scholarly journals Peripheral Blood Cell Sorting Strategies for Transcriptomic Analysis in Chronic Myelomonocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4232-4232
Author(s):  
Moritz Binder ◽  
Ryan Carr ◽  
Nathalie Droin ◽  
Abhishek A Mangaonkar ◽  
Giacomo Coltro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Sorting ◽  
Peripheral Blood ◽  
Housekeeping Genes ◽  
Median Number ◽  
Chronic Myelomonocytic Leukemia ◽  
Transcriptomic Analysis ◽  
Strong Positive Correlation ◽  
Primary Cells ◽  
Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by sustained peripheral blood (PB) monocytosis and an inherent risk for leukemic transformation. Clonal origins of the disease can be detected in hematopoietic progenitor cells (CD34+/CD38-), while the complete spectrum of mutational evolution can be seen in circulating monocytes (CD14+). Cell sorting strategies have been employed to select cells in CMML, and while there are adequate monocyte numbers in the PB, there are very few circulating progenitor cells. In addition, attrition related to the selection process significantly depletes primary cells available for biological experiments and multiomics studies such as RNA-seq, ChIP-seq, ATAC-seq, and DIP-seq. While single-cell methods may be able to overcome this challenge, bulk sequencing methods remain a robust and cost-effective approach. We hypothesized that, secondary to the stem cell origin of this disease and significant myeloproliferation, PB mononuclear cells (MNC) would provide comparable results with regards to transcriptomic analysis, in comparison to cell selection procedures. Methods: Peripheral blood obtained from 15 molecularly annotated patients with WHO-defined CMML was ACK-lysed and subjected to a Ficoll procedure for collection of MNC. MNC were left unsorted (n=5) or further selected for CD34+/CD38- (n=5) and CD14+ (n=5) using a fully automated RoboSep-S (StemCell Technologies) protocol. All samples were then subjected to bulk whole transcriptome shotgun sequencing (using Illumina TruSeq and an Illumina HiSeq 4000). After data quality control, counts of detectable transcripts were log2-normalized and Pearson's product-moment correlation coefficients were calculated to evaluate the correlation between the two cell-sorting strategies and unsorted cells in terms of detectable transcripts. To visualize sample differences log2-normalized transcripts counts were centered and scaled per gene for a select number of genes relevant to myeloid biology as well as a number of housekeeping genes. Results: Fifteen patients with WHO-defined CMML, median age 69 years (55-73 years), 66% male, were included. Next generation sequencing for somatic mutations was performed on PB MNC obtained at CMML diagnosis (Figure 1, top heatmap). Considering the small sample size, mutations were evenly distributed among groups with the exception of ASXL1 (higher frequency in CD14+ and CD34+/CD38- cells), ZRSR2 (higher frequency in unsorted cells), and TET2 (lower frequency in CD14+ cells). The three groups were also well matched with regards to other CMML-related variables such as WHO and FAB morphological subtypes, cytogenetic abnormalities, and risk stratification by the Mayo Molecular Model. Transcriptomic analysis revealed a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD34+/CD38- cells (ρ = 0.96, p < 0.001, top scatterplot). Likewise, there was a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD14+ cells (ρ = 0.91, p < 0.001, bottom scatterplot). The latter correlation was marginally lower, which was explained by increased global gene expression in 3 of the 5 CD14+ samples (bottom heatmap). Increased gene expression in these 3 samples involved key myeloid genes and housekeeping genes known to have stable expression across human tissues alike. In comparison to PB MNC, both cell sorting strategies resulted in significant depletion of primary cells required for other experiments, and for procedures such as ChIP-seq, DIP-seq and ATAC-seq (CD34+/CD38- had greater depletion than CD14+). Additional experiments to assess this strategy for the above mentioned epigenetic studies are currently being planned. Conclusions: Accounting for sample differences, different cell sorting strategies (unsorted, CD34+/CD38- selection, and CD14+ selection) yielded similar results when performing bulk transcriptomic assessments on PB MNC from patients with CMML. For the purpose of gene expression profiling there was no clear advantage with CD34+/CD38- or CD14+ selection. These results support the use of unsorted cells for bulk transcriptomic analysis in CMML. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Gene Body Methylation and Transcriptional Activity in ASXL1-Mutant Chronic Myelomonocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Moritz Binder ◽  
Ryan M. Carr ◽  
Terra Lasho ◽  
Christy Finke ◽  
Abhishek A. Mangaonkar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Promoter Region ◽  
Transcriptional Activity ◽  
Median Number ◽  
Chronic Myelomonocytic Leukemia ◽  
Gene Body ◽  
Gene Body Methylation ◽  
Differential Gene ◽  
Myelomonocytic Leukemia

Introduction: Truncating mutations in Additional Sex Combs-Like 1 (ASXL1) are associated with a high-risk disease phenotype in myeloid malignancies. In chronic myelomonocytic leukemia (CMML), truncating ASXL1 mutations are known to increase transcriptional activity of leukemic driver genes and have been associated with gene body hypermethylation. We interrogated the transcriptome and methylome of patients with ASXL1-mutant (MT) and -wildtype (WT) CMML using a multi-omics approach to test the hypothesis that gene expression is mediated through gene body methylation. Methods: Bone marrow mononuclear cells from patients with ASXL1 WT (n=8) and MT (n=8) CMML were subjected to targeted NGS of DNA, whole transcriptome shotgun sequencing (RNA-seq), and immunoprecipitation of DNA methyl residues (DIP-seq). After quality control all samples were sequenced on an Illumina HiSeq 4000 before further processing and data analysis. Differential gene expression analysis was performed to identify genes up-regulated in MT CMML. The samples in the two groups were treated as biological replicates and subjected to a consensus peak calling strategy requiring an overlap of at least 30% between samples and an adjusted p-value &lt; 5x10-5 for a methylation peak to be considered statistically significant. For validation purposes methylation analysis was performed on 3 ASXL1 MT and 3 WT CMML patients using Illumina Infinium MethylationEPIC microarrays and differentially methylated regions were identified using a bump hunting strategy. Gene body methylation was defined as methylation in gene bodies (outside the promoter region, i.e. transcription start site ±2kb). Gene body methylation was compared between WT and MT CMML for the up-regulated genes and correlated with expression of all genes in MT CMML. Results: Sixteen WHO-defined CMML patients were included, median age 69 years (48-77), 63% male, 50% had truncating ASXL1 frame shift mutations. Abnormal karyotypes were observed in the same number of patients and the burden of co-mutations was similar between the two groups (median number per group 3 vs. 3, p=0.508). This included several modulators of DNA methylation including TET2, DNMT3A, and IDH2 (median number per group 1 vs. 1, p=0.699). There was a predominant up-regulation of gene expression in MT CMML: 707 genes up- and 124 down-regulated (FDR&lt;0.050) without evidence of differential methylation in promoter regions of the differentially expressed genes. There was no difference in the number of genes with consensus methylation peaks in the gene body or the extent of gene body methylation (area under the consensus peaks) between MT and WT CMML for the differentially expressed genes (Figure 1a). We further validated these results using methylation microarrays (Figure 1b). Among the MT CMML patients, there was a curvilinear relationship between gene body methylation and gene expression across all genes with intermediate gene body methylation being associated with the highest gene expression (Figure 1c). As an alternative unbiased approach, we identified 1595 differentially methylated regions (DMR, regions with FDR&lt;0.05) in the validation data set. We mapped all hypermethylated regions to gene bodies requiring that there was no concurrent hypermethylation of the promoter region of the same gene. With this approach we identified one of the 707 up-regulated genes (0.14%) with evidence of isolated gene body hypermethylation (HBZ, log2-fold change in gene expression 4.25, FDR=0.0008, DMR area=1.61, DMR FDR=0.018). Representative examples of up-regulated mitotic kinases without evidence of differential gene body methylation are shown in Figure 1d (pie charts represent the mean β-values of microarray probes, p-values represent the comparisons of mean β-values between MT and WT CMML per gene). Conclusions: Gene body methylation was positively associated with gene expression in MT CMML. However, the lack of differential gene body methylation between WT and MT CMML for the up-regulated genes make it an unlikely explanation for the observed increase in transcriptional activity among patients with MT CMML. Figure 1 Disclosures Ordog: Millipore Sigma: Patents & Royalties.

389 Combining transcriptomic- and tissue-based immune biomarkers to evaluate GB1275, a CD11b modulator, as a single agent or with pembrolizumab in patients with advanced solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A414-A414
Author(s):  
Wells Messersmith ◽  
Drew Rasco ◽  
Johann De Bono ◽  
Andrea Wang-Gillam ◽  
Wungki Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Solid Tumors ◽  
Peripheral Blood ◽  
Post Treatment ◽  
Transcriptomic Analysis ◽  
Gene Set Enrichment Analysis ◽  
Cancer Center ◽  
Advanced Solid Tumors ◽  
Core Tissue ◽  
Dose Dependent

BackgroundGB1275 is a first-in-class CD11b modulator in development as monotherapy and in combination with pembrolizumab or chemotherapy for the treatment of advanced solid tumors. Nonclinical data show that GB1275 reduced influx of tumor-associated myeloid-derived suppressor cells (MDSCs) and macrophages (TAMs), and repolarized M2 immuno-suppressive TAMs towards an M1 phenotype. We hypothesize that GB1275 administration can alleviate myeloid cell-mediated immunosuppressive effects and improve cancer treatment outcomes. A phase 1 trial evaluating GB1275 as monotherapy and in combination with pembrolizumab in specified advanced tumors in ongoing (NCT04060342).MethodsBlood gene expression variations as well as core tissue biopsies pre- and post-treatment were assessed following GB1275 monotherapy and combination with pembrolizumab. After obtaining informed consent, peripheral blood for MDSCs was collected from 21 patients pre- and two weeks post-treatment; core tissue biopsies were collected from 13 patients pre- and post-treatment. The frequency of MDSCs in whole blood was measured using the Serametrix MDSC FACS Assay. Gene expression transcriptome profiles were generated using NovaSeq platform. CD8 staining was performed at Neogenomics, and tumor infiltrating lymphocyte (TIL) quantification was performed by an independent pathologist.ResultsPreliminary statistical analysis of MDSC immunophenotyping pre- and post- treatment is consistent with the proposed mechanism of GB1275, showing modulation of peripheral blood MDSCs in some patients. Preliminary gene expression analysis in the blood showed dose-dependent clusters following treatment with GB1275 alone. Moreover, the transcriptomic analysis revealed two unique expression patterns for patients treated with GB1275 monotherapy or in combination with pembrolizumab. Gene Set Enrichment Analysis showed that the CD11b pathway is downregulated in patients treated with GB1275. Analyses of TIL count revealed an increase in lymphocyte trafficking into the tumor after treatment with GB1275 alone or in combination with pembrolizumab. CD8 expression and transcriptomic analysis are underway and will be presented.ConclusionsGB1275 alone or in combination with pembrolizumab demonstrates biological activity, which may be dose dependent. The observed increase in TILs after treatment is supportive of the mechanism of action of GB1275. Further biomarker analyses in blood and tissues are ongoing and will be correlated with clinical activity in a larger number of patients.Ethics ApprovalThis ongoing study is being conducted in accordance with the the Declaration of Helsinki and Council for International Organizations of Medical Sciences (CIOMS) International Ethical Guidelines. The study was approved by the Ethics Boards of University of Colorado Hospital, Washington University School of Medicine - Siteman Cancer Center, Memorial Sloan Kettering Cancer Center, The Sarah Cannon Research Institute/Tennessee Oncology, South Texas Accelerated Research Therapeutics, and The Royal Marsden NHS Foundation Trust.

Alpha-defensins secreted by dysplastic granulocytes inhibit the differentiation of monocytes in chronic myelomonocytic leukemia

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 78-88 ◽  
Author(s):  
Nathalie Droin ◽  
Arnaud Jacquel ◽  
Jean-Baptiste Hendra ◽  
Cindy Racoeur ◽  
Caroline Truntzer ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Chronic Myelomonocytic Leukemia ◽  
Cellular Heterogeneity ◽  
Uridine Diphosphate ◽  
Blood Monocytes ◽  
Healthy Donors ◽  
Leukemic Clone ◽  
Macrophage Colony ◽  
Myelomonocytic Leukemia

Abstract Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder that occurs in elderly patients. One of the main diagnostic criteria is the accumulation of heterogeneous monocytes in the peripheral blood. We further explored this cellular heterogeneity and observed that part of the leukemic clone in the peripheral blood was made of immature dysplastic granulocytes with a CD14−/CD24+ phenotype. The proteome profile of these cells is dramatically distinct from that of CD14+/CD24− monocytes from CMML patients or healthy donors. More specifically, CD14−/CD24+ CMML cells synthesize and secrete large amounts of alpha-defensin 1-3 (HNP1-3). Recombinant HNPs inhibit macrophage colony-stimulating factor (M-CSF)–driven differentiation of human peripheral blood monocytes into macrophages. Using transwell, antibody-mediated depletion, suramin inhibition of purinergic receptors, and competitive experiments with uridine diphosphate (UDP)/uridine triphosphate (UTP), we demonstrate that HNP1-3 secreted by CD14−/CD24+ cells inhibit M-CSF–induced differentiation of CD14+/CD24− cells at least in part through P2Y6, a receptor involved in macrophage differentiation. Altogether, these observations suggest that a population of immature dysplastic granulocytes contributes to the CMML phenotype through production of alpha-defensins HNP1-3 that suppress the differentiation capabilities of monocytes.

Clinical Experience with Azacitidine In Chronic Myelomonocytic Leukemia (CMML) in Spain

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5040-5040
Author(s):  
Pablo Gonzalez Navarro ◽  
Regina García Delgado ◽  
Alicia Bailén Garcia ◽  
Juan Antonio Múñoz Múñoz
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Clinical Trials ◽  
Clinical Experience ◽  
Peripheral Blood ◽  
Progression Free Survival ◽  
Complete Response ◽  
Chronic Myelomonocytic Leukemia ◽  
Free Survival ◽  
Myelomonocytic Leukemia

Abstract Abstract 5040 Clinical Experience with Azacitidine In Chronic myelomonocytic leukemia (CMML) in Spain Pablo González Navarro 1*, Regina García Delgado 2*, Alicia Bailén Garcia 3*, Juan Antonio Muñoz Muñoz 4* 1MD, PhD. Hospital San Cecilio, 18014 Granada, Spain, Teléfono: 958023600 [email protected]; 2Hospital Virgen De La Victoria, Málaga, Spain; 3Hospital Carlos Haya, Málaga, Spain; 4MD, PhD. Hospital Universitario Puerta del Mar, Cádiz, Spain Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal disorder of hematopoietic stem cells often occurring in elderly patients. In the new WHO classification, CMML has been reclassified as a myelodysplastic/myeloproliferative disease. CMML has been subdivided in two subclasses: CMML-1:<5% blasts in peripheral blood and 5–9% blasts in bone marrow, and CMML-2: <10% blasts in peripheral blood and 10–19% blasts in bone marrow (Greco et al. Mediterr J Hematol Infect Dis.2011). Azacitidine (AZA) is an hypomethylating agent approved in Europe for the treatment of myelodysplastic syndromes, with an intermediate to high risk of progressing to AML or death; chronic myelomonocytic leukemia (CMML) and AML that has developed from a myelodysplastic syndrome (prescribing information EMEA 2011). Until its approval in May 2009, AZA was used in Spain under compassionate use in clinical trials. AZA produce a direct decrease of DNA methyltransferase activity, reverting aberrant DNA methylation and increasing the expression of silenced genes, leading to celular differentiation and/or apoptosis (Greco et al. Mediterr J Hematol Infect Dis. 2011). Materials and Methods: We report the results of a retrospective, longitudinal, multicenter Spanish study of 27 patients to assess the effectiveness of AZA to treat CMML. We present results of: Response, Overall Response, Overall Survival and Progression Free Survival. Results: Eighteen of the patients (69.23%) had Chronic Myelomonocytic Leukemia (CMML) type 1 and nine (30.77%) CMML type 2. Median age at diagnosis was 69 years. Male/female ratio: 19/8. ECOG performance status score 1–2 was 78%, twenty patients (74%) received an initial dose of 75 mg/m2 of AZA, whereas three patients (11%) received 50mg/ m2. The mean number of cycles received was 8.32, 95%IC (5.91; 10.73). Overall response to treatment was 53% (CR+PR+HI+mCR): 14.81% complete response, 7.4% partial response, 3,7% Medular complete response and 29,62% Hematological Improvement. In addition, 18,51% had stable disease. Thirty-six percent of patients were alive at the end of treatment with AZA. Median Overall Survival and Progression Free Survival were 17.47 months (95%CI 9.33, upper limit not reached) and 10.97 (95%IC 3.97, 17.47) respectively (Figure 1, 2). Conclusion: Our results show that AZA is an active drug in the treatment of patients with CMML, with similar response rates in the published literature. More data from this study and further investigation with different clinical trials are needed to confirm these outcomes as well as safety and effectiveness of this treatment. Disclosures: García Delgado: Celgene and Novartis: Speakers Bureau.

Efficiency of Leukemic Stem Cell Separation From Patients with Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4997-4997
Author(s):  
Lu Zhang ◽  
Susanne Hofmann ◽  
Lars Bullinger ◽  
Marlies Goetz ◽  
Markus Wiesneth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Sorting ◽  
Peripheral Blood ◽  
Recovery Rate ◽  
Mononuclear Cells ◽  
Separation Efficiency ◽  
Conflicts Of Interest ◽  
Cell Number ◽  
Hematopoietic Stem

Abstract Abstract 4997 Leukemic stem cells (LSC) are the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSCs probably represent a critical target for therapeutic options. Xenograft models confirmed repeatedly that LSCs from AML patients reside mainly in CD34+CD38- compartment of leukemic blasts which makes the pure and efficient separation of this population mandatory to identify new therapeutic drugs to target LSC in different AML subtypes. We separated this subpopulation out of primary AML peripheral blood mononuclear cells (PBMC) samples with fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) and compared the efficiency of both methods. In order to profile gene expression of LSCs and hematopoietic stem cells (HSC) MicroArrays were performed using GeneChip Human Genome U133 Plus 2.0 from Affymetrix. The CD34+CD38- subpopulation was separated from PBMCs of 12 AML patients and 5 healthy volunteers using FACS. Concerning the 12 primary AML samples, the ratio of CD34+CD38- cells ranges between 0.79% and 86.2% using 1–5×107 PBMC for separation. After sorting, the purity of those AML samples increased to 88.4–98.4% while 2×104-3.6×106 cells were obtained. MACS was used to separate 2 representative samples, in which the CD34+CD38- subpopulation was rather small (sample1: 0.78%) or large (sample2: 86.1%). Those sorted subpopulations were compared to the samples sorted via FACS. In order to evaluate separation efficiency in a standardized manner, we defined the recovery rate: (CD34+CD38- cell number obtained /total CD34+CD38- cell number) × 100%. The total CD34+CD38- cell number was calculated through a pre-sorting FACS analysis. For sample 1, MACS resulted in a recovery rate of 4.2–6.4% with a purity of 86.6–90.3%, which is inferior to the recovery rate of 17% and the purity of 92.1% using FACS. For Sample 2, MACS resulted in a recovery rate of 0.4% with a purity of 98.8%, compared to the recovery rate of 11.6% with a purity of 98.1% by FACS. Comparing both methods it is obvious that the purity doesn't differ a lot, but the yield is much higher using FACS. This could represent a powerful tool, when managing rare samples. Finally, by comparing purity and yield, we showed that FACS is the adequate separation method. At the moment MicroArrays are being performed in order to investigate the gene expression profile for 12–15 AML patients and 5 HVs. Taken together, we showed a widely efficient method to routinely separate LSCs from patients with different subtypes of AML. Microarrays, that have been performed, represent a method that allows the comparison of the characteristics of LSCs in different AML subtypes and also of LSCs from bone-marrow with LSCs from peripheral blood and with HVs. These array data analyses are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

5-Azacitidine Therapy in Patients with Myelodysplastic Syndromes, Chronic Myelomonocytic Leukemia and Acute Myeloid Leukemia: a Single Institution Experience

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4963-4963
Author(s):  
Alessandra Freyrie ◽  
Gianluigi Reda ◽  
Daniele Vincenti ◽  
Mariarita Sciumé ◽  
Francesca Binda ◽  
...  
Keyword(s):  
High Risk ◽  
Myeloid Leukemia ◽  
Response Rate ◽  
Median Number ◽  
Observation Time ◽  
Chronic Myelomonocytic Leukemia ◽  
Female Ratio ◽  
Number Of Cycles ◽  
Male To Female ◽  
Myelomonocytic Leukemia

Abstract Abstract 4963 Overall survival (OS) is significantly improved by 5-azacitidine in intermediate-2 (int-2) and high risk myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) with 10–29% marrow blasts, and acute myeloid leukemia (AML) with 20–30% marrow blasts, compared with conventional treatments, and currently appears as the standard of care, at least in patients who are not candidates to allogeneic stem cell transplantation (alloSCT). We retrospectively evaluated the efficacy and tolerability of 5-azacitidine in 25 patients treated at our institution from 2009 to 2012, outside of clinical trial. Our series was composed by 17 cases of MDS with IPSS risk int-2 or high, 6 AML with marrow blasts between 20% and 30% and 2 CMML. Patients were treated with 5-azacitidine at a dosage of 75 mg/m2/d subcutaneously for 7 days every 28 days (schedule 5 day on, 2 day off and 2 day on). Median age of our cohort was 72 years (range 37–81 y), male to female ratio was 0. 6 and the median number of cycles received was 7 (range 1–26). According to the MDS-specific comorbidity index 9 pts (53%) were classified as low-risk, 7 pts (41%) as intermediate risk and 1 pt (6%) as high risk. Seventeen (68%) patients (13 MDS, 3 AML, 1 CMML) who had received at least 4 cycles of therapy were evaluable. Median age of these 17 patients was 71 years (range 37–81 y), male to female ratio was 0. 8 and median number of cycles administered was 8 (range 4–26). The overall response rate (ORR) was 59% (10/17 patients). According to International Working Group (IWG) 2006 criteria, five patients (29%) reached complete remission (CR) after a median of 5 cycles of therapy (range 4–6), two patients (12%) obtained hematologic improvement with bone marrow complete remission (marrow CR) after 6 and 11 cycles of therapy respectively, three patients (18%) showed hematologic improvement (HI) after 5 cycles (range 4–6), while stable disease (SD) and progressive disease (PD) were observed in 4 (23%) and in 3 patients (18%) respectively after 5 cycles (range 4–7). Median duration of response was 12 months (range 6–26 mo); median overall survival from the beginning of 5-azacitidine, for all patients treated, was 14. 4 months (range 7–33 mo). We did not observe any differences in response rate according to age, bone marrow fibrosis, cytogenetics and transfusion requirements. In the responder group (10 patients) we did not observe grade 3 or 4 non-hematologic toxicity after a median observation time of 10 months (range 5–33 mo). Among non-responding patients, four (57%) recurred to hospitalization due to infectious or hemorrhagic complications (median observation time 15 months, range 7–33). 5-azacitidine confirmed to be an active therapy for patients with int-2 and high risk MDS and AML with low marrow blast counts not candidate to high intensity treatment for age and or comorbidities, showing high response rate and good tolerability. The low rate of serious adverse events and need of hospitalization improved patient's quality of life and reduced the utilization of medical resources. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular Alterations in Chronic Myelomonocytic Leukemia Monocytes: Transcriptional and Methylation Profiling

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3889-3889
Author(s):  
Anca Franzini ◽  
Jamshid S Khorashad ◽  
Hein Than ◽  
Anthony D. Pomicter ◽  
Dongqing Yan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Damage ◽  
Transcription Factors ◽  
Chronic Myelomonocytic Leukemia ◽  
Healthy Controls ◽  
Age Related ◽  
Dna Methylation Profiling ◽  
Myelomonocytic Leukemia ◽  
Methylation Profiling

Abstract Chronic myelomonocytic leukemia (CMML) is a genetically heterogeneous hematopoietic stem cell disorder that combines features of a myelodysplastic syndrome and a myeloproliferative neoplasm and exhibits a strong bias towards older age. The prognosis of CMML is poor, with overall survival of less than 3 years in most studies, however recurrent somatic mutations explain only 15-24% of the clinical heterogeneity of CMML (Elena C. et al. Blood 128:1408-17, 2016). The extreme skewing of the CMML age distribution suggests that CMML reflects the malignant conversion of the myelomonocytic-biased differentiation characteristic of an aged hematopoietic system. We hypothesized that separating the contribution of the normal aging process from bona fide CMML-specific alterations will improve the molecular characterization and biological understanding of CMML. We decided to focus on monocytes as the phenotypic minimal common denominator of genetically heterogeneous diseases. CD14+ monocytes were sorted from the blood of untreated CMML patients (N=12, median age 77 years, range 61-90), age-matched healthy controls (old controls: N=12, median age 68 years, range 62-74) and young healthy controls (young controls: N=16, median age 29 years, range 24-44) and subjected to RNA sequencing and DNA methylation profiling. Differentially expressed genes in CMML monocytes compared to healthy controls were identified with DESeq2 using a 1% false discovery rate (FDR) and a fold-change cutoff set at >│2│ (Figure 1A). We identified the 2480 CMML-specific genes by subtracting all genes with significant differences in the young controls vs. old controls comparison from the CMML vs. old controls comparison. The top-25 most significantly upregulated genes (Figure 1B) included transcription factors, TNFα signaling genes, genes that regulate genomic stability, and genes involved in apoptosis. The most significantly downregulated transcripts were genes involved in response to DNA damage, RNA binding, monocyte differentiation and mediators of inflammatory process. To link these observations to function, we imputed the 2480 CMML-specific differentially expressed genes into the ingenuity pathway analysis (IPA) application. This analysis uncovered significant enrichment of pathways involved in: mitotic roles of Polo-like kinase, G2/M DNA damage checkpoint regulation, lymphotoxin β receptor signaling, IL-6 signaling and ATM signaling (Figure 1C). DNA methylation profiling revealed 909 differentially methylated regions (DMRs) between CMML and age-matched controls, with most regions being hypermethylated in CMML monocytes. Of these, 37% of the DMRs were intronic, 22% were exonic, 14 % were in the promoter region (Figure 1D), 10% were downstream, 10% were upstream, the remainder were 3' and 5'-overlaps. We also performed integrated analysis using the promoter DMRs and the gene expression profile to identify CMML-associated genes that are likely to be regulated by specific changes in methylation. We observed concomitant changes in CMML-specific mRNA transcripts and DNA methylation promoter regions in the CMML vs. old controls contrast for 10 genes (Figure 1E). AOAH, SERINC5, TAF3 and AHCYL1 were downregulated and hypermethylated; MS4A3, TNF, VCAM1, and IFT80, were upregulated and hypermethylated; TUBA1B was upregulated and hypomethylated and PITPNA was downregulated and hypomethylated. Our study is the first to combine transcriptional and methylation profiling for molecular characterization of CMML monocytes. Conclusions: (i) age-related gene expression changes contribute significantly to the CMML transcriptome; (ii) the CMML-specific transcriptome is characterized by differential regulation of transcription factors, inflammatory response genes and anti-apoptotic pathway genes; (iii) differences in promoter methylation represent only a small proportion of overall differences in methylation, suggesting that intragenic or intronic methylation is a major contributor to the leukemic phenotype; (iv) age-related changes may be necessary, but are not sufficient to realize the CMML phenotype. Figure 1. Figure 1. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

scholarly journals Deubiquitnase BAP1 Downregulation in Myeloid Malignances: A New Pathogenic Mechanism, Dominant in Chronic Myelomonocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5594-5594
Author(s):  
Ana Maria Hurtado ◽  
Eva Caparrós ◽  
Jose Miguel Torregrosa ◽  
Ginés Luengo ◽  
Mara Toderici ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Chronic Myelomonocytic Leukemia ◽  
Common Mechanism ◽  
Regulation Of Transcription ◽  
Brca1 Protein ◽  
Protein Levels ◽  
Increased Risk ◽  
Myelomonocytic Leukemia

Abstract Background: De-ubiquitinating enzyme BAP1, a fundamental deubiquitinase in the epigenetic regulation of transcription factors and functionally related to ASXL1, is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. In a recent murine study, absolute BAP1 depletion generated specimens with similar characteristics to myelodysplastic / myeloproliferative syndromes in humans (ineffective hematopoiesis and myeloproliferation), mainly to chronic myelomonocytic leukemia (CMML) (Dey, et al. Science 2012). Aim: The aim of this study was to quantify BAP1 gene expression in patients diagnosed with a variety of myeloid neoplasms, and compared it with healthy donors. We furthermore explored the possible association of BAP1 low expression level and the presence of ASXL1 mutations or BRCA1 protein levels. In addition, a regression analysis to determine the possible correlation of peripheral blood and bone marrow expression levels was performed. Methods: We included patients diagnosed between 2008-2014 of CMML, myelodysplastic sydrome (MDS) chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), of whom bone marrow DNA and RNA were available at diagnosis. As controls, 6 healthy bone marrow donors were used. BAP1 and BRCA1 expressions levels were quantified by RT-qPCR, using the same healthy bone marrow donor sample as an inter-assay normalizing- calibrator. The study of somatic ASXL1 mutations was carried out by the Sanger method. For statistical studies, the T-Student, Pearson correlation and/or U Mann-Whitney test, were used when needed. For survival analysis COX regression and the ROC curves were used. A two-side P<0.05 was used as statistical significance threshold. Results: Samples of 116 patients were included in the study: CMML=26; MDS=15; AML=50; CML=25 and 6 controls. This study shows that levels of BAP1 expressions are decreased when compared to controls along the spectrum of myeloid diseases. In the comparison among entities, CMML shows the lowest values (percentage respect to the calibrator), significantly lower than the other groups, except for CML patients: CMML vs MDS, p=0.001; CMML vs AML, p<0.001; CMML vs Controls, p<0.001; LMMC vs CML, p=0.346. No differences were found between CMML patients with dysplastic and myeloproliferative variant, WHO types I and II or according to the presence of ASXL1 mutations (33% CMML patients were mutated). Of potential clinical interest, BAP1 expression in bone marrow and peripheral blood showed a direct and significant correlation ( r=0.884, p= 0.001). BRCA1 expression were decreased uniformly through the different myeloid diseases, suggesting that the heterogeneous BAP1 expression could be responsible for different BRCA1 protein levels by posttranslational regulation. Conclusion:In summary; this study shows that BAP1 decreased expression is a common mechanism among the myeloid malignances, being CMML mainly affected. This mechanism is independent of the presence of ASXL1 mutations, and it could constitute a new therapeutic target in chronic myelomonocytic leukemia. Disclosures No relevant conflicts of interest to declare.

Blastic transformation in Mexican population with chronic myelomonocytic leukemia treated in a tertiary referral center.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e18566-e18566
Author(s):  
Alfonso Alfredo Rivera Duarte ◽  
Alejandra Armengol Alonso ◽  
Elena Tuna-Aguilar
Keyword(s):  
Overall Survival ◽  
Acute Leukemia ◽  
Peripheral Blood ◽  
Chronic Myelomonocytic Leukemia ◽  
Tertiary Referral ◽  
Blastic Transformation ◽  
Tertiary Referral Center ◽  
Short Overall Survival ◽  
Blast Count ◽  
Myelomonocytic Leukemia

e18566 Background: Chronic myelomonocytic leukemia (CMML) is the most aggressive of chronic leukemias, with a short overall survival and a high transformation rate to acute leukemia. We investigated factors related to blastic transformation in Mexican population treated in a tertiary referral center Methods: Records of patients diagnosed with CMML between 2000-2015 were reviewed; patients with incomplete data were excluded. IBM SPSS Statics 21.0 software was used to perform statistical analysis. Results: 54 patients were included, with a median age of 71 years and an overall survival of 16 months. The rate of blastic transformation found was 33% (18 patients), with a time to progression of 9 (0-87) months. Comparing patients who didn’t underwent blastic transformation to those who did, those who progress to acute leukemia tend to be younger (58 vs. 71 years, p = 0.001), have a higher peripheral blood blast count (2% vs. 0%, p = 0.003), where more likely to have immature myeloid precursors circulating in peripheral blood (94% vs. 64%, p = 0.02). In multivariate analysis, age continued to be statistically significant (HR 0.97, 95% IC = 0.929-0.987). There where no statistical difference in the two groups regarding hemoglobin levels (8.2g/dL vs. 10.1g/dL) platelets count (115 X 109 vs. 93 X 109), absolute neutrophil count (5.83 X 109 vs. 5.18 X 109), absolute monocyte count (3.09 X 109 vs. 2.680 X 109), and bone marrow blast count (0 vs. 2%). Cytogenetic considered as high risk was not predictor of blastic progression. Intensive chemotherapy was offered to 7(38.9%) patients, with a complete response rate of 0%, the overall survival was 1.4 months (0-9). Conclusions: Contrary to other reported series;Mexican patients with CMML that progresses to overt acute leukemia were considerably younger, with a higher tumor burden and a very short overall survival. In this population, it is important to consider more aggressive treatments at diagnosis, focusing in high dose chemotherapy and hematopoietic stem cell transplantation in a short term ratter than watching and waiting or using agents that do not impact in disease natural history.

PD-1 and PD-L1 Are Overexpressed in the "Intermediate CD14+CD16+" and "Non Classical CD14lowCD16+" but Not in the "Classical CD14+CD16-" Monocytes in the Peripheral Blood of Chronic Myelomonocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1694-1694
Author(s):  
Hae Tha Mya ◽  
Maria Diez Campelo ◽  
Sandra Valle Herrero ◽  
Agustin Díaz-Alvarez ◽  
Domingo Bustos ◽  
...  
Keyword(s):  
Internal Control ◽  
Peripheral Blood ◽  
Research Funding ◽  
Chronic Myelomonocytic Leukemia ◽  
Programmed Death ◽  
Cd16 Expression ◽  
Myelomonocytic Leukemia ◽  
Classical Monocytes ◽  
Intermediate Monocytes ◽  
Normal Controls

Abstract Chronic myelomonocytic Leukemia (CMML) is a heterogeneous clonal disorder highly resistant to the few therapies that are available nowadays. There is increasing evidence to suggest that Programmed Death-1 (PD-1), and its major ligand Programmed Death Ligand-1 (PD-L1), are involved in immune suppression and disease progression, are highly expressed in many hematological malignancies, and can be involved in MDS pathogenesis and resistance mechanisms to hypomethylating agents. However, the expression of PD-1 and PD-L1 is not widely explored in CMML. Different types of monocytes based on CD14 and CD16 expression show different genetic profiles and functions, having different distribution in several conditions, including malignancy. In our study, we studied the expression of PD-1 and PD-L1 in the peripheral blood (PB) monocytic compartment of patients with CMML using flow cytometry , to better understand their potential role in the pathogenesis of the disease, and as a basis for the evaluation of this pathway for the development of future immunotherapy strategies. Peripheral blood samples from 16 CMML, and age matched normal (n=10) and reactive (n=9) monocytosis (>1 x109 /L) were studied. Two hundred µl of each PB sample were stained with an 8-color panel of monoclonal antibodies (CD16-FITC, CD64-PE, PD1-PCP5.5, PDL1-PC7, CD300-APC, CD14-APCH7, HLADR-V450 and CD45-OC515). A minimum of 1x 106 events were acquired by FACSCanto II (BD Biosciences, San Jose, USA) and the data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytic population was selected first on the automated population separator plot and confirmed by the expression of CD64 and HLADR expression. Lymphocytes were used as the internal control. Three types of monocytes were defined based on the CD14 and CD16 expression, as previously described. As expected, CMML type 1 patients had higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (0.001). The distribution (median) of the monocytic subpopulations based on CD14 and CD16 expression among the monocytic compartment in PB of CMML, reactive and normal cases, respectively, was as follows: "classical"(CD14+CD16-) were of 98%, 90% and 85% (p<0.000); "intermediate" (CD14+CD16+) 1.4%, 3.7% and 2.6% (p=0.01); and "non-classical" (CD14lowCD16+) monocytes 1%, 5% and 12% (p<0.001). The expression of PD-1 in the major population ("classical" monocyte) was similar among CMML (Median MFI 370), reactive (Median MFI 403), and normal cases (Median MFI 265). However, in the "intermediate CD14+CD16+" and in the "non-classical CD14lowCD16+" monocytes, PD-1 was overexpressed in CMML and reactive cases, compared to normal controls. Reactive cases had even a higher overexpression of PD1 in both "intermediate" and "non-classical" monocytes compared to CMML (Median MFI of 312, 529, and 398 for "intermediate" and Median MFI of 185, 465, and 271 for "non- classical" in normal, reactive and CMML cases, respectively-p=0.01, and p<0.01). For PDL1, we did not find differences in their expression in "classical" nor "intermediate" monocytes among CMML, reactive and normal cases (Median MFI in "classical" monocytes of 2415 vs 2086 vs 2003 for CMML, reactive and normal cases -p>0.05-; and Median MFI in "intermediate" monocytes of 3803 vs 2737 vs 3200 for CMML, reactive and normal cases -p>0.05-). However, in the "non-classical" monocytic population, PD-L1 was clearly overexpressed in CMML (Median MFI of 1782) compared to normal controls (Median MFI of 699), and this was also significantly higher than in reactive cases (Median MFI of 1040) (p=0.002). We found that PD-1 and PD-L1 were overexpressed in CMML, but not in the main "classical" monocyte population of the PB, but in the less represented "intermediate" and "non classical" monocytic compartment. Interestingly, the CD16+ monocytes (intermediate and non-classical) were proposed to have a more important role in inflammation and immunomodulation. Therefore, these populations could have an important function in the pathogenesis of the CMML, and the overexpression of PD-1 and PD-L1 could be investigated as a target for immunotherapy in the development of new therapeutical strategies to improve the adverse prognosis of the CMML. Disclosures Diez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Puig:The Binding Site: Consultancy; Janssen: Consultancy.

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