scholarly journals Mesenchymal Stromal Cell-Derived S100A8 Promotes Acute Myeloid Leukemia Progression Via ROS Signaling

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4300-4300
Author(s):  
Jinxian Wu ◽  
Xiaoyan Liu ◽  
Fuling Zhou
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Mesenchymal Stromal Cell ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Control Group ◽  
Lentivirus Vector ◽  
Apoptosis Rate ◽  
Acute Myeloid

Abstract Introduction: Mesenchymal stromal cell (MSC) is an important cell component in the bone marrow microenvironment. MSC-derived inflammatory factors regulate the progression of acute myeloid leukemia (AML) by regulating the signaling pathways in hematopoietic cells. S100A8 is an inflammatory factor which belong to the calcium-binding protein S100 family. In vivo animals experiments showed that increased expression of S100A8 in MSC was accompanied by increased proliferative MSCs, and decreased mature osteoblasts. MSC-derived S100A8 can also cause mitochondrial dysfunction in hematopoietic stem progenitor cells, induce oxidative stress response and DNA damage repair, that promotes the progression of myeloid dysplastic syndromes (MDS). However, whether MSC-derived S100A8 involved in AML development have not been reported. In this study, we attempted to elucidate the regulation of MSC-derived S100A8 on MSC itself as well as leukemia cells. Methods: Human MSCs were isolated from AML patients samples by whole bone marrow adherent culture, and the third to fifth passage cells were collected for analysis. The lentivirus vector carrying cDNA of S100A8 gene and the empty lentivirus vector were constructed and infected into MSCs,respectively. Cell cycle and apoptosis of MSCs were analysed by flow cytometry. Acute myeloid leukemia cell line Kasumi-1 was co-cultured with the two groups of mesenchymal stem cells in vitro for 3 days, respectively.cell cycle and apoptosis were analysed, and the cell proliferation was detected by Edu. The ROS levels of co-cultured Kasumi-1 cells were detected by flow cytometry. The apoptosis of kasumi-1 co-cultured cells treated with VP-16 for 48 hour was detected by flow cytometry. Results: The rate of G0 phase cell in S100A8-overexpressed MSCs was higher than in control group.The proliferation rate of Kasumi-1 cells was significantly increased S100A8 overexpressed group than in control after 72-h co-culture, while the apoptosis rate of Kasumi-1 cells was significantly decreased in S100A8 overexpressed group. Futhermore, the apoptosis rate of Kasumi-1 cells co-cultured with S100A8-overexpressed MSCs was markedly lower than in control group after exposed in vp-16 for 48 hour.The ROS level of Kasumi-1 cells co-cultured with S100A8-overexpressed MSCs were significantly increased than those of the control group. Conclusion: S100A8 derived from MSCs plays a critical role in progression and drug resistance of acute myeloid leukemia, by increasing the ROS levels of AML cells, that indicates S100A8 may serve as a potential novel therapeutic target in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2228-2228
Author(s):  
Xin Long ◽  
Laszlo Perlaky ◽  
Tsz-Kwong Chris Man ◽  
Michele S. Redell
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Chemotherapy Resistance ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment

Blood ◽  
2019 ◽  
Vol 133 (5) ◽  
pp. 446-456 ◽  
Author(s):  
Amina M. Abdul-Aziz ◽  
Yu Sun ◽  
Charlotte Hellmich ◽  
Christopher R. Marlein ◽  
Jayna Mistry ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Cell Senescence ◽  
Senescent Phenotype ◽  
Age Related ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. With increasing age, tissues accumulate senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of a set of proinflammatory cytokines, chemokines, and growth factors, collectively known as the senescence-associated secretory phenotype (SASP). Here, we report that AML blasts induce a senescent phenotype in the stromal cells within the BM microenvironment and that the BM stromal cell senescence is driven by p16INK4a expression. The p16INK4a-expressing senescent stromal cells then feed back to promote AML blast survival and proliferation via the SASP. Importantly, selective elimination of p16INK4a+ senescent BM stromal cells in vivo improved the survival of mice with leukemia. Next, we find that the leukemia-driven senescent tumor microenvironment is caused by AML-induced NOX2-derived superoxide. Finally, using the p16-3MR mouse model, we show that by targeting NOX2 we reduced BM stromal cell senescence and consequently reduced AML proliferation. Together, these data identify leukemia-generated NOX2-derived superoxide as a driver of protumoral p16INK4a-dependent senescence in BM stromal cells. Our findings reveal the importance of a senescent microenvironment for the pathophysiology of leukemia. These data now open the door to investigate drugs that specifically target the “benign” senescent cells that surround and support AML.

Study on mechanism of Tim-3 on immune escape in benzene-induced acute myeloid leukemia

2020 ◽  
Author(s):  
qiong Ning ◽  
xiangxin li ◽  
Xiangdong Jian ◽  
Xiaopeng He
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Immune Escape ◽  
Serum Levels ◽  
Control Group ◽  
M2 Macrophages ◽  
Experimental Group ◽  
Group Serum ◽  
Acute Myeloid

Abstract To study the mechanism of Tim-3 on immune escape in benzene-induced acute myeloid leukemia (AML), to provide potential targets of clinical monitoring and intervention of hematological toxicity in benzene-induced AML . C3H/He mice were randomly divided into control group and experimental group. Serum levels of IL-12 in the experimental group were significantly lower than that in the control group. Serum levels of TGF-β1 in the experimental group were significantly higher than that in the control group( p <0.05). The proportion of Tim-3 positive CD14 + monocytes of bone marrow and spleen in the experimental group were both significantly higher than that in the control group ( p <0.05) by Flow cytometry (FCM). Compared with the control group, the expression of Tim-3 on (M1+M2) macrophages of bone marrow in the experimental group significantly increased by immunofluorescence assay. The expression of type M2 macrophages in (M1+M2) macrophages of bone marrow and spleen tissues in the experimental group were both higher than that in the control group. The expression levels of p-PI3K, p-AKT and p-mTOR in the experimental group were all significantly higher than that in the control group. Tim-3 was highly expressed in macrophages in benzene-induced AML. It promoted the activation of PI3K/AKT/mTOR signaling pathway, stimulated the secretion of anti-inflammatory cytokines, and inhibited the secretion of pro-inflammatory cytokines. High expression of Tim-3 changed the phenotype and function of macrophages by promoting the macrophages polarization, thus inducing negative immune response in the tumor microenvironment and tumor immune escape.

The Growth Inhibition and Apoptosis Induction of Acute Myeloid Leukemia Blast Population by the Blocking IGF-IR Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4515-4515
Author(s):  
Si Sun ◽  
Yanli He ◽  
Xingbing Wang ◽  
Wei Liu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Pi3k Pathway ◽  
Leukemia Cell Line ◽  
Free System ◽  
Acute Myeloid ◽  
Marrow Cells

Abstract The insulin-like growth factor-1receptor (IGF-1R) is overexpressed in a variety of tumors and has been associated with cancer development. Here, we analysis the IGF-IR expression on the bone marrow cells from 45 newly diagnosed patients with acute myeloid leukemia (AML) by flow cytometry. IGF-1R universally expressed on AML blasts and the leukemia cell line HL-60, did not show significant correlation with FAB subtypes. However, the bone marrow cells from AML patients with high myeloblast counts (&gt;80%) generally showed brighter IGF-IR expressions, which indicated the IGF-IR pathway might play an important role for AML blast proliferation and survival. Indeed, blocking the IGF-1R pathway by neutralizing monoclonal antibodies could reduce the proliferation of HL-60 cells by 38.28% at 48 hr. This inhibitory effect on blast growth was observed in 4 of 5 AML samples. In the same IGF-1R blocking treatment, the apoptosis of HL-60 cells was significantly induced, resulting in apoptosis of 57% of the cell population with the measurement of Annexin V vs PI staining by flow cytometry. The control contained only 20% apoptotic cells. We also demonstrated that the blockade of the IGF-1R pathway inhibited the phophorylation of the PI3K pathway component Akt in HL-60 cells when cultured in a serum free system with a supplement of 50ng/ml exogenous IGF. Since PI3K pathway activation greatly contributes to the proliferation, survival and drug resistance of AML, it is of interest to study whether blockading IGF-IR could also inhibit the PI3K pathway in primary AML blasts and synergize other anti-leukemia agents to improve the therapeutic effectiveness. Conclusions: IGF-IR may play an important role in the proliferation and survival of the AML blast population; Blocking the IGF-IR pathway could significantly inhibit the growth of AML blasts and considerably induce the apoptosis of AML blasts; IGF-IR could become a critical molecular target in anti-leukemia drug discovery.

Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3513-3513
Author(s):  
David B. Sykes ◽  
Mark K Haynes ◽  
Nicola Tolliday ◽  
Anna Waller ◽  
Julien M Cobert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Prostacyclin Receptor ◽  
Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

Molecular Mechanism Of Regulation Of Extramedullary Infiltration In AML1/ETO Positive Acute Myeloid Leukemia By APP/ERK/MMP-2

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3769-3769
Author(s):  
Guopan Yu ◽  
Fan Yi Meng ◽  
Ling Jiang ◽  
Changxin Yin ◽  
Zhixiang Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Molecular Mechanism ◽  
Myeloid Leukemia ◽  
High Expression ◽  
Control Group ◽  
Expression Levels ◽  
High Expression Group ◽  
App Gene ◽  
Acute Myeloid

Abstract Amyloid precursor protein (APP) has been reported to be highly expressed in AML1/ETO positive acute myeloid leukemia (AML1/ETO+ AML), and we found it express even higher in those with extramedullary infiltration in our previous study. But it’s still unknown what role APP plays and how it works in AML1/ETO+ AML. This study was designed to investigate the effect of APP gene on the prognosis and its molecular mechanism of extramedullary infiltration in the patients with AML1/ETO+ AML. 44 cases of AML1/ETO+ AML patients with median age of 29 years old, who were admitted to our hospital from February, 2006 to February, 2012 and made the diagnosis according to WHO2008 diagnosis standard, and had completed conventional induction, consolidation and intensive therapy, were investigated in this study. They were divided into high expression group (n=22) and low one (n=22) according to APP mRNA median expression level from bone marrow cells before the first chemotherapy by QRT-PCR. Some of bone marrow samples were checked by Western Blot, and 5 biopsy specimens from extramedullary infiltration were tested by APP antibody immunohistochemistry staining. Incidence of extramedullary leukemia (EML), complete response (CR), overall survival (OS), and recurrence free survival (RFS) was differentiated between the two groups. Differences of cell ultrastructure, migration, proliferation, apoptosis and expression of ERK, MMP-2, MMP-9 and CXCR4 were studied on Kasumi-1 cell line between wild, negative control (NC) and si-APP group in which the expression levels of APP gene were down regulated with application of siRNA technology.Çå The incidence of EML was significantly different (45.5% versus 9.1%) in the two groups (P=0.007) and it was positively correlative with the expression levels of APP mRNA (rp=0.435, P=0.004). Extramedullary infiltration site also showed high expression of APP by immunohistochemistry, while the control group was negative. Not only CR rate after two courses of chemotherapy, but also OS and RFS with median follow-up of 28(4-70) months, of high expression group was all significantly lower than that of low expression group (Table 1). Compared with the wild and NC group, cell apoptosis of si-APP group was significantly increased (12.33 ± 0.75 vs 19.80 ± 1.51, P=0.000); the number of microvilli on the surface of the cell membrane significantly reduced; the ability of the cell migration by Tanswell chamber migration assay significantly decreased (P=0.004); and expression of P-ERK, c-MYC, MMP-2 decreased significantly which was confirmed by ERK and c-MYC blocker treatment (Figure 1). In sum, incidence of EML is significantly higher and the prognosis is poor in the patients with AML1/ETO+ AML with high expression of APP gene. We first describe that APP gene may mediate AML1/ETO+ leukemia cells in the development of extramedullary infiltration by up-regulation of the ERK/MMP-2 pathway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Impact of Minimal Residual Disease and Chimerism Monitoring at Different Timepoints after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Reyes María Martín-Rojas ◽  
Gillen Oarbeascoa ◽  
Rebeca Bailén ◽  
Ignacio Gómez-Centurión ◽  
Luis Miguel Juarez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Cumulative Incidence ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Minimal Residual ◽  
Risk Of Relapse ◽  
Acute Myeloid

¶ Martin-Rojas RM and Oarbeascoa G contributed equally to this work. INTRODUCTION Relapse is the main cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). The evaluation of minimal residual disease (MRD) could provide a more accurate assessment of the depth of response, and therefore identify patients with higher risk of relapse. AIMS The aim of our study was to analyze the impact of pre-HSCT flow cytometry (FCM) and molecular MRD together with chimerism and MRD in the early post-HSCT period in patients with AML. METHODS We conducted a retrospective study in patients with complete remission AML who underwent a HSCT between 2008 and 2019 in our center. MRD was analyzed by flow cytometry in bone marrow aspirates and by quantitative RT-PCR (NMP1, RUNX1-RUNX1T1, CBFB-MYH11, KMT2A-MLLT3, WT1) in bone marrow aspirates and/or peripheral blood. MRD was determined within the 30 days preceding the HSCT and at day +30 and +90 post-HSCT. Bone marrow and selected CD34+ lineage chimerism was analyzed by STR (AmpFISTR SGM Plus, Thermo Fisher) at days +30 and +90 post-HSCT. This study was approved by our Institutional Ethics Committee. Data were analyzed using IBM SPSS Statistics version 24 and R version 3.5.1. RESULTS A total of 115 patients were analyzed. Pre-HSCT MRD was negative in 58 patients (50.4%) and positive in 57 patients (49.6%). We found no statistically significant differences in the characteristics between the two groups (Table 1). Median follow up was 39 months (IQR 10.4-55.8). 3-year overall survival (OS) for patients with pre-HSCT negative MRD was 72.5% versus 70.3% in patients with positive MRD (p=0.41), with an event free survival (EFS) of 66.9% versus 66.1 (p=0.48) respectively (Figure 1). Median time to the beginning of immunosuppression withdrawal was 82.5 days (IQR 59-93) for patients with negative MRD and 68 days (IQR 55.3-85.3) for patients with positive MRD (p&lt;0.001). The cumulative incidence of grade II-IV acute graft versus host disease (aGVHD) and moderate-severe chronic GVHD did not show statistically significant differences based on the MRD status. Similarly, the cumulative incidence of relapse and the 2-year mortality was not significantly different between the two groups. Patients with negative MRD at day +30 showed a 2-year OS of 83.5% versus 58.1% in patients with positive MRD (p=0.03) and a EFS of 79.9% versus 48.6% (Figure 2). The cumulative incidence of relapse was more elevated in patients with positive MRD (29.8% versus 13.6%) at day +30. Patients with mixed chimerism (MC) at day +30 showed a significantly lower 3-year OS and EFS than patients with complete chimerism (CC). Likewise, the cumulative incidence of relapse was significantly higher in patients with MC, both if detected in bone marrow aspirate and in CD34+ cells. The multivariate analysis revealed that MRD status at day +30 post-HSCT was an independent prognostic factor for EFS (HR 3.74; 95% CI 1.38-10.1; p=0.009). CONCLUSIONS Patients with AML presenting a positive MRD in the early post-HSCT period and those who show a MC at day +30 post-HSCT have lower EFS, with positive MRD at day +30 being an independent prognostic factor for EFS. The evaluation of MRD and chimerism in the early post-HSCT period is useful to identify patients with higher risk of relapse, who may take advantage of preemptive measures. Disclosures Kwon: Gilead, Novartis, Pfizer, Jazz: Consultancy, Honoraria.

scholarly journals Development of Somatic NRAS Mutation Associated with Rapid Transition from Germline GATA2 Mutation Associated Myelodysplastic Syndrome to Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3616-3616 ◽  
Author(s):  
Yanqin Yang ◽  
Yubo Zhang ◽  
Jun Zhu ◽  
Catherine E. Lai ◽  
Jingrong Tang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Marrow Aspirate ◽  
Nras Mutation ◽  
Trisomy 8 ◽  
Acute Myeloid

Abstract There is increasing recognition of the role of inherited germline predisposition for myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The additional somatic genetic events required for development of a malignant phenotype are however poorly understood. A 25 year old woman was referred to the NHLBI hematology branch in March 2014 for a seven year history of pancytopenia. Her medical history included recurrent pneumonias, oral ulcers, severe varicella infection and arthralgias. Prior bone marrow examinations at ages 21 and 23 at outside institutions reported normocellular marrow, tri-lineage hematopoiesis and mild dyspoiesis. Cytogenetics were remarkable for trisomy 8 in 80% (aged 21) or 90% (aged 23) of metaphases. Previously unrecognized lymphedema was noted on examination. Peripheral blood counts showed WBC 2.28 K/ul [normal range: 3.98-10.04], HGB 9.9 g/dL [11.2-15.7], PLT: 67 K/ul [173-369], ALC: 0.36 K/ul [1.18-3.74] and AMC: 0.06 [0.24-0.86]. Peripheral blood flow cytometry demonstrated decreased CD3+ CD4+ (T) cells, CD19+ (B) cells and NK cells. HLA-DR15 negative. Bone marrow examination showed trilineage hematopoiesis, 50-60% cellularity, mild erythroid predominance and mildly increased, mildly atypical megakaryocytes. Blasts less than 5%. Bone marrow flow cytometry revealed severely decreased B-cells and monocytes, absent B-cell precursors, absent dendritic cells, inverted CD4:CD8 ratio, and atypical myeloid maturation pattern. Cytogenetics demonstrated stable trisomy 8 in 90% of metaphases. On the basis of this assessment the diagnosis of MDS was confirmed. Sanger sequencing revealed a GATA2 L375S mutation in the second zinc finger of known pathogenic significance. Four months later she developed increased fatigue and easy bruising with worsening thrombocytopenia (PLT: 10K/ul). Bone marrow was dramatically changed; now markedly hypercellular (90-100%) with diffuse sheets of immature cells consistent with blasts having fine chromatin, distinct or prominent nucleoli, and visible cytoplasm. Blasts were positive for CD33, CD56, CD64, CD123, and CD163; and were negative for CD34, CD14, and myeloperoxidase. Cytogenetics showed a new trisomy 20 in 65% of metaphases, in addition to previously seen trisomy 8 in 100%. A diagnosis of acute monoblastic leukemia (M5a subtype) was made. At both clinic visits bone marrow aspirate was collected on an IRB approved research sample acquisition protocol. Whole exome sequencing of 1ug DNA was performed using Agilent SureSelect v5 Exome enrichment Kits on an Illumina HiSeq 2000 with 100-bp paired-end reads (Macrogen, Rockville, MD). Data was mapped to hg19 (BWA) and processed using an in-house pipeline (Samtools/Picard/GATK/VarScan/Annovar). Mean read depth of target regions was 157 and 149. There was high correlation between both samples with the exception of a NRAS:NM_002524:exon3:c.C181A:p.Q61K mutation (57 of 180 reads) seen only in the later sample. Confirmatory ultra-deep sequencing for NRAS was performed using Illumina TruSight Myeloid Sequencing Panel on an Illumina MiSeq. No evidence of the NRAS Q61K mutation was found in the earlier March MDS bone marrow sample even when sequenced to a depth greater than 1750 reads (see figure). The mutation was confirmed in the August AML sample at a variant allele frequency of 35%. If heterozygous this would reflect a clone size of 70%, consistent with data from both cytogenetics (new trisomy 20 in 65% of metaphases) and the 76% blasts documented by bone marrow aspirate smear differential. We report here the rapid progression to AML in a patient with germline GATA2 MDS associated with development of a new trisomy 20 karyotype and a NRAS Q61K mutation. The NRAS mutation was not detectable after the patient achieved a complete remission following induction chemotherapy further supporting this association. This NRAS mutation has been implicated in the pathogenesis of multiple cancers by constitutive activation of proliferative signaling. GATA2 associated MDS is a high-risk pre-leukemic condition with the potential for rapid evolution to AML. This is the first report of acquired somatic mutations in the RAS/RTK signaling pathway in the context of germline GATA2 insufficiency associated with acute leukemic transformation. Figure 1. Figure 1. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding.

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