Dynamics of Bcl-2, Bax and ACE Expression In CD34+ Cells of Peripheral Blood and Bone Marrow In Patients with Acute Leukemia During Induction Therapy

Abstract Abstract 4840 The causes of drug resistance in acute leukemias (AL) have been studied very intensively and the key research was done on Bcl-2 family proteins. Last studies have showed that high level Bcl-2 expression in acute leukemia is really associated with drug resistance andpoor prognosis [Haematologica 2007, U. Testa]. It was demonstrated that lower Bax/Bcl-2 ratio (<0,3) was associated with FAB M0-M1 classes (p=.00001), poor-risk cytogenetics and poor prognosis [Blood 2003, G. Poeta]. But there were no studies on the dynamic evaluation of Bcl2 and Bax expression on CD34+ cells during chemotherapy. Renin-angiotensin system and angiotensin concertin enzyme (ACE) influence on leukogenesis is extensively investigated. It was reported that ACE expression on blast cells is high [Leuk Lymphoma 2006, S. Aksu]. Recent publications indicate that primitive hematopoietic precursors have different characteristics regarding ACE: CD34+ACE+cells transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population [Blood 2008, V. Jokubaitis]. But it's still unknown how CD34+ACE+ cells in AL behave on and after chemotherapy. We have studied the dynamics of Bcl-2 and Bax expression by flow cytometry in CD34+ cells of peripheral blood (PB) and bone marrow (BM) in pts with AL. PB and BM samples were collected before treatment, on days +8, +36, only PB - on day + 21. Bcl-2 and Bax were detected on CD34+ cells by flow cytometry using specific monoclonal antibodies: CD34 (8G12, BD), Bcl-2 (100, BD), Bax (2D2, Santa Cruz). ACE (9B9, BD) expression was also evaluated. We calculated 10 000 cells in each sample. 10 pts were included in the study: 4 AML, 6 ALL. The control group comprised 4 healthy donors. At time of diagnosis CD34+ cells number in BM was 38,7%± 9,75, in PB - 38,3%± 8,14 in AL pts, not differing much in AML and ALL, and indicating blast cells population. CD34+ cells numbers in BM and PB of healthy donors were 1,35% and 0,23%, respectively. After induction therapy and WBC recovery (days +36-38) CD34+ cells number in AL pts decreased dramatically in BM to 3,83%±1,51 (p=0,001) and in PB to 0,98%± 0,29 (p=0,0001), indicating the efficacy of chemotherapy. The dynamics of Bcl-2, Bax and ACE expression on CD34+ cells of BM and PB in AL pts are presented in fig.1-6 As seen in the fig.1,2 CD34/Bcl-2 expression in BM is significantly higher (p=0,04) and in PB is similar in AL pts at the diagnosis comparing with donors. It's also worth to note that BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 demonstrating much higher level of antiapoptotic marker in PB cells. On the contrast CD34+ AL cells in BM and PB had similar characteristics regarding CD34/Bcl-2 expression. This expression level decreased substantially in BM at day +36 comparing with day 0 (p=0,04), but it never reached the donors level remaining extremely high and supposing the persistence of antiapoptotic activity in CD34+ cells in AL pts. It did not change at all during chemotherapy in PB cells, being identical to donors characteristics. The fig.2,3 demonstrate that, CD34/Bax expression in BM is almost 3-times higher (p=0,14) and in PB is twice lower (p=0,02) in AL pts in comparison with donors. It's interesting that CD34/Bax expression in leukemic BM and PB cells looks very similar, when in donors we had very low expression in BM and high - in PB. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar in Bax expression. Chemotherapy caused the significant augmentation of CD34/Bax expression in PB on day +8 (p=0,01) and near significant on day +21 (p= 0,09) showing the increased level of “dieing” cells in PB after cytostatic influence. The fig. 5,6 show that CD34/ACE coexpression in BM cells of AL pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AL pts was much lower (p=0,02) than in donors and substantially increased at day +36 almost reaching the donor level. We may conclude that Bcl-2, Bax, ACE expression on CD34+ cells in AL pts and donors significantly differs, the dynamics of expression in AL while chemotherapy shows critical changes in CD34/Bcl-2 expression in BM, CD34/Bax and CD34/ACE in PB. Disclosures: No relevant conflicts of interest to declare.

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Pro- and Antiapoptotic Proteins Expression on Bone Marrow and Peripheral Blood CD34+Cells in Acute Leukemia (AL) Patients

Blood ◽

10.1182/blood.v118.21.4996.4996 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4996-4996

Author(s):

Elena E. Khodunova ◽

Elena N Parovichnikova ◽

Irina V. Galtzeva ◽

Sergey M. Kulikov ◽

Valeri G Savchenko

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Expression Level ◽

Leukemia Cells ◽

Control Group ◽

Healthy Donors ◽

Cd34 Cells ◽

Significant Difference ◽

Blast Cells

Abstract Abstract 4996 It was shown that drug resistance, poor-risk cytogenetics and poor prognosis in AL is associated with high level of Bcl-2 expression and low Bax/Bcl-2 ratio (<0,3). Fas-antigen (CD95) as a protein triggering the extrinsic apoptotic pathway is differently expressed on hematopoietic precursors. More immature CD34+/CD38- AML blast cells have lower expression of Fas/Fas-L and lower Fas-induced apoptosis than CD34+/CD38+cells. CD34+/CD38− leukemia precursors also have a reduced sensitivity to daunorubicin in vitro and increased expression of multidrug resistance genes (mrp/lrp). CD34+ leukemia cells have not yet been properly characterized regarding the expression of angiotensin converting enzyme (ACE) which regulatory influence on hematopoiesis is now beeing extensively investigated. ACE expression on blast cells is high, but it's still unknown how CD34+ACE+ leukemia cells behave after chemotherapy. Recent publications indicate that CD34+ACE+ hematopoietic precursors transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts. We have studied the dynamics of Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in peripheral blood (PB) and bone marrow (BM) in AL pts during treatment. PB and BM samples were collected before and on +36 day after chemotherapy. The antigens were detected by flow cytometry using monoclonal antibodies. We calculated 10 000 cells in each sample. 19 pts were included in the study: 10 - AML and 9 - ALL. The control group comprised 8 healthy donors. At time of diagnosis there were 40±5,7% of CD34+ cells in BM and 26±4,9% - in PB. There was no significant difference between AML and ALL. CD34+ cells in BM and PB of healthy donors constituted 1,6% and 0,27%, respectively. After induction therapy (+36 day) CD34+ cells decreased in BM to 6,1%±3,3 (p=0,0001), in PB to 3,7%± 2,7 (p=0,0008) in all pts. The data on antigens expression on CD34+ cells of BM and PB are presented in table 1 CD34+/Bcl-2+ CD34+/Bax+ CD34+/CD95+ CD34+/ACE+ BM PB BM PB BM PB BM PB AML pts n=10 0 day 38±11,6* 41±14 24,4±7,9 29,2±7,6* 16,4±8,5 23,2±7,8 21,7±9,5 20,8±8,7* 36 day 13,5±3,4** 23,7±5** 46,2±11,5 50,3±11 19,9±5,5 36,4±10 34±6,6 35±9,2** ALL pts n=9 0 day 36±11 33,7±12 46,2±9,4 37,4±3,7* 3,4±1,1* 7,1±2,5* 41±10,9 33,2±9,7* 36 day 18,4±5,8 26±8,9 38±11,8 40,5±10 26,2±9,1** 40,9±9,2** 34±10 62,8±10** Donors n=8 11,7±1,6 26,1±5,9 22,8±4 67,8±6,7 13,4±3,2 47,7±11,6 28±5,3 68,2±10,2 * − p<0.05 compare with donors ** − p<0.05 compare with day 0 CD34/Bcl-2 expression in BM in AML pts is significantly higher (p=0,04) at the diagnosis comparing with donors. CD34/Bcl-2 expression in PB in AML pts and in BM and PB in ALL pts is higher too, but not significantly. This expression level decreased substantially in BM and PB in AML pts on +36 day comparing with day 0 (p<0,05). We did not found significant changes in ALL pts. CD34/Bax expression in PB is significantly lower (p=0,003) both in AML and ALL pts in comparison with donors. In AML, not in ALL, chemotherapy caused augmentation of Bax expression in CD34+ BM and PB cells on +36 day. BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 and Bax, demonstrating much higher level of pro- and antiapoptotic markers in PB cells. On the contrast CD34+ leukemia cells in BM and PB had similar characteristics regarding CD34/Bcl-2 and CD34/Bax expression. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar. CD95 expression on CD34+ BM and PB before treatment is significantly lower (p=0,01, p=0,008) in ALL pts in comparison with donors, and this expression level increased after chemotherapy (p<0,05). CD34/CD95 expression in AML pts is similar with donors, and we didn't find changes after treatment. CD34/ACE coexpression in BM cells of leukemia pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AML and ALL pts was much lower (p<0,05) than in donors and substantially increased on the day 36. So, our data demonstrate that Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in AL pts and donors significantly differs. The chemotherapy provokes critical changes in CD34/CD95 expression in BM and PB in ALL pts, CD34/Bcl-2 expression in AML pts and ÑÂ34/ACE expression in PB in all AL pts. Disclosures: No relevant conflicts of interest to declare.

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Immunophenotypic Aberrancies in Acute Leukemia: A Tertiary Care Centre Experience

Oman Medical Journal ◽

10.5001/omj.2021.03 ◽

2021 ◽

Vol 36 (1) ◽

pp. e218-e218

Author(s):

Monika Gupta ◽

Lovekesh Monga ◽

Dimple Mehrotra ◽

Sonia Chhabra ◽

Shivani Singhal ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Acute Leukemia ◽

Peripheral Blood ◽

Tertiary Care ◽

Flow Cytometric Analysis ◽

Antigen Expression ◽

Molecular Techniques ◽

Acute Leukemias ◽

Simultaneous Application

Objectives: Acute leukemias (AL) are a heterogeneous group of hematological malignancies with the presence of 20% or more blasts in the peripheral blood or bone marrow. Malignant cells display characteristic patterns of surface antigenic expression. Aberrant phenotypes are defined as patterns of antigen expression on neoplastic cells different from the process of normal hematopoietic maturation. We sought to evaluate the occurrence of aberrant phenotypes in newly diagnosed cases of AL. Methods: The study included 100 patients in whom both bone marrow aspiration and flow cytometry were performed. Patients with blasts > 20% of all ages were included in the study. Flow cytometric analysis was done using the monoclonal antibody panel of peripheral blood/bone marrow. Results: Out of 100 cases, 53 were categorized as acute myeloid leukemia (AML), 43 as acute lymphoid leukemia (ALL), and four cases of mixed phenotypic acute leukemia (MPAL). ALL were subcategorized based on immunophenotyping into B-ALL and T-ALL, which comprised 88.4% and 11.6%, respectively, of total ALL (43.0%) cases. Cluster of differentiation 33 (CD33) and CD13 were the most commonly expressed antigens in AML, with CD7 being the most common aberrancy. CD19 was expressed in all B-ALL cases followed by cCD79a, CD10, Tdt (86.8%) with CD13 being the most common aberrancy. cCD3, CD7, and CD5 were expressed in all T-ALL cases with aberrant antigen expression in 80.0% of T-ALL cases. MPAL cases showed expression of B/myeloid antigens. Conclusions: The diagnosis and classification of leukemia rely on the simultaneous application of cytomorphology, cytochemistry, flow cytometry, cytogenetics, and molecular techniques. Flow cytometry is of great help in the diagnosis of AL, particularly in ALL for lineage assignment and in classifying MPAL. It also helps in detecting aberrant antigen expression and assisting in minimal residual disease detection.

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Absolute Lymphocyte Count Is a Novel Prognostic Indicator in Acute Leukemia: Implications for Risk Stratification and Future Studies.

Blood ◽

10.1182/blood.v108.11.2276.2276 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2276-2276

Author(s):

Guillermo R. De Angulo ◽

Carrie Yuen ◽

Shana Palla ◽

Peter M. Anderson ◽

Patrick A. Zweidler-McKay

Keyword(s):

Bone Marrow ◽

Young Adults ◽

Multivariate Analysis ◽

Risk Stratification ◽

Acute Leukemia ◽

Induction Therapy ◽

Age At Diagnosis ◽

Acute Leukemias ◽

Children And Young Adults ◽

Current Risk

Abstract Background: Despite improving outcomes, 25–50% of children and young adults with acute leukemia still relapse and most salvage rates are discouraging. Additional prognostic factors, particularly those that represent host factors, may further stratify patients and decrease relapse rates. Purpose: To determine if absolute lymphocyte counts (ALC) during induction chemotherapy can improve current risk stratification and predict relapse-free survival (RFS) and overall survival (OS) in children and young adults with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Methods: We analyzed 160 consecutive cases of de novo ALL and AML patients 1–21 years of age, treated at the University of Texas M. D. Anderson Cancer Center from 1995–2005. Age at diagnosis, initial WBC, bone marrow blast % on days 0 and 7, were analyzed with ALC on days 0, 15, 21 and 28 of induction therapy. Results: ALC during induction therapy is a significant independent predictor of RFS and OS in young adults and children with either ALL or AML. Specifically, an ALC <350 cells/mcL on day 15 of induction therapy for ALL significantly predicts poor 6-year OS (52% vs. 87%, p=0.015; HR=4.2, Figure 1A) and RFS (46% vs. 80%, p=0.001; HR=4.8, Figure 1B). Similarly, an ALC of <350 cells/mcL on day 15 of induction therapy for AML predicts poor 6-year OS (35% vs. 86%, p=0.033; HR=4, Figure 1C). ALC-15 remains a significant predictor of OS and RFS after adjusting for age at diagnosis, initial WBC and bone marrow response on day 7 (p=0.013; HR=6.3, and p=0.003; HR=6.3, respectively) in multivariate analysis (Table 1). Importantly, ALC-15 defines a subgroup of half of our AML patients and predicts an excellent 5-year OS of 86% (p=0.033, Figure 1C). Conversely, prolonged lymphopenia predicts that 16% of young AML patients will have a dismal 5-year RFS of 14% (p=0.004, Figure 1D). Finally, ALC-15 <350 cells/mcL is able to predict 70% of relapses in both ALL and AML patients. One possible algorithm could identify half of AML patients with a predicted OS of 86% simply by measuring the ALC-15. Those patients with a low ALC on day 15 would be assessed at day 21 and 28 and those with persistent lymphopenia would be predicted to to have an RFS of 14% and would be stratified to receive intensified and/or experimental therapy. Conclusion: We demonstrate that ALC can identify patients at high and low risk for relapse early in the course of treatment for ALL or AML. Our data indicates that ALC is both independent of and a more powerful predictor than age at diagnosis, initial WBC and bone marrow response on day 7. This routine measurement could enhance current risk-stratification and lead to improved outcomes in young patients with acute leukemias. Figure 1 Figure 1. Table 1 Multivariate Analysis of ALC, Age, WBC, Bone marrow response and Survival

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The Expression and Clinical Significance of PRAME Gene in Acute Leukemia.

Blood ◽

10.1182/blood.v110.11.4225.4225 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4225-4225

Author(s):

Rong Fu ◽

Kai Ding ◽

Zonghong Shao

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Clinical Significance ◽

Blood Count ◽

Mononuclear Cells ◽

Residual Disease ◽

White Blood Count ◽

Gene Expressions ◽

Healthy Donors ◽

Blast Cells

Abstract Objective To investigate the expression of PRAME (preferentially expressed antigen of melanoma) gene in acute leukemia and its clinical significance in monitoring prognosis, detecting minimal residual disease (MRD) and gene immunotherapy. Methods The expression of PRAME gene mRNA in bone marrow mononuclear cells is measured by reverse transcriptase polymerase chain reaction in 34 patients with acute leukemia and 12 bone marrow samples of health donors. The relationships between PRAME gene expressions and some clinical data, such as gender, age, white blood count, leukemic immunophenotype, the percentage of blast cells, and the karyotype of chromosome, were also estimated. Results PRAME gene was expressed in 38.2% of all the patients, 40.7% of all the AML patients, which was higher than the 28.6% of ALL patients (p >0.05). There was no expression of PRAME gene in healthy donors. In all the sub phenotypes of AML, the expressive rate of PRAME gene in M3 patients is 80%, which is higher than that in M2 (33.3%) and in M5 (28.6%). The expressive rate of PRAME gene was also positively correlated with the expression of CD15, CD33, and the abnormality in the karyotype of chromosome, but not correlated with age, gender, white blood count and percentage of blast cell in bone marrow. Conclusion PRAME gene is highly expressed in acute leukemia, and could be regarded as a useful tool for monitoring MRD. Differential expression in acute leukemia patients vs. healthy donors suggests that the immunogenic antigens PRAME are potential candidates for immunotherapy in acute leukemia.

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Circulating Megakaryocyte-Derived Cells Detected by Flow Cytometry As Marker of Aggressive Neoplasms of Megakaryocytic Lineage in Acute Megakaryoblastic Leukemia and Allied Malignancies Presenting As Primary Myelofibrosis

Blood ◽

10.1182/blood.v120.21.1461.1461 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1461-1461

Author(s):

Serena Marotta ◽

Giovanna Giagnuolo ◽

Giulia Scalia ◽

Maddalena Raia ◽

Santina Basile ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Differential Diagnosis ◽

Cell Population ◽

Peripheral Blood ◽

Primary Myelofibrosis ◽

Giant Platelets ◽

Megakaryoblastic Leukemia ◽

History Of ◽

Blast Cells

Abstract Abstract 1461 The differential diagnosis of myelofibrotic disorders encompasses chronic primary myelofibrosis (PMF), myelodysplastic syndromes with fibrosis (MDS-F), acute panmyelosis with myelofibrosis (APMF) and acute megakaryoblastic leukemia (AMKL). Most of these conditions are recognized as distinct entities by the WHO 2008 revised classification of myeloid neoplasms; however, the WHO admits that often a definitive diagnosis is problematic, mostly because of specimens with insufficient cellularity (e.g., “dry tap”). Nevertheless, the correct identification of the most aggressive fibrotic disorders (APMF and AMKL) remains crucial, given their poor prognosis and subsequent need of intensive treatment (including transplantation). Even the most recent molecular studies did not result in any contribution in the differential diagnosis. Here we report our experience on a cohort of about 300 patients who were admitted in our bone marrow failure unit because of cytopenia in the last 7 years. All these patients were evaluated by standard peripheral blood and bone marrow cytology, karyotype analysis and bone marrow thephine biopsy, aiming to a definitive hematological diagnosis. Flow cytometry analysis was performed at initial presentation and then serially during the follow up on both peripheral blood and bone marrow aspirate. All patients were classified according to the WHO 2008 revised classification of myeloid neoplasms, and received the best standard treatment based on the specific disease, age and comorbidities. This report focuses on 8 patients who shared a unique flow cytometry finding of an aberrant megakaryocyte-derived cell population, which seems associated with a distinct disease evolution. Two of these patients received the diagnosis of AMKL according to bone marrow aspirate and trephine biopsy; the karyotype was complex in one case (monosomal karyotype, including a 5q-), whereas no Jak-2 mutation or any other genetic lesions could be demonstrated. Their blast cells were CD34+, CD38+, CD45+, CD117+, CD33+, CD13+; in addition, in the peripheral blood, we detected the presence of an aberrant cell population which was CD45-, CD42b+ (CD34+ in one case and CD34- in the other one). In the blood smear, we observed megakaryocyte fragments which likely correspond to this aberrant cell population, as identified by flow cytometry. Other three patients presented with a severe pancytopenia: all of them had a dry tap, and their trephine biopsies documented a massive fibrosis. They had no previous hematological disorder (one suffered from Behcet syndrome), normal karyotype and absence of any typical genetic lesion (i.e., wild-type Jak-2). All of them did not show splenomegaly, increased LDH or leukoerythroblastosis; their peripheral blood smear showed abnormal giant platelets, often resembling megakaryocyte fragments. Flow cytometry documented in the peripheral blood the presence of a distinct population of CD45-, CD42b+, CD61+ cells, which was also CD34+ in one case. These 3 patients were initially classified as PMF, even if APMF could not be ruled out; however, within 6 months they all progressed to AMKL. At this stage, typical CD34+, CD45+ blast cells were accompanied by a progressive increase of CD45+, CD42b+, CD61+ cells. This aberrant megakaryocyte-derived cell population (which could not be demonstrated in patients with thrombocytopenia) was also identified in 3 additional patients, who have a previous history of hematologic disorders: two had a history of pure red cell aplasia (successfully treated by immunosuppressive therapy), and one a 5q- melodysplastic syndrome (responding to lenalidomide, even with transient cytogenetic remission). In all of them we observed the appearance of CD45-, CD42b+ cells in the peripheral blood, which appeared as giant platelets/megakaryocyte fragments in the blood film; this finding within a few weeks was followed by progression to AMKL (5q- was detected in 2 of 3 cases). In conclusion, we demonstrate that aberrant circulating megakaryocyte-derived cells detected by flow cytometry may be useful in the differential diagnosis of myelofibrotic disorders. These giant platelets or megakaryocyte fragments, regardless the initial diagnosis, were associated with early evolution into AMKL, likely representing a surrogate marker for aggressive neoplasms of the megakaryocytic lineage. Disclosures: Risitano: Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Expression of MPL in Leukemia Stem Cells and Its Role in Stemness Maintainance

Blood ◽

10.1182/blood.v128.22.1723.1723 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1723-1723 ◽

Cited By ~ 1

Author(s):

Huan Li ◽

Qing Rao ◽

Pei Yu ◽

Shuying Chen ◽

Zheng Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Drug Resistance ◽

Colony Formation ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Cd34 Cells ◽

Clonogenic Potential

Abstract Osteoblast cells play an important role in bone marrow niche. The interaction between osteoblast and leukemia cells promotes leukemia development, which is mediated by some cytokines including TPO. It has become evident that TPO-MPL signaling is essential for the quiescence and self-renewal of hematopoietic stem cells, however, its expression pattern and the role in leukemia stem cells have not been reported. This study was aimed to determine the expression of MPL in acute myeloid leukemia (AML) and investigate the role of MPL in the leukemia stem cells' quiescence, drug resistance and self renewal. The expression levels of CD34, CD38 and MPL were detected by flow cytometry in bone marrow cells from 57 newly diagnoses AML patients. The correlation between MPL and CD34, CD38 expression in AML patients were analyzed. The results showed that expression of MPL in AML patients was higher significantly than that in 13 normal donors (P<0.05). Expression of MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P<0.01). MPL was higher expressed in CD34+ cells than that in CD34- cells significantly (P<0.0001). We also detected the expression of MPL in different populations of leukemia cells in AML1-ETO9a mouse leukemia model established in our lab. We found that the ratios of MPL positive cells in Lin-c-kit+ and Lin-c-kit+sca-1+ populations were significantly higher than that in total leukemia cells. In addition, in chemotherapy treated AML1-ETO9a mice, the proportion of Lin-c-kit+MPL+ leukemia cells were increased 23.5 folds than that in untreated leukemia mice, which indicates that MPL+Lin-c-kit+ LSCs population could be enriched by chemotherapy. Furthermore, MPL+ and MPL- cells in Lin-c-kit+ leukemia population were sorted by flow cytometry and the colony formation and quiescence state were determined. The results showed that MPL+Lin-c-kit+ cells produced significantly more colonies in the second round of colony formation (p<0.05) than MPL-Lin-c-kit+ cells. The G0 phase accumulation of MPL+Lin-c-kit+ cells was significantly higher than that of MPL-Lin-c-kit+ cells (p<0.01). Above results indicate that MPL+ leukemia cells display more clonogenic potential and maintain quiescence. These data demonstrate that as a receptor of TPO, MPL is highly expressed in leukemia stem cells and MPL positive leukemia stem cells could be enriched by chemotherapy. MPL positive leukemia stem cells exhibit more clonogenic potential, quiescence and drug resistance. It suggests TPO-MPL mediated interaction of osteoblast and leukemia cells take a role in the stemness of leukemia stem cells. Disclosures No relevant conflicts of interest to declare.

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AML1-ETO Collaborates with the Homeobox Gene Meis1 in Inducing Acute Leukemia in the Mouse Bone Marrow Transplantation Model.

Blood ◽

10.1182/blood.v112.11.928.928 ◽

2008 ◽

Vol 112 (11) ◽

pp. 928-928 ◽

Cited By ~ 3

Author(s):

Vegi M. Naidu ◽

Vijay P.S. Rawat ◽

Christina Schessl ◽

Konstantin Petropoulus ◽

Monica Cusan ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Insertional Mutagenesis ◽

Fusion Gene ◽

Genetic Alterations ◽

Control Group ◽

Mouse Bone Marrow ◽

Acute Leukemias ◽

Hox Gene ◽

Blast Cells

Abstract AML1-ETO is the most frequent fusion gene in human AML. Previously, we and others have demonstrated that the fusion is not able to cause leukaemia on its own in experimental murine models, but that it needs collaborative partners. However, although mutations such as the FLT3-length mutation and C-KIT mutations were defined as important collaborative genetic events in AML1-ETO positive AML, most human AML1-ETO cases do not carry these mutations, indicating the presence of unkown collaborative partners in these patients. On the other hand Meis1, a HOX gene co-factor, belonging to the TALE family of homeodomain proteins, has a well established function as a protooncogene with a strong collaborative potential in Hox gene associated AML in mice. First we confirmed expression of MEIS1 in some patients with AML1-ETO positive AML by real-time PCR. Based on this we sought to determine if AML1-ETO can collaborate with Meis1 in inducing acute leukemias: single constructs or both genes were co-transfected in 5-FU treated primary murine bone marrow cells by retroviral gene transfer, using MSCV retroviral constructs with an IRES–GFP or YFP cassette. Mice were transplanted with BM cells expressing Meis1 alone (n=10), with BM cells solely expressing the fusion gene (n=10) or EGFP (n=7, control) or with BM expressing both genetic alterations (n=14). None of the mice in the Meis1 and AML1-ETO as well as in the control group developed disease. In contrast, 14 mice transplanted with BM co-expressing AML1-ETO and Meis1 developed lethal disease after a median latency of 102 days. Three mice succumbed to a myeloproliferative syndrome and nine mice died by acute leukemia (6 mice developed AML, 3 mice ALL), which was serially transplantable into secondary recipients (median = 57 days). Immunohistochemistry of various organs of leukemic mice showed massive infiltration with blast cells. In MPS and AML 85 ± 9.3 % of the blast cells co-expressed Gr-1+ and Mac1+. In ALL cases 40 ± 19.9 % of the malignant cells co-expressed Mac1 and the lymphoid-associated B220 antigen. Analysis of retroviral integration did not reveal recurrent integration sites as an indication for insertional mutagenesis. In summary, our data demonstrate for the first time that AML1-ETO can collaborate with Meis1 and identify a novel collaborative partner in t(8;21) positive AML. Furthermore, our analyses demonstrate that Meis1 can collaborate with non-homeobox genes in inducing acute leukemia.

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Comparison of CXCR-4 and Adhesion Molecule Expression in Healthy Bone Marrow with Expression in Bone Marrow and Peripheral Blood of Patients Receiving G-CSF Plus AMD3100.

Blood ◽

10.1182/blood.v106.11.1974.1974 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1974-1974

Author(s):

Uta Oelschlaegel ◽

Martin Bornhaeuser ◽

Frank Kroschinsky ◽

Gerhard Ehninger ◽

Uwe Platzbecker

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Adhesion Molecules ◽

Peripheral Blood ◽

Surface Expression ◽

Stem Cell Mobilization ◽

Qualitative And Quantitative ◽

Healthy Donors ◽

Cd34 Cells ◽

Cell Mobilization

Abstract It is known that the crosstalk between adhesion molecules, bone marrow microenvironment, and cytokines facilitates the multi step process of stem cell mobilization from bone marrow to peripheral blood. A combination of G-CSF plus AMD3100 - a CXCR-4 antagonist - has been shown to be safe and efficient in stem cell mobilization of healthy donors and cancer patients. Nevertheless, data predicting the efficacy of this approach are still missing. The present study investigated the correlation of the expression of CXCR-4 (CD184) and adhesion molecules with the kinetics and efficacy of stem cell mobilization in nine patients with Multiple Myeloma (MM) or NHL, respectively. Steady-state mobilization was performed using a combination of G-CSF (Filgrastim, 10μg/kg/d, 8 am) for 4 days followed by AMD3100 (240μg/kg) on day 4 at 10pm. Autologous aphereses were started on day 5. Bone marrow and peripheral blood (PB) before AMD3100 application (day 4) and PB on day 5 were investigated with a 4-color flow cytometric procedure. Bone marrow aspirates of healthy donors (n=20) served as control. The qualitative (%) and quantitative (mean fluorescence intensity, [MFI]) antigen expression of CXCR-4 in relation to CD34 was assessed as well as the expression of certain adhesion molecules including LFA-1, PECAM-1, VLA-1, L-selectin and CD44. First, the median percentage of CXCR-4 surface expression in healthy bone marrow was significantly higher (92%; range: 52 – 99%) than in patients bone marrow (70%; 30 – 88%; p=0.002), PB before AMD3100 (87%; 35 – 97%; p=0.050) and on day 5 (17%; 2 – 74%; p<0.001), whereas cytoplasmic expression was comparable (91%; 53 – 95%) in all cell compartments. The median quantitative CXCR-4 surface expression was significantly decreased in PB on day 5 compared to pre AMD3100 (14 vs. 95; p=0.003). Furthermore, the qualitative expression of LFA-1 and the quantitative expression of LFA-1, PECAM-1, VLA-1, and CD44 were also downregulated in response to AMD3100 (p<0.010). Second, a median of 63/μl (range: 15 – 132/μl) CD34+ cells was measured in the PB on day 5. Thus, a high absolute count of CD34+ cells in the PB on day 5 significantly correlated with lower qualitative and quantitative CXCR-4 expression in the same material (r=0.833; p=0.015). Evaluating CXCR-4 expression in bone marrow, PB before AMD3100 and on day 5 no significant correlation to CD34+ counts could be detected. However, there was one very poor mobilizing patient (15/μl CD34+ cells on day 5) in whom the quantitative CXCR-4 expression in the bone marrow was significantly higher than the median of all patients (MFI 95 vs. 26). Furthermore, some of the adhesion molecules (L-selectin, VLA-4, and CD44) showed a rather positive correlation with CD34 count. In summary, these preliminary data suggest that the amount of CD34+ cells in the peripheral blood after G-CSF plus AMD3100 application seems to be negatively correlated with CXCR-4 expression. A higher quantitative CXCR-4 expression in the bone marrow pre AMD3100 might predict a lower mobilization efficacy.

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Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽

10.1182/blood.v106.11.5195.5195 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5195-5195

Author(s):

Lulu Lu ◽

Yongping Song ◽

Baogen Ma ◽

Xiongpeng Zhu ◽

Xudong Wei ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Cord Blood ◽

Peripheral Blood ◽

Lymphocyte Subsets ◽

Color Flow ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

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Aberrant Megakaryocyte Gene Expression Contributes to Primary Myelofibrosis.

Blood ◽

10.1182/blood.v120.21.2867.2867 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2867-2867

Author(s):

Laure Gilles ◽

Christy Finke ◽

Terra L Lasho ◽

Animesh Pardanani ◽

Ayalew Tefferi ◽

...

Keyword(s):

Gene Expression ◽

Clinical Trial ◽

Bone Marrow ◽

Flow Cytometry ◽

Primary Myelofibrosis ◽

Gene Set Enrichment Analysis ◽

Gene Expression Program ◽

Healthy Donors ◽

Cd34 Cells ◽

Expression Program

Abstract Abstract 2867 Primary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML. We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease. Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks. We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi. GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks. We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures: Pardanani: Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.

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