Ruxolitinib Treatment Induces a Transition of Classical to Non-Classical Monocytes in Patients with Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2838-2838 ◽  
Author(s):  
Sanja Prijic ◽  
Taghi Manshouri ◽  
Ying Zhang ◽  
Ivo Veletic ◽  
Xiaorui Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Size Reduction ◽  
Spleen Size ◽  
Healthy Individuals ◽  
Normal Individuals ◽  
Monocyte Subpopulations ◽  
Classical Monocytes

Abstract Introduction: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm that develops de novo (primary myelofibrosis) or transforms from polycythemia vera or essential thrombocytosis. MF is characterized by stem cell-derived clonal myeloproliferation, abnormal cytokine expression, bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis, constitutional symptoms, cachexia, leukemic progression, and shortened survival. Several studies suggested that clonal monocytes play a role in the pathobiology of MF. However whether a specific monocyte subpopulation is predominantly present in MF is largely unknown. Traditionally, three subpopulations of CD14+ monocytes have been identified: classical (CD14++/CD16-), intermediate (CD14++/CD16+), and non-classical (CD14dim+/CD16++). Whether MF patients' monocyte subpopulations are different from those of normal individuals and how ruxolitinib treatment affects them has not been elucidated. Methods: Using flow cytometry we first assessed the distribution of the three monocyte subpopulations in the bone marrow (BM) of healthy individuals and MF and then assessed their distribution in BM samples from phase I/II clinical trial of ruxolitinib in patients with primary or secondary MF (Verstovsek S. etal. N Engl J Med 12:1117, 2010). Results: Using BM samples from 7 healthy individuals and 12 untreated MF patients we found a significant decrease in the percentage of monocytes (p =0.0061) in the mononuclear gate of untreated MF patients compared to normal individuals. However, the distribution of classical vs. non-classical monocyte subpopulations in MF was similar to that of normal BM (p =0.3, p =0.3, respectively). Remarkably, ruxolitinib treatment significantly altered the distribution of classical vs. non-classical monocyte subpopulations. During treatment (years 0-3, 3-6, 6-8) we identified a progressive increase in the percentage of monocytes in the mononuclear gate (p =0.1; p =0.04, and p =0.03, respectively) with a substantial increase in the non-classical monocyte subpopulation in years 0-3 and 3-6 of treatment (p= 0.04, p= 0.005, respectively) and a decrease in the classical monocytes (p =0.07, p =0.008, respectively). This trend reversed after 6-8 years of therapy (p =0.3, p =0.2, respectively). Importantly, in ruxolitinib-treated patients with a ≥50% spleen size reduction highest percentage of non-classical monocytes was observed during the first 3 years and years 3-6 of treatment (p =0.01, p =0.01, respectively). However during years 6-8 this difference was no longer detected and the percentage of non-classical monocytes was similar to the percentage detected in the pre-treatment BM samples (p =0.4). These changes correlated with response to ruxolitinib treatment. In patients with a <50% spleen size reduction the percentage of non-classical monocytes in years 0-3 of ruxolitinib treatment was significantly lower compared to patients with ≥50% spleen reduction (p =0.005), and patients with ≥50% spleen reduction show correlation between post-treatment spleen size and percentage of non-classical monocytes (p <0.0001, r=−0.4). Conclusions: Taken together, our results suggest that ruxolitinib induces a transition of classical to non-classical monocyte subpopulation during the first years of ruxolitinib treatment and that this effect correlates with the patients' clinical response. Further studies aimed at exploring the role of monocytes and their subpopulations in the pathobiology of MF are warranted. Disclosures No relevant conflicts of interest to declare.

p15INK4b Methylation Is Related to Blast Percentage, Thrombocytopenia, the Number of Lineages Involved in Cytopenia and Survival in Myelodysplastic Syndrome at the Time of Diagnosis: a Pyrosequencing Study.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3793-3793
Author(s):  
Miyoung Kim ◽  
Bora Oh ◽  
Song-yee Kim ◽  
Hyun-Kyung Park ◽  
Sang Mee Hwang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Cytosine Methylation ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Bone Marrow Examination ◽  
Specific Pcr ◽  
Hypomethylating Agent ◽  
Kaplan Meier ◽  
System Variables

Abstract Abstract 3793 Poster Board III-729 Introduction The inactivation of tumor suppressor genes by promotor hypermethylation, particularly p15INK4b, is believed to contribute to the initiation and the progression of myelodysplastic syndromes (MDS). p15INK4b methylation was found to be more frequent in high-risk MDS (RAEB) at the time of diagnosis, and to be associated with progression to AML in the studies using methylation-specific PCR (MSPCR). We investigated if the quantity of p15INK4b methylation is related to International Prognosic Scoring System variables (IPSS) and survival in the myelodysplastic syndrome (MDS) patients and if the quantity of p15INK4b methylation reflects the bone marrow status (the percentage of blasts or the degree of dysplasia) of the patients under treatment with or without hypomethylating agents. Materials and Methods The study included 74 de novo MDS patients including 24 with follow-up bone marrow examination: 10 under treatment with hypomethylating agent and 14 under treatment with other than hypomethylating agent including stem cell transplantation (SCT). We pyrosequenced 63 base pairs of p15INK4b including 11 CpGs using PSQ96MA system (Biotage, Uppsala, Sweden), with the extent of CpG cytosine methylation assessed in terms of methylation index (MtI) and methylation level (MtL). Result Patients with >5% bone marrow blasts had higher MtI and MtL (58.8% and 10.1%, respectively) than patients with <5% blasts (44.0% and 6.1%, p = 0.031 and p = 0.030, respectively). Methylation quantity was not associated with chromosomal aberrations. The MtI and MtL of patients with thrombocytopenia were higher than patients without thrombocytopenia (62.1% vs. 44.1%, p = 0.009, and 11.2% vs. 6.2%, p = 0.036, respectively); they were higher in patients with cytopenias in °Ã2 lineages than in patients with either unilineage or no cytopenia (56.5% vs. 39.4%, p = 0.055, and 9.8% vs. 4.1%, p = 0.036, respectively). The survival of patients with >50% MtI or >7% MtL was worse than patients with <50% MtI or <7% MtL (p = 0.023 and p = 0.031, respectively). Methylation quantity of p15INK4b did not correlate with the bone marrow status of the patients under treatment regardless of using hypomethylating agent or not. Conclusion Using pyrosequencing, we showed that the quantity of p15INK4b methylation correlates with thrombocytopenia and blast percentage in MDS patients. Heavy p15INK4b methylation in MDS is associated with adverse survival. Methylation quantity of p15INK4b did not appear to reflect the bone marrow status of the patients under treatment regardless of the use of hypomethylating agent in this study, however, further study with controlled patient group is required to clarify this issue. Fig. 1. Heavy methylation of the p15INK4b promotor region is a prognostic factor for poor survival in MDS patients. (a) Kaplan-Meier curves of MDS patients with MtI >50% and patients with MtI <50% (mean survival time: 1118 days vs. 1864 days, respectively). (b) MDS patients with MtL >7% and patients with MtL <7% (738 days vs. 1804 days, respectively). Disclosures: No relevant conflicts of interest to declare.

Egr1 and Apc Haploinsufficiency Cooperate in the Pathogenesis of Myeloid Neoplasia: A Mouse Model for Therapy-Related Myeloid Neoplasms with a Del(5q)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 117-117 ◽  
Author(s):  
Angela Stoddart ◽  
Jianghong Wang ◽  
Anthony Fernald ◽  
John Anastasi ◽  
Michelle Lebeau
Keyword(s):  
Bone Marrow ◽  
Mouse Models ◽  
Median Survival ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Macrocytic Anemia ◽  
Complete Inactivation ◽  
Myeloid Neoplasms ◽  
Single Allele

Abstract Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS). To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P<0.0001). Although disease latency was significantly shorter for Apcdel/+Egr1+/− mice, their phenotype was similar to Apcdel/+ mice, with only two exceptions. For both cohorts, mice typically developed splenomegaly and a lethal macrocytic anemia with monocytosis. Anemic mice had an increased proportion of CD71+Ter119+ erythroblasts, indicating a block in erythroid development between the early and late basophilic erythroblast stage. Two mice displayed anemia and leukocytosis (WBC 51–72 k/mL) with an increased proportion of Mac1+ cells in the spleen and Kit+ cells in the bone marrow (1 mouse). As anticipated, mice with wild type levels of Apc (Mx1-Cre-Apcfl/+) or with loss of one allele of Egr1 showed no signs of anemia. Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure. It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures: No relevant conflicts of interest to declare.

EZH2 Mutations Are Associated with Low Blast Percentage in Bone Marrow and −7/Del(7q) Karyotype in De Novo Acute Myeloid Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2544-2544
Author(s):  
Xiuli Wang ◽  
Haiping Dai ◽  
Qian WANG ◽  
Qinrong Wang ◽  
Yang Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Univariate Analysis ◽  
Myelogenous Leukemia ◽  
Prognostic Impact ◽  
Coding Region ◽  
Acute Myeloid

Abstract Abstract 2544 Somatic mutation of the EZH2 gene is seen in myelodisplastic syndrome, myelofibrosis, and chronic myelomonocytic leukemia patients. The prevalence and prognostic impact of somatic mutations of EZH2 in patients with acute myelogenous leukemia (AML) remains unknown. In this study, we sought to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo AML by PCR amplification of the entire coding region followed by direct bidirectional DNA sequencing. EZH2 mutations were identified in 13/714 (1.8%) of AML patients and occurred almost exclusively in males (11/13, P=0.033). In univariate analysis, the presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P=0.0001) and −7/del(7q) (P=0.025). There was no difference in the incidence of mutations in 13 genes, including ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. Complete remission, event-free survival or overall survival was similar between AML patients with and without EZH2 mutation (p>0.05). These results demonstrated EZH2 mutation as a recurrent genetic abnormality associated with lower blast percentage in BM and −7/del(7q) in de novo acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Canonical NF-κB Signalling Is a Potential Target in FLT3/ITD AML.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2447-2447 ◽  
Author(s):  
Jing Zhang ◽  
Li Li ◽  
Alan D. Friedman ◽  
Donald Small ◽  
Ido Paz-Priel
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Myeloproliferative Neoplasm ◽  
Initial Response ◽  
Wild Type ◽  
Normal Numbers ◽  
Toxic Dose ◽  
Marrow Cells

Abstract Abstract 2447 Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor is common in acute myeloid leukemia (AML) and is associated with a dismal outcome. Despite initial response, FLT3/ITD AMLs often relapse early, suggesting a residual population of resistant leukemia stem cells (LSC). Clinically, FLT3 inhibitors asmonotherapy have yet to improve outcome significantly and therefore, targeting additional pro-survival pathways may be necessary for this group of AML patients. Mice genetically egineered to express a hemizygous FLT3/ITD mutation develop a progressive, fatal, myeloproliferative neoplasm. Lin−cells isolated from the bone marrow of FLT3/ITD or control mice were subjected to gel shift analysis using a radio-labeled NF-kB binding site. This analysis demonstrated high levels of nuclear activation of NF-kB in the FLT3/ITD-expressing cells, suggesting its activation downstream of mutant FLT3 signaling. MV4–11 is a human AML-derived cell line harboring a homozygous FLT3/ITD mutation. Cells expressing high levels of aldehyde dehydrogenase (ALDH) have been shown to be enriched for LSC in primary AML samples and cell lines. High ALDH expressing MV4–11 cells were isolated using FACS and analyzed for NF-kB activation. Western blot analysis demonstrated preferential phosphorylation of NF-kB p65 by activated IKK on Ser536 in this subpopulation, compared with cells with low ALDH activity. These findings indicate activation of NF-kB in MV4–11 LSCs. We wanted to next test the requirement for NF-kB signaling in transformation by FLT3/ITD mutations. NF-kB p65 null mice die in utero. We therefore established C57BL/6 p65(flox/flox);Mx1-Cre mice. Intra-peritoneal injection of pIpC every other day for 7 doses efficiently deletes the RelA/p65 gene, resulting in expression of <1% of the corresponding RNA or protein. Despite effective excision of p65, the mice survive. Bone marrow cells harvested from control or p65(del/del) mice were transduced with a FLT3/ITD-expressing lentivirus and seeded in methylcellulose without cytokines. Equal transduction rate was verified by measurement of GFP expression by flow cytometry. Reproducibly, p65(del/del) marrow transduced with FLT3/ITD was ineffective in forming cytokine independent colonies, in contrast to wild-type marrow (5 +/− 0.6 vs. 55 +/− 6 colonies per 1E5 cells, P<0.001), and the few p65(del/del) colonies that resulted were smaller than those from p65 expressing wild-type marrow cells. Cells transduced with a lentiviral vector expressing GFP but not FLT3/ITD did not form colonies without cytokines, and p65(del/del) marrow formed normal numbers of colonies of normal size and distribution in the presence of IL-3, IL-6, and SCF. Sorafenib inhibits FLT3 signaling and kills MV4–11 cells with an IC50 of approximately 10 nM. Reproducibly, a sub-toxic dose of sorafenib (5 nM) combined with sub-toxic levels of the IKKb inhibitor IMD-0354 (400 nM) resulted in synergistic cell killing as indicated by the calculated combination index of 0.55. Currently, clinical efforts in FLT3/ITD leukemia concentrate on FLT3 inhibition alone. Our data suggest that canonical NF-kB may be an important pathway in FLT3/ITD AML and that simultaneously targeting FLT3 and NF-kB in this disease may be an effective approach. Disclosures: No relevant conflicts of interest to declare.

Tet2 Loss Accelerates The Myeloproliferative Neoplasm (MPN) Phenotype Of Jak2V617F Knockin Mice But Is Insufficient To Cause Leukemic Transformation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4095-4095
Author(s):  
Edwin Chen ◽  
Lawrence J Breyfogle ◽  
Rebekka K. Schneider ◽  
Luke Poveromo ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Conditional Knockout ◽  
The Other ◽  
Myelogenous Leukemia ◽  
Leukemic Transformation ◽  
The Impact

Abstract TET2 mutations are early somatic events in the pathogenesis of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) and are one of the most common genetic lesions found in these diseases. In MPN, TET2 mutations are enriched within more advanced disease phenotypes such as myelofibrosis and leukemic transformation and often co-occur with the JAK2V617F mutation, which is present in the majority of MPN patients. We have developed and characterized a Jak2V617F conditional knockin mouse (Jak2VF/+), the phenotype of which closely recapitulates the features of human MPN. To determine the impact of Tet2 loss on Jak2V617F-mediated MPN, we crossed Tet2 conditional knockout mice with Jak2VF/+ knockin and Vav-Cre transgenic mice and backcrossed the compound mutant animals. We then characterized the effects of heterozygous and homozygous loss of Tet2 on the phenotype of Jak2VF/+ mice. We assessed peripheral blood counts, histopathology, hematopoietic differentiation using flow cytometry, colony formation and re-plating capacity. We also evaluated the effects of Tet2 loss on the transcriptome of the HSC compartment using gene expression microarrays and on HSC function using competitive bone marrow transplantation assays. Similar to Jak2VF/+/VavCre+ mice, Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice develop leukocytosis, elevated hematocrits (HCT) and thrombocytosis. Tet2-/-/Jak2VF/+/VavCre+ mice demonstrate enhanced leukocytosis and splenomegaly compared to the other groups. All groups demonstrate myeloid expansion, erythroid hyperplasia and megakaryocytic abnormalities consistent with MPN in the bone marrow and spleen, while more prominent myeloid expansion and megakaryocytic morphological abnormalities are observed in Tet2-/-/Jak2VF/+/VavCre+ mice as compared to the other groups. Notably, we do not see the development of acute myelogenous leukemia (AML) in Tet2-/-/Jak2VF/+/VavCre+ mice at 6 months. We see enhanced expansion of lineagelowSca1+cKithigh (LSK) cells (enriched for HSC) most prominently in the spleens of Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice as compared to Jak2VF/+/VavCre+ mice. In colony forming assays, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced re-plating activity compared to Jak2VF/+/VavCre+ LSK cells and that Tet2-/-/Jak2VF/+/VavCre+ LSK cells form more colonies that Tet2-/-/Jak2+/+/VavCre+ cells. Gene expression analysis demonstrates enrichment of a HSC self-renewal signature inTet2-/-/Jak2VF/+/VavCre+ LSK cells. Concordant with this, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced competitive repopulation at 16 weeks as compared to Jak2VF/+/VavCre+ and Tet2+/-/Jak2VF/+/VavCre+ LSK cells. In aggregate these findings demonstrate that Tet2 loss promotes disease progression in MPN but is insufficient to drive full leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

ASXL1 Mutations in AML: Molecular Biomarker for Secondary AML?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2343-2343
Author(s):  
Jingmei Hsu ◽  
Anita J. Kumar ◽  
Martin P. Carroll ◽  
Noelle V. Frey ◽  
Nirav N. Shah ◽  
...  
Keyword(s):  
Overall Survival ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Myeloproliferative Neoplasm ◽  
Molecular Characteristics ◽  
Intermediate Risk ◽  
Kaplan Meier ◽  
The Impact

Abstract Background: Additional sex combs like transcription factor 1 (ASXL1) is a member of the polycomb group protein. ASXL1 mutation has been implicated in myeloid malignancy transformation. It is hypothesized that mutated ASXL1 leads to the loss of polycomb repressive complex 2 (PRC2) mediated gene repression and subsequent transforming events. Recent studies identify ASXL1 mutation as a poor prognostic marker in patients (pts) with de novo acute myeloid leukemia (AML) who present with intermediate–risk cytogenetic lesions (Patel, NEJM 2012; Schnittger, Leukemia2013). To study the impact of ASXL1 mutations in an unselected AML population, we analyzed clinical and molecular characteristics of patients with untreated AML who express ASXL1 mutation at presentation. Methods: Using next generation sequencing, 254 adult patients with AML seen at the Hospital of the University of Pennsylvania were analyzed for mutations, including ASXL1, using a 33-gene hematologic malignancy panel. Clinical characteristics were obtained from retrospective chart review. Kaplan-Meier estimates were used to calculate overall survival (OS) from time of diagnosis. Living patients were censored at date last seen. Results: ASXL1 mutations were detected in 36/254 (14%) AML pts. There were 29 known pathologic mutations, 1 benign, 1 probable pathologic, and 9 variants of unknown clinical significance (VUS). In 6/36 (16.7%) pts, ASXL1 was the sole mutation identified. Of the 30 pts with additional mutations (Figure 1), 6/30 (20%) pts harbored 2 independent ASXL1 mutations. When the 27 patients with pathologic ASCL mutations were analyzed for co-mutations, TET2 (13/27, 48%) was the most frequent ASXL1 co-mutation. FLT3 (0/27, 0%) and NPM1 (1/27, 3.7%) were notable for their absence. Median age of pts at diagnosis was 69 years (range 23-80). Prior myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN) was noted in 9/36 (25%) and 11/36 (30.6%) pts, respectively. Four pts (11.1%) had received chemotherapy and/or radiation therapy for a prior non-myeloid neoplasm. Karyotype was normal in 18/36 (50%) pts, and 7 additional pts had intermediate cytogenetic lesions. There were 7 pts (19.4%) with unfavorable cytogenetics (complex karyotype (3 pts), 7q- (3 pts), and 5q- (1 pt)). Four pts (11.1%) had a favorable karyotype, with t(8;21) in 3 pts and t(15;17) in 1 pt. At presentation, median white blood cell count (WBC) was 6.4x103/uL (1.0 x -103). In pts whose AML transformed from prior MPN, median WBC was 50 X103/uL (3.3-140). Standard induction chemotherapy with an anthracycline and cytarabine was given to 17/36 (47%) pts. An additional 3/36 (8.3%) pts underwent induction therapy with clofarabine. Complete remission (CR) was documented in 14/20 (70%) evaluable pts. Of the remaining pts, 11 received a hypomethylating agent, and 5 received other therapies. Thirty-day treatment mortality for all 36 pts and for 27 pts with known ASXL1 pathologic mutation was 13.4% and 18.5% respectively. Kaplan-Meier estimate showed a median overall survival of 349 days (median follow up of 107 days (range 15-1570)). For the 27 pts with a pathologic ASXL1 mutation, the OS was 276 days (Figure 2, median follow up of 145 days (range 18-1570)). Conclusion: ASXL1 mutations in de novo AML with intermediate-risk cytogenetics is associated with poor clinical outcome in cooperative group trials. Strikingly we demonstrate in a single institution, retrospective analysis that 66.7% of pts who present with ASXL1 mutations in the setting of previously untreated AML had documented MDS, MPN and/or prior chemotherapy/radiation. Further studies are necessary to evaluate if ASXL1 mutation has independent prognostic significance in AML or if it is primarily a marker for secondary leukemia. Figure 1: ASXL1 and co-mutations Figure 1:. ASXL1 and co-mutations Figure 2: Overall survival for AML patients with ASXL1 pathologic mutation Figure 2:. Overall survival for AML patients with ASXL1 pathologic mutation Disclosures No relevant conflicts of interest to declare.

Gemtuzumab Ozogamicin (GO) in Infants with Acute Myeloid Leukemia (AML) – Combined Results from the Children’s Oncology Group (COG) Trials, AAML03P1 and AAML0531

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 377-377
Author(s):  
Erin M. Guest ◽  
Richard Aplenc ◽  
Lillian Sung ◽  
Susana C. Raimondi ◽  
Betsy A. Hirsch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Early Death ◽  
Gemtuzumab Ozogamicin ◽  
Juvenile Myelomonocytic Leukemia ◽  
Cell Transplant ◽  
Intensive Chemotherapy

Abstract Background: Infants <1 year of age with AML are at high risk of early death during induction, pulmonary and infectious toxicities, and treatment related mortality (TRM). Compared with older children, infants are more likely to have higher risk clinical and cytogenetic features. Targeted therapy is needed to improve the outcomes for infants with AML while minimizing toxicities. Objective: To determine if the addition of gemtuzumab ozogamicin (GO) to standard chemotherapy is safe, tolerable, and improves event free survival (EFS) in infants <1 year of age with AML. Methods: Infants >/= 1 month to <1 year of age with de novo AML or <1 month of age with progressive AML were eligible for enrollment on the COG trials AAML03P1 and AAML0531. Infants with acute promyelocytic leukemia, juvenile myelomonocytic leukemia, bone marrow failure syndromes, or Down syndrome were not eligible. The 5-course chemotherapy backbone was identical in both trials. GO 3 mg/m2/dose (0.1 mg/kg for patients with a body surface area <0.6 m2) was given on day 6 of Induction (Ind) I and day 7 of Intensification (Int) II to all patients on AAML03P1 and to randomized patients on the experimental arm of AAML0531. Patients on the control arm of AAML0531 were treated with standard therapy without GO (noGO). Stem cell transplant (SCT) was given following IntI to patients on AAML03P1 with a matched family donor (MFD) and to patients on AAML0531 with high risk (HR) disease or with intermediate risk (IR) disease and a MFD. SCT recipients only received one dose of GO. Patients were removed from protocol if the bone marrow had >/= 20% blasts after IndI on AAML03P1 or >/= 5% blasts after IndII on both studies. Early death (ED) was defined as death during IndI. Results: AAML03P1 enrolled 39 infants from 2003-2005 and AAML0531 enrolled 103 infants from 2006-2010 (Table 1). Median follow up was 5.02 years. The demographics and disease characteristics of infants enrolled on both studies were similar. Compared with enrolled children >/= 1 year of age, infants had higher frequencies of hepatomegaly (p<0.001), splenomegaly (p<0.001), hyperleukocytosis (WBC>100x103/µL, p=0.003) and French-American-British (FAB) M5 (p<0.001), and M7 (p<0.001) AML. Infants had less FAB M1 (p<0.001), M2 (p<0.001), and M4 (p=0.029) AML. There was no difference in CNS disease by age. The frequency of 11q23/MLL rearrangement was highest in infants 0-179 days of age (44%) and significantly decreased with increasing age (p<0.001). Favorable cytogenetics were rare in infants: t(8;21) was not found and inv(16)/t(16;16) was only found in 4 patients. FLT3-ITD HAR was absent in infants. The majority of infants (89%) fell into the IR group and infants were less likely to have had high (HR) or low risk (LR) disease when compared with children >/= 1 year. Table 1: Combined characteristics of infants on AAML03P1 and AAML0531 Table 1:. Combined characteristics of infants on AAML03P1 and AAML0531 The ED rate was higher in younger infants (7 ED, infants 0-179d) when compared with older infants (1 ED, infants 180-364d) (p=0.013). EDs were not increased in infants who received GO vs. noGO (5 vs. 3 ED, p=0.730). The complete remission (CR) rate for infants was 68% at the end of IndII. The 5 year EFS was lower in infants than children >/= 1 year (42 vs. 50%, p=0.001). The 5 year OS for infants was 62%, relapse risk (RR) was 44%, and TRM was 10%. Infants who received GO had improved OS, EFS, and RR, though the differences were not statistically significant (Table 2). Table 2: Combined outcomes of infants on AAML03P1 and AAML0531: noGO vs. GO Conclusion: GO in combination with intensive chemotherapy is tolerable in infants with AML, does not increase early deaths, and is associated with trends toward improved EFS, OS, and RR. Table 2:. Combined outcomes of infants on AAML03P1 and AAML0531: noGO vs. GO Conclusion: GO in combination with intensive chemotherapy is tolerable in infants with AML, does not increase early deaths, and is associated with trends toward improved EFS, OS, and RR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Monocyte subpopulations of blood and bone marrow in patients with chronic heart failure

2018 ◽  
Vol 17 (4) ◽  
pp. 16-22
Author(s):  
M. V. Vins ◽  
S. P. Chumakova ◽  
O. I. Urazova ◽  
D. A. Azarova ◽  
V. M. Shipulin ◽  
...  
Keyword(s):  
Heart Failure ◽  
Bone Marrow ◽  
Chronic Heart Failure ◽  
Ischemic Cardiomyopathy ◽  
Venous Blood ◽  
Relative Content ◽  
Red Bone Marrow ◽  
Healthy Donors ◽  
Monocyte Subpopulations ◽  
Classical Monocytes

The aim of the investigationwas to evaluate the ratio of classical (CD14++CD16-), intermediate (CD14++CD16+), nonclassical (CD14+CD16+) and transient (CD14+CD16–) monocytes in the blood and bone marrow in patients with chronic heart failure (CHF) against ischemic cardiomyopathy (ICMP).Materials and methods. 17 patients with ICMP and 14 practically healthy donors were observed. The material of the study was venous blood (in patients and healthy donors) and red bone marrow (in patients). In the materials the relative content of different monocytes subpopulations was determined by flow cytometry. The obtained results were analyzed by statistical methods.Results. It is shown that in the blood of patients the proportion of monocytes with the phenotype CD14++CD16- is 57.77 [of 46.35; 79.76]%, CD14++CD16+ – 25.06 [4.96; 42.31]%, CD14+CD16+ 5.05 [4.08; 6.58]% and CD14+CD16- – 6.03 [3.58; 10.89]%; in the bone marrow – 43.44 [40.54; 44.68]%, 0.16 [0; 1.07]%, 0,54 [0.35; 1.07]% and 54,32 [52.83; 56.08]%, respectively, which is different from the content of the data cells subpopulations in the blood (p < 0.05). At the same time, the content of non-classical monocytes in the patients’ blood is 2 times lower than in healthy donors, and the number of other cells varies within the norm.Conclusion. The differentiation of monocytes into 4 subpopulations in patients with CHF occurs directly in the bloodstream, since mainly the classical and transitional monocyte fractions with the prevalence of the latter are present in the bone marrow. Deficiency of non-classical monocytes of blood in CHF is probably associated with a disruption of their extramedullary differentiation.

scholarly journals Differential Diagnosis of Hereditary and Acquired Thrombocytopenia By the Immature Platelet Fraction and Thrombopoietin Levels

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1049-1049 ◽  
Author(s):  
Fabio Luiz Bandeira Ferreira ◽  
Marina Pereira Colella ◽  
Samuel de Souza Medina ◽  
Maiara Marx Luz Fiusa ◽  
Loredana Nilkenes Gomes da Costa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Differential Diagnosis ◽  
Platelet Count ◽  
Healthy Volunteers ◽  
Complete Blood Count ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Healthy Individuals ◽  
Technical Limitation ◽  
Immature Platelet Fraction

Abstract Introduction: The differential diagnosis of hereditary and acquired thrombocytopenias can be challenging, especially when between immune thrombocytopenia (ITP) and less well characterized hereditary thrombocytopenias (HT) such as MYH9-related disorders. Fundamental differences in the management of these two conditions add clinical relevance to the search for novel parameters that differentiate these conditions. The immature platelet count (IPF) represents the fraction of platelets with higher RNA content, and in analogy to the reticulocyte count for erythropoiesis is a biomarker of thrombopoietic activity. In a recent report (Miyazaki et al, 2015), IPF values that were more than 5-fold higher than those observed in ITP patients were reported in a population of 15 patients with HT. However, whether this increased values represented a real increase in thrombopoietic activity, or reflected a technical limitation of IPF determination in large platelets could not be clarified. Here, we aimed to evaluate the role of IPF determination in the differential diagnosis between HT and several forms of acquired thrombocytopenia in a larger and more diverse population of patients. We also evaluated thrombopoietin (TPO) levels in HT compared to ITP, to further investigate the mechanisms by which extremely large IPF values are observed in HT. Methods: IPF and mean platelet volume (MPV) were prospectively determined using a Sysmex XE5000 hematologic analyzer (as part of the complete blood count) in a cohort of patients with post-chemotherapy thrombocytopenia (n=56), bone marrow failure (myelodysplastic syndromes and aplastic anemia; n=22), ITP (ITP; n=105) and inherited thrombocytopenias (n=27). The latter population consists of a well-defined cohort of individuals with HT thrombocytopenia characterized by clinical, familial, laboratory and molecular data. TPO levels were determined by ELISA (R&D Systems) in 21 HT patients and 22 ITP patients matched for platelet count and age. A group of 178 healthy volunteers were used to determine normal IPF and MPV values. Results: Median platelet counts were similar in post-chemotherapy patients (CTx) (32.0*109/L), bone marrow failure (BMF) (33.5*109/L), ITP (52.0*109/L) and HT (52.0*109/L) (P=0.15). Similar IPF levels were observed in CTx and BMF patients (5.6%; IQR 3.4-8.8% and 6.5%; IQR 3.5-13.7%. Compared to these two groups, higher IPF values were observed in both ITP (12.3%; 7.0-21.0%) and HT patients (29.8%; 17.5-56.4%) (both P values < 0.05). In addition, IPF were significantly higher in HT compared to ITP (Kruskall-Wallis test and Dunn's post test,P=0.001). MPV values were different between HT and CTx/BMF groups, but could not differentiate ITP from HT. TPO levels. The accuracy of IPF to discriminate HT from all other causes of thrombocytopenia estimated by ROC analysis was 0.88 (CI95%0.8-0.96, p<0.0001). Similar TPO levels were observed in platelet count-matched ITP, HT patients and healthy volunteers without thrombocytopenia. Interestingly, TPO presented marked correlations with both platelet count (Rs = - 0.61, P=0.002) and IPF (Rs= 0.59, P=0.003), even with TPO levels in the same range of healthy individuals. In contrast, no significant correlation could be observed between TPO and IPF or platelet count in HT patients. Conclusions: IPF represents an informative biomarker for the differential diagnosis of hereditary and acquired thrombocytopenias, and accurately differentiates ITP from the most common HT. As expected, TPO levels in patients with ITP were not higher than in individuals with normal platelets counts. The inverse correlation between TPO and platelet count in these patients confirm a blunted TPO response to thrombocytopenia in these patients. Similarly, patients with HT did not present increased TPO levels compared to healthy individuals. Accordingly, increased IPF levels in these patients cannot be attributed to higher TPO levels. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Unique Case of CSF3R-Mutated Chronic Neutrophilic Leukemia with Thrombocytosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Parastou Tizro ◽  
Melissa Yacur ◽  
Min-Ling Liu ◽  
Victor E. Nava ◽  
Anita Aggarwal
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Myeloproliferative Neoplasm ◽  
Gene Mutations ◽  
Normal Karyotype ◽  
Chromosome Analysis ◽  
Peripheral Blood Smear ◽  
Chronic Neutrophilic Leukemia ◽  
Activating Mutations

Chronic neutrophilic leukemia (CNL) is a well-recognized, extremely rare myeloproliferative neoplasm (MPN) with only ~200 reported cases until 2017. The current WHO diagnostic criteria include leukocytosis of ≥ 25 x 109/L with ≥ 80% neutrophils/bands, &lt; 10% circulating neutrophil precursors and &lt;1% blasts (&lt;5% bone marrow blasts). Exclusion criteria includes dysplasia, or clinical/molecular diagnosis of other MPN. The WHO classification was updated in 2016 to include CSF3R T618I and other activating mutations in CSF3R as diagnostic. SETBP1 and ASXL1 mutations are frequent but unspecific in CNL and appear to be of prognostic significance. Hepatosplenomegaly and a hypercellular bone marrow with marked granulocytic hyperplasia are common in CNL, but thrombocytosis has not been reported previously. We report a 72-year-old man with past medical history of hypertension and hepatitis C status post-treatment, who presented with WBC 38.4k/cmm, Hgb 6.3g/dl, MCV 102 fl and platelet count of 2601k/cmm. Reactive etiology of thrombocytosis was ruled out. Peripheral blood smear revealed rare blasts. Bone marrow biopsy was hypercellular (60-80%) with M:E ratio of 10:1, granulocytic and megakaryocytic hyperplasia, clustering of dyspoietic megakaryocytes, no erythroid or myeloid dysplasia and minimal patchy reticulin fibrosis suggestive of primary MPN. Chromosome analysis showed normal karyotype of 46 XY. Molecular studies were negative for BCR-ABL, JAK-2 V617F, CALR and MPL, Next Generation Sequencing revealed mutations in the CSF3R gene (Q781* and T618I) and concurrent inactivating ASXL1-G646fs*12 and activating U2AF1-Q157P mutations. These findings were compatible with a diagnosis of CNL. We describe an unusual case of CNL confirmed by CSF3R gene mutations (T618I and Q781*) with significant thrombocytosis and without splenomegaly. Although the T618I mutation has commonly been reported in CNL, to the best of our knowledge, the Q781* mutation is novel in CNL, and has only been reported before in severe congenital neutropenia (SCN). Association of Q781* mutation in CSF3R gene with significant thrombocytosis in our patient needs further investigation. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document