AMD3100 Mobilizes Acute Promyelocytic Leukemia Cells from the Bone Marrow into the Peripheral Blood and Sensitizes Leukemia Cells to Chemotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 246-246 ◽  
Author(s):  
Bruno Nervi ◽  
Matthew Holt ◽  
Michael P. Rettig ◽  
Gary Bridger ◽  
Timothy J. Ley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Acute Promyelocytic Leukemia ◽  
Reporter Gene ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells

Abstract CXCR4/SDF-1 axis regulates the trafficking of normal stem cells to and from the bone marrow (BM) microenvironment. SDF-1 is a chemokine widely expressed by many tissues especially BM stromal cells and osteoblasts. AMD3100 (AMD) is a novel bicyclam molecule that is a competitive inhibitor of SDF-1/CXCR4 binding and has been used to enhance stem cell mobilization when combined with G-CSF in mouse, dog and man. We are interested in evaluating whether leukemic cells “mobilize” similar to normal stem cells after treatment with AMD, and if so, whether this mobilization increases the efficacy of chemotherapy. Therefore, we utilized a mouse model of human acute promyelocytic leukemia (APL) in which the PML-RARα transgene was knocked into a single allele of the murine cathepsin G locus. To more efficiently track the leukemic cells, we transduced banked APL tumors with a dual function reporter gene that encodes a fusion protein comprised of click beetle red (CBR) luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (CBR/EGFP). We generated large numbers of CBR/EGFP+ APL cells by isolating EGFP+ cells using a MoFlo cell sorter, and passaging them in secondary syngeneic recipients. Importantly, the secondary recipients developed a rapidly fatal acute leukemia after intravenously (iv) or intraperitoneal injection, which displayed an APL phenotype (CD34/GR1 co-expression) and exhibited luciferase activity. Upon iv injection into syngeneic recipients, the CBR/EGFP+ APL cells rapidly migrated to the BM microenvironment, as evidenced by the significantly increased BLI signal in the femurs, spine, ribs, and skull of recipients at 4 days after injection. Over the next 2–3 days the CBR/EGFP+ cells migrated to the spleen followed rapidly by widespread dissemination and death due to leukostasis by 14–16 days. To our knowledge, this represents the only mouse leukemia model in which leukemia cells home preferentially to the BM microenvironment in a manner that is similar to what is seen in human AML. Therefore, we used this model to study the effect of AMD on the “mobilization” of APL cells into the peripheral blood (PB) and on their sensitivity to chemotherapeutic agents that are known to affect the proliferation of these cells. Surprisingly, injection of AMD (5 mg/kg) immediately at the time of APL infusion had no impact on the engraftment (short term or long term) of either normal BM stem cells or the leukemic cells. However, we observed rapid mobilization of the leukemic cells when AMD was administered 11 days after APL injection. In fact, 40% of mice that received a single dose of AMD on day +11 after APL injection died 2 to 4 hours after AMD injection as a result of the rapid and massive mobilization of blasts. Overall, we found that AMD treatment on day +11 induced a 3-fold increase in total WBC counts with a 10-fold increase in the leukemic blasts into PB. Interestingly, the administration of AMD concomitant with cytarabine (AraC) (200 mg/kg) on day +11 significantly prolonged the overall survival of mice, compared with mice treated only with AraC. In summary, we developed a mouse model to study the APL cell trafficking, and we have shown leukemia cell mobilize from the BM into PB after AMD administration. We propose that CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. In these preliminary results, we observed that AMD sensitizes APL cells to AraC.

Comparison of angiopoietin-1 and -2 and VEGF expression in bone marrow and peripheral blood leukemic cells of patients with acute promyelocytic leukemia.

2014 ◽  
Vol 32 (15_suppl) ◽  
pp. 7079-7079
Author(s):  
Mariana Scaranti ◽  
Jose Mauricio Segundo Correia Mota ◽  
Ana Silvia Gouveia de Lima ◽  
Barbara Amelia Santana Lemos ◽  
Antonio Roberto Lucena-Araujo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Promyelocytic Leukemia ◽  
Peripheral Blood ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Vegf Expression ◽  
Angiopoietin 1

New Approach to the Mechanism of Hyperleukocytosis in Patients with APL After Treatment with ATRA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3585-3585
Author(s):  
Guosheng Jiang ◽  
Kehong Bi ◽  
Chuanfang Liu ◽  
Peie Wen
Keyword(s):  
Bone Marrow ◽  
Acute Promyelocytic Leukemia ◽  
Mtt Assay ◽  
Peripheral Blood ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Malignant Cells ◽  
Remarkable Feature ◽  
New Approach ◽  
Mitotic Cells

Abstract Abstract 3585 Poster Board III-522 Objective To detect the new approach to the mechanism of hyperleukocytosis during induction therapy with ATRA in patients with acute promyelocytic leukemia. Methods Diagnosis of acute promyelocytic leukemia (APL) was performed according to the FAB cytological classification criteria and cytogenetic criteria. The collected leukemia cells were re-suspended in ATRA at 37°C for 96 h. Differentiation of leukemia cells was assessed by NBT reduction assay and indicated as percent of CD11b positive cells. Constant MTT assay and cell number count were used to detect the proliferation of leukemia cells after treatment with ATRA. For FACS analysis, cells were incubated with CD11b, CD54, CD106 and Ki67 mAb or isotypic control IgG1 antibody. L-CFU assay was taken to measure the colony formation. GM-CFU assay was used to detect the granulocyte-macrophage colony-forming unit. The mRNA expression of MMP-9, TIMP-1 was detected by RT-PCR. RESULTS (1) The results showed that the total number of WBC was up-regulated after treatment with ATRA in patients with APL, which was chiefly due to the up-regulation of myelocytes and more matured granulocytes. (2) The primary APL cells, unlike normal promyelocyte, have little requirement for survival factors, Its plating efficiency was also lower and could be up-regulated by exposure to ATRA. MTT assay showed that the OD value of primary APL cells was lower than that of NB4 clone cells or normal hematopoitic cells. Otherwise, the proliferation of primary APL cells, post exposure to ATRA, was up-regulated and more cytokine-dependent or more sensitive to G-CSF stimulation. (3) Most importantly, the division hypothesis should be taken into consideration, because the leukocytosis chiefly contributed to the up-regulation of differentiated myelocytes and more matured neutrophils, which should be the key proliferation pool as the normal myelocytes. The primary APL cells had a low proliferation potential, the mitotic cells are often scarcely detected even for 4 d in culture. Most of primary APL cells had a growth arrest in G0/G1 or G0 phase. The APL cells occur to switch from G0 into cell cycle that was indicated by the up-regulation of Ki67 antigens. In our opinion, most of APL cells enter into cell cycles after exposure to ATRA or arsenic trioxide in vivo, with more mitotic cells. (4) The rheological hypothesis was taken into consideration. The up-regulation of adhessive ability of APL cells, which resulted in more release from bone marrow into peripheral blood. The results indicated that ATRA can induce differentiation of the malignant cells, most remarkable feature was the progressive change of malignant cells with signs of their terminal differentiation and with Auer rods being sometimes observed in mature cells. Usually, the percent of promyelocyte was down-regulated and the percent of myelocyte, metamyelocyte and more matured myeloid cells was obviously up-regulated, especially for the percent of myelocyte or myelocyte-like cells. These results suggest that an asynchronism between rheological and morphological maturation in each APL cell might explain the occurrence of hyperleukocytosis in some patients during ATRA therapy. For example, the serum sICAM1 and sVCAM1level was up-regulated, the secretion up-regulation of adhesive molecules ICAM1 and VCAM1 in primary APL cells was also observed, as inagreement with the up-regulation of CD11b, CD54 and CD106. The adhessive coefficiet was also up-regulated. Conclusion The leukocytosis or hyperleukocytosis chiefly contributed to the up-regulation of myelocyte-like cells, which chiefly contributed to the more myelocytes divisions and more sensitive to cytokine stimulation. Partly due to the up-regulation of adhesive index and adhesion molecules of differentiationed leukemia cells, which could easily resulted in the release of APL cells from bone marrow to peripheral blood. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CXCR4 Has a CXCL12-Independent Essential Role in MLL-AF9 Driven Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 774-774
Author(s):  
Ramprasad Ramakrishnan ◽  
Pablo Peña-Martínez ◽  
Puneet Agarwal ◽  
Carl Högberg ◽  
Marion Chapellier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Fold Increase ◽  
Drop Out ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Cxcl12 Expression

Abstract Acute Myeloid Leukemia (AML) is a clonal hematological disorder associated with poor prognosis, and there is a strong need to develop new therapeutic strategies. AML is propagated by a small population of leukemic cells in the bone marrow with self-renewal capacity, a population termed leukemia stem cells (LSC). To identify in vivo dependencies of LSCs, we conducted a CRISPR/Cas9 drop-out screen targeting cell surface receptors on murine LSCs. To this end, we generated a pooled lentiviral CRISPR library targeting 96 cell surface receptors that are upregulated on murine MLL-AF9 LSC compared to normal granulocyte and macrophage progenitor (GMP) cells based on global gene expression data. The CRISPR library was transduced into Cas9-expressing murine c-Kit+MLL-AF9 AML cells and subsequently injected into sublethally irradiated (600 cGy) C57BL/6 mice. Two weeks later when the mice had developed leukemia, the bone marrow was harvested, and sgRNAs sequenced. A high reproducibility in sgRNA representation was observed between five biological replicates, demonstrating that the screen was robust. The screen ranked CXCR4 as the most critical cell surface receptor on AML stem cells with all five sgRNAs targeting Cxcr4 depleting more than 4-fold in vivo. Cxcr4 has previously been shown to be critical in several types of cancers including hematological malignancies. Moreover, CXCR4 has been shown to be important for homing of normal and leukemia stem cells in the bone marrow. To validate the role of CXCR4 in a microenvironment and cell intrinsic manner in AML, we disrupted Cxcr4 in c-Kit+MLL-AF9 AML cells using lentiviral vectors expressing sgRNAs targeting Cxcr4 and GFP as a marker gene. Whereas Cxcr4 disruption did not affect the growth and survival of the AML cells in in vitro cultures with standard cytokines, a 15-fold depletion (p = 0.006) of Cxcr4 sgRNA-expressing AML cells was observed 12 days post-transplantation in vivo. We next studied whether the loss of CXCR4 on the surface of leukemic cells would translate into a survival difference in vivo. Notably, the mice receiving sorted leukemia cells expressing Cxcr4 sgRNAs did not develop AML, demonstrating that CXCR4 is essential for LSCs in vivo. To investigate if this effect was due to a homing defect, we transplanted Cxcr4 sgRNA-expressing cells into sublethally irradiated mice and measured the number of AML cells in the bone marrow after 24 hours. A 2.5-fold reduction (p = 0.03) of leukemia cells expressing Cxcr4 sgRNA compared to the control sgRNA was observed in the bone marrow, suggesting that the depletion of leukemic cells in vivo was partially due to a homing defect. CXCL12, the most studied ligand of CXCR4, is a homeostatic chemokine widely expressed by several cell types in the bone marrow and exists both in a membrane and soluble form. Although the CXCR4-CXCL12 interaction is critical for the retention of normal hematopoietic stem and progenitor cells in the bone marrow, the role of CXCL12 in AML is less characterised. To study the role of different CXCL12-expressing bone marrow cell populations in AML development, we first transplanted c-Kit+MLL-AF9 leukemia cells into Cxcl12f/f-Tek-Cre+ mice, which are devoid of Cxcl12 expression in endothelial cells, and in Cxcl12f/f-Prx1-Cre+ mice, which lack expression of Cxcl12 in mesenchymal stem cells. No reduction in leukemia levels in the blood and bone marrow was observed with either of these mouse strains as recipients, indicating that Cxcl12 expression by endothelial cells and MSC is not critical for AML development. Next, we repeated the experiment using Cxcl12f/f-Ubc-Cre+mice, which lack Cxcl12 expression in all cells. Intriguingly, there was no reduction in leukemia levels in these mice. Instead, a significant 2-fold increase in leukemia levels in the peripheral blood (p=0.011) and a 1.5-fold increase in leukemia levels in the bone marrow (p=0.043) were observed 12 days after transplantation. These findings suggest that the CXCR4-CXCL12 interaction is dispensable for AML development and progression. Taken together, we here established an in vivo pooled CRISPR/Cas9 drop-out screen using murine LSCs and identified Cxcr4 as an essential regulator for LSCs. Further validations identified that CXCR4 has a previously uncharacterized CXCL12-independent essential role for MLL-AF9 AML cells. Ongoing studies are aimed at identifying the ligand that is essential for CXCR4 in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection of residual leukemic cells in patients with acute promyelocytic leukemia by the fluorescence in situ hybridization method: potential for predicting relapse

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 495-499 ◽  
Author(s):  
L Zhao ◽  
KS Chang ◽  
EH Estey ◽  
K Hayes ◽  
AB Deisseroth ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Low Frequencies

Abstract The translocation between chromosomes 15 and 17, t(15;17)(q22–24;q11– 21), is present in the bone marrow cells of most patients with acute promyelocytic leukemia (APL). Although conventional cytogenetic methods are useful for diagnosing this disease, difficulties are experienced in detecting residual disease among those patients who have achieved remission. In this study, we used the fluorescence in situ hybridization (FISH) method to attempt to detect residual leukemic cells in 10 APL patients in clinical remission. The duration of remission ranged from 2 to 93 months at the time of study. Multiple bone marrow samples were analyzed by FISH in most patients. In 6 patients, no cell with t(15;17) was found. These patients remain in complete remission at present (approximately 25 to 33 months since first studied by FISH). In 4 patients, low frequencies of cells with t(15;17) were observed in at least one bone marrow sample examined. All of these patients relapsed within 1 to 14 months. No cell with t(15;17) was identified by the conventional G-banding method in any sample. The FISH results correlated well with that of a two-round nested reverse transcription polymerase chain reaction assay that was performed on the same samples. Thus, our study suggests that FISH is potentially a useful tool for detecting residual APL cells and for identifying patients at high risk of relapse.

Nucleostemin Gene Expression in Acute Promyelocytic Leukemia Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4430-4430
Author(s):  
Farzaneh Ashrafi ◽  
Fatemeh Nadali ◽  
Ardeshir Ghavamzadeh ◽  
Kamran Alimoghaddam ◽  
Shahrbano Rostami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Normal Bone Marrow ◽  
Newly Diagnosed ◽  
Normal Bone ◽  
Ns Gene ◽  
Significant Difference

Abstract Abstract 4430 Background Nucleostemin (NS), a novel p53-binding protein has been shown essential for stem and cancer cell proliferation and implicated in oncogenesis. Nucleostemin expression had been shown in gastric cancer (SGC-7901) cells, human hepatocarcinoma (HepG2) cells, human cervical cancer (Hela) cells, human osteosarcoma (OS-732) cells. Aim This work designed to study the NS gene expression in bone marrow cells in acute promyelocytic leukemia (APL) patients and in normal bone marrow specimens. Materials &Methods We examined NS gene expression by Quantitative Real Time PCR in bone marrow specimens of 15 cases of APL patients, before treatment and in 4 bone marrow specimens of healthy donors of bone marrow transplantation. In the same samples of bone marrow aspiration morphology of smears was evaluated. Diagnosis of APL was based on morphology and positive PML/RARA in PCR. RT-PCR used to amplify the NS mRNA, and the GAPDH primer sets used for normalizing. For comparison of NS gene expreesion in 2 groups Mann-Whitney U test was used. Results 15 patients enrolled in this study, 11(73%) newly diagnosed APL and 4(27%) relapsed cases. Mean age of patients was 28.67±9.56 year. NS gene expressed in all bone marrow samples of APL patients. NS gene expressed in normal bone marrow specimens too. NS gene expression in bone marrow of APL patients was significantly higher than normal bone marrows(p value =0.002) Fig 1. There was no significant difference in NS gene expression between newly diagnosed and relapsed APL cases. Discussion According to the results of this study it seems that NS gene expressed in normal marrow. NS expression in adult bone marrow hematopoietic stem cells had been reported in previous reports and it had been shown that NS does not express in granulocytes and B lymphocytes. It seems that stem cells and proliferating cells in the normal marrow are the source of NS expression detected in normal marrow. NS expreesion in bone marrow of APL patients was significantly higher than normal marrow. In these patients before treatment marrow is replaced by undifferentiated blasts and promyelocytes. We concluded that NS expression in these cells were high. It had been shown that NS down regulation may lead to cell cycle exit. High expression of NS in APL patients can be used in future researches for finding new targeted therapies in this disease. Disclosures: No relevant conflicts of interest to declare.

Molecular Characterization of the Leukemic Niche in Chronic Myeloid Leukemia (CML) and Evaluation of a Leukemia / Niche Cross-Talk

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 908-908
Author(s):  
Djamel Aggoune ◽  
Nathalie Sorel ◽  
Sanaa El Marsafy ◽  
Marie Laure Bonnet ◽  
Denis Clay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Myeloid Leukemia ◽  
Gene Expression Pattern ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Cd34 Cells

Abstract Abstract 908 There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software. Results: TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

scholarly journals Detection of residual leukemic cells in patients with acute promyelocytic leukemia by the fluorescence in situ hybridization method: potential for predicting relapse

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 495-499
Author(s):  
L Zhao ◽  
KS Chang ◽  
EH Estey ◽  
K Hayes ◽  
AB Deisseroth ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Acute Promyelocytic Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Low Frequencies

The translocation between chromosomes 15 and 17, t(15;17)(q22–24;q11– 21), is present in the bone marrow cells of most patients with acute promyelocytic leukemia (APL). Although conventional cytogenetic methods are useful for diagnosing this disease, difficulties are experienced in detecting residual disease among those patients who have achieved remission. In this study, we used the fluorescence in situ hybridization (FISH) method to attempt to detect residual leukemic cells in 10 APL patients in clinical remission. The duration of remission ranged from 2 to 93 months at the time of study. Multiple bone marrow samples were analyzed by FISH in most patients. In 6 patients, no cell with t(15;17) was found. These patients remain in complete remission at present (approximately 25 to 33 months since first studied by FISH). In 4 patients, low frequencies of cells with t(15;17) were observed in at least one bone marrow sample examined. All of these patients relapsed within 1 to 14 months. No cell with t(15;17) was identified by the conventional G-banding method in any sample. The FISH results correlated well with that of a two-round nested reverse transcription polymerase chain reaction assay that was performed on the same samples. Thus, our study suggests that FISH is potentially a useful tool for detecting residual APL cells and for identifying patients at high risk of relapse.

scholarly journals Complete Remission of t(11;17) Positive Acute Promyelocytic Leukemia Induced by All-trans Retinoic Acid and Granulocyte Colony-Stimulating Factor

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 39-45 ◽  
Author(s):  
J.H. Jansen ◽  
M.C. de Ridder ◽  
W.M.C. Geertsma ◽  
C.A.J. Erpelinck ◽  
K. van Lom ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Stimulating Factor

The combined use of retinoic acid and chemotherapy has led to an important improvement of cure rates in acute promyelocytic leukemia. Retinoic acid forces terminal maturation of the malignant cells and this application represents the first generally accepted differentiation-based therapy in leukemia. Unfortunately, similar approaches have failed in other types of hematological malignancies suggesting that the applicability is limited to this specific subgroup of patients. This has been endorsed by the notorious lack of response in acute promyelocytic leukemia bearing the variant t(11;17) translocation. Based on the reported synergistic effects of retinoic acid and the hematopoietic growth factor granulocyte colony-stimulating factor (G-CSF), we studied maturation of t(11;17) positive leukemia cells using several combinations of retinoic acid and growth factors. In cultures with retinoic acid or G-CSF the leukemic cells did not differentiate into mature granulocytes, but striking granulocytic differentiation occurred with the combination of both agents. At relapse, the patient was treated with retinoic acid and G-CSF before reinduction chemotherapy. With retinoic acid and G-CSF treatment alone, complete granulocytic maturation of the leukemic cells occurred in vivo, followed by a complete cytogenetical and hematological remission. Bone marrow and blood became negative in fluorescense in situ hybridization analysis and semi-quantitative polymerase chain reaction showed a profound reduction of promyelocytic leukemia zinc finger–retinoic acid receptor- fusion transcripts. This shows that t(11;17) positive leukemia cells are not intrinsically resistant to retinoic acid, provided that the proper costimulus is administered. These observations may encourage the investigation of combinations of all-trans retinoic acid and hematopoietic growth factors in other types of leukemia.

The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4694-4694
Author(s):  
Hong Xu ◽  
Ziqiang Zhu ◽  
Yiming Huang ◽  
Suzanne T. Ildstad
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Dose Titration ◽  
Great Promise ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Abstract 4694 Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF. A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB. We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

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