high incidence (16.2%) of ASXL2mut in t(8;21) AML and the exclusivity to this subgroup of core-binding factor AML implies a peculiar role of ASXL2 in the leukemogenesis of t(8;21) AML providing a basis for further studies. Disclosures Horst: MSD: Research Funding; Pfizer: Research Funding; Amgen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Boehringer Ingleheim: Research Funding. Schlenk:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Honoraria; Arog: Honoraria, Research Funding; Teva: Honoraria, Research Funding.

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Circulating Plasminogen Activator Inhibitor-1 (PAI-1) Is Reduced By In Vivo Thrombin Generation and Subsequent Formation of Activated Protein C (APC)

Blood ◽

10.1182/blood-2019-124731 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2389-2389

Author(s):

Sara Reda ◽

Franziska Isabelle Winterhagen ◽

Christina Berens ◽

Jens Müller ◽

Johannes Oldenburg ◽

...

Keyword(s):

Board Of Directors ◽

Plasma Levels ◽

Research Funding ◽

The Other ◽

Healthy Controls ◽

Advisory Committees ◽

Pai 1 ◽

D Dimer ◽

Thrombin Formation

Background: APC has been suggested to contribute to a hyperfibrinolytic state in various acquired coagulation disorders, including coagulopathies induced by trauma and sepsis. However, in vivo evidence for the proposed underlying mechanism of proteolytic degradation of PAI-1 by APC remains inconclusive. Recently, we have shown increased APC generation in response to in vivo thrombin formation in carriers of the factor V Leiden mutation (FVL). In this approach of stimulated hemostasis activity pattern evaluation (SHAPE), in vivo thrombin formation was triggered by low-dose administration of recombinant activated factor VII (rFVIIa). Aim of the present study was to investigate the resulting effects on the fibrinolytic system. Methods: The study population consisted of 30 FVL carriers (thereof 13 with a history of venous thromboembolism) and 15 healthy controls. Blood samples were collected immediately before and during a period of 8 hours following injection of 15 µg/kg rFVIIa. Plasma levels of APC were quantified using an oligonucleotide-based enzyme capture assay (OECA). Other monitored parameters included plasma levels of prothrombin activation fragment 1+2 (F1+2), tissue-type plasminogen (t-PA), PAI-1, plasminogen, α2-antiplasmin, plasmin-α2-antiplasmin complexes (PAP), soluble fibrin monomers, d-dimer, and thrombin-activatable fibrinolysis inhibitor (TAFI). Results: At baseline, FVL carriers showed higher median levels of APC in comparison to the controls (1.39 vs. 0.86 pmol/L, P=.001), higher PAI-1 levels (30.1 vs. 15.3 ng/mL, P=.002) and lower plasminogen levels (94.8 vs. 110.4%, P=6·10-4). The other baseline parameters did not differ significantly. In both cohorts a comparable increase of F1+2 was observed after administration of rFVIIa, whereas APC increased more (P=.004) in FVL carriers (by 6.40 pmol/L) than in healthy controls (by 2.14 pmol/L), Concurrently, median PAI-1 levels decreased more (P=.007) in FVL carriers (by 19.8 pmol/L) than in healthy controls (by 8.0 pmol/L) (Figure 1). TAFI levels decreased temporarily, from 104.8 to 94.4% (P=.007) in FVL carriers and from 105.0 to 92.1% (P=2·10-4) in healthy controls. PAP levels increased from 164 to 209 ng/mL (P<10-4) in FVL carriers and from 154 to 189 ng/mL (P=.023) in the control group. The extent of these changes did not differ between the two cohorts. D-dimer level increased only in FVL carriers, from 0.34 to 0.41 mg/L (P=.008). t-PA and the other parameters did not show significant changes after rFVIIa administration. Conclusion: Increased APC formation rates in FVL carriers were associated with a greater decline of PAI-1 levels in the absence of interfering changes in t-PA levels. These data provide further in vivo evidence that APC down-regulates PAI-1. Overall, the SHAPE approach utilized here does not induce a significant profibrinolytic response, even in patients with thrombophilia. Disclosures Reda: Grifols: Honoraria; Shire: Honoraria. Winterhagen:SOBI: Honoraria. Berens:Pfizer: Honoraria; Shire: Honoraria; Biotest: Honoraria; CSL Behring: Honoraria; Novo Nordisk: Honoraria; Baxter: Honoraria. Müller:NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Siemens: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Oldenburg:CSL Behring: Consultancy, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Research Funding, Speakers Bureau; Swedish Orphan Biovitrum: Consultancy, Speakers Bureau; Grifols: Consultancy, Speakers Bureau; Takeda (Shire): Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Biotest: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau. Rühl:CSL Behring: Honoraria; Bayer: Honoraria; Shire: Honoraria; SOBI: Honoraria; Sanofi Genzyme: Honoraria; Grifols: Honoraria.

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In-Vivo Purging with Rituximab (R) Followed By Z/BEAM Vs BEAM/R Autologous Stem Cell Conditioning for Relapsed Diffuse Large B-Cell Lymphoma (DLBCL) Patients (pts): Mature Results from a Combined Analysis of 3 Trials

Blood ◽

10.1182/blood.v126.23.3192.3192 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3192-3192 ◽

Cited By ~ 1

Author(s):

Issa F. Khouri ◽

Dawen Sui ◽

Francesco Turturro ◽

William D Erwin ◽

Roland L Bassett ◽

...

Keyword(s):

Stem Cell ◽

Randomized Trial ◽

Board Of Directors ◽

Research Funding ◽

Ibritumomab Tiuxetan ◽

Advisory Committees ◽

Significant Difference ◽

Conditioning Regimens

Abstract Background: The addition of R has been shown to improve results for pts with relapsed DLBCL who undergo BEAM (carmustine, etoposide, cytarabine, melphalan) high-dose chemotherapy followed by an autologous stem cell transplantation (ASCT) (Khouri IF, J Clin Oncol 2005;23;2240). The incorporation of radiolabeled antibodies such as yttrium-90 ibritumomab tiuxetan to conditioning regimens has also been evaluated without additional toxicity (Khouri IF, ASH 2007, abstract 620). Subsequently, a randomized trial which was undertaken at our center to compare these 2 regimens was closed early because of slow accrual. Herein, we compare the long-term outcome in pts treated in these trials. Methods: A combined analysis was carried out from 113 pts. Between 1999 and 2003, 57 pts with relapsed DLBCL were enrolled on a protocol with BEAM conditioning plus R at 1000 mg/m2 on days +1 and +8 after ASCT (Group A). Between 2004 and 2006, a similar group of 26 patients were entered onto a trial consisting of ibritumomab tiuxetan plus BEAM (Z/BEAM). Ibritumomab tiuxetan was given at the fixed doseof 0.4 mCi/Kg on day -21 followed by BEAM(days -7 to -1) (Group B). In 2007-2010, a randomized trial was undertaken comparing BEAM/R (Group C, n=16) and Z/BEAM (Group D, n=14). All pts received R during stem cell collection, administered at 375 mg/m2 on the day before initiating chemotherapy for stem cell mobilization, and again at 1000 mg/m2, 7 days later. The same eligibility criteria were used for all groups. In addition, pts who were enrolled on the randomized trial (Groups C and D) underwent FISH analysis for -5 and -7 and cytogenetic analysis by G-banding pre-enrollment; those who had a clonal abnormality were excluded. We also retrospectively evaluated the histologic subtypes of mediastinal, transformed and de novo DLBCL. We determined the cell-of-origin of the latter using the Visco/Young and Choi W, et al. immunohistochemical algorithms. A univariate analysis was conducted for factors of interest: this included the group conditioning, age, sex, number of prior treatments, disease status at transplantation (CR/PR), IPI (0 vs >0), LDH, β2-microglobulin, PET status, and histology subtype. Multivariate survival analysis was then conducted using backward elimination on the basis of the likelihood ratio test and including the conditioning regimens and all the factors with P < 0.05 in the univariate analyses. Patients: There was no significant difference in the prognostic factors described above between the 4 groups with the exception of prior treatments. More pts in the BEAM/R groups received > 2 prior chemotherapies than the Z/BEAM groups (32%, 11.5%, 69% and 50% in Groups A, B, C and D, respectively; P < 0.001). Non-GCB histology within the 4 groups was present in 39%, 26%, 31% and 21%, respectively (P=0.31). Results: The median follow-ups times for Groups A, B, C and D were 11.8, 8.1, 4.2 and 4.9 years, respectively. The 5-year overall survival (OS) rates for these groups were 74%, 73%, 69% and 86%, respectively (P = 0.46) and the disease-free survival (DFS) rates were 62%, 65%, 63%, and 63%, respectively (P = 0.99) (Figure 1). Multivariate analysis at 5-year showed that previous exposure to >3 prior chemotherapies and IPI were predictors for OS (P= 0.003 and 0.005, respectively), and DFS (P = 0.003 and 0.006, respectively). There was no significant difference in OS or DFS rates between GCB, non-GCB, mediastinal and transformed DLBCL (Figure 2). The 5-year non-relapse mortality for all pts was 4.7% and the 5-year relapse rate was 32.5% (34.0%, 30.8%. 25% and 37.5% for Groups A, B, C and D, respectively). The 5-year risk of secondary myelodysplasia (MDS) for all pts was 6.2%. None of the pts in Groups C and D who had pre-transplant cytogenetic testing developed MDS at 5-year. Conclusions: Long-term follow-up results show no difference in OS, DFS, relapse rate, or risk of MDS between Z/BEAM and BEAM/R after in-vivo purging with R during stem cell collection. Our results were reproducible in 3 trials and appear to be superior to published reports in the literature of similar trials without in-vivo purging. This highlights the importance of this procedure for ASCT in DLBCL. Figure 1. Figure 1. Disclosures Off Label Use: The use of Zevalin and rituximab in transplantation. Alousi:Therakos, Inc: Research Funding. Fanale:Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Research Funding; Infinity: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Research Funding; Genentech: Research Funding; Medimmune: Research Funding; Novartis: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Molecular Templates: Research Funding; ADC Therapeutics: Research Funding; Onyx: Research Funding; Gilead: Research Funding. Nastoupil:AbbVie: Research Funding; Janssen: Research Funding; Celgene: Honoraria; TG Therapeutics: Research Funding; Genentech: Honoraria. Westin:Spectrum: Research Funding.

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Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Blood ◽

10.1182/blood-2021-146349 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2414-2414

Author(s):

Xiaoguang Wang ◽

Vi Lam ◽

Dan Vuong ◽

Tingting Liu ◽

Olga Danilova ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Tumor Immunity ◽

Research Funding ◽

Checkpoint Inhibitors ◽

Dependent Manner ◽

Advisory Committees

Abstract Introduction: Immune checkpoint inhibitors have limited single agent activity in B-cell non-Hodgkin lymphoma (NHL). Hence, it is important to develop strategies which will thwart immune evasion in this disease. Neddylation is a sequential enzyme-based process which ultimately regulates protein turnover. In the initial step, NAE activates NEDD8 in an ATP-dependent reaction in which a high-energy thioester bond is formed between NEDD8 and the catalytic cysteine of NAE. Active NEDD8 is then transferred to the NEDD8-specific E2 conjugating enzyme (UBE2M) and is conjugated to cullin proteins which are part of the Cullin-RING E3 ubiquitin ligases (CRL). Pevonedistat (pevo) forms a covalent adduct with NEDD8, thereby inhibiting NAE and thus reduces CRL activity and diminishes ubiquitination and proteasomal degradation of CRL substrates (IκB, HIF-1α, etc). We have recently reported that neddylation regulates T cell activation and polarization (Best et al, Leukemia 2021). Here we investigate how pharmacologic targeting of neddylation modulates anti-tumor immunity using NHL models. Methods: Peripheral blood mononuclear cells were isolated from patients with NHL and T cells were purified using Dynabeads. A20 cells were transplanted into flanks of syngeneic BALB/c mice. When tumors reached 100 mm 3, mice were randomized into groups and treated with pevo 60 mg/kg subcutaneously daily for 10 days or vehicle control. Once moribund, mice were sacrificed, tumors were processed into single-cell suspension and analyzed by flow cytometry. Pevo was provided by Takeda Development Center Americas Inc. (Cambridge, MA). Results: Primary patient-derived CD3/28-stimulated CD3 + T cells exhibited upregulation of TNFα and IFNγ in vitro in the presence of pevo. Concurrently, we observed increased expression of PD-1 and CTLA-4. Pre-treatment of T cells with pevo enhanced killing of NHL cell lines (JeKo-1, Mino, Maver-1 and VAL) in allogeneic cytotoxicity assays. Expectedly, treatment with pevo resulted in increased expression of HIF-1α in TCR-stimulated T cells. shRNA-mediated knockdown of HIF-1α abrogated the pevo effect, suggesting that NAE inhibition modulates T cell function in HIF-1α-dependent manner. While A20 cells showed resistance in vitro, treatment with pevo delayed lymphoma progression in A20 mice in vivo (Fig 1A). This was accompanied by an increase of tumor-infiltrating lymphocytes (TILs; Fig 1B). CD8 + TILs from pevo-treated mice exhibited activated phenotype as manifested by increased secretion of IFNγ (Fig 1C). Meanwhile, expression of the exhaustion molecules CTLA-4 and PD-1 by CD4/CD8 + TILs remained unchanged. To further investigate the role of T-cell immunity in this setting, we employed 1) CD8 depletion by pre-treatment with 12.5 mg/kg anti-CD8 antibody (IV); or 2) CRISPR/Cas9-mediated knockout of β2-microglobulin (MHC class I protein) in A20 cells. Either approach led to a partial decrease of pevo efficacy in vivo compared with respective controls. To exclude tumor-intrinsic effect of NAE inhibition, we knocked down UBE2M in A20 cells. Loss of UBE2M had no effect on growth of control tumors, or pevo therapeutic effect, implying that the anti-tumor efficacy of NAEi was T cell-mediated in this model. Since pevo modulates PD-1 on human T cells, we explored its effect on PD-L1 expression. Treatment with pevo upregulated PD-L1 expression in A20 cells in a MYC-dependent manner. Hence, we explored a combination of pevo and αPD-1 blockade in A20 model. Combination treatment significantly increased the CD4 + and CD8 + TILs. A decrease in tumor growth was significantly more pronounced than with either drug alone (Fig 1A). The combination benefit was fully reversed by loss of B2M, again highlighting the importance of immune mechanism . We observed expansion of IL-2, IL-4 and IL-17-secreting CD4+ TILs following the combined treatment, compared with either drug alone. In addition, CD4+ and CD8+ TILs derived from these mice secreted high levels of IFNγ (Fig. 1C). Conclusions: NAE inhibition enhanced T cell-mediated cytotoxicity in vitro. Treatment with pevo promoted activation of TILs and restricted tumor growth in an A20 mouse lymphoma model. Pevo-treated tumors were sensitized to αPD-1 . Thus, targeting NAE enhances anti-tumor immunity. Our data provide a strong rationale for future studies of pevo in combination with immune checkpoint inhibitors in lymphoma and other tumors. Figure 1 Figure 1. Disclosures Siddiqi: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncternal: Research Funding. Berger: Takeda Development Center Americas, Inc.: Current Employment. Danilov: Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Takeda Oncology: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding; Rigel Pharm: Honoraria.

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Favorable Long-Term Outcomes after Daratumumab Discontinuation in AL Amyloidosis Patients Achieving Deep Responses

Blood ◽

10.1182/blood-2019-124981 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1884-1884 ◽

Cited By ~ 1

Author(s):

Alfred Chung ◽

Gregory P. Kaufman ◽

Surbhi Sidana ◽

David Iberri ◽

Erik Eckhert ◽

...

Keyword(s):

Disease Progression ◽

Board Of Directors ◽

Median Duration ◽

Research Funding ◽

Control Group ◽

Al Amyloidosis ◽

Cell Transplant ◽

Advisory Committees ◽

Free Interval ◽

Significant Difference

Daratumumab (DARA) is a CD38-targeted antibody FDA-approved for the treatment of multiple myeloma (MM) and its efficacy has recently been demonstrated in the treatment of AL amyloidosis. DARA is conventionally given indefinitely until evidence of disease progression or intolerance for the treatment of MM. In AL amyloidosis, the optimal duration of therapy is not known, and patients may be treated indefinitely on maintenance, extrapolating from MM data. However, the plasma cell burden observed in AL amyloidosis is often lower than in MM, and thus certain patients achieving deep responses may have durable responses with time-limited treatment. Outcomes for patients who are observed after DARA discontinuation are not known. We report the outcomes of patients at our institution who received time-limited DARA. A retrospective analysis of AL amyloidosis patients treated at Stanford University from 2016 to 2019 with DARA monotherapy and dexamethasone for at least 2 months was performed, and patients who subsequently had DARA discontinued for reasons other than disease progression or lack of response were selected for the study. Hematologic responses were assessed by consensus guidelines. Duration on and off therapy were explored, along with time-to-next treatment or death (TTNT), defined as the time from DARA initiation to restarting/switching therapy or death. An exploratory analysis comparing TTNT between the study population and a control cohort who achieved hematologic CR and were maintained on DARA was conducted with the Kaplan-Meier method and log-rank testing. 67 patients received at least 2 months of DARA monotherapy and dexamethasone; among these, 15 patients discontinued therapy for reasons other than disease progression and were included. Median age was 66 years old and median lines of prior therapies was 4 (range: 1 - 6). Baseline difference between involved and uninvolved free light chains (dFLC) prior to DARA initiation was 2.6 mg/dL (range: 0 - 16.8 mg/dL). 10 of 15 patients had cardiac involvement with median NT-proBNP of 1982 pg/mL and 9 of 15 patients had renal involvement with median 24-hour proteinuria of 6.2 g and eGFR of 32 mL/min/1.73m2 at DARA initiation. Median duration from starting to stopping DARA was 7.8 months (range: 2 - 21 months). Median duration from achieving best hematologic response to stopping DARA was 3 months (range: 0 - 17 months). Reasons for discontinuation included: patient preference (5), fatigue/body aches (4), infection (2), other active medical comorbidities (3), and lack of perceived further benefit (1). At DARA discontinuation, median dFLC was 0.1 mg/dL (range: 0 - 2.2 mg/dL) and there were 12 hematologic CR, 1 VGPR, 1 PR, and 1 not assessable for response. Outcomes for all 15 patients are shown in Figure 1. The median treatment-free interval was 17.5 months (range: 5 - 34 months); estimated 2-year TTNT-free survival was 83% (95% CI: 61 - 100%). All 14 evaluable patients eventually achieved CR. 3 patients restarted DARA for rising dFLC, and all 3 patients demonstrated response to retreatment (2 achieving CR and 1 near PR with ongoing follow-up). There were 2 deaths. One patient with severe baseline cardiac amyloidosis developed sudden rise in dFLC after treatment-free interval of 21 months; although he rapidly achieved hematologic CR on retreatment, he died of heart failure within 2 months of restarting DARA. The other patient developed therapy-related AML while off therapy and underwent allogenic stem cell transplant but died of leukemia (censored for AL amyloidosis outcomes at transplant). There was no significant difference in the TTNT between the study group and a control group of 16 patients who achieved CR and were on continuous maintenance (Figure 2; p=0.807). AL amyloidosis patients achieving deep responses with DARA can have favorable outcomes after treatment discontinuation, including a long treatment-free interval. Although our sample size is small, the outcomes of these patients appeared comparable to those achieving CR on continuous DARA maintenance, and patients were able to regain responses when retreatment was necessary. These results suggest that DARA may be safely discontinued in patents achieving deep hematologic responses, which has significant implications for quality of life and financial burden of treatment. Future studies evaluating time-limited versus continuous DARA maintenance after achievement of deep responses are warranted. Disclosures Kaufman: Janssen: Other: travel/lodging, Research Funding. Liedtke:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; IQVIA/Jazz: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Roche: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celator: Research Funding; Caelum: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Agios: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Daratumumab for treatment of AL amyloidosis

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Outcomes of Patients with Relapsed or Refractory Acute Myeloid Leukemia Receiving Hypomethylating Agent and Venetoclax

Blood ◽

10.1182/blood-2019-128958 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1357-1357 ◽

Cited By ~ 2

Author(s):

Hannah Asghari ◽

Dasom Lee ◽

Yehuda E. Deutsch ◽

Onyee Chan ◽

Najla Al Ali ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Risk Groups ◽

Initial Therapy ◽

Intermediate Risk ◽

Advisory Committees ◽

Significant Difference ◽

Frontline Therapy ◽

Refractory Aml

Background: Patients with acute myeloid leukemia (AML) have dismal overall outcomes and survival is exceptionally poor in patients who experience relapse or are refractory (R/R) to frontline therapy. Since December 2018, combination therapy with hypomethylating agents (HMA) and venetoclax (HMA+Ven) has become standard frontline therapy for older patients or younger unfit patients. Moreover, it has been routinely utilized in patients experiencing relapsed or refractory AML yet response and outcome data is limited in patients with R/R disease. Thus, we investigated outcomes after HMA+Ven in patients with relapsed or refractory AML. Methods: We retrospectively annotated 72 patients who received treatment with HMA+Ven at Moffitt Cancer Center and Memorial Healthcare System between 2017 and 2019. Patients were divided into two subgroups: 1) initial remission therapy and 2) salvage therapy. Clinical and molecular data were abstracted in accordance with the Institutional Review Board approved protocol. Overall response rate (ORR) included patients achieving complete remission (CR), CR with incomplete count recovery (CRi), and morphologic leukemia free state (MLFS). Patients achieving CR, CRi, or MLFS were termed as responders (RES) and patients without CR, CRi, or MLFS were nonresponders (NRES). Fisher's Exact method was used to determine significance for categorical variables. Kaplan-Meier analysis was performed to determine median overall survival (mOS) and log-rank test was utilized to determine significance. All p-values are two-sided. Results: Out of 72 patients, 41 received HMA+Ven as initial therapy and 31 received it in the R/R setting. Baseline characteristics are outlined in Table 1. Median age was 63 years for patients with R/R AML with 58% female. In the R/R cohort, ORR was 34.5% with 0 (0%) patients achieving CR, 8 (27.6%) patients achieving CRi, and 2 (6.9%) achieving MLFS (Table 2). When compared to patients receiving HMA+Ven as initial therapy, ORR was significantly lower in the R/R cohort (64.1% vs. 34.5%, p=0.03). Among 31 patients in the R/R cohort, 6.5% (n=2) proceeded to allogeneic stem cell transplant (allo-SCT) after achieving CRi. European LeukemiaNet (ELN) risk stratification was known in 22 patients in the R/R cohort and ORR were similar in patients in the favorable/intermediate risk group (n=8) compared to adverse risk group (n=14) (37.5% vs. 28.6%, p=1.0). When compared to HMA+Ven used as initial therapy, ORR among the R/R cohort were not different among adverse risk groups (58.3% vs. 28.6%, p=0.10); however, ORR were significantly lower among patients with favorable/intermediate risk (100% vs. 37.5%, p=0.009). At a median follow-up of 7.6 months (mo), mOS was 4.9mo in the R/R cohort with mOS among RES superior to NRES (not reached vs. 2.4mo, p=0.0009) (Figure 1). Moreover, mOS was inferior in R/R patients compared to initial therapy (4.9mo vs. 13.8mo, p=0.0013) (Figure 2). A total of 15 (48.4%) patients had HMA exposure prior to receiving HMA+Ven without apparent impact on mOS (3.7mo (prior HMA) vs. 4.9mo (no prior HMA), p=0.97). The median duration of CR/CRi was 5.2mo and the median time to CR/CRi was 2.4mo. Based on ELN risk groups, mOS was not statistically different among patients with favorable/intermediate risk disease compared to adverse risk disease (8.6mo (fav/int) vs. 2.8mo (adverse), p=0.07). Responses were also analyzed based upon somatic mutations (Figure 2). In patients with isocitrate dehydrogenase 1 and 2 mutations (IDH1/IDH2) compared to patients without IDH1/2, ORR were 60% vs. 25%, respectively (p=0.28) with no significant difference in mOS (7.2mo (IDHmut) vs. 3.1mo (IDHwt), p=0.38). Comparing patients with TP53 mutation to those without TP53 mutations, no significant difference in ORR (25% vs. 33%, p=1.0) or mOS (4.4mo vs. 6.9mo, p=0.0.84) was noted. Conclusion: Although combination therapy with HMA+Ven has yielded impressive responses as frontline therapy, response rates with this combination in the salvage setting are less encouraging with the possible exception of those patients with IDH1/IDH2 mutations. Nevertheless, responders to salvage HMA+Ven had a significant survival benefit compared to nonresponders, suggesting that this combination is a reasonable salvage option in patients with relapsed or refractory AML. Disclosures Padron: Incyte: Research Funding. Kuykendall:Incyte: Honoraria, Speakers Bureau; Celgene: Honoraria; Janssen: Consultancy; Abbvie: Honoraria. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lancet:Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services . Sallman:Celyad: Membership on an entity's Board of Directors or advisory committees. Komrokji:JAZZ: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; DSI: Consultancy; pfizer: Consultancy; celgene: Consultancy; Novartis: Speakers Bureau; Incyte: Consultancy. Sweet:Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Jazz: Speakers Bureau; Incyte: Research Funding; Pfizer: Consultancy; Stemline: Consultancy. Talati:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Daiichi-Sankyo: Honoraria; Astellas: Honoraria, Speakers Bureau; Pfizer: Honoraria; Celgene: Honoraria; Agios: Honoraria. OffLabel Disclosure: Venetoclax is approved in combination with hypomethylating agents (azacitidine or decitabine) or low dose cytarabine for treatment of newly diagnosed AML in adults aged 75 years or older, or those who have comorbidities that preclude the use of induction chemotherapy.

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Genomic and Transcriptomic Differences of Myelodysplastic Syndrome/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis (MDS/MPN-RS-T) and Myelodysplastic Syndrome with Ring Sideroblasts (MDS-RS)

Blood ◽

10.1182/blood-2020-139983 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Guillermo Montalban Bravo ◽

Rashmi Kanagal-Shamanna ◽

Faezeh Darbaniyan ◽

Irene Ganan-Gomez ◽

Koji Sasaki ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Myeloproliferative Neoplasm ◽

Enrichment Analysis ◽

Cytokine Signaling ◽

Healthy Controls ◽

Kinase Signaling ◽

Advisory Committees ◽

Ring Sideroblasts

INTRODUCTION: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a rare hematological disorder characterized by anemia, bone marrow dysplasia with ring sideroblasts and persistent thrombocytosis, and high frequency of SF3B1 and JAK2 mutations. Despite clinical, histological and molecular similarities with MDS with ring sideroblasts (MDS-RS), the clinical outcomes of these entities are diverse. To date, there is no data evaluating specific functional pathways which might explain phenotypic and clinical differences beyond diverse frequencies of JAK2 mutation. METHODS: We evaluated a total of 24 patients (pts) with MDS/MPN-RS-T and 27 pts with MDS-RS. Diagnosis was based on WHO 2017 criteria and confirmed by two independent hematopathologists. Whole bone marrow DNA was subject to 81 gene targeted next-generation sequencing (NGS) analysis. CD34+ cells from bone marrow samples of 4 pts with MDS/MPN-RS-T, 7 pts with MDS-RS and 17 healthy individuals obtained from AllCells (Emeryville, CA) were isolated using the CD34 MicroBead Kit and RNA was isolated using the PicoPure RNA isolation kit. Fastq files were mapped to the human genome (build GRCh38) in TopHat2 using the default options. Differential gene expression analysis was conducted using DESeq2 in R version 3.6.2. Pathway enrichment analysis was performed using gene set enrichment analysis, with the fgsea library in R. RESULTS: Patients with MDS/MPN-RS-T had higher median bone marrow ring sideroblast percentage (47% vs 32%, p=0.04) and absolute neutrophil count (4.34x109/L vs 2.99x109/L, p=0.001). Frequency of identified mutations and their VAFs compared to MDS-RS are shown in Figure 1A. The median number of mutations was higher in MDS/MPN-RS-T than in MDS-RS (3 vs 2, p<0.001). SF3B1 mutations were the most frequent in both entities (MDS/MPN-RS-T: 92%, MDS-RS: 82%), had similar median VAF (34% vs 32%, p=0.619), and involved the hot spot codon K700E in 64% and 43% of MDS-RS and MDS/MPN-RS-T (p=0.227), respectively. As expected, 58% of pts with MDS/MPN-RS-T had JAK2 V617F mutations but were also more likely to have mutations in kinase signaling genes (NF1, SETBP1, CBL, CBLB, FLT3 TKD, MPL) compared to MDS-RS (29% vs 4%, p=0.019). Four (40%) of JAK2 negative MDS/MPN-RS-T had mutations in kinase signaling genes. There were no differences in frequency of TET2 mutations between both entities. However, there was a trend for the median VAF of TET2 mutations in MDS/MPN-RS-T to be lower than in MDS-RS (1.5% vs 21.1%, p=0.177) suggesting a likely subclonal nature of these mutations compared to MDS-RS in which they appeared as dominant events. MDS/MPN-RS-T showed distinct transcriptomic profile compared to both healthy controls and MDS-RS. Compared to healthy controls, a total of 2 pathways were significantly upregulated and 58 were downregulated in MDS/MPN-RS-T while 5 pathways were upregulated and 69 were downregulated in MDS-RS. Compared to MDS-RS, a total of 29 pathways were significantly upregulated and 26 were downregulated in MDS/MPN-RS-T. The most significantly upregulated pathways in MDS/MPN-RS-T included genes involved in platelet activation and aggregation, cytokine signaling, and signaling through GPC receptors (Figure 1C). Compared to both healthy control and MDS-RS, MDS/MPN-RS-T was characterized by downregulation of genes involved in DNA damage response, regulation of apoptosis, telomere maintenance and RNA synthesis (Figure 1D). MDS-RS was characterized by downregulation of genes involved in signaling by GPC receptors and MAPK signaling, mRNA splicing, cytokine signaling and signaling through interleukins compared to both control and MDS/MPN-RS-T (Figure 1C). CONCLUSIONS: MDS/MPN-RS-T is characterized by co-dominance of SF3B1 and JAK2 mutations and presence of minor kinase signaling mutations not observed in MDS-RS. Upregulation of cytokine and interleukin signaling mediated through GPC receptors, and downregulation of genes involved in apoptosis and DNA damage are unique transcriptomic features of MDS/MPN-RS-T likely driven by genotype. These unique genomic and transcriptomic characteristics of MDS/MPN-RS-T supports the classification of MDS/MPN-RS-T based on genomic features beyond presence of SF3B1 mutation, and might represent potential therapeutic avenues for this rare disease. Figure Disclosures Sasaki: Otsuka: Honoraria; Pfizer Japan: Consultancy; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy. Kantarjian:Sanofi: Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria; BMS: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotechnologies: Honoraria; Aptitute Health: Honoraria; Immunogen: Research Funding; Jazz: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BioAscend: Honoraria; Novartis: Honoraria, Research Funding; Delta Fly: Honoraria; Pfizer: Honoraria, Research Funding; Oxford Biomedical: Honoraria; Ascentage: Research Funding. Garcia-Manero:Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding; Onconova: Research Funding; Merck: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy.

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Iron Overload Diminishes the Effectiveness of the Innate Immune Response in Thalassemia Major: a Possible Mechanism for Increased Infection Risk.

Blood ◽

10.1182/blood.v114.22.4071.4071 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4071-4071

Author(s):

Patrick B Walter ◽

Paul R Harmatz ◽

Annie Higa ◽

David Killilea ◽

Nancy Sweeters ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Response ◽

Iron Overload ◽

Board Of Directors ◽

Innate Immune Response ◽

Research Funding ◽

Innate Immune ◽

Healthy Controls ◽

Advisory Committees ◽

Fluorescent Intensity

Abstract Abstract 4071 Poster Board III-1006 Introduction Infection is the second most common cause of death in thalassemia. The innate immune system provides a first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) are important for transducing the signal for bacterial Lipopolysaccharide (LPS), resulting not only in cytokine production, but also in the control of extracellular iron levels through production of neutrophil gelatinase associated Lipocalin (NGAL). However, the exact role that NGAL plays and the expression level of PRRs are unknown in thalassemia. Thus, the goal in these studies is to investigate the relationship of iron overload to the innate immune cell expression of PRRs and NGAL in thalassemia. Patients and Methods Fifteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox (an oral iron chelator) and deferoxamine were enrolled (Novartis sponsored CICL670AUS24T). Fasting blood samples were obtained i) at baseline after a 72 hr washout of chelator, and ii) at 6 and 12 months on study. Five healthy controls (13 - 18 yrs old) were also enrolled. Fresh monocytes were isolated using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes were verified by flow cytometry. The expression of TLR4, also examined by flow cytometry is reported as the mean fluorescent intensity (MFI). In patients with thalassemia, liver iron concentration (LIC) was analyzed by biomagnetic susceptibility (“SQUID”, Ferritometer®). The plasma levels of NGAL were analyzed by ELISA. Results At baseline the expression of monocyte TLR4 (mean 18.8 ± 3.5 MFI) was reduced 30% compared to the healthy controls (mean 26.9 ± 7.6 MFI, p<0.05). The expression of TLR4 over the follow-up period of 52 weeks in patients receiving intensive combination chelator therapy significantly increased 27% / year (7 MFI / year, p=0.005). Interestingly the expression of monocyte TLR4 was negatively correlated with LIC (r=-0.6, p=0.04). Finally, thalassemia patients at baseline have significantly higher levels of NGAL (80 ± 20 ng/ml) compared to controls (42 ± 15 ng/ml, p=0.01). Conclusions These preliminary studies support the hypothesis that iron burden has a negative impact on the innate immune response in thalassemia as demonstrated by the decreased expression of TLR4. After intensive chelation, the levels of TLR4 increased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. Finally, the iron transport protein NGAL is significantly elevated in thalassemia possibly acting to prevent essential iron uptake by pathogenic bacteria. Disclosures: Harmatz: Novartis: Research Funding; Apotex : Membership on an entity's Board of Directors or advisory committees; Ferrokin: Membership on an entity's Board of Directors or advisory committees. Vichinsky:Novartis: Consultancy, Research Funding.

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