CD34 Percentage Research Articles

Comparison of Biosimilar Filgrastim (Nivestym) Versus Originator Filgrastim (Neupogen) for Peripheral Blood Stem Cell Mobilization for Allogeneic Hematopoietic Stem Cell Transplantation

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is an optimal and potentially curative therapy for patients with hematologic malignancies. Granulocyte colony-stimulating factor (G-CSF) is used for the mobilization of peripheral blood stem cells (PBSCs) in allogeneic donors, and an optimal CD34 cell dose plays a critical role in successful transplantation. Nivestym, a biosimilar G-CSF to the originator Filgrastim (Neupogen), is now used by multiple institutions as it has become more available; however, there is a lack of data in this regard among healthy donors for allogeneic HSCT mobilization. In this study, we aim to compare the efficacy of Nivestym compared to Neupogen for allogeneic donor PBSC mobilization. Methods: For this retrospective single-center study, we included all (n=541) adult allogeneic HSCT donors at the University of Kansas Medical Center receiving Nivestym (January 2013-July 2020) or Neupogen (July 2020-June 2023) for donor PBSC mobilization. We conducted a bivariate analysis utilizing the ANOVA test. Statistical analyses were performed using SPSS version 28. Statistical significance was determined at p<0.05. Results: Our study included 541 allogeneic HSCT donors who received Neupogen (n=345, 64%) or Nivestym (n=196, 36%) for PBSC mobilization. The median age was 47 (range 17-76) years. The median donor weight was 86 (95% CI 87-91) kilograms. Donors receiving Neupogen had similar pre-GCSF WBC count (median 45 vs 47 K/uL, p=0.739), pre-GCSF CD34 percentages (median 17% vs 17%, p=0.527), and pre-GCSF circulating CD34 count (median 74 vs 74 million cells/kg, p=0.842) compared to donors receiving Nivestym. Neupogen recipients had similar median PBSC product total neutrophil count (TNC, 6.8 vs 6.5 K/uL, p=0.347), product CD34 percentage (75% vs 79%, p=0.068), absolute CD34 count (503 vs 533 million cells, p=0.636), and final CD34 dose (6.1 vs 6.3 million cells/kg, p=0.365) compared to Nivestym recipients. Seven percent (n=28) of donors needed two days of PBSC collection to achieve the target PBSC dose, with a statistically similar proportion noted in Neupogen (n=25, 7.3%) and Nivestym (n=13, 6.6%) cohorts. In a subgroup analysis stratified by age, donors aged 35 or older (n=154) had similar responses in all mentioned variables (p>0.05); however, for donors under the age of 35 years, the CD34 dose was higher in donors receiving Neupogen compared to Nivestym (median 6.9 vs 6.3 million cells/kg, p=0.044). Most of the PBSC collections (98%) were completed in one day, except for five donors in both Neupogen (5%) and Nivestym (8.5%) cohorts. Conclusions: Biosimilar G-CSF (Nivestym) demonstrated similar efficacy for peripheral blood stem cell mobilization compared to the originator G-CSF (Neupogen) among allogeneic HSCT donors. In donors aged 35 years or younger, a slightly lower PBSC product CD34 count was noted with Nivestym compared to Neupogen.

Effect of Duration of Root Canal Infection on the Ability of Dentin-Pulp Complex Regeneration of Immature Permanent Teeth: An Animal Study

IntroductionPersistent infection is always considered the most important reason for the failure of dentin-pulp complex regeneration. The present study aimed to evaluate the effect of the duration of root canal infection (from 1–12 weeks) on the ability of dentin-pulp complex regeneration. MethodsIn this animal study, 64 roots of immature premolar teeth of 4 dogs were randomly divided into the following groups: the positive control group, 8 root canals treated with the regenerative endodontic procedure (REP); the negative control group, 12 infected root canals; the intervention groups, 36 root canals infected with supragingival plaque (1, 3, 6, and 12 weeks) and treated with REP; and an additional positive control group, 8 normal roots. After 3 months, the teeth were investigated by radiographic images and immunohistochemical staining (CD31, CD34, and S100 markers). In addition, DSPP gene expression was assessed using a real-time polymerase chain reaction technique. ResultsBased on radiologic evaluation among the intervention groups, the highest root canal development (length and width) occurred in the intervention group of 1 week, and the lowest radiologic results were in the intervention groups of 6 and 12 weeks (1-way analysis of variance, P-value < .05). There was a significant difference between the groups in terms of CD31, CD34, S100, and DSPP expression percentage (1-way analysis of variance, P-value < .05); the highest and lowest expression percentages belonged to the 1- and 12-week groups, respectively, among the intervention groups. ConclusionsThis study demonstrated that long root canal infection decreased the ability of the body to regenerate the dentin-pulp complex.

Impact of Umbilical Cord Milking on Hematological Parameters in Preterm Neonates With Placental Insufficiency.

BackgroundData is still lacking about the expediency of umbilical cord milking (UCM) in preterm neonates born to mothers with placental insufficiency (PI).ObjectiveTo study the effect of UCM in preterm neonates who had ante-natal evidence of placental insufficiency on peripheral blood cluster of differentiation 34 (CD34) percentage, hematological indices, and clinical outcomes.MethodsPreterm neonates, <34 weeks' gestation, born to mothers with evidence of placental insufficiency that underwent UCM (PI+UCM group) were compared with historical controls whose umbilical stumps were immediately clamped [PI+ICC (immediate cord clamping) group] in a case-control study. Peripheral blood CD34 percentage as a measure of hematopoietic stem cell transfusion was the primary outcome. Early and late-onset anemia; polycythemia; frequency of packed red blood cells (PRBCs) transfusion during NICU stay; peak total serum bilirubin (TSB); incidence of phototherapy, admission rectal temperature; first 24 h hypothermia and hypoglycemia; episodes of hypotension and need for volume expander boluses and inotropic support during the first 24 h of age; duration of oxygen therapy; bronchopulmonary dysplasia (BPD); severe intra-ventricular hemorrhage (IVH); necrotizing enterocolitis (NEC); culture-proven late-onset sepsis; length of hospital stay; and in-hospital mortality were secondary outcomes.ResultsIn preterm infants with placental insufficiency, umbilical cord milking was associated with greater peripheral blood CD34 percentage, hemoglobin levels initially and at postnatal age of 2 months, alongside significantly shorter duration of oxygen therapy compared with ICC group. Frequency of packed RBCs transfusion during hospital stay was comparable. Neonates in UCM group had a greater peak TSB level during admission with significantly higher need for phototherapy initiation compared with ICC. Logistic regression, adjusted for gestational age, revealed that UCM resulted in greater CD34 percentage, higher initial hemoglobin level, higher peak serum bilirubin, significant increase of phototherapy initiation, and higher hemoglobin level at 2 months.ConclusionsUCM in preterm neonates born to mothers with placental insufficiency was feasible and resulted in greater CD34 percentage, higher initial hemoglobin level, higher peak serum bilirubin, significant increase of phototherapy initiation, and higher hemoglobin level at 2 months.

Effect of delayed cord clamping on stem cell transfusion and hematological parameters in preterm infants with placental insufficiency: a pilot randomized trial.

The feasibility of delayed cord clamping (DCC) in preterm infants with placental insufficiency (PI) is questionable. We aimed to study the effect of DCC on stem cell transfusion, hematological parameters, and clinical outcomes in preterm infants born to mothers with PI. Preterm infants, < 34weeks' gestation, born to mothers with PI were randomized based on the timing of umbilical cord clamping into delayed clamping for 60s (DCC group) or immediate cord clamping (ICC group) groups at time of birth. CD34 percentage as a marker of stem cell transfusion, early and late-onset anemia, hypothermia, hypotension, polycythemia, hyperbilirubinemia, duration of oxygen therapy, bronchopulmonary dysplasia, intra-ventricular hemorrhage, necrotizing enterocolitis, sepsis, mortality, and length of hospital stay were compared between studied groups. We found that peripheral blood CD34 percentage was significantly higher in DCC compared with that in the ICC group (median (IQR) of 0.5 (0.40-0.7) versus 0.35 (0.20-0.5), p = 0.004). Infants in the DCC group had significantly lower episodes of anemia of prematurity at 2months, red blood cell transfusion, and shorter duration of oxygen therapy compared with those in the ICC group.Conclusion: In conclusion, DCC compared with ICC increased stem cell transfusion and decreased early- and late-onset anemia in preterm infants with placental insufficiency.Trial registration: NCT03731546 www.clinicaltrials.gov What is Known: • Delayed cord clamping has been recommended by the American Academy of Pediatrics as a standard of care practice during delivery of preterm infants. • The feasibility of DCC in preterm infants with placental insufficiency (PI) is uncertain. What is New: • This randomized controlled trial demonstrated that DCC in the delivery room care of preterm infants born to mothers with placental insufficiency increased stem cell transfusion and decreased early- and late-onset anemia.

Detailed analysis of bone marrow from patients with ischemic heart disease and left ventricular dysfunction: BM CD34, CD11b, and clonogenic capacity as biomarkers for clinical outcomes.

Bone marrow (BM) cell therapy for ischemic heart disease (IHD) has shown mixed results. Before the full potency of BM cell therapy can be realized, it is essential to understand the BM niche after acute myocardial infarction (AMI). To study the BM composition in patients with IHD and severe left ventricular (LV) dysfunction. BM from 280 patients with IHD and LV dysfunction were analyzed for cell subsets by flow cytometry and colony assays. BM CD34(+) cell percentage was decreased 7 days after AMI (mean of 1.9% versus 2.3%-2.7% in other cohorts; P<0.05). BM-derived endothelial colonies were significantly decreased (P<0.05). Increased BM CD11b(+) cells associated with worse LV ejection fraction (LVEF) after AMI (P<0.05). Increased BM CD34(+) percentage associated with greater improvement in LVEF (+9.9% versus +2.3%; P=0.03, for patients with AMI and +6.6% versus -0.02%; P=0.021 for patients with chronic IHD). In addition, decreased BM CD34(+) percentage in patients with chronic IHD correlated with decrement in LVEF (-2.9% versus +0.7%; P=0.0355). In this study, we show a heterogeneous mixture of BM cell subsets, decreased endothelial colony capacity, a CD34+ cell nadir 7 days after AMI, a negative correlation between CD11b percentage and postinfarct LVEF, and positive correlation of CD34 percentage with change in LVEF after cell therapy. These results serve as a possible basis for the small clinical improvement seen in autologous BM cell therapy trials and support selection of potent cell subsets and reversal of comorbid BM impairment. http://www.clinicaltrials.gov. Unique identifiers: NCT00684021, NCT00684060, and NCT00824005.

Myeloid Derived Suppressor Cells in Acute Leukemia and Its Association with Conventional Cytogenetic and Molecular Risk Factors

AbstractAbstract 1446Myeloid derived suppressor cells (MDSC) are a relatively novel population of myeloid derived immunomodulatory cells which have been reported to be associated with poor prognosis in solid tumors. Preliminary data suggest that this population is also increased in high risk newly diagnosed patients with chronic myeloid leukemia. There is no reported data evaluating the proportion of these cells in newly diagnosed acute leukemia and their association with conventional risk factors in acute leukemia. We undertook a prospective study and evaluated peripheral blood (PB) samples of healthy controls (HC; n=10) and newly diagnosed leukemia patients (B-ALL=49, T-ALL=17, APL= 47 and AML=137) for presence of MDSC. To detect MDSC, PB mononuclear cells were isolated and labeled with a panel of monoclonal antibodies to CD14, CD33, HLADR and CD11b directly conjugated with FITC, PE, PerCP and APC respectively. Cells were then washed and analyzed using FACSCalibur and CellQuestPro software. MDSC subset was defined as previously reported by the phenotype of CD14-HLADR-CD33+ CD11b+ (Nature Reviews Immunol, 2009; Figure 1A summarizes the gating strategy). The median percentage of MDSC in PB samples of HC and newly diagnosed B-ALL, T-ALL, APL and AML was 0.7% (range: 0.11–0.97), 0.14% (range: 0.01–2.76 ), 0.05% (range: 0.01–0.9 ), 0.58% (range: 0.04–5.94) and 1.07 % (range:0.01–31.1% ) respectively (Figure 1B). The median values were significantly lower in ALL and APL when compared with AML (P=0.001 and 0.005 respectively). There was no significant association among cases with ALL and APL with conventional risk factors. Among newly diagnosed cases of AML there is significant heterogeneity in the proportion of MDSC with 77 (56%) having values above the median of HC (Figure 1B). There was weak but significant positive correlation of MDSC with WBC counts at diagnosis (Figure 1C) and a significant negative correlation with CD34 percentage at diagnosis (Figure 1D). Comparison of AML cases in the highest quartile (MDSC>3.74%; n=34) with the remaining patients (MDSC≤3.74%; n=103) is summarized in Table 1. AML patients in the highest quartile of MDSC had significantly higher WBC count and a significantly lower CD34 count at diagnosis; there were also a significantly higher proportion of cases that were NPM1 and FLT3 mutation positive in this quartile as was the high risk NPM1+and FLT3 mutation+ subset (Table 1). The clinical follow up in this series of AML patients was short with too few events to analyze the impact of this population on clinical outcomes.In summary MDSC at diagnosis in PB samples is low in ALL and APL. There is significant heterogeneity among cases with AML and a significant association with WBC count at diagnosis, NPM1 and FLT3 mutations and a significant negative correlation with CD34 percentage at diagnosis. The biological relevance and impact of this population on clinical outcomes warrants a more detailed study. [Display omitted] Table 1Comparison of conventional risk factors among cases in the highest quartile of MDSC expression (>3.74%) versus the rest in newly diagnosed AMLVariableMDSC > 3.74% N=34 n(%)/median (range)MDSC ≤ 3.74% N=103 n(%)/median (range)P-valueAge39 (2-76)42 (1-78)0.67WBC25.2 (2.2-269.6)15.4 (0.6-298.9)0.04CD3446 (0.6-94.5)70 (0.1-99.5)0.009Blast index3.3 (0.03-256.12)1.3 (0.01-225.3)0.83CTG high risk group3 (8.8%)13 (12.6%)0.55NPM1 positive6 (17.6%)6 (5.8%)0.034FLT3 positive5 (14.7%)5 (4.9%)0.056NPM1 and FLT3 positive5(14.7%)2 (1.94%)0.003 Disclosures:No relevant conflicts of interest to declare.

Thawed autologous peripheral blood stem cells require modified quantification methods for hematopoietic progenitor cell evaluation

The aim of this study was first, to analyze the post-thaw progenitor assays usually performed on peripheral blood stem cell autografts and second, to achieve standardization with improved flow cytometric and CFU-GM assays. In the first part of the study (n=79), recovery and Intraclass Correlation Coefficient (ICC) of total nucleated cells, CD34 and CFU-GM were analyzed before and after cryopreservation. In the second part (n=20), evaluation methods were modified : the washing step was suppressed in the flow cytometric method and 500 CD34 were plated compared to 4×10(4) total nucleated cells in the CFU-GM assay. The recovery rates were analyzed and the CFU-GM results were regarded as reliable when 30-100 colonies were observed, according to the manufacturer recommendation. The analysis of the first part showed an ICC that was perfect for total nucleated cells (0.93), substantial for CD34 (0.67) and fair for CFU-GM (0.25). Median CD34 recovery was 112.6% (29.9-222%). The CFU-GM median recovery was 31.7% (0.19-142%) leading to reliable results for 27 grafts. In the second part, the median CD34 recovery was 85.75% (54-99%). No recovery over 100% was observed. The CFU-GM assay led to 18 out of 20 evaluable autografts when 500 CD34 were seeded, compared to 10 out of 20 when total nucleated cell were seeded. Avoiding cell washing in the flow cytometric method limited the overestimate of the CD34 percentage. Plating 500 thawed CD34 improved reliability of the results and allowed a better standardization of the assay.

The Prognostic Value of CD34 Expression In Acute Myeloid Leukemia. A Mystery Solved

AbstractAbstract 2725Already for decades, controversy exists as to whether CD34 expression predicts for poor prognosis (Kanda et al, Cancer 88:2529, 2000). One of the complicating factors between studies was the use of different cut-off values, ranging from 5% to 25% CD34 (% of WBC). Such cut-offs usually result in arbitrary boundaries between positive (+) and negative (-) cells. Earlier, we have identified a particular subtype of AML with a small (≤1%) CD34+ population present, which was homogeneous in the flowcytometry properties forward- and sideward scatter and CD34 expression (Figure A) and was of non-malignant origin (van der Pol et al, Haematol. 88: 983, 2003). We considered this type of AML as truly negative for CD34+ AML cells (called: CD34- AML). With this definition, patients with >1% CD34+ cells thus have CD34+ AML. In a retrospective analysis of 98 patients with long-term follow up (median= 14.8 months; range 0.4–169), CD34- patients (n=24) had a much better prognosis compared to CD34+ AML patients (n=74): median OS not reached (>160 months) versus 13 months (p<0.005; figure B). For RFS this was >160 months versus 10 months (p=0.009). Importantly, when applying the historical cut-offs of 5%, 10%, 15% and 20%, no significant prognostic value was found, with only trends in some cases. When excluding all CD34- patients, no prognostic impact al all was left for the 4 cut-offs of 5%-20%. This proves that, when applying the cut-off strategy (5%-20%) in the whole patient group (including the CD34- patients) it is the CD34- sub-group herein that is responsible for the better prognosis of patients below these (5-20%) other cut-off levels. The difference in prognosis implies differences in therapy resistance. P-glycoprotein activity is one of the functional factors that best predict therapy resistance and prognosis in AML. Almost all CD34- patients (22/24) had P-glycoprotein activity lower than the median of normal bone marrow (nBM) CD34+ cells. Above 1% CD34 level, increasing CD34 percentages were accompanied by increasing P-glycoprotein activity (38/74 higher than nBM CD34+), as is known from literature too. The prognostic value of CD34-negativity was confirmed in a retrospective setting in a large (n= 394; 85 CD34- and 309 CD34+) group of uniformly treated patients (<60 yrs). The results confirmed the retrospective results: Figure C shows that median OS is not reached (> 41 months) for CD34- patients and 19 months for CD34+ patients (p=0.006). For RFS (n=225) median RFS was not reached but differences were significant ((p=0.003). However, in contrast to the retrospective study, now also the cut-offs of 5–20% had significant, be it low, prognostic impact both for OS and RFS (not shown). This could be explained by the impact of CD34- patients within the patient groups below the cut-offs: when applying the cut-offs of 5%-20% after exclusion of the CD34- group, no prognostic impact at all was left. Altogether these results show that finding a prognostic impact for CD34 percentage using the historical cut-offs strongly depends on the size of the patient group studied and likely also on the size of the CD34- group herein. These phenomena fully explain the discrepancies in literature up to date. NPM1 mutated leukemias are reported as CD34 negative, although without using our strict definition of CD34 negativity. With our definition, 46/68 evaluable CD34- cases were NPM1 mutated, 31/46 with single mutation (group 1) and 15/46 with an additional FLT3-ITD (group 2); 22 patients had neither NPM1 mutations nor FLT3-ITD (group 3) [only 1 patient with cytogenetic good risk aberrancy t(8;21)]. All these 3 CD34 negative subgroups showed similar good prognosis (70% survival after 30–40 months follow up). For comparison, in the CD34+ group, NPM1 single mutation (n=19/260) had good prognosis; NPM1 mutation+FLT3-ITD (n=17/260), similar to FLT3-ITD alone (n=22/260), had a very poor prognosis. In conclusion, we show that a particular group of AML patients, who completely lack malignant CD34 positive cells, have CD34 percentages ≤1 %, with a specific profile on flowcytometer, have a good prognosis compared to CD34+ patients. Cells from CD34- patients show therapy sensitive characteristics and contain both NPM1 wild type and NPM1 mutated cases. The global way in which prognostic impact of CD34 percentage has been applied for decades, thus has prevented the clear statement that CD34 has indeed strong prognostic impact in AML. [Display omitted] [Display omitted] [Display omitted] Disclosure:No relevant conflicts of interest to declare.

AML FAB Subtypes Shows Different Proportion of Leukemia Initiating Cell Which Has Lower Single Cell Dividing Property.

Abstract 4568 BackgroundAcute myeloid leukemias (AML) are maintained by a subpopulation of leukemia initiating cell that can regenerate themselves as well as give rise to more differentiated and less proliferative cells that constitute the bulk of the disease. Recent studies discovered both the nature of leukemia stem cells and their origin. Immunophenotype of AML stem cell (ASC) is known as CD34+CD38– leukemic cells. Yet, despite their critical importance, much remains to be learned about the frequency, proportion and dividing activity of ASC in a single cell levels. To address these issues, the current study investigated the frequency, proportion according to AML FAB subtypes and single cell plating efficiency of ASC from bone marrow (BM) at the initial diagnosis. Materials and methodsThe proportion and frequency of ASC were examined on BM samples of twenty AML patients using single cell sorter (BD FACS Aria cell sorter, BD Biosciences, USA). Growth and proliferation capacities of single normal hematopoietic stem cells and LSC were determined using plating efficiency (number of the wells in which more than two cells grew / total number of cells in 96-well plate culture x 100). Individual single cells were cultured in 96-well plates with each well containing 100 uL of serum media with each 100 ng/mL of stem cell factor, Flt-3, thrombopoietin and G-CSF for five days. ResultsThe frequency and proportion of ASC varied according to FAB subtypes. The mean proportion of ASC from AML M2, M4 and M3 showed 25.1±22.5% (mean±SD), 15.1±16.6% and 6.5±3.3%, respectively. The mean proportion of remaining other subtypes was 13.6±16.2%. CD34 percentage in BM at the initial diagnosis closely correlated with the proportion of ASC. ASC proportion was statistically significant different between patient's group with more than 20% of CD34 and those with less than 20% (P=0.003). Single cell plating efficiency of ASC was markedly reduced compared with non-ASC (CD34+CD38+ leukemic cell population) (7.2 ± 4.7% in ASC vs. 12.6 ± 7.6% in non-ASC). ConclusionASC proportion and frequency in BM samples were very diverse according to FAB subtypes. This study directly proved lower dividing activity of single ASC, and also implicated strategies for single ASC cloning to develop new therapeutics targeting ASC. Disclosures:No relevant conflicts of interest to declare.

MiRNA Expression Signatures in Acute Myeloid Leukemia Are Predictors for Patient Outcome.

Only recently, a class of gene regulatory molecules (miRNA's) has been described that have the potential for posttranscriptional and translational gene regulation. MiRNA's can influence critical cellular properties like proliferation and differentiation. Since these properties vary substantially in genetically defined AML subsets we have chosen to analyze AML patients with inv(16) (n=12); standard risk cytogenetic classification (mainly comprising the normal karyotype) (SR) (n=21) and with monosomy 7 or deletion7q (n=17) using DNA oligonucleotide arrays which are employing the mirVANA miRNA probe set. Principal Component Analysis (PCA) of AML samples from patients with inv(16) showed only minor differences to normal bone marrow (BM). In contrast AML patients with SR could be clearly divided in two subgroups - one subgroup that showed miRNA expression pattern close to BM and a distinctly different subgroup. Comparison of SR patient samples with BM using ANOVA tests revealed 9 differentially expressed miRNA's (mir-223; mir-15a; mir-16; mir-203; mir-103; mir-23b; mir-107, mir-17–5p and mir23a). AML patients with aberrations of chromosome 7 showed highly distinct PCA pattern as compared to BM samples. 64 miRNA's were found to be differentially expressed in those patients as compared to BM. All miRNA's located on chromosome7 that were included in the array were found downregulated in AML patients with aberrant chromosome 7 (e.g.mir-335).To further substantiate the microarray data we analyzed a total of 108 AML patients with inv(16), SR cytogenetics and aberrations of chromosome 7 for mir-23a, mir-223, mir-16 and mir-335 expression by qRT-PCR. Supporting the microarray data it could be shown that the median expression levels of mir-23a, mir-223 and mir-16 were lowest in patients with SR cytogenetics, whereas AML patients with chromosome 7 aberrations showed lowest median expression for mir-335 transcripts. This was in clear contrast to the expression of mir-150, a miRNA which could be previously shown to associate with immature T-cells and which had the highest expression in AML samples with chromosome 7 aberrations. Expression of mir-150 was correlated with a high CD34 percentage in the investigated AML samples (p<0.001) whereas mir-223 did not correlate with CD34 but was strongly associated with the surface expression of CD14 (p<0.001). In an in-vitro model of G-CSF induced CD34+ stem cell differentiation mir-223 and mir-16 were slightly upregulated whereas mir-150 disappeared during the process of differentiation. Finally, the investigated miRNA were entered in a model for outcome prediction in AML samples with SR cytogenetics including known risk factors. Within this model the ratio of mutant vs. wt FLT-3 was the strongest predictor of overall survival (p<0.01). Additonally, mir-23a expression proved to be an independent negative prognostic factor for AML patients with SR cytogenetics (p<0.05).In conclusion, the presented data demonstrate specific expression profiles of miRNA's in AML patients with different cytogenetic risk profiles. As it could be shown for patients with SR cytogenetics the observed expression profiles are likely to contribute to a deregulated differentiation and may influence therapeutic outcome.

Progressive Emergence of a Dominant Unit during Dual Unit Umbilical Cord Blood (UCB) Culture.

Umbilical cord blood (UCB) as a stem cell source for allogeneic transplantation is limited by the cell dose a single unit can provide to adult recipients. One approach to overcoming the cell dose limitation is the use of two UCB units. However, murine and human clinical studies have shown that after infusion of two UCB units, one unit dominates as early as 3 weeks post engraftment. To understand the biology that determines the engraftment of each donor, we investigated the effect of dual donor competition on hematopoietic progenitors from 3 unrelated units and 2 units from dizygotic twins during methylcellulose and LTC-IC co-culture of equal numbers of mononuclear cells from each unit pair. Donor chimerism was evaluated in primary CFU (t=0) and secondary CFU from LTC-IC cultures (weeks 4 and 5) by PCR of informative polymorphic short tandem repeats (STR analysis). In 10 units analyzed, the CFU frequency per 100,000 mononuclear cells was 152 ± 18 and the LTC-IC frequency was 1 in 20,000 ± 5.81. The mononuclear cell composition was CD34: 1.07 ±.29%; CD3: 56.3 ± 4.1%; CD33: 17.4 ± 3.4%; and CD19: 8.42 ± 1.1%. In 4 UCB pairs, the unit with the higher primary CFU frequency dominated by an average ratio of 2:1 based on STR analysis of the primary CFU co-culture. In secondary CFU derived from the LTC-IC cultures, the skewing progressively became more pronounced with the same unit identified in primary culture predominating by an average ratio of 7:1. In mixed cultures, the absolute number of primary CFU from the dominant units increased by 15 ± 2% over the number from individual units while CFU from the non-dominant units decreased by 37 ± 4%. Interestingly, there was no dominant unit in primary CFU cultures from one set of dizygotic twins and the CFU number from each unit increased by 30% in the mixed culture. This set of twins emerged with a dominant unit during LTC-IC culture, yielding a donor ratio of 2:1. We found no correlation between the predominating unit and the CD34 percentage or LTC-IC frequency. However, in all 5 dual UCB experiments, the unit with the higher CD3 percentage dominated in primary CFU cultures and was intensified during LTC-IC culture. These results demonstrate an early and progressive dominance of one donor based on CFU analysis during mixed UCB culture. The correlation with CD3 content suggests both an immediate and protracted T-cell effector mechanism even though the LTC-IC culture does not promote T-cell proliferation. Additionally, the increase in the absolute cell number of the dominant unit may suggest a graft vs. graft progenitor cell growth-stimulatory effect which may explain the observed more rapid engraftment of the dominant unit after dual UCB transplantation. Thus, immediate UCB competition predicts the dominant unit during in vitro culture and presumably mimics the process occurring after clinical dual UCB transplantation.

Prognostic Impact of Percentage CD34 Expression in Bone Marrow Trephines of Patients with Chronic Myeloid Leukaemia in Chronic Phase Treated with Imatinib.

CD34 is a surface glycophosphoprotein expressed on developmentally early lymphohematopoietic stem and progenitor cells, small-vessel endothelial cells, and embryonic fibroblasts. We studied the prognostic value of presence of CD34 expressing cells in the marrow trephine biopsies of 58 patients with chronic myeloid leukemia in chronic phase treated with imatinib (21 newly diagnosed and 37 IFN failures). Briefly, patients received 400 mg daily and the dose was then adjusted according to tolerance and response. Bone marrow examinations were performed before starting therapy with imatinib and then every 3–6 months. All patients achieved complete haematological remission and 48% achieved complete cytogenetic remission. Chronic, accelerated and blastic phases were defined as described by Kantarjian 1988. Paraffin embedded sections were stained with the monoclonal antibody QBEND10. A minimum of 500 nucleated cells were counted per trephine section. Results were expressed as percentage of CD34+ vs all nucleated marrow cells. The expression of CD34 by the endothelial cells was used as an internal control. The median CD34 percentage before the onset of the imatinib therapy was 0.8% (25 and 75 percentiles 0.8 and 2%). We performed univariate and multivariate analysis for PFS with variables defined at the time of starting imatinib. Sokal risk group (defined at diagnosis), presence or absence of additional cytogenetic abnormalities, percentage of blasts in the bone marrow aspirate and the percentage of CD34+ cells in the trephine (considered as a continuos variable) were significant in the univariate analysis. Only CD34 expression and Sokal risk group were significant in the multivariate model. Sokal risk group lost significance when the percentage of CD34 cells was categorized as <=2% and >2% (RR for PFS=11.7, 95 CI=1.9–70.4, p=0.007; RR for survival = 10.5, 95CI= 1.01–80.3, p=0.05). The 3 years PFS for patients with a CD34 percentage <= 2% before the onset of the imatinib therapy (n= 44) was 77% while for patients with a CD34 >2% (n=14) it was 36% (p=0.006), similarly the three year overall-survival was 80 and 45% respectively (p=0.009). The median value of CD34 in the first bone marrow examination performed after the start of imatinib (typically performed at 3–6 months) was 0.8 (25 and 75 percentiles 0.8 and 1.5%). The degree of reduction from the baseline level achieved in the first bone marrow examination had favourable prognostic significance (as a continuos variable) with a RR for PFS of 0.88, 95CI 0.53–0.93 (p= 0.006) and RR for overall survival of 0.93, 95CI 0.19–0.998 (p=0.05). During follow up, an increment of 2 percentage points over the pre therapy CD34 value in patients in otherwise complete haematological remission had an adverse prognostic implication with a RR for PFS of 10.2 95 CI 2.1–17, (p=0.006) and a RR for survival of 6.8 95 CI 1.01–101-(p=0.05). We conclude that the use of CD34 staining in bone marrow trephines is a valuable tool for both diagnosis and monitoring in patients with CML treated with imatinib.

Myelodysplastic syndromes: A study of surface markers and in vitro growth patterns

A study of surface markers and in vitro growth in semi-solid and liquid medium was performed in 35 patients with newly diagnosed myelodysplastic syndrome (MDS). Surface markers were studied by CD34, CD13, CD14, CD15, and CD33 monoclonal antibodies. There was no strict correlation with the FAB typing, but CD34 was expressed only in refractory anemia with excess of blasts (RAEB) or RAEB in transformation (RAEB-t). CD14 was markedly positive in the 4 cases of chronic myelomonocytic leukemia. Colony-forming cells were assessed by culture in semi-solid medium in the presence of HTB9 as growth factor. Four growth patterns were identified: a) normal growth (6 cases); b) no growth or low plating efficiency (10 cases); c) low colony and high cluster number (15 cases); and d) normal or high colony number with high number of clusters (4 cases). Expression of CD34 was associated with low colony and high cluster number. Finally we studied the proliferation and differentiation capacities in liquid culture without stimulating factor. Fifteen patients had a spontaneous proliferation. This was not correlated with any surface marker. Differentiation assessed by the loss of CD34 and/or the increase of CD15 by more than 20% at day 7 was observed in 21 cases. None of the surface markers or growth patterns was associated with a specific chromosomal abnormality, except the lack of growth in liquid culture observed in all 5q deletion cases. In univariate analysis, RAEB and RAEB-t FAB subtypes, percentage of blasts higher than 5%, staining by CD33 and CD34, and lack of differentiation in liquid culture were significantly associated with progression to leukemia and shorter survival. In multivariate analysis, only CD34 expression (P = .002) and percentage of blasts (P = .05) remained independent significant variables. CD34 was the only significant variable for prediction of survival (P = .05). It is concluded that surface marker analysis at diagnosis and after liquid culture may be a useful tool for the initial evaluation of MDS.