CD34 Blasts Research Articles

Study of significance of bone marrow microvessel density in myeloproliferative neoplasms in correlation with CD34 blasts, mast cell count and fibrosis.

Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell diseases characterised by myeloid cell growth from one or more lineages. Angiogenesis, in contrast to other subtypes, plays a substantial role in the pathophysiology of primary myelofibrosis (PMF). Research expressing the correlation of microvessel density (MVD), blasts, fibrosis and mast cell count in MPN cases are rarely conducted. We aimed to study the significance of MVD in correlation with CD34 blasts, mast cells and fibrosis in bone marrow biopsies of MPN patients. Methods: The current research was a cross sectional study conducted on 66 cases diagnosed as MPN during a six-year period. This comprised of 32 chronic myeloid leukemia (CML), 31 PMF and three essential thrombocythemia (ET) cases. Routine staining along with reticulin stain to look for fibrosis and immunohistochemistry (IHC) using CD34 and mast cell tryptase (MCT) were performed. Results: We found increased MVD in PMF, when compared to CML and ET (p = 0.042). Further, mean MVD was observed to be increased with high blast counts (p = 0.036). On follow up, raised mean MVD was seen in those cases with relapse/deceased as compared to disease-free patients, which was highly significant (p = 0.000). Conclusions: Increased MVD score was mostly associated with PMF subtype among all the MPNs. Further, higher MVD was observed to be associated with increased blast count and poor prognosis. With angiogenesis playing a critical role in disease outcome, we now have drugs to regulate angiogenesis that are supported by contemporary research. However, further studies with larger cohorts to establish the theranostic role of MVD in MPNs is recommended.

Study of significance of bone marrow microvessel density in myeloproliferative neoplasms in correlation with CD34 blasts, mast cell count and fibrosis

Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell diseases characterised by myeloid cell growth from one or more lineages. Angiogenesis, in contrast to other subtypes, plays a substantial role in the pathophysiology of primary myelofibrosis (PMF). Research expressing the correlation of microvessel density (MVD), blasts, fibrosis and mast cell count in MPN cases are rarely conducted. We aimed to study the significance of MVD in correlation with CD34 blasts, mast cells and fibrosis in bone marrow biopsies of MPN patients. Methods: The current research was a cross sectional study conducted on 66 cases diagnosed as MPN during a six-year period. This comprised of 32 chronic myeloid leukemia (CML), 31 PMF and three essential thrombocythemia (ET) cases. Routine staining along with reticulin stain to look for fibrosis and immunohistochemistry (IHC) using CD34 and mast cell tryptase (MCT) were performed. Results: We found increased MVD in PMF, when compared to CML and ET (p = 0.042). Further, mean MVD was observed to be increased with high blast counts (p = 0.036). On follow up, raised mean MVD was seen in those cases with relapse/deceased as compared to disease-free patients, which was highly significant (p = 0.000). Conclusions: Increased MVD score was mostly associated with PMF subtype among all the MPNs. Further, higher MVD was observed to be associated with increased blast count and poor prognosis. With angiogenesis playing a critical role in disease outcome, we now have drugs to regulate angiogenesis that are supported by contemporary research. However, further studies with larger cohorts to establish the theranostic role of MVD in MPNs is recommended.

Myeloid Leukemias Directly Suppress T Cell Proliferation Through STAT3 and Arginase Pathways

▪The immune-editing effect of myeloid leukemia has recently been reported in several studies. We previously demonstrated that the K562 leukemia-derived cell line suppresses T cell proliferation, which suggests that myeloid leukemia may function in a similar way to myeloid derived suppressor cells (MDSC). While the mechanism of suppression in leukemia is not fully understood, recent murine and human studies suggest that the STAT3 and arginase pathways play a key role in the immunosuppressive function of MDSC. We hypothesized that myeloid leukemia utilizes the MDSC STAT3 and arginase pathway to evade immune control, and block anti-leukemic immune responses.To evaluate the suppressive capacity of myeloid leukemia on T cell proliferation, we isolated CD34+ blasts and myeloid derived suppressor cells (MDSC: CD11b+CD14+) from blood of primary leukemia samples by FACS sorting (n=5). These cells were co-cultured with CFSE-labeled CD4+ T cells (n=9), previously isolated from healthy donor PBMCs using an automated cell separator (RoboSep). After stimulating with CD3/CD28 Dynabeads (Invitrogen, New York, USA) for 72 hours, proliferation was measured by CFSE dilution of the viable cell population. In three myeloid leukemias studied, CD4+ T cell proliferation was significantly suppressed in the presence of primary CD34 blasts and MDSC cells (p<0.001). Interestingly, CD34 blasts demonstrated a greater suppressive effect on T cells compared to MDSC cells for these samples (not statistically significant p=0.61). Next we repeated the proliferation assay using five leukemia cell lines: THP-1 and AML1 (derived from AML), K562 and CML1 (derived from CML), and the Daudi lymphoid-derived leukemia cell line. After staining with cell tracer dye and irradiating 100Gy, the cells were co-incubated with CFSE-labeled CD4+ T cells from healthy volunteers (n=6). We found that CD4+ T cell proliferation in the presence of the myeloid leukemia cell lines was significantly suppressed (mean proliferation 5.7±0.9% to 26.1±10.7%: p<0.0001 to 0.05) compared to lymphoid cell lines (mean proliferation 76.3±8.2%: p>0.05), consistent with the results obtained with the primary leukemia samples.To evaluate the impact of STAT3 and arginase on the immunosuppressive function of myeloid leukemia, the five cell lines were primed overnight with either arginase inhibitor (N(ω)-Hydroxy-nor-L-arginine; EMD Biosciences, Inc., California, USA) or two STAT3 inhibitors (STAT3 Inhibitor VI or Cucurbitacin I; EMD Millipore, Massachusetts, USA). Then, CD4+ T cells from healthy donors (n=3) were cultured with either (1) leukemia without any inhibitor (2) leukemia in the presence of inhibitor (3) leukemia primed with inhibitor. Priming leukemia with arginase inhibitor and STAT3 inhibitors almost completely abrogated their suppressive effect of T cell proliferation (p<0.001).We conclude that myeloid leukemia, like MDSC, directly immunosuppresses T cells, through STAT-3 and arginase. This finding may underlie the immune-editing of T cells by myeloid leukemia. Our results suggest that STAT3 inhibitors could be used to augment leukemia-targeted immunotherapy. Further investigation of T cell biology within the leukemia microenvironment is needed to further define immune editing mechanisms in myeloid leukemia. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

AC133 Expression in Egyptian Children With Acute Leukemia: Impact on Treatment Response and Disease Outcome

AC133 antigen is expressed restrictively in the immature subset of the CD34 cells. Hence, it is expected to be a valuable prognostic marker in acute leukemia. Sixty Egyptian children with acute leukemia were enrolled into this prospective study divided into 2 groups: 30 acute myeloblastic leukemia (AML) and 30 acute lymphoblastic leukemia (ALL) patients. Flow cytometric assessment of AC133 expression was performed on CD34 blast cells. AC133 was expressed in 66.7% and 40% of AML and ALL patients, respectively. AC133-positive expression was not associated with any of the studied standard prognostic factors. In AML, 80% of patients with poor clinical outcome (relapse or death) were positive for AC133 expression, whereas, all ALL patients who developed resistance as well as those who displayed poor clinical outcome had AC133-positive expression (P<0.05). Patients with positive AC133 expression had significantly shorter overall and disease-free survival times compared with AC133-negative patients in both ALL (P<0.001) and AML (P<0.05) groups. AC133 expression percentage was a reliable poor prognostic marker in ALL patients (P<0.0001). AC133-positive expression is an independent poor prognostic factor in childhood acute leukemia and could characterize a group of patients with resistance to standard chemotherapy, as well as high incidence of relapse and death.

WT1 monitoring in core binding factor AML: Comparison with specific chimeric products

Minimal residual disease may help to establish clinical decisions in patients with AML. WT1 offers the possibility to analyze those cases without currently known underlying genetic abnormalities. To compare the value of chimeric specific quantitative PCR with WT1 PCR in CBF acute leukemia, 445 samples from 96 AML (49 AML1-ETO+ and 47 CBFB-MYH11+) cases were included in the study. For each sample AML1-ETO or CBFB-MYH11 levels obtained using the conditions of the BIOMED group were compared with the results of WT1 levels using sensitive primers and conditions. Simultaneously, normal range expression of WT1 was established using RNA obtained from eight healthy donors. WT1 mutations were also investigated both at RNA and at the genomic level.The majority of CBF samples showed rises in WT1 levels (88.7%) at diagnosis. However, 18% of AML1-ETO showed WT1 levels below 250 copies in contrast with 5% CBFB-MYH11 cases. WT1 mutation was not detected in any case (70 diagnostic samples). We found correlation between WT1 levels at diagnosis and the CD34 blast population estimated by flow cytometry in CBFB-MYH11+ cases. We found no association between WT1 levels and clinical outcome. There was a high concordance between chimeric transcript analysis and WT1 levels in CR patients. Concordance was also high in relapsed patients (78% in AML1-ETO and 98% in CBFB-MYH11+ cases). Both WT1 and specific chimeric transcript identified and rescued false negative results of the other test.Additional studies are needed to determine whether the rare discrepancies are a reflection of the cooperative nature of WT1 overexpression or a consequence of the uneven distribution in the leukemic population. WT1 is a powerful MRD tool even in cases with currently available molecular targets.

Global Methylome of Normal and Malignant Hematopoietic Stem Cells.

Methylation of eukaryotic DNA is a major mechanism by which epigenetic changes regulate gene expression. Imprinting patterns associated with ontogenetic changes and epigenetic silencing are cell-type specific and can lead to profound functional consequences. Aside from physiologic methylation, altered methylation patterns have been found in malignant cells and during aging. It is likely that specific methylation patterns critical for self-renewal/pluripotence exist in hematopoietic stem cells. Due to technical limitations, most studies focused on a narrow empiric selection of promoters. However, new technologies have been developed to assess the “methylome” of a given cell/tissue. We stipulated that if we applied such a global technology to normal and malignant hematopoietic stem cells, we would discover a physiologic methylation pattern distinct from stem cell-derived hematologic malignancies. Here, genome-wide DNA methylation was determined using ChIP-DSL promoter array technology. This entailed using a 5-methyl-cytosine mAb to immunoprecipitate methylated chromatin fragments, which were annealed to a pool of 40K paired oligo probes corresponding directly to 20,000 human promoter regions, PCR amplified, and used to probe the promoter arrays to identify methylated promoters allowing for analysis of the global methylome. Previously, we validated the results of this technology through analysis of individual promoters by bisulfite sequencing. First, the array method was applied to analysis of highly purified CD34 cells from controls to establish a normal CD34-specific methylation pattern and compared to that of CD34 blasts derived from AML and CD34-expressing AML cell lines (KG1 and TF1) and more differentiated AML cell lines (K562, HL60, U937). Globally, methylation in AML patients (2.1% of all promoters studied) and malignant cell lines (2.5%) was greater than CD34 cells (0.6%). Overall, in comparison to CD34 cells, AML promoters demonstrated 1.4% and 0.9% hyper- and hypomethylation, respectively. Using ≥2.5 fold enrichment as a parameter, we identified 64 (0.33%) promoters concordantly methylated in normal CD34 cells, constituting a cellular ”methylation signature”. The methylomes of AML and CD34 cells showed concordant methylation levels in 26 (0.13%) promoters. Methylation profiles of the AML patients differed greatly; only 24 promoters demonstrated similar enrichment, suggesting that AML epigenotypes are variable, consistent with the heterogeneity of phenotype and clinical features. Examples of these hypermethylated promoters in AML include tumor suppressor genes (ETF1, Loc284948), apoptotic and stress-response genes (MAP2K7, PRKC1), and various loci previously demonstrated to play a role in AML (MAN1A1, PH2k-08, BCL11A, PAK1). The methylome of cell lines was remarkably dissimilar to normal CD34 cells, AML blasts and each other. Of the distinctively hypermethylated promoters in primary AML cells only 54% demonstrated concordant hypermethylation in KG1, 38% in TF1, 66% in U937, 43% in K5262 and 72% in HL60. Primary AML cells, KG1 and TF1 cells shared hypermethylation at 34% of loci. To our knowledge, our studies represent the first comprehensive report of global promoter methylation profiles of CD34 cells. Our results indicate the need for study of primary cells in study of disease-associated methylation patterns and point towards a variety of genes aberrantly silenced upon AML transformation.

Immunophenotypic characterization of myelopoiesis in early and late myelodysplastic syndromes: use of CD44 as an aid in early diagnosis.

We investigated the possibility that myeloid cells from the bone marrow (BM) of myelodysplastic patients differ in their expression of CD44 antigen compared with expression of the antigen in normal controls. In addition, two triple-surface marker assays incorporating, respectively, CD44/CD33/CD66 and CD33/CD34/HLA-DR were used to evaluate the degree of myeloid maturation and assess the number of blasts in BM by flow cytometry. Patients with early-stage myelodysplastic syndrome (MDS; RA [FAB classification]) have significantly decreased expression of CD44 on gated myeloid cells. In contrast, patients with late-stage MDS (RAEB and RAEB-T [FAB classification]) showed an elevated expression of CD44 and an increased number of CD34 blasts compared with early-stage MDS patients and normal controls. Late-stage MDS patients also had an increase in the immature myeloid compartment (CD66 weak expression) compared with early-stage MDS patients and normal controls. We have already included this assay as part of our MDS evaluation protocol alongside BM morphology and cytogenetics.