Bone Marrow Blast Cell Counts Research Articles

A Prediction Model for Early Recurrence after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with CMML: A Nationwide Representative Multicenter Study

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell malignancy. Some patients experience prolonged remission after aggressive chemotherapy, but the only current therapy with proven curative potential is allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, not all CMML patients benefit from allo-HSCT, and many experience relapse. Relapse is the leading cause of treatment failure for CMML treated with allo-HSCT, and treatment options for patients relapsing after allo-HSCT are limited. This study conducted a nationwide multicenter real-world study to identify risk factors and develop a novel prediction model for early recurrence in CMML patient posttransplantation. Clinicians can improve the survival of CMML patients with a high risk of early recurrence by intervening as early as possible. Methods: Patients ≥18 years old with CMML treated with allo-HSCT for the first time between 2005 and 2022 were included in this nationwide, multicenter, real-world study. Twenty-seven medical centers participated in this analysis, including the derivation cohort from 26 centers and the validation cohort from another center. Information collected included demographic and clinical characteristics, previous treatments and responses, and allo-HSCT treatment details. We first calculated the univariable association of each variable with 1-year relapse in the derivation cohort. Second, the variables with p values less than 0.1 in the univariate analysis were further included as candidate predictors in the multivariate analysis using a backward stepwise logistic regression model. The variables that remained in the final model based on the outcomes of the multivariate analysis in the derivation cohort were identified as independent prognostic factors. A scoring system to predict early recurrence after allo-HSCT was also established, and scores were assigned to the prognostic factors based on the regression coefficient. Results: Using multivariable logistic regression methods with stepwise variable selection, two highly significant independent prognostic factors for the early recurrence of CMML posttransplantation were identified: bone marrow blast cell count at diagnosis (p=0.026; odds ratio [OR], 1.149; 95% confidence interval [CI], 1.02-1.30), and leukocyte count pretransplantation (p=0.005; odds ratio [OR], 1.212; 95% confidence interval [CI], 1.06-1.39). A risk grading model was constructed according to the regression coefficients. Bone marrow blast cell count 0-5% is 0 point, 5%-10% is 1 point; 10-20% is 2 points, and >20% is 3 points. Leukocyte count 0-10×10 9/L is 0 point, 10-20×10 9/L is 2 points, and >20 ×10 9/L is 4 points. The points scored for each of these two factors were added to yield the overall risk score, which ranged from zero to seven. Patients were stratified into a low-risk group (0-1 point), a intermediate-risk group (2-3 points) and a high-risk group (4-7 points). The validated internal c-statistic was 0.790 (95% CI, 0.657-0.923), and the external c-statistic was 0.710 (95% CI, 0.580-0.840). The early recurrence rates of the derivation cohort in the low-risk, intermediate-risk, and high-risk groups were 5.9%, 17.9% and 31.8% (p<0.05). The Kaplan‒Meier estimations of relapse-free survival revealed good separation between these risk groups, and 91.9%, 79.4% and 66.1% (p<0.05), respectively. According to the calibration plots, the model-predicted probabilities showed a good correlation with the actual observed frequencies. Decision curve analysis indicated that the clinical implementation of the prognostic model could benefit early recurrence CMML patients. Conclusion: CMML patients who relapse after allo-HSCT have a poor prognosis. An integrated prediction model based on clinical biomarkers was developed and externally validated, and this is the first straightforward scoring model that incorporates clinical and laboratory risk factors to evaluate the early recurrence of CMML patients after receiving allo-HSCT. This model can be effectively utilized to help improve the survival and prognosis of CMML patients by accelerating the early identification of patients at a high risk of relapse and contributing to the appropriate implementation of urgent medical support.

Impact of a novel prognostic model on allogeneic hematopoietic stem cell transplantation outcomes in patients with CMML.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell malignancy, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curable treatment. The outcomes after transplant are influenced by both disease characteristics and patient comorbidities. To develop a novel prognostic model to predict the post-transplant survival of CMML patients, we identified risk factors by applying univariable and multivariable Cox proportional hazards regression to a derivation cohort. In multivariable analysis, advanced age (hazard ratio [HR] 3.583), leukocyte count (HR 3.499), anemia (HR 3.439), bone marrow blast cell count (HR 2.095), and no chronic graft versus host disease (cGVHD; HR 4.799) were independently associated with worse survival. A novel prognostic model termed ABLAG (Age, Blast, Leukocyte, Anemia, cGVHD) was developed and the points were assigned according to the regression equation. The patients were categorized into low risk (0-1), intermediate risk (2, 3), and high risk (4-6) three groups and the 3-year overall survival (OS) were 93.3% (95%CI, 61%-99%), 78.9% (95%CI, 60%-90%), and 51.6% (95%CI, 32%-68%; p < .001), respectively. In internal and external validation cohort, the area under the receiver operating characteristic (ROC) curves of the ABLAG model were 0.829 (95% CI, 0.776-0.902) and 0.749 (95% CI, 0.684-0.854). Compared with existing models designed for the nontransplant setting, calibration plots, and decision curve analysis showed that the ABLAG model revealed a high consistency between predicted and observed outcomes and patients could benefit from this model. In conclusion, combining disease and patient characteristic, the ABLAG model provides better survival stratification for CMML patients receiving allo-HSCT.

Clinical Value of Serum miRNA in Patients with Acute Promyelocytic Leukemia.

Objective To explore the clinical value of specific miRNA in patients with acute promyelocytic leukemia. Methods 129 patients with acute promyelocytic leukemia diagnosed in our hospital from January 2015 to January 2020 were selected as the observation group. At the same time, 74 patients with nonacute promyelocytic leukemia who underwent bone marrow aspiration were included as the control group. The expression levels of miR-126-5p and miR-13, different characteristic parameters, and prognosis were compared between the two groups, and the clinical significance of miR-126-5p and miR-13 in acute promyelocytic leukemia was analyzed. Results The expression of miR-126-5p (12.31 ± 2.25 versus 17.30 ± 3.28) and miR-13 (16.05 ± 3.47 versus 21.66 ± 2.18) in the observation group was significantly lower than that in the control group (P < 0.05). The expression level of miR-126-5p was significantly correlated with lactate dehydrogenase level, HGB level, NPM1 mutant type, and complete remission (P < 0.05). The expression level of miR-13 was significantly correlated with HGB level, NPM1 mutant type, and complete remission (P < 0.05). Both expression levels of miR-126-5p and miR-13 were not correlated with sex, age, WBC, PLT, proportion of bone marrow primordial cells, hepatomegaly, splenomegaly, lymph node enlargement, and FLT3-ITD (P > 0.05). Cox multivariate regression analysis showed that peripheral blood WBC, bone marrow blast cell count, and miR-126-5p and miR-13 were prognostic factors in patients with acute promyelocytic leukemia (P < 0.05). The sensitivity, specificity, accuracy, and AUC of serum miR-126-5p prediction were 75.83%, 84.56%, 82.17%, and 0.729, respectively. The sensitivity, specificity, accuracy, and AUC of serum miR-13 prediction were 78.64%, 88.49%, 86.20% and 0.882, respectively. Conclusion Serum miR-126-5p and miR-13 are closely related to the prognosis of patients with acute promyelocytic leukemia. Serum miR-126-5p and miR-13 can be used as reliable indexes to predict the prognosis of patients.

Prognostics and Clinical Outcomes in Patients Diagnosed With Acute Myeloid Leukemia (AML) in a Teaching Hospital.

Background: Acute myeloid leukemia (AML) is a heterogeneous disease. Prognosis and survival depend on several factors that determine tumor behavior and response to therapy. AML has a poor prognosis that depends on several factors: patient's age, gender, body mass index (BMI), baseline white blood cells count, and bone marrow blast (BMB) cell count at the time of diagnosis. Therefore, this study aimed to determine the prognostic role of these factors and their impact on outcomes, and how these prognostic factors may affect AML patients before and after induction chemotherapy.Methods: The study design is an observational, retrospective record review. We included records of patients diagnosed with primary and secondary AML who received chemotherapy between 2013 and 2019 at King Abdulaziz University in Jeddah, Saudi Arabia. Data were extracted from medical records, entered into an Excel sheet (Microsoft Corp., Redmond, WA), and analyzed using SPSS Statistics, version 25 (IBM Corp., Armonk, NY).Results: Forty-two AML patients who were started on chemotherapy were analyzed. The mean age at diagnosis was 35 ± 22.2 years; 52.4% were male. The ability to achieve the first remission varied according to age group; the 21-45 age group had the higher ability and survival rate of 75.0%. On the other hand, the mortality incidence was higher (at 70.0%) in both the 11-20 and the 46-70 age groups. A strong negative correlation was observed between age and survival duration after treatment (SDAT) (r = - 0.618, p = 0.004). The death incidence was increased in the BMI ranges that were under and above the normal weight range. SDAT differed significantly between the three groups in favor of the normal-weight patients (p = 0.019). We found that patients with BMB < 5 had the most deaths. There was a significant negative association between BMB and days to achieve the first remission after treatment (p = 0.033). Conclusion: Age, BMI, and BMB are considered effective prognostic factors for AML patients.

Role of matrix metalloproteinase MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1) in the clinical progression of pediatric acute lymphoblastic leukemia

ABSTRACT Background Matrix metalloproteinases (MMPs) play a crucial role in cancer progression and metastasis, however their role in pediatric Acute lymphoblastic leukemia (ALL) is still unrevealed. Methods The diagnostic, prognostic and predictive value of tissue inhibitor of metalloproteinase (TIMP-1), MMP-2, MMP-9 and CD34+CD38− cancer stem cells (CSCs) were assessed in bone marrow (BM) samples of 76 ALL children using Flow Cytometry analysis. Results There was a significant increase in TIMP-1 [1.52 (0.41–10) versus 0.91(0.6–1.12); respectively, p < 0.001], and CSCs CD34+CD38− [1 (0.03–18.6) versus 0.3 (0.01–1.1), p < 0.001] expression in ALL patients compared to controls. While there were no significant differences regarding MMP-2 and MMP-9 expression between the two groups. The sensitivity, specificity, area under curve (AUC) of MMP-2 were (80.3%, 53.3% and 0.568, p = 0.404), and of MMP-9 were (53.9%, 40% and 0.660, p = 0.053). While that of TIMP-1 were (78.9%, 100% and 0.892, p < 0.001), and that of CD34+CD38− CSCs were (78.9%, 73.3% and 0.855, p < 0.001). Increased TIMP-1 expression associated with the high-risk disease (p < 0.001). CD34+CD38− CSCs and MMP-2 overexpression associated with MRD at day-15, increased BM blast cell count at diagnosis and at day-15 ( p < 0.05). TIMP-1 overexpression is associated with shorter DFS and OS rates ( p = 0.009 and p = 0.048). Multivariate logistic regression analysis showed that both TIMP-1 [OR: 4.224, p = 0.046], and CD34+CD38− CSCs [OR: 6.873, p = 0.005] could be potential independent diagnostic factors for pediatric ALL. Conclusion TIMP-1 and CD34+CD38− CSCs could be possible useful diagnostic markers for pediatric ALL. Also, TIMP-1 is a promising prognostic marker for poor outcome of the patients.

The Association of FLT3-ITD Gene Mutation with Bone Marrow Blast Cell Count, CD34, Cyclin D1, Bcl-xL and hENT1 Expression in Acute Myeloid Leukemia Patients.

Background & Objective: FLT3-ITD has been recently used as a molecular prognostic marker for risk classification in acute myeloid leukemia (AML) therapy. In this study we aimed to investigate the association of FLT3-ITD gene mutation with bone marrow blast cell count, CD34 expression as malignant cell burden, cyclin D1 and Bcl-xL expressions as indexes of cell proliferation and anti-apoptosis and human equilibrative nucleoside transporter 1 (hENT1) expression as cytarabine transporter during AML treatment.Methods:We investigated FLT3-ITD mutations, bone marrow blast cell count, CD34, cyclin D1, Bcl-xL and hENT1 expression in bone marrow aspirates from 22 de novo AML patients in a cross sectional study.Results: FLT3-ITD mutations were observed in 5 out of 22 de novo AML patients (22.7%). Patient with FLT3-ITD mutations had higher blast cell counts (79.5% vs 56.1%, P=0.004). In patients with FLT3-ITD mutations, CD34 and cyclin D1 expressions were higher (MFI 328.80 vs 25.78, P=0.003 and MFI 74.51 vs 57.15 P=0.005) than the patients without mutations. hENT1 expression in AML with FLT3-ITD mutation was lower (MFI 29.64 versus 56.32, P=0.0000) than in mutation-free AML. There was no significant difference in Bcl-xL expression between patients with and without mutations (P=0.61).Conclusion:A significant association was found between FLT3-ITD gene mutations in AML patients with bone marrow blast cell count, CD34, cyclin D1 and hENT1 expressions, however no association was obtained with Bcl-xL expression. These findings support the role of such mutation in pathogenesis of AMLand its contribution in rearrangement of standard therapy with cytarabine in management of AML.

Interobserver concordance of assessments of dysplasia and blast counts for the diagnosis of patients with cytopenia: From the Japanese central review study

The diagnosis of myelodysplastic syndromes (MDS) is based on morphology and cytogenetics. However, limited information is currently available on the interobserver concordance of the assessment of dysplastic lineages (<10% or ≥10% in bone marrow (BM)). The revised International Prognostic Scoring System (IPSS-R) described a new threshold (2%) for BM blasts. However, the interobserver concordance of the categories (0–≤2% and >2–<5%) has limited data. The purpose of the present study was to investigate the assessment of dysplastic lineages and IPSS-R reproducibility. Our study was divided into two Steps. In each Step, the microscopic examinations were performed separately by two morphologists. Regarding the category of BM blasts ≤2% and >2–<5%, interobserver agreement was more than ‘moderate’ in all pairs (kappa test: 0.43–0.90). Regarding dysgranulopoiesis (dysG) and dyserythropoiesis (dysE) in BM, interobserver agreement was more than ‘moderate’ in all pairs (kappa test, dysG: 0.45–0.96, dysE: 0.45–0.81). Regarding the category of dysmegakaryopoiesis (dysMgk) in BM, interobserver agreement was more than moderate in 4 out of 5 pairs (kappa test: 0.58–1.00), and was fair for one pair (kappa test: 0.37). We consider that high interobserver concordance may be possible for the BM blast cell count (≤2% or >2–<5%) and dysplasia (<10% or ≥10%) of each lineage.

The Prognostic Value of D-Dimer in De Novo Acute Myeloid Leukemia

BackgroundThe aim of this study was to evaluate the prognostic value of D-dimer in patients with newly diagnosed de novo non-M3 subtypes of acute myeloid leukemia (AML). Materials and MethodsWe retrospectively analyzed the clinical data from 245 patients with newly diagnosed de novo non-M3 subtypes of AML at the Tongji Hospital from January 2010 to December 2014. ResultsThe comparison results indicated that the D-dimer values were higher in patients with AML with the following characteristics: WBC count ≥ 20 × 109/L (2.20 versus 6.00, P = 0.001), percentage of bone marrow (BM) blasts ≥ 60% (2.06 versus 5.69, P = 0.003), and poor-risk stratification (P < 0.001). Cox univariate regression analysis showed that overall survival was negatively affected by the following factors: age > 60 years, poor-risk stratification, BM blast cell count ≥60%, and D-dimer ≥1μg/mL. Multivariate analysis showed that only age > 60 years (P < 0.001), BM blast cell counts ≥60% (P = 0.001) and D-dimer values ≥1μg/mL (P = 0.014) were independent adverse prognostic factors. ConclusionsD-dimer ≥1μg/mL is related to high tumor burden and can be considered as an independent prognostic factor in patients with de novo non-M3 AML.

Outcome of myeloablative allogeneic peripheral blood hematopoietic stem cell transplantation for refractory/relapsed AML patients in NR status

To further find effective method to improve the long term survival of refractory or relapsed acute myeloid leukemia (AML) patients, we retrospectively analyzed the outcomes of myeloablative hematopoietic stem cell transplantation (HSCT) for 133 consecutive patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) therapy related AML(t-AML) in not remission status. The overall 3-year OS and DFS were 40.9% and 35.6% respectively. The variables associated with improved long term DFS were a bone marrow blast cell count less than 20% and an intensified conditioning regimen. In addition, the t-AML group had higher rates of relapse and III–IV acute GVHD than the primary AML group. The unrelated donor group had similar OS and DFS with sibling groups.Our study suggested that decreasing bone marrow blast cell counts before HSCT and strengthening the conditioning regimen may improve long-term DFS for refractory/relapsed AML patients, and unrelated donor group can get similar effect when compared to the sibling group.

Clinical Features and Prognostic Assessment in 233 Patients with Therapy-Related Myelodysplastic Syndromes: The IPSS-R Is a Powerful Predictor of Outcome

▪BackgroundTherapy-related Myelodysplastic Syndromes (t-MDS) are those MDS occurring after cytotoxic and/or radiation therapy administered for a prior neoplastic or non-neoplastic disorder. Their prognosis is generally very poor. The commonly used risk prognostic models for MDS (IPSS and IPSS-R) are not validated in this entity as they were developed after the exclusion of therapy-related cases (Greenberg et al. Blood 1997; Greenberg et al. Blood 2012).AimsThe main aims of this study are: a) to report clinical findings and overall survival on 233 patients with t-MDS, and to compare them with a large series of de novo cases; b) to test if IPSS-R is applicable to t-MDS patients.Patients and methodsThe study is based on the Spanish Registry for MDS, a retrospective database that includes more than 10000 cases. The investigators were asked to fill in a questionnaire regarding prior disease (PD) and prior therapy in those cases reported to be t-MDS. Herein are described the clinical features and overall survival of the first 233 cases with the required information, and compared with patients with de novo MDS from a single center series (n=725). Log Rank test was applied to asses IPSS-R in t-MDS group.ResultsThe 233 reported patients were diagnosed between January 1993 and February 2014. The series includes 104 women (44,6%) and 129 men (55,4%). One hundred and two patients (43.9%) had a primary hematologic malignancy, 119 (51%) had a solid tumor, and 12 (5.1%) received cytotoxic therapy for autoimmune disorders. Ninety eight patients (42.6%) received only chemotherapy (CT), 45 (19.6%) received only radiotherapy (RT), 44 (19.1%) received combined modality treatment (CMT), and 43 (18.6%) received an autologous stem cell transplantation (ASCT). The median time of latency between PD and diagnosis in t-MDS group was 4.56 years (range: 0.03-29.63) in patients previously treated with CT or CMT, significantly lower than the observed after RT (8.54; range 0.83-23.02) or ASCT (8.64; range 2.87-28.32) groups (p=0.023 and p<0.0001, respectively). Median age, hemoglobin concentration, platelet count and absolute neutrophil count at diagnosis were significantly lower in t-MDS compared with de novo MDS, while bone marrow blast cell count was significantly higher. A higher proportion of high risk karyotypes (intermediate, poor and very poor categories of R-IPSS for cytogenetics) in t-MDS than in de novo MDS was observed (45.5% versus 25.6% respectively, p<0.0001). Within t-MDS cases, those patients who had received CT, CMT or ASCT also presented a higher proportion of high risk karyotypes compared with t-MDS after RT (52.1 vs 24.1% respectively, p=0.006). There was a significantly shorter median overall survival in patients treated with CT/CMT or ASCT in comparison with de novo patients or t-MDS patients treated with RT (14.88 versus 25.06 versus 47.18 months, respectively (log rank test < 0, 0001). In contrast, no difference between de novo and t-MDS after RT was found (median survival 53.6 months vs. 47.17 months, respectively, n.s). These findings are in concordance with previously published data (Nardi et al, JCO 2012) suggesting that some t-MDS after RT may not be truly t-MDS, but just coincidental. The IPSS-R prognostic score could separate t-MDS patients into five risk groups in terms of overall survival, with a median survival of 76.68 months, 38.73 months, 16.59 months, 13.3 months and 6.37 months, respectively (p<0.0001; Figure 1). A shorter overall survival for each category was found in t-MDS in comparison with de novo MDS, reaching statistical significance in the intermediate and high risk category and showing a trend towards significance in the very low and low risk categories (Figure 2).ConclusionsIn conclusion, our data supports the notion that t-MDS may contain entities with heterogeneous prognosis due to a diverse biological basis. The capacity of the IPSS-R for separating good prognostic from bad prognostic cases shown in this study could be very useful in the clinical setting in order to offer risk-adapted treatments to patients, although the development of a specific prognostic score for these entities would be needed. [Display omitted] [Display omitted] <![if !vml]><![endif]> [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interobserver variance in myelodysplastic syndromes with less than 5 % bone marrow blasts: unilineage vs. multilineage dysplasia and reproducibility of the threshold of 2 % blasts.

Previous studies have shown the reproducibility of the 2008 World Health Organization (WHO) classification in myelodysplastic syndromes (MDS), especially when multilineage dysplasia or excess of blasts are present. However, there are few data regarding the reproducibility of MDS with unilineage dysplasia. The revised International Prognostic Scoring System R-IPSS described two new morphological categories, distinguishing bone marrow (BM) blast cell count between 0-2 % and >2- < 5 %. This distinction is critical for establishing prognosis, but the reproducibility of this threshold is still not demonstrated. The objectives of our study were to explore the reliability of the 2008 WHO classification, regarding unilineage vs. multilineage dysplasia, by reviewing 110 cases previously diagnosed with MDS, and to study whether the threshold of ≤2 % BM blasts is reproducible among different observers. We used the same methodology as in our previous paper [Font et al. (2013) Ann Hematol 92:19-24], by encouraging investigators to include patients with <5 % BM blasts. Samples were collected from 11 hospitals and were evaluated by 11 morphologists. Each observer evaluated 20 samples, and each sample was analyzed independently by two morphologists. Discordance was observed in 36/108 suitable cases (33 %, kappa test 0.503). Diagnosis of MDS with unilineage dysplasia (refractory cytopenia with unilineage dysplasia (RCUD), refractory anemia with ring sideroblasts (RARS) or unclassifiable MDS) was assessed in 33 patients, by either of the two observers. We combined this series with the cases with RCUD or RARS included in our 2013 paper, thus obtaining 50 cases with unilineage dysplasia by at least one of the observers. The whole series showed very low agreement regarding RCUD (5/23, 21 %) and RARS (5/28, 18 %). Regarding BM blast count, the threshold of ≤2 % was not reproducible (discordance rate 32/108 cases, kappa test 0.277). Our study shows that among MDS WHO 2008 categories, interobserver discordance seems to be high in cases with unilineage dysplasia. We also illustrate that the threshold of ≤2 % BM blasts as settled by the R-IPSS may be not easy to reproduce by morphologists in real practice.

The Challenge Of Quantifying CD34+ Myeloid Cells In Myelodysplastic Syndromes With Less Than 5% Bone Marrow Blasts. Reproducibility Among 6 Flow Cytometry Observers

IntroductionIn low-risk myelodysplastic syndromes (MDS), the morphological bone marrow (BM) blast cell count between 0 and 2%, and >2 - <5%, has demonstrated prognostic value and is critical for R-IPSS. Flow cytometry immunophenotyping (FCI) provides an accurate way for quantification of the immature BM cell compartment through the identification of CD34+ cells and may contribute to a better characterization of these MDS low-risk categories. However, there is a wide variety of FCI strategies to study the CD34+ cells, without a universal consensus on how many and which markers should be used to reach their best identification. There are some studies evaluating the correlation between FCI and the morphological blast cells count, but it is not clear if FCI allows a good concordance among several observers when the morphological blast count is <5%. Objectives1-To explore the concordance among 6 FCI observers to quantify the CD34+ myeloid BM cells from patients diagnosed with MDS with <5% BM blasts 2- To study the correlation between FCI and morphology for detecting <5% BM blast cells. 3- To determine if the mophological threshold of 2% is reproducible by FCI. MethodsFCI data files from 48 MDS BM samples with <5% blasts were simultaneously and independently evaluated by 6 FCI observers from 6 different Spanish hospitals. According to the WHO criteria patients were distributed as follows: 3 refractory cytopenia with unilineage dysplasia; 13 refractory anemia with ring sideroblasts; 25 refractory cytopenia with multilineage dysplasia; 1 unclassifiable MDS; 2 chronic myelomonocytic leukemia and 4 therapy-related myeloid neoplasms. Each participant contributed with 8 samples and all files were exchanged among them. All of them used the INFINICYTTM software program for analysis according to their usual strategies. The morphological quantification of BM blast cells was provided by each centre and was blinded to the others. Each centre processed the samples for FCI according to their usual strategies in their clinical practice, without previous agreement on standardization of the protocols used. All centres used the stain-lyse-wash protocol but panels of monoclonal antibodies were different: combinations of 4, 6 and 8 fluorochrome–conjugated monoclonal antibodies were included in 28, 8 and 12 files respectively. Median number of events recorded per file was 157,200 (range 10,000-500,000). The combination CD34/CD45/CD117 was included in 38 files and 20 also associated HLA-DR. The fluorochrome attached to CD34 was PerCP-Cy5 in 26 samples. 8G12 was the clone used in 40 samples.The degree of agreement among the 6 observers for quantification of CD34+ myeloid cells was evaluated using the intraclass correlation coefficient (ICC). The generalized kappa statistic for multiple rates (κ) calculated the concordance among observers after categorization of quantitative variables. Both the ICC and the generalized κ statistic were interpreted as follows: 0-0.2 poor; 0.3-0.4: fair; 0.5-0.6 moderate; 0.7-0.8 strong; >0.8 almost perfect agreement. ResultsFinally, 47 samples could be evaluated by the FCI observers. The ICC showed a strong agreement among observers (0.720), and also a good concordance on the quantification of CD34+ cells at the critical level of 2% (k=0.587). Regarding the comparison between FCI and morphology, only one participant counted >5% CD34+ cells in a sample. However, the absolute quantification of BM blasts <5% by FCI showed poor agreement with morphology (ICC ranged from 0.106 to 0.458). Indeed, none of the FCI observers could reproduce the new morphological categories using the threshold of 2% BM blasts (k= 0.320). ConclusionsIn our study, FCI seems a reproducible tool to quantifying CD34+ cells in MDS patients with <5% BM blasts, despite of the great heterogeneity of the protocols used. A FCI threshold of 2% CD34+ cells was also reproducible among observers. However, the lack of a precise correlation between the morphological blast cell count and the number of CD34+cells by FCI, illustrates the importance of considering each value independently. Probably, homogenization of the FCI protocols will contribute to improve the correlation between the 2 techniques. Disclosures:No relevant conflicts of interest to declare.

Combined flow cytometric assessment of CD45, HLA‐DR, CD34, and CD117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes

Quantification of bone marrow (BM) blasts by cytomorphology is essential for the diagnosis of myelodysplastic syndromes (MDS). Owing to its subjectivity and the potential impact of dysplastic features on accurate identification of blast cells, more objective approaches are required, multiparameter flow cytometry (MFC) being a particularly promising approach in this regard. However, no consensus exists about the optimal combination of markers and strategy to be used. BM blast counts from 74 MDS patients were evaluated by morphology versus four different MFC phenotypic criteria: "CD34⁺", "CD34⁺ and/or CD117⁺", "CD34⁺, and/or CD117⁺ HLA-DR⁺", and "CD34⁺ and CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-/lo) " cells. For each criterium, the percentage of blasts was calculated using either all BM nucleated cells or non-erythroid CD45⁺ cells as denominator. RESULTS.: The number of "CD34⁺ and/or CD117⁺ HLA-DR⁺"cells showed the highest correlation and agreement with morphological counts, only a minor proportion of cases being misclassified by MFC vs. morphology for the >5% and >10% classification thresholds. In turn, a CD34⁺ phenotype was insufficient to correctly identify and quantify blasts. Conversely, usage of non-erythroid BM cells as denominator, or inclusion of "CD34⁺ and/or CD117⁺ HLA-DR⁺ plus CD64⁺ CD14(-lo") cells were both associated with overestimated blast counts. Quantification of "CD34⁺ and/or CD117⁺ HLA-DR⁺" cells (from all nucleated BM cells) by MFC is an efficient method for the enumeration of blasts in MDS. However, caution should be taken with replacing morphology by MFC blast counts; its combined use may rather provide complementary information increasing the accuracy and reproducibility of BM blast cell counts in these patients.

Usefulness of Peripheral Blood Circulating DNAs As an Alternative to DNA From Bone Marrow Cells to Detect CpG Global Methylation Status and Genetic Mutations in Patients with Myelodysplastic Syndromes

Abstract 3854 Background:Several genetic/epigenetic abnormalities are associated with the pathogenesis of myelodysplastic syndromes (MDS). DNA methyltransferase inhibitors (DNMTi), azacitidine and decitabine, have recently come to be considered as standard therapeutics for patients with MDS. However, biomarkers that predict the effectiveness of DNMTi before and/or during treatment are still lacking. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic status, repeated sampling is difficult because of pain and safety concerns. Therefore, alternatives are required. One possibility is to use circulating cell-free DNA in the plasma and serum of peripheral blood (PB); a technique previously used for the detection of genetic/epigenetic abnormalities in solid tumor cells. Aims:Assess the quality of PB circulating DNA from patients with MDS, and investigate its usefulness for analyzing serial genetic/epigenetic changes following treatment. Methods:DNA from BM, PB mononuclear cells (MNC), plasma and serum were obtained repeatedly from MDS patients and visualized via agarose gel electrophoresis. Bisulfite conversion of genomic or circulating DNA was performed and then pyrosequencing performed for the 4 CpG sites of long interspersed nuclear elements-1 (LINE-1) elements to detect global methylation. Bisulfite pyrosequencing for the 5 CpG sites of p15INK4B gene promoter was also performed. Genetic mutations were screened firstly by single nuclear polymorphism (SNP) array analysis and confirmed by DNA sequencing for the specific gene mutations. BM cells were sorted into CD34(+)/CD38(-), CD34(+)/CD38(+), and CD34(-) subpopulations, and the percentage of mutated DNA confirmed by pyrosequencing analysis. Results and Discussion:Circulating DNA from both healthy volunteer donors and MDS patients showed a ladder pattern of DNA fragments 160∼180 base pairs apart, suggesting their accumulation from mono-/di-nucleosomes. The plasma DNA concentration was relatively higher in patients with higher BM blast cell counts. Plasma DNA concentration had been changed even in the same patients according to the disease status. CpG methylation status of the LINE-1 promoter after treatment with azacitidine (days 1 to 28) was analyzed by pyrosequencing. Although the methylation status of 1 patient did not show any significant change, other 2 patients showed a serial decrease of the methylation percentage confirmed at days 3, 6, and 9 using DNA from plasma, serum, and PBMNC. Plasma DNA showed more rapid and significant changes at days 3 (p<0.001) and 6 (p<0.05) compared with serum DNA (not significant (N.S.) at day 3 and 6) and PBMNC (N.S. at day 3 and p<0.05 at day 6). These data suggest that circulating DNA from plasma can be used for analysis of the LINE-1 promoter as a measure of global methylation as an alternative strategy to using MNC in PB and/or BM. The changing ratio is different among patients and it may reflect the efficacy of DNMTi or the amount of MDS clones. p15INK4B gene promoter CpG methylation status was analyzed using BM cells, PBMNC, and circulating DNA. The methylation percentage was not significantly different among whole BM cells and 3 BM subpopulations, and plasma and serum DNA showed similar methylation pattern as whole BM DNA. Next, PB circulating DNA was utilized for the detection of genetic mutations in MDS cells. TET2 mutation (Y1245Term; TAC to TAG) was confirmed in a patient showing chromosome 4q uni-parental disomy (UPD), and the existence ratio of the mutation was significantly higher in plasma and serum DNA than in CD34(-) BM cells (p<0.05 and p<0.01, respectively) and almost equivalent to that in the CD34(+)/38(-) BM stem cell population. The high percentage of mutated genes in circulating DNA may result from abnormal DNA being released from fragile MDS clones, enriching the circulating DNA compared with DNA from BM or PBMNC but further molecular analyses and higher patient numbers are required to confirm this. Conclusions:PB circulating DNA can be reliably and sensitively used to detect epigenetic changes on genomic DNA after treatment with DNMTi. Genetic mutations in MDS clones can also be detected sensitively, at a level almost equivalent to that in BM CD34+/38- stem cells. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a better, less painful and safer alternative to using BM aspiration. Disclosures:No relevant conflicts of interest to declare.

Analysis of clinical outcomes of 63 children with acute monocytic leukemia

To evaluate the outcomes of childhood acute monocytic leukemia (AML-M5) and explore the indications of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for children with AML-M5. Seventy-five AML-M5 patients and 201 non-AML-M5 AML patients were enrolled in this retrospective analysis. Event-free survival (EFS) and overall survival (OS) rates were estimated by Kaplan-Meier method and prognostic factors were evaluated by COX regression with SPSS. (1) Twelve patients gave up treatment after confirmed diagnosis. Two patients died on the second day after chemotherapy. Of the 61 patients, 73.8% (45/61) achieved complete remission (CR) after two courses of chemotherapy. The 5-year EFS rate was 34.5% ± 6.8%. But of the 117 non-AML-M5/M3 AML patients, the 5-year EFS rate was 51.0% ± 4.9%. (2) Multivariate analysis showed that age ≥ 10 y, the proportion of bone marrow blast cell counts ≥ 15% after the first induction therapy, not CR after two courses of chemotherapy were risk factors for the long-term prognosis. (3) Of the 20 patients whose bone marrow blast cell counts ≥ 15% after the first induction therapy, 5 patients who choose allo-HSCT had a better OS than the other 15 patients who choose chemotherapy only (60.0% ± 21.9% vs. 7.3% ± 7.1%, P = 0.024). Children with AML-M5 had a poorer prognosis than the other AML patients; patients whose bone marrow blast cell counts ≥ 15% after the first induction therapy chose allo-HSCT had a better prognosis. At present, there is no enough evidence to support that patients whose bone marrow blast cell counts < 15% after the first induction therapy should choose unrelated donor for allo-HSCT.

Expression of FLT3 Internal Tandem Duplication in Pediatric Patients with Acute Myeloid Leukemia and Its Correlation with Multidrug Resistance.

Abstract 4427FLT3 Internal Tandem Duplication is a kinase mutation frequently observed in cases of adult acute myeloid leukemia (AML). This study was aimed to investigate the expression of FLT3 internal tandem duplication (FLT3-ITD) in pediatric patients with acute myeloid leukemia (AML) and to analyse the clinical features of patients with mutations and the relation of FLT3-ITD with multidrug resistance gene 1 (mdr1). Samples were analysised from eighty-one new diagnosed pediatric patients with AML. The diagnosis was based on French-American-British (FAB) criteria. Immunophenotyping was done by flow cytometry. For each patient, several clinical and biological characteristics were analyzed (age, WHO performance status, WBC count at diagnosis, bone marrow blasts, FAB subtypes, immunologic phenotype, and cytogenetics). The expressions of FIT3-ITD and mdr1 gene in bone marrow samples was determined by RT-PCR. patients were treated with uniform chemotherapy. Statistical analysis were carried by SAS. The results indicated that the FLT3-ITDs were detected in 8 out of 81 pediatric patients (9.88%) and all mutations detected were hybrid, while less frequently this mutation was detected in adult patients. Although they were irrelevant with sex and immunophenotypes, the mutations seemed predominant in older pediatric patients. The average age is 14-year-old in the patients with mutations, but 9-year-old in those without mutation (P=0.039). The leukocyte counts and bone marrow blast cell counts in pediatric patients with FLT3-ITD at diagnosis were higher than those in pediatric patients without FLT3-ITD (median of 58.4×10∧9/L versus 19.49 ×10∧9/L, p = 0.001 and median of 78.5% versus 54.4%,p = 0.041 respectively). The normal chromosomes were found in most pediatric patients with FLT3-ITD, although no significane in the stastistics. The patients with FLT3-ITD had lower induction remission rate (only 25%), but the patients without FLT3-ITD had higher remission rate (76.1%). According results detected by RT-PCR, the mdr1 gene was found in 27 pediatric patients, but only 3 out of 8 pediatric patients with FLT3-ITD were detected to express both FLT3-ITD and mdr1. It is concluded that the FLT3-ITD is a frequent mutation in pediatric patients with AML, especially in the elder pediatric patients. It is related with higher leukocyte counts and bone marrow blast cell counts, which may due to the continual activation of Flt3 tyrosine kinase. The prognosis of these patients with mutations is worse and the induction remission rate is lowe. But the FLT3-ITD not relates with the mdr1, which suggests that the low induction rate is not resulted from the multidrug resistance, at least not from the P-glycoprotein (Pgp) activity. So the common MDR modulators may be uneffective for therapy of the patients with FLT3-ITD. Disclosures:No relevant conflicts of interest to declare.

Elevated serum endostatin levels are associated with favorable outcome in acute myeloid leukemia

Background: Endostatin is the C-terminal antiangiogenic fragment of the extracellular matrix protein collagen XVIII, and is generated by tumor-derived proteases. The levels and the prognostic relevance of serum endostatin in acute myeloid leukaemia (AML) patient are not fully clear.Objective: The aim of this study was to evaluate serum levels of endostatin in acute myeloid leukemia patients before chemotherapy and after achieving complete remission and to correlate endostatin levels with patient outcome.Materials and methods: Serum samples from 30 adult patients (22 males and eight females, median age 37, range 19–66 years) with AML had been taken before chemotherapy was administered. In addition 25 out of 30 patients were reinvestigated again at complete remission (CR). Ten samples from healthy normal persons of matched age and sex were evaluated as a reference control group. Serum endostatin levels were determined using enzyme linked immune sorbent assay (ELISA).Endostatin serum levels were not significantly different in the pre-treatment AML patients as compared to that in normal controls (p>0·05). In AML patients the baseline endostatin levels were significantly lower than at CR (p=0·001).Results: No significant relation were detected between pre-treatment serum endostatin levels and age, peripheral blood white cell counts, platelet counts, bone marrow blast cell counts, blast cell distribution ratio or cytogenetic findings. The prognostic value of serum endostatin (sE) was also evaluated by dividing AML patients into high and low sE groups using the 75 percentile sE levels of the patients group as cutoff. The authors found that patients group in the high sE group survived for significantly longer time than those patients in the low sE group.Conclusion: Elevated endostatin levels at AML diagnosis is a good prognostic marker for patients' outcome. Wide scale study is recommended in order to establish the clinical value of this study.

Elevated serum endostatin levels is associated with favorable outcome in acute myeloid leukemia

Endostatin is the C-terminal antiangiogenic fragment of the extracellular matrix protein collagen XVIII, and is generated by tumor-derived proteases. The levels and the prognostic relevance of serum endostatin in AML patient is not fully clear. To evaluate serum levels of endostatin in acute myeloid leukemia patients before chemotherapy and after achieving complete remission and to correlate endostatin levels with patients outcome. Serum samples from 30 adult patients (22 males and 8 females, median age 37, range 19-66 years) with AML had been taken before chemotherapy was administered. In addition 20 out of 30 patients were reinvestigated again at complete remission (CR). Ten samples from healthy normal persons of matched age and sex were evaluated as a reference control group. Serum endostatin levels were determined using enzyme linked immune sorbent assay (ELISA). Endostatin serum levels were not significantly different in the pretreatment AML patients as compared to that in normal controls (P>0.05). In AML patients the baseline endostatin levels were significantly lower than at CR (P=0.001). No significant correlation were detected between pretreatment serum endostatin levels and age, peripheral blood white cell counts, platelet counts, bone marrow blast cell counts, blast cell distribution ratio. The prognostic value of sE was also evaluated by dividing AML patients into high and low sE groups using the 75 percentile sE levels of the patients group as cutoff. The authors found that patients group in the high sE group survived for significantly longer time than those patients in the low sE group. Elevated endostatin levels at AML diagnosis is a good prognostic marker for patients' outcome. Wide scale study is recommended in order to establish the clinical value of this study.

Angiogenesis factor pattern differs in acute lymphoblastic leukemia and chronic lymphocytic leukemia

Angiogenesis is an important event in the survival and progression of solid tumors. The angiogenic status and the exact role of the angiogenic cytokines in lymphoid leukemia has not been fully elucidated.We have investigated the profile of the systemic components of angiogenic regulation in B-lineage acute lymphoblastic leukemia (B-ALL) and B-chronic lymphocytic leukemia (B-CLL), namely vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), endostatin and matrix metalloproteinase-9 (MMP-9) using enzyme-linked immunosorbent assay (ELISA).In B-ALL patients, sVEGF, and MMP-9 were significantly lower than control levels at diagnosis (p < 0.001) and increased to near control levels in remission (p > 0.05). Both serum TNF-α and endostatin levels showed no significant difference at diagnosis (p > 0.05) and in remission (p > 0.05) compared to control levels. VEGF, TNF-α, MMP-9 and endostatin levels were not significantly correlated with peripheral white cell count or bone marrow blast cell count, but were positively correlated with platelet count.In B-CLL patients, serum VEGF, MMP-9 and TNF-α were significantly higher (p < 0.001 = 0.009, 0.007, respectively) and decreased to near control levels in remission (p > 0.05 for all). Serum endostatin levels showed no significant difference at diagnosis and in remission compared to control levels (p > 0.05). A significant positive correlation between VEGF, TNF-α, MMP-9 and peripheral white cell counts, bone marrow lymphocytic count and platelets count were found.In conclusion, our data suggest that the driving forces of angiogenic factors (VEGF, TNF-α and MMP-9) in adult B-ALL appears different from that in B-CLL patients.

Prognostic relevance of circulating matrix metalloproteinase‐2 in acute myeloid leukaemia patients

Matrix metalloproteinases (MMPs) were postulated to have important implication in progression and invasiveness of many malignant disorders. On the other hand the biological role of MMP-2 in acute myeloid leukaemia (AML) is not fully clear. Serum samples from 37 adult patients with AML had been taken before chemotherapy was administered. In addition 20 out of the 37 patients were analysed again after achieving complete remission (CR). Ten samples from healthy volunteers were evaluated as the control. Total MMP-2 levels were measured using ELISA Kit obtained from R&D system. MMP-2 serum levels were significantly lower in pretreatment AML patients than that in the normal controls (p = 0.000) and in CR (p = 0.007). No significant correlations were detected between pretreatment sMMP-2 levels and FAB subtypes, peripheral blood blast cell counts, peripheral blood WBCs, bone marrow blast cell counts or blast cell distribution ratio. The prognostic value of MMP-2 was evaluated by dividing AML patients into high and low MMP-2 groups using the pretreatment median MMP-2 level of the AML group as the cut-off. The authors found that patients in the high group survived for a significantly shorter time than those patients in the lower MMP-2 group. High pretreatment levels of sMMP-2 among AML patients were associated with poor survival. Prospective studies are recommended to establish the clinical value of longitudinal sMMP-2 measurement.