scholarly journals Flow Cytometric Detection of the Ki-67 Proliferation Index and the Bcl-2 Anti-Apoptotic Index in Myelodysplastic Syndromes and Acute Myeloid Leukemia, and Their Clinical Implications

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3444-3444
Author(s):  
Stefan G.C. Mestrum ◽  
Roanalis B.Y. Vanblarcum ◽  
Roosmarie J.M. Drent ◽  
Norbert C.J. De Wit ◽  
Bert T. Boonen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Cells ◽  
Ki 67 ◽  
Monocytic Cells ◽  
Proliferation Indices ◽  
Flow Cytometric ◽  
Apoptotic Markers ◽  
Blast Cells ◽  
Proliferative Indices

Abstract Introduction: Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry in classification of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To further advance the integration and objectification of flow cytometry for characterization of these malignancies, more dynamic parameters assessing cell behavioral characteristics could prove useful, such as proliferative and (anti-)apoptotic markers. Proliferation and (anti-)apoptosis are processes that are tightly related to the pathogenesis, progression and chemo-/immunotherapy response of cancers. As a result, proliferation and (anti-)apoptotic markers have proven their value as objective parameters in the field of histopathology for diagnostic and prognostic applications in solid tumors and lymphoma. Although use of proliferative and (anti-)apoptotic markers as objective parameters in the diagnostic process of MDS and AML was studied in the past decades, this did not result in the incorporation of these biomarkers in their clinical diagnosis. The recent developments in flow cytometric analyses now allow the quantification of proliferative and (anti-)apoptotic fractions at the level of individual maturing bone marrow cells. Therefore, we aim to determine the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices in maturing bone marrow cells in order to assess whether these parameters could have future clinical implications for the diagnostic work-up of MDS and AML. Methods: Fifty bone marrow aspirates from femoral heads of non-malignant cases, 20 aspirates of MDS patients and 20 aspirates of AML were included in this study. Ten-color flow cytometry in combination with a software-based maturation tool was used for analysis of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices of blast cells and during the erythro-, myelo-, and monopoiesis. Results: Ki-67 proliferative indices of blast cells and immature erythroid, myeloid and monocytic cells were significantly lower in MDS patients compared to the non-malignant cases, while the Bcl-2 anti-apoptotic indices were significantly elevated in these cells. Furthermore, the Bcl-2 anti-apoptotic indices were also increased in mature erythroid, myeloid and monocytic cells of MDS patients. The decreased Ki-67 proliferative indices and increased Bcl-2 anti-apoptotic indices in blast cells and erythroid, myeloid and monocytic cells were even more prominently observed in AML patients. Conclusions: The lowered Ki-67 proliferative indices and elevated Bcl-2 anti-apoptotic indices in blast cells and immature progenitor cells led to a better understanding of the pathophysiology of MDS and AML, and explained the low chemotherapy response of these patients. Side-effects of such therapies can also be explained by the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices. Moreover, the increase of the Bcl-2 anti-apoptotic fraction is an important factor in the progression of MDS to AML. Future studies on the clinical applications of these parameters for MDS and AML are necessary and can include many applications, such as prediction of chemo-/immunotherapy response, diagnostic and prognostic applications. Disclosures Ramaekers: Nordic-MUbio: Current Employment.

Using Multiparametric Flow Cytometry to Analysis the Immunophenotypic Abnormalities of Bone Marrow Cells in Patients with MDS-RAEB.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4856-4856
Author(s):  
Jing Huang ◽  
Xin Du ◽  
Suxia Geng ◽  
Maohua Zhou ◽  
Jianyu Weng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Surface Marker ◽  
Multiparametric Flow Cytometry ◽  
Hla Dr ◽  
Significant Difference ◽  
Blast Cells ◽  
Marrow Cells

Abstract Abstract 4856 Objective The multiparametric flow cytometry is becoming a very useful tool of the diagnosis and prognostication for patients with Myelodysplastic Syndrome(MDS). This study was aimed at using multiparametric flow cytometry to explore the immunophenotypic abnormalities of bone marrow cells from patients with RAEB. Methods We collected BM samples from 12 MDS-RAEB patients (6 male, 6 female, Median Age 67.5) and 20 non-MDS patients (11 male, 9 female, median age 32.5, 7 AA, 5 PNH, 3 IDA, 1 ALL, 2 CML, 2 MM). The multiparametric flow cytometric analysis was performed using an extensive panel of monoclonal antibodies. We used the conventional and secondary gating strategies to analysis the BM cells compartments such as the percents of blast cells and the expression of lineage and maturation-associated antigens of BM hemopoietic cells quantified. Results Compared with the non-MDS group, the proportion of blast cells increased significantly in the MDS-RAEB group (P=0.001), but the percentages of nucleated erythrocyte, lymphocyte, monocyte and granulocyte were no significant difference (P=0.954, P=0.893, P=0.730 and P=0.182). As the percentage of blasts cells increasing, the survival time became shorter (13 ± 6 vs 35 ± 15 months; P=0.02). The expressions of haemopoietic stem/progenitor cell surface marker CD34+ and T lymphocyte surface marker CD7+ on blast cells were much higher by secondary gating method than non-MDS group (P=0.009, P=0.002, respectively), while no significant difference of the expression of CD56 (P=0.375). The expressions of CD7+ and CD56+ on lymphocyte were no significant difference between the two groups (P=0.195, P=0.369, respectively), however the expression of CD19+ may be different (P=0.039). The expressions of CD33+ and CD13+ on granulocyte were no significant difference between the two groups (P=0.289, P=0.744, respectively). However, the expression levels of CD15+CD11b+, CD15+CD11b-, CD10+, HLA-DR, CD56+ in the MDS-RAEB group were significantly higher than those in the non-MDS group, specially, the levels of CD10+, HLA-DR and CD56+ were much higher (P=0.016, P=0.011, P=0.005, P=0.005 and P=0.005, respectively) and these patients showed a shorter median overall survival (15 ± 5 vs 36 ± 10 months; p = 0.03). Conclusions The percentage of blast cells increased in MDS-RAEB patients and the expressions of CD34+, CD7+ on blast cells and the expressions of CD10+, HLA-DR and CD56+ on granulocyte is higher than non-MDS group. It will be necessary to increase the number of cases (MDS-RAEB) to confirm our findings. Using multiparametric flow cytometry can find the immunophenotypic abnormalities more sensitively, accurately and objectively, and this new approach could provide much more useful information in the diagnosis and prognosis of MDS-RAEB patients. Disclosures No relevant conflicts of interest to declare.

Human Primary Mantle Cell Lymphoma Can Be Established in NOD/SCID/IL2Rγ-Null Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1565-1565
Author(s):  
Sunil Iyengar ◽  
Linda Ariza-McNaughton ◽  
Andrew James Clear ◽  
Amy Roe ◽  
Debra Lillington ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cyclin D1 ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Xenograft Model ◽  
Scid Mice ◽  
Ki 67 ◽  
Nsg Mice ◽  
Novel Drugs

Abstract Abstract 1565 Background: The relatively low incidence of MCL (0.55 per 100,000) poses a challenge for effective evaluation of novel therapies in patients affected by this aggressive and incurable lymphoma. A xenograft model of human MCL provides a useful model for pre-clinical evaluation of novel drugs, rational drug combinations and biomarkers. The only mouse xenograft model of primary human MCL reported was established by injecting CD19 selected PBMCs in a subcutaneously implanted human embryonic bone graft in SCID mice and SCID mice without subcutaneous bone grafts did not show engraftment. NOD/SCID/IL2Rγ chain null (NSG) mice, which lack mature T or B cells and are also deficient in NK cells, permit engraftment of a wider range of primary human cells compared to SCID mice. In view of recent reports of successful engraftment of human CLL in NSG mice, we hypothesised that primary human MCL can be established in these mice. Methods: We initially introduced luciferase transduced Jeko-1 cells at 2 concentrations – 0.5 and 2 million cells by tail vein injection of 8 to 12 week old γ-irradiated (3.75 Gy) NSG mice in an attempt to track the kinetics and distribution of MCL cells. Bioluminescent imaging (BLI) following injection of luciferin was performed weekly for 4 weeks. We then injected NSG mice (4 replicates) with 107, T-cell depleted, previously cyropreserved human MCL cells from 3 patients. Mice were bled at 3, 6 and 12 weeks and flow cytometry was performed on PBMCs for human CD45, CD5 and CD20. Mice were sacrificed at 20 weeks and immunohistochemistry (IHC) for human CD20 and cyclin D1 was performed on formalin fixed paraffin embedded spleen, ileo-caecal junction and liver while FACS for human CD45, CD5 and CD20 was performed on bone marrow cells flushed from the femur. Tissues harbouring CD20 and cyclin D1 positive cells were stained with Ki-67 to assess proliferation and FISH for t(11;14) was performed on fresh cells isolated from the spleen to further confirm engraftment. Results: NSG mice injected with Jeko-1 cells showed rapid engraftment at both concentrations (0.5 and 2 million) on assessment with BLI, with a more rapid progression after 3 weeks in mice injected with 2 million cells. Mice had to be sacrificed at 4 weeks because of illness. Bioluminescence was seen primarily in the bone marrow, spleen and along the spine. Consistent engraftment was also seen in all mice injected with sample 1 - PBMCs from a patient in 1st relapse with blastoid morphology, classic immunophenotypic features and the IgH:CCND1 translocation. CD5 and CD20 double positive cells were consistently detected at 6 and 12 weeks in the peripheral blood of all 4 mice examined. Mice were not visibly ill at 20 weeks but had gross splenomegaly at sacrifice. No lymph node or abdominal masses were found. A clear human CD5/CD20 population was found on flow cytometry of bone marrow cells. The splenic architecture was disrupted in mice that engrafted, compared to those that did not and a heavy infiltration of CD20 and cyclin D1 positive cells was found with proliferation estimated at 35–40% by Ki-67 staining. Interphase FISH on fresh cells derived from the spleen showed the IGH/CCND1 [t(11;14)] rearrangement in all cells examined. Scattered CD20 positive cells were observed in the liver but no polyps or submucosal infiltration was found in the ileo-caecal regions of these mice. NSG mice injected with samples 2 and 3 had no evidence of engraftment in peripheral blood, bone marrow or other tissues. Conclusion: Our studies demonstrate that a mouse model of human MCL can be established in NSG mice and are encouraging for developing this model for pre-clinical evaluation of novel drugs. The lymphoma cells that engrafted were from a patient with relapsed, blastoid MCL suggesting that a more aggressive phenotype may favor engraftment as seen with AML xenografts in NSG mice. Ongoing studies are examining additional patient samples and the need for T cell depletion. This model will also provide an opportunity to investigate the role of tumor-initiating side populations in this disease. Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

scholarly journals Bone Marrow Immunophenotyping By Flow Cytometry in Refractory Cytopenia of Childhood

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1916-1916
Author(s):  
Anna M. Aalbers ◽  
Marry M. van den Heuvel-Eibrink ◽  
Irith Baumann ◽  
Michael Dworzak ◽  
Henrik Hasle ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Pediatric Patients ◽  
Healthy Controls ◽  
Monosomy 7 ◽  
Flow Cytometric Immunophenotyping ◽  
Flow Cytometric ◽  
Blast Cells ◽  
B Cell Precursors

Abstract Refractory cytopenia of childhood (RCC) is the most common type of childhood myelodysplastic syndrome (MDS). Because the majority of patients with RCC have a normal karyotype and a hypocellular bone marrow, differentiating RCC from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia ((v)SAA) can be challenging. The histopathological differentiation between RCC and (v)SAA is mainly based on the presence of patchy erythropoiesis with defective maturation and/or the presence of micromegakaryocytes in RCC, and the absence of erythropoiesis and megakaryopoiesis in (v)SAA. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable additional diagnostic tool in differentiating MDS from non-clonal cytopenias in adults, but in childhood MDS, only a limited number of flow cytometric immunophenotyping studies have been reported. Here, we performed the first comprehensive flow cytometric analysis of myeloid and lymphoid progenitor cells, maturing granulocytes, monocytes and erythrocytes in bone marrow aspirates obtained from a large prospective cohort of 81 RCC patients, collected by the European Working Group of MDS in Childhood (EWOG-MDS). RCC was diagnosed according to WHO criteria and confirmed by central review of bone marrow morphology and histology. Bone marrow aspirates obtained from healthy adult individuals (n=9) and pediatric patients with (v)SAA (n=17) or advanced MDS (n=7) were used as controls. We employed a 7-tube, 6-color flow cytometry protocol, and data analysis was performed largely in line with European LeukemiaNet recommendations for flow cytometry in MDS. RCC patients had a strongly reduced myeloid compartment compared to healthy controls, but not as severe as (v)SAA patients. In the majority of RCC patients, immature myeloid and/or lymphoid cells were reduced in numbers, but still detectable, while in the vast majority of (v)SAA patients, myeloid blast cells and CD34+B-cell precursors were absent: both cell types were absent in 27 of 81 RCC patients (33%) and in 15 of 17 (v)SAA patients (88%). Furthermore, the number of flow cytometric abnormalities was significantly higher in RCC patients than in healthy controls and in pediatric patients with (v)SAA, but lower than in advanced MDS. The most frequently occurring flow cytometric abnormalities in RCC were the heterogeneous expression of CD71 and CD36 on immature erythroid cells. Two or more flow cytometric abnormalities were detected in 49 of 81 RCC patients (60%), and in 2 of 17 (v)SAA patients (12%). If a diagnosis of RCC was considered if myeloid blast cells and/or CD34+ B-cell precursors were present, or if two or more immunophenotypic abnormalities were detected, 61 of 81 RCC patients (84%) could be correctly classified, using histopathology as gold standard, whereas the specificity of this combination, using (v)SAA as a control group, was 76% (13 of 17 (v)SAA patients). No significant associations were detected between the presence of flow cytometric abnormalities (defined as two or more abnormalities) in RCC patients and age or sex, the presence of HLA-DR15, bone marrow cellularity, transfusion dependency at diagnosis, the presence of a PNH clone, or skewing of the T-cell receptor Vβ chain. Of interest, although only 5 patients with monosomy 7 were included in the present study, all of them displayed at least 2 flow cytometric abnormalities. RCC with monosomy 7 confers a high risk of progression to AML, but histopathologically, no differences can be detected between RCC cases with or without monosomy 7. In conclusion, our results indicate that, although flow cytometric abnormalities in RCC patients are present at a relatively low frequency, flow cytometric immunophenotyping might be a relevant addition to histopathology and cytogenetic analysis in the diagnostic work-up of RCC. Disclosures No relevant conflicts of interest to declare.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

scholarly journals Aberrant Ki-67 expression in normal bone marrow revealed by multiparameter flow cytometric analysis

Cytometry ◽  
1991 ◽  
Vol 12 (1) ◽  
pp. 50-63 ◽  
Author(s):  
Dirk R. Van Bockstaele ◽  
Jar Lan ◽  
Hans-W. Snoeck ◽  
Marcel L. Korthout ◽  
Robrecht F. De Bock ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometric Analysis ◽  
Normal Bone Marrow ◽  
Ki 67 ◽  
Normal Bone ◽  
Flow Cytometric

scholarly journals Simultaneous flow cytometric DNA and volume measurements of bone marrow cells as sensitive indicator of abnormal proliferation patterns in rat leukemias.

1979 ◽  
Vol 27 (1) ◽  
pp. 398-403 ◽  
Author(s):  
G Valet ◽  
B Fischer ◽  
A Sundergeld ◽  
G Hanser ◽  
V Kachel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Parameter Analysis ◽  
Alpha Cell ◽  
Brown Norway ◽  
Normal Limits ◽  
Flow Cytometric ◽  
Volume Measurements ◽  
Marrow Cells

Simultaneous flow cytometric DNA and volume analysis of normal rat bone marrow cells shows three populations of nucleated cells with different mean volume. Each of these populations proliferates in a distinct cell cycle (alpha, beta, gamma). Normally the alpha-cell cycle has the highest amplitude, the beta-cell cycle is intermediate, and the gamma-cell cycle is low. The alpha-cell cycle was very significantly depressed and the beta + gamma-cell cycle was increased in three different rat leukemias (L5222, Shay, BNML), growing on three different rat strains (BDIX, Holtzmann, Brown Norway). The two parameter analysis further revealed that cells of the beta + gamma-cell cycle were slightly hyperdiploid and hypertetraploid in leukemic animals. The decrease of the alpha-cell cycle and the hyperploidies were more sensitive indicators for the abnormal proliferation pattern than the analysis of one parameter DNA distributions which remained within normal limits in all three leukemias.

Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽  
2017 ◽  
Vol 27 (1) ◽  
pp. 49-59 ◽  
Author(s):  
X Yang ◽  
J Yang ◽  
X Li ◽  
W Ma ◽  
H Zou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Lupus Nephritis ◽  
Bone Marrow Cells ◽  
Tfh Cells ◽  
Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

scholarly journals Reutilization of Thymidine in Various Groups of Rat Bone Marrow Cells

Blood ◽  
1971 ◽  
Vol 37 (3) ◽  
pp. 340-348 ◽  
Author(s):  
H. J. HEINIGER ◽  
L. E. FEINENDEGEN ◽  
K. BüRKI
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Single Cells ◽  
Marrow Cell ◽  
Lymphoid Cells ◽  
Cell Populations ◽  
Salvage Pathway ◽  
Blast Cells ◽  
Autoradiographic Technique ◽  
Rat Bone

Abstract Thymidine reutilization was studied in single cells of the rat bone marrow. Using 3H-TdR in parallel with 125I-UdR in conjunction with the autoradiographic technique, cells of the erythrocytic series, the megakaryocytic group, and the lymphoid cells were analyzed. Reutilization of thymidine was observed only in those cells known to synthesize DNA. An estimate of the amounts of the thymidine reutilized by the salvage pathway indicated that approximately 40-60 per cent of the thymidine in the blast cells is supplied from DNA of dead cells. This value is similar to that reported previously for whole bone marrow cell populations, suggesting the presence of a common thymidine pool within the bone marrow.

Mutant CXCR4 Identified in Neutropenic Patients with Myelokathexis Impairs Survival of Human Myeloid Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3071-3071
Author(s):  
Vahagn Makaryan ◽  
David C. Dale ◽  
Andrew A. Aprikyan
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Survival ◽  
Mutational Analysis ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Receptor Internalization ◽  
Morphological Examination ◽  
Exon 2 ◽  
Whim Syndrome

Abstract Myelokathexis (WHIM syndrome) is a very rare hematopoietic congenital disorder that is characterized by extremely low level of circulating neutrophils in peripheral blood. It is inherited as an autosomal dominant disease and is diagnosed in early childhood. These patients may have hypogammaglobulinemia and suffer from recurrent infections associated with warts. The hallmark of myelokathexis is a hyperplastic bone marrow and hypersegmented neutrophils with nuclear lobs connected with thin filaments. Myelokathexis is due to a characteristic retention of mature neutrophils in bone marrow, which are not being released to peripheral circulation. We and others reported abnormal cell survival characteristics and impaired bcl-x expression in bone marrow myeloid cells of myelokathexis patients that was partially restored by G-CSF treatment. Recently, it has also been reported that heterozygous truncation mutations in the carboxyterminal domain of the CXCR4 gene, a sole receptor for SDF-1 chemokine, were observed in most, but not all of the families with WHIM syndrome. Subsequently, an impaired receptor internalization and increased chemotaxis towards SDF-1 have been observed in cells expressing truncated CXCR4. Nevertheless, the mechanism of mutant CXCR4 induced myelokathexis remains largely unknown. We performed mutational analysis of the CXCR4 gene in 3 unrelated families with myelokathexis and identified a previously reported R334ter truncation mutation in exon 2 in two of the families. In addition, two silent polymorphisms have been identified in exon 2 of the CXCR4 gene in one of these patients. The third family with afflicted mother and son had a new mutation in the CXCR4 carboxyterminal domain, which resulted in deletion of the last 16 amino acids and subsequent frame shift. None of these mutations were observed in healthy volunteers examined. Since the morphological examination by electron microscopy and flow cytometry analysis of bone marrow cells from some of these patients revealed characteristic apoptotic features, we examined the effect of mutant CXCR4 gene expression on survival of human promyelocytic HL-60 cells. Preliminary data demonstrated that human promyelocytic cells transfected with truncated CXCR4 exhibited impaired cell survival characteristics compared with control HL-60 cells transfected with intact CXCR4. The truncated, but not wild type CXCR4 also increased apoptosis in HL-60 cells induced to differentiate along the granulocytic pathway as determined by flow cytometry of annexin V labeled cells. Thus, these data link together the abnormal survival of proliferating and differentiating myeloid cells in WHIM syndrome with mutant CXCR4 expression. Current studies are focused on elucidation of specific signaling pathways mediating mutant CXCR4-triggered myelokathexis.

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