A Five Parameter Based Flow Cytometric Scoring System Refines the Revised International Prognostic Scoring System for Myelodysplastic Syndromes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3849-3849
Author(s):  
Canan Alhan ◽  
Theresia M. Westers ◽  
Eline M.P. Cremers ◽  
Claudia Cali ◽  
Gert J Ossenkoppele ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Multivariate Analysis ◽  
Myelodysplastic Syndromes ◽  
Scoring System ◽  
Risk Group ◽  
Myeloid Cells ◽  
Low Risk ◽  
Added Value ◽  
Flow Cytometric ◽  
Myeloid Progenitors

Abstract Abstract 3849 The International Prognostic Scoring System (IPSS) has found widespread application in the clinical practice of myelodysplastic syndromes (MDS) for estimation of prognosis and treatment decision making. Increasing knowledge on components that encompass the IPSS has led to the design of the revised IPSS (IPSS-R, Greenberg, Blood 2012). Furthermore, it was recognized that techniques such as flow cytometry and molecular analyses should be validated for their prognostic value in MDS; once proven the added value, they might be part of future prognostic scoring systems. Over the past years, flow cytometry based scoring systems have been proposed to refine prognostication of MDS (Wells, Blood 2003; Matarraz, Cytometry 2010). The aim of our study was to construct a simple and widely applicable hazard-adapted flow cytometry based scoring system for the prognosis of myelodysplastic syndromes and investigate whether it is of added value to the IPSS-R. Bone marrow of 96 patients with MDS was analyzed by flow cytometry. Patients were assigned to an IPSS-R category based on bone marrow blast count, cytogenetics and depth of cytopenias. Patients that received a hematopoietic stem cell transplantation or chemotherapy were censored from start of treatment. All samples were processed according to European LeukemiaNet guidelines (van de Loosdrecht, Haematologica 2009 and Leukemia 2012). Aberrancies with regard to count, expression level of markers and lineage infidelity marker expression on myeloid progenitors, B cell progenitors, maturing erythroid, monocytic and myeloid cells were analyzed. The optimal cut-off level per variable was determined based on significance and hazard ratio (HR) of overall survival (OS) and univariate analysis. In the second step, multivariate analysis was performed with the variables that had a p-value ≤0.1. The parameters that were most predictive of OS from multivariate analysis were increased CD45 expression of myeloid progenitors, asynchronous expression of CD11b on myeloid progenitors, percentage of CD34negCD117pos mature myeloid cells, percentage of mature myeloid cells with CD14 expression and CD45 expression on monocytes. In the next step, a flow cytometric scoring system based on these five parameters was constructed. Each variable was scored for in a weighed manner and the weight was determined by the size of the HR. From this a MDS flow cytometric scoring system (MFCS) was designed that identified four risk groups. i.e. patients with a normal flow score (0–0.5 points), low (1 point), intermediate (>1–2 points) and patients with a high score (>2–4.5 points). The MFCS identified patients with significantly different OS, normal score, median OS 60.6 months (range 0.6–198.6 months), vs. low score, median OS 26.8 months (range 2.5–89.2 months), vs. intermediate score, median OS 19.1 months (range 1.6–62.3 months), vs. high score, median OS 7.9 months (range 0.5–15.1 months), p<0.001. In concordance with the IPSS-R cohort, the largest number of patients was within the low risk group, 41.7% (40/96). Interestingly, within this IPSS-R risk group, the MFCS was able to identify prognostic subgroups. The median OS of IPSS-R low risk patients with a normal MFCS score was not reached, compared with low risk patients with a low MFCS, median OS 40.6 months (range 22.5–89.2 months) and compared with intermediate MFCS, median OS 19.1 months (range 12.5–43.6 months), p<0.001. In a multivariate analysis the MFCS combined with the IPSS-R were best predictive for OS rather than the scores alone (p=0.002 and p=<0.001, respectively). In conclusion, we designed a simple, five parameter MDS flow cytometric (MFCS) score based on hazard. The MFSC score was able to identify prognostic subgroups within a well defined IPSS-R risk group. Furthermore, the MFCS combined with the IPSS-R offered refined prognostication for MDS. This implies that flow cytometric analysis has an added value for prognostication and should be part of a prognostic scoring system for MDS. In a currently ongoing prospective multi center study, these new scoring models will be validated. Disclosures: Alhan: Celgene: Honoraria. Ossenkoppele:Celgene: Consultancy, Honoraria. van de Loosdrecht:Celgene: Consultancy, Honoraria.

Hesk Ratio: A Complementary Tool for the Diagnosis of Myelodysplastic Syndromes and a Novel Method for Flow Cytometry Analysis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4946-4946
Author(s):  
Evgenia Verigou ◽  
Georgia Kolliopoulou ◽  
Nikoleta Smirni ◽  
Elisavet Hala ◽  
Polixeni Lampropoulou ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Myelodysplastic Syndromes ◽  
Myeloid Cells ◽  
Antigen Expression ◽  
Low Risk ◽  
Empirical Parameter ◽  
Analytical Approaches

Abstract Abstract 4946 Establishing the diagnosis of Myelodysplastic Syndromes (MDS) is a challenging task for hematologists due to the heterogeneity of this clinical entity. Several attempts have been made to include findings from advanced technologies to the diagnostic criteria of MDS, but still in the majority of cases, morphology of peripheral blood and bone marrow remains the cornerstone for the diagnosis. Flow cytometry(FC) can identify abnormal antigen expression on myeloid cells. FC has been proposed as a complementary method in the diagnosis of low and intermediate risk MDS, particularly for patients not exhibiting characteristic karyotype abnormalities. On the other hand, recent literature suggests that these findings are not MDS-related, questioning the specificity of immunophenotyping for the diagnosis of MDS. The aim of the present study is to maximize the utility of FC data and simplify their interpretation for the diagnosis of MDS, by developing new analytical approaches of digital data, other than the conventional sequential biparametric analysis. The applied methodology was based on a mathematical model of scale analysis. Bone marrow(BM) samples from 50 subjects were analysed for the expression of CD45PC7, CD11bPC5, CD16FITC and CD13PE (antigens by Beckman Coulter, FC500 flow cytometer Beckman Coulter). 36 patients were diagnosed with MDS (23 low risk, 13 high risk) and 14 patients had other than an MDS diagnosis (ITP, chronic idiopathic neutropenia, systemic lupus erythematosus, LGL leukemia, age-related cytopenias, aplastic anemia, myelofibrosis etc). Additionally, 3 BM samples of patients with post-MDS acute myeloid leukemia(AML) were analysed. The data used for the development of the mathematical model were the following: two populations (neutro1, neutron2) were gated according to their CD45 and CD13/CD16 antigen expression (Figure 1i-1v).Seven subpopulations of Neutrophils were defined on CD11b/CD16 density plot N=g+h+i and O=k+j (Figure 1vi). In an attempt to identify correlations between data that cannot be routinely revealed by sequential biparametric analysis, we have developed the HeSK* ratio, which is given by: where x is the median of CD11b in gate O, y is the median of CD16 in gate O, z is the median of CD45 in gate neutro, pO is the percentage of gate O in the total CD11b/CD16 diagram gated in neutro, pN is the percentage of gate N in the total CD11b/CD16 gated in neutro and 1000 is an empirical parameter. The HeSK ratio combines fluorescence levels of CD16, CD11b and CD45 with the percentage of two distinct neutrophil populations (N and O), which differ in their maturation and differentiation stage. The ratio can quantify the abnormal differentiation profile of mature myeloid cells and thus distinguish MDS from non-MDS samples with statistical significance P<0. 0001 (Kruskal Wallis test) as indicated in graph 1. Descriptive statistics are shown in table 1. · HeSK ratio is based upon a novel FC analysis method that could change the conventional biparametric routine FC analysis and quantify patterns that are not evaluated properly. Mathematical modeling of antigen expression patterns optimizes the interpretation of single immunophenotype findings. · The present study proposes HeSK as a complementary diagnostic tool for MDS and a strong indicator for the classification of the patients according to their prognosis as well. *the name HeSK comes from the initials of the 4 main authors (H=Hala, e=Evgenia, S=Smirni, K=Kolliopoulou). Table 1 non MDS low risk MDS high risk MDS Number of values 14 23 13 Minimum 50,76 4,789 0,2850 25% Percentile 304,8 26,11 17,05 Median 2133 92,52 47,64 75% Percentile 10650 228,9 144,3 Maximum 55040 3043 671,7 Mean 10320 316,1 122,7 Std. Deviation 17860 647,9 185,1 Std. Error 4773 135,1 51,33 Figure 1 Figure 1. Disclosures: No relevant conflicts of interest to declare.

Validation of a Flow Cytometric Scoring System for the Prognostication of the Myelodysplastic Syndromes: a Retrospective Cohort Study

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4012-4012
Author(s):  
Canan Alhan ◽  
Theresia M. Westers ◽  
Claudia Cali ◽  
Gert J. Ossenkoppele ◽  
Arjan A. Van de Loosdrecht
Keyword(s):  
Healthy Volunteers ◽  
Board Of Directors ◽  
Myelodysplastic Syndromes ◽  
Scoring System ◽  
Research Funding ◽  
Classification Systems ◽  
Prognostic Information ◽  
Advisory Committees ◽  
Flow Cytometric ◽  
Myeloid Progenitors

Abstract Abstract 4012 The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders, characterized by cytopenia(s), dysplasia and a propensity to evolve into acute myeloid leukemia. The International Prognostic Scoring System (IPSS) and WHO-based prognostic scoring system provide prognostic information. However, even if patients are allocated in the same risk category their clinical course remains heterogeneous. Recent developments in the treatment of MDS require refinement of prognostication and identification of patients who might benefit from treatment with potentially disease modifying agents such as lenalidomide or azacitidine. Flow cytometry (FC) is emerging as a valuable technique for the diagnosis and prognosis of MDS. Recently, we demonstrated that flow cytometric analysis of BM in low and int-1 risk MDS is instrumental to identify clinically relevant subgroups. (Westers et al, Blood 2010) Previously, it was reported that a flow cytometric scoring system (FCSS) is predictive for worse outcome in MDS. (Wells et al, Blood 2003, van de Loosdrecht et al, Blood 2008) The FCSS is a scoring system that allows for a numerical display of immunophenotypic aberrancies in the (im)mature myelo-monocytic lineage. Scores are generated by enumerating abnormalities; a high score reflects a high number of aberrancies. The current study aimed to validate the FCSS for identification of prognostic subgroups in MDS. We analyzed aberrancies in (im)mature myelo-monocytic cells by FC in BM of 102 MDS patients, including 48 MDS patients from the previous cohort. The diagnoses according to WHO 2001 classification were RA(RS) n=19, RCMD(RS) n=54, RAEB-1 n=11, RAEB-2 n=13, MDS-U n=5 and also age-matched healthy volunteers (n=39) were included. The median age of MDS patients was 66 and of healthy volunteers 57. The FCSS in RA(RS) (median=3, range 1–6) patients was significantly higher compared with healthy controls (median=1, range 0–2, p<0.001). In contrast to our previous results the FCSS for RA(RS) and RCMD(RS) patients did not differ. This is a remarkable finding, since by morphology RA(RS) patients have unilineage dysplasia, in contrast to flow cytometric findings, where 84% (16/19) of the RA(RS) patients had two or more aberrancies in the (im)mature myelomonocytic compartment. The FCSS was higher in RAEB-1 (median=6, range 2–7) and RAEB-2 (median=6, range 4–8) compared with RCMD(RS) (median=3, range 0–6, p=0.02 and p<0.0001, respectively). Overall, the FCSS correlated significantly with WHO 2001 classification (p<0.0001). The FCSS showed a significant correlation with IPSS categories low, int-1, int-2 and high (p<0.0001). Remarkably, the FCSS was not correlated with cytogenetic risk categories low, intermediate and poor. The new German-Austrian Cytogenetic Prognostic Scoring System for MDS was also not correlated with the FCSS. This indicates that the FCSS and cytogenetics might provide separate prognostic information in MDS. Neutrophil granularity corresponding with side scatter by FC was significantly decreased in MDS patients compared with healthy volunteers (p<0.0001). In the RA(RS) and RCMD(RS) category, 40% (29/73) of patients expressed an aberrant marker such as CD5, CD7 and/or CD56 on myeloid progenitors. Transfusion data was available of 51 patients. Interestingly, the majority of MDS patients who were transfusion dependent or progressive, had aberrant expression of CD5, CD7 and/or CD56 on myeloid progenitors compared with MDS patients without aberrant marker expression (64% (16/25) vs 31% (8/26), respectively p=0.04). When the cumulative amount of all aberrancies in the (im)mature myelo-moncytic cells were taken into account, transfusion dependent patients had significantly more aberrancies than transfusion independent MDS patients, (median 6.5 vs 4, respectively, p=0.006). In conclusion, we here confirmed our previous findings in a larger cohort. The majority of RA(RS) patients already has multilineage dysplasia as detected by FC, which might be of prognostic relevance. Although the FCSS correlates with current prognostic systems, a striking heterogeneity remains within prognostic subgroups. Therefore, the FCSS and detection of aberrant myeloid progenitors can provide refined prognostication by identification of patients at risk for transfusion dependency and adverse clinical outcome, independent of current classification systems. Disclosures: Ossenkoppele: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Degree of Aberrant Antigen Expression in Myelodysplastic Syndromes Correlates with the Number of Molecular Mutations

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2856-2856
Author(s):  
Wolfgang Kern ◽  
Manja Meggendorfer ◽  
Seishi Ogawa ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Molecular Genetics ◽  
Myelodysplastic Syndromes ◽  
Antigen Expression ◽  
Erythroid Cells ◽  
Employment Equity ◽  
Cell Compartment ◽  
Flow Cytometric ◽  
Equity Ownership ◽  
Myeloid Progenitors

Abstract Introduction: The diagnostic approach for suspected myelodysplastic syndromes (MDS) is evolving and flow cytometry and molecular genetics are increasingly considered to be applied in addition to cytomorphology and cytogenetics. While reports on comparisons of flow cytometric findings with results of cytomorphology and cytogenetics are available, data on comparisons between results obtained by flow cytometry and molecular genetics, however, have not yet been presented in detail. Aims: 1) To assess the correlation between flow cytometric findings on MDS-specific aberrant antigen expression and the presence of molecular mutations in patients with cytomorphologically proven MDS. 2) To determine the respective impact of flow cytometric findings and of molecular mutations on survival in patients with MDS. Patients and methods: In 256 patients (male/female, 161/95; median age 72 years, range 24-90) with proven MDS (137 low-risk MDS, 119 RAEB1/2) we compared data on aberrantly expressed antigens (AEA) determined according to ELN guidelines (Westers, Leukemia 2012) to the previously published mutational status of 104 genes (Haferlach, Leukemia 2014). Results: Median numbers (ranges) of AEA were 0 (0-3) in myeloid progenitors, 2 (0-4) in granulocytes, 1 (0-5) in monocytes and 0 (0-1) in erythroid cells. Median number of mutation was 2 (0-7). The number of AEA in myeloid progenitors, granulocytes and monocytes increased with increasing number of mutations (r=0.257, p<0.001). Accordingly, in cases with ≥3 mutations the number of AEA in myeloid progenitors, granulocytes and monocytes was higher than in cases with ≤2 mutations (mean±SD, 3.9±1.9 vs. 3.0±2.0, p=0.001). This correlation was significant also when considering granulocytes as a single cell compartment (r=0.308, p<0.001) but non-significant trends only for myeloid progenitors and monocytes. No such correlation was observed for erythroid cells. Specifically, mutations in each of the genes TET2, ASXL1, SRSF2, STAG2, ZRSR2 or NF1 were associated with significantly higher numbers of AEA in ≥1 cell compartment. Cases with mutations in ≥1 of these genes (n=145), as compared to those without these 6 mutations (n=111), had higher numbers of AEA in myeloid progenitors (0.4±0.7 vs. 0.2±0.5, p=0.037), granulocytes (2.0±1.1 vs. 1.4±1.1, p<0.001) and monocytes (1.5±1.3 vs. 1.0±1.0, p=0.002). Consequently, the difference in the total of AEA was even larger (3.9±2.0 vs. 2.7±1.9, p<0.001). Regarding scoring points according to IPSS-R, there was a significant correlation with the number of AEA in granulocytes (r=0.189, p=0.004) as well as with the number of AEA in monocytes (r=0.159, p=0.017). Consequently, there was also a significant correlation between the IPSS-R scoring points and the number of AEA in myeloid progenitors, granulocytes and monocytes (r=0.227, p=0.001). Overall survival was impacted by the presence of mutations in ≥1 of the genes TP53, EZH2, ETV6, RUNX1 and ASXL1 (p<0.001, HR 2.9) published by Bejar (NEJM 2011) as well as by the presence of ≥3 AEA in myeloid progenitors, granulocytes and monocytes (p=0.015, HR 1.7) and by IPSS-R (p<0.001, HR 1.4). Multivariate analysis considering mutations and AEA revealed an independent significance for both of them (mutations, p<0.001, HR 2.9; AEA, p=0.017, HR 1.7). However, inclusion of also IPSS-R as a covariate resulted in a trend only for AEA (p=0.16, HR 1.4) and independent significance for mutations (p<0.001, HR 2.3) and IPSS-R (p<0.001, HR 1.3). Conclusions: This data demonstrates that the degree of flow cytometric findings on MDS-related aberrant antigen expression correlates with the number of molecular mutations as well as with the IPSS-R. The present result therefore further support the consideration of both flow cytometry and molecular genetics for the diagnostic work-up of MDS in an integrated approach in combination with cytomorphology and cytogenetics. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Flow Cytometric Score Correlates with Clinical Response to Azacitidine In Intermediate-2 and High Risk Myelodysplastic Syndrome Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 441-441
Author(s):  
Canan Alhan ◽  
Theresia M. Westers ◽  
Corien Eeltink ◽  
Claudia Cali ◽  
Gert J. Ossenkoppele ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Risk ◽  
Scoring System ◽  
Response To Treatment ◽  
Advisory Committees ◽  
Flow Cytometric ◽  
Marker Expression ◽  
Pre Treatment ◽  
Myeloid Progenitors

Abstract Abstract 441 New treatment strategies that potentially change the natural course of intermediate (int)-2 and high risk myelodysplastic syndromes (MDS), such as azacitidine, are emerging. Recently, we reported that flow cytometric analysis of bone marrow (BM) in low and int-1 risk MDS is instrumental to identify clinically relevant subgroups. (Westers et al, Blood 2010) Moreover, it was reported that a flow cytometric scoring system (FCSS) is predictive for worse outcome in MDS. (Wells et al, Blood 2003, van de Loosdrecht et al, Blood 2008) The FCSS is a scoring system that allows for a numerical display of immunophenotypic aberrancies in the (im)mature myelo-monocytic lineage. Scores are generated by enumerating abnormalities; e.g. high scores reflect a high number of aberrancies. The current study aimed to investigate the role of this flow cytometry-based scoring system to assess and monitor response to treatment in int-2 and high risk MDS patients treated with azacitidine. Bone marrow aspirates were analyzed by flow cytometry in 18 MDS patients who were treated with azacitidine. Aspirates were drawn before treatment and after every third cycle of azacitidine. Response to treatment was evaluated using IWG-2006 criteria. The median age was 71 (range 50–78). Distribution over WHO 2001 categories was RCMD-RS n=2, RAEB-2 n=7, AML with 20–30% blasts n=6 and MDS/MPD n=3. International prognostic scoring system (IPSS) categories comprised int-2 n=8 and high n=5. In 5 patients the IPSS score could not be assessed due to lack of cytogenetics, these patients were at least int-2 MDS patients. Flow cytometric follow up was available in 12 patients due to short follow up, i.e. in 4 responders, 4 progressive disease (PD) and 4 stable disease (SD) patients; 3 patients stopped due to non-hematologic toxicity, 4 patients died of PD. Median follow up was 7 months (range 3–12). Median pre-treatment Hb was 6.7 mmol/L, platelets 35.5*10e9/L and absolute neutrophil count (ANC) 0.82*10e9/L. Responders had a significant increase in Hb (median 7.8 mmol/L, p=0.04), platelets (291.5 *10e9/L, p=0.03) and ANC (1.4*10e9/L, p=n.s. compared with baseline values). SD and PD patients had a median Hb of 6.5 mmol/L, platelets 69*10e9/L and ANC 0.77*10e9/L. The median pre-treatment flow score was 6 (range 3–8). Interestingly, responders had a significant decrease in flow score from median 5 to median 2 (range 1–3, p=0.005) after 3 months of treatment. No change in flow scores was seen in PD and SD patients after 3 months of treatment (median=6, range 4–8 and pre-treatment FCSS median=6.5, range 3–8). A sustained decrease in flow score was seen in 4 responders after 6 months of treatment (median 2, range 1–3) parallel to a further increase in median Hb (9.0 mmol/L), platelets (278.5 *10e9/L) and ANC (1.7*10e9/L). After 9 cycles of azacitidine, 3 patients were still responsive to treatment (median Hb 9.9 mml/L, platelets 169, ANC 4.07, median flow score 5, range 2–4). The majority of SD and PD MDS patients had aberrant marker expression on myeloid progenitors, such as CD5, CD7 and/or CD56 compared with responsive MDS patients, (63% (5/8) vs 25% (1/4). Moreover, initial loss of aberrant marker expression on myeloid progenitors was detected in one patient who responded to azacitidine treatment. At 9 months, this initially responsive patient and a patient from the stable disease group developed progressive disease. Interestingly, the initially responsive patient showed an increase in the percentage of myeloid progenitors with an aberrant immunophenotype at 6 months; of note, the total percentage of CD34+ cells was less than 3% by flow cytometry. The initial loss of aberrant marker expression on myeloid progenitors might be caused by a relative increase of normal progenitors and decrease of malignant progenitors caused by azacitidine treatment. In conclusion, our data indicate that flow cytometry identifies MDS patients who may benefit from azacitidine treatment by detection of aberrant marker expression on myeloid progenitors. Moreover, the data indicate that the FCSS may be instrumental in selection of SD patients who may benefit from prolonged treatment with azacitidine. Disclosures: Ossenkoppele: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Flow Cytometric Detection of Human Telomerase Reverse Transcriptase Expression in CD34 Positive Precursor Fraction in Myelodysplastic Syndrome Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4300-4300
Author(s):  
Hiroshi Handa ◽  
Takafumi Matsushima ◽  
Norifumi Tsukamoto ◽  
Masamitsu Karasawa ◽  
Hiroyuki Irisawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Disease Progression ◽  
Risk Group ◽  
Telomerase Activity ◽  
Low Risk ◽  
Htert Expression ◽  
Trap Assay

Abstract Telomerase activity has been found in most common cancers indicating that telomerase detection may be a useful marker in cancer diagnosis. For detection of telomerase activity and the expression of associated genes in cells, TRAP assay and RT-PCR are customarily used. Immunohistochemical detection of hTERT is useful to detect telomerase-positive cells in a background of non- cancerous cells. We developed a method for the detection of intra-nuclear hTERT protein, in a sub-population of hematopoietic cells, using concurrent staining cell surface antigen and multi color flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas neutrophils of normal subjects showed 0% positivity, it is consistent with telomerase activity assessed by TRAP assay (r=0.71, p&lt;0.0001) and previous observations. Then we applied this method to analyze hTERT expression in myelodysplastic syndrome (MDS). Forty MDS patients samples were obtained, 36 patients were diagnosed as low risk MDS (RA), 14 patients were diagnosed as high risk MDS (RAEB or RAEB-t) according to FAB classification. All samples were acquired after informed consent was obtained from the patients. Expression of hTERT protein was higher in CD34-positive blast-gated cells than CD34-negative blast-gated cells. The percentage of the CD34+ cells expressing hTERT ranged from 9.66% to 90.91% in low risk MDS patients, whereas from 50.46% to 97.68% in high risk MDS. The expression level was higher in the high risk group compared to that in the low risk group in MDS (p=0.0054, p=0.0084). This observation implied that telomerase up-regulation and hTERT expression were important for disease progression and could be a marker of more advanced disease. In subsets of MDS and AML bone marrow specimens obtained from these patients, we examined the hTERT expression in CD34+/CD38 high cells and CD34+/CD38 low cells containing stem cell fraction. Of interest, some of the patients showed higher expression of hTERT in CD34+/CD38 low cells than in CD34+/CD38 high cells. This observation is inconsistent with previous reports describing normal bone marrow hematopoietic cell findings. We speculated that this phenomenon could be a marker of MDS abnormality and that telomerase up-regulation may be initiated in the more primitive precursor fraction containing hematopoietic stem cells during the disease progression. Telomerase studies may be useful for definition of the risks associated with disease severity. Multi-parameter nature of flow cytometry and its ability to identify cellular sub-populations will facilitate a fuller understanding of the mechanisms of activation of telomerase.

Independent Validation of the 2008 World Health Organization (WHO) Reclassification of Myelodysplastic Syndromes (MDS) Associated with Ring Sideroblasts

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2686-2686 ◽  
Author(s):  
David P. Steensma ◽  
Curtis A Hanson ◽  
Ayalew Tefferi
Keyword(s):  
High Risk ◽  
Median Survival ◽  
Scoring System ◽  
Who Classification ◽  
Risk Group ◽  
Low Risk ◽  
Risk Category ◽  
Intermediate Risk ◽  
Multilineage Dysplasia ◽  
Ring Sideroblasts

Abstract Background: The 2001 WHO classification of myeloid neoplasms distinguished 2 forms of MDS associated with &gt;=15% ring sideroblasts and &lt;5% marrow blasts: refractory cytopenia with multilineage dysplasia and with ring sideroblasts (RCMD-RS) vs. refractory anemia with ring siderblasts (RARS, erythroid-restricted dysplasia). However, the real prognostic value of separating RCMD-RS from RCMD with &lt;15% ring sideroblasts and from RARS is uncertain, and the WHO has proposed merging RCMD-RS and RCMD in the 2008 classification revision. Furthermore, the WHO-based Prognostic Scoring System (WPSS), proposed by Malcovati and colleagues in 2005 as a dynamic system that overcomes some of the limitations of the 1997 International Prognostic Scoring System (IPSS), has undergone limited independent external validation to date and its applicability to sideroblastic MDS in particular is unclear. We assessed the validity of the 2008 WHO reclassification and the WPSS for MDS cases associated with &gt;=15% ring sideroblasts and a normal blast proportion. Methods: We reviewed WPSS and IPSS component parameters at diagnosis and the clinical outcomes of 465 patients (68% males, median age 72) evaluated at our institution over a 13-year period: 140 with RARS, 114 with RCMD-RS, and 211 with RCMD. Patients were assigned a WPSS score and risk category (very low-risk group=0 points; low=1; intermediate=2, high=3 or 4) by summing 3 subscores: 2001 WHO classification (0 for RARS, 1 point for RCMD or RCMD-RS), IPSS cytogenetic risk group (0=good, 1=indeterminate, 2=poor), and red cell transfusion dependence (0=no, 1=yes). Survival was assessed by Kaplan-Meier estimates, and prognostic factors examined by proportional hazards analysis. Results: The median time until death or last followup was 26 months, and 70% of patients were known to have died. The median survival by WHO MDS subtype was 75 months for RARS, 25 months for RCMD-RS, and 26 months for RCMD (Log-Rank p&lt;0.0001 for RARS vs. either RCMD-RS or RCMD; p=0.60 for RCMD vs. RCMD-RS ). Both the WPSS and IPSS predicted overall survival in patients with ring sideroblasts. Median survival for the patients grouped by WPSS risk category was 89 months for very low risk (n=95), 41 for low risk (n=198), 31 for intermediate risk (n=82), and 11 for high risk (n=91) (p&lt;0.0001, except for low risk vs. intermediate risk, p=0.31). (Very high risk WPSS scores cannot be achieved without excess marrow blasts, and such patients were excluded from this analysis.) Median survival by IPSS was 73 months for low-risk, 33 months for intermediate-1, and 8 months for intermediate 2 (p&lt;0.0001). The IPSS’ predictive power was unchanged if patients with secondary MDS were included or excluded (the IPSS was based on a review of 816 patients with apparently de novo MDS). Conclusions: These data support the WHO’s proposal to merge RCMD and RCMD-RS, and suggest that the adverse prognostic significance of multilineage dysplasia renders the presence of ring sideroblasts unimportant. The WPSS is a valid prognostic tool in patients with MDS associated with ring sideroblasts, but in this subgroup both the WPSS and IPSS stratify patients into 3 risk groups, and the WPSS does not offer additional value over the IPSS. Figure Figure

An Automated and Quantitative Protein Expression-Based Classification System to Identify High Risk Multiple Myeloma Patients

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 735-735
Author(s):  
Alex Klimowicz ◽  
Paola Neri ◽  
Adnan Mansoor ◽  
Anthony Magliocco ◽  
Douglas A. Stewart ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
High Risk ◽  
Protein Expression ◽  
Classification System ◽  
Risk Group ◽  
Univariate Analysis ◽  
High Risk Group ◽  
Group Classification ◽  
Low Risk ◽  
High Expression

Abstract Background: Autologous stem cell transplantation (ASCT) has dramatically improved the survival of myeloma patients; however, this approach has significant toxicities and nearly 25% of MM patients progress within one year from their transplant. While gene expression profiling-based (GEP) molecular classification has permitted the identification of unresponsive high-risk patients, these approaches have proven too costly and complex to translate into clinical practice. Less expensive and more readily available methods are needed clinically to identify, at the time of diagnosis, MM patients who may benefit from more aggressive or experimental therapies. While protein-based tissue arrays offer such alternative, biases introduced by the “observer-dependent” scoring methods have limited their wide applicability. Methods: We have designed a simplified, fully automated and quantitative protein expression based-classification system that will allow us to accurately predict survival post ASCT in a cost effective and “observer-independent” manner. We constructed tissue microarrays using diagnostic bone marrow biopsies of 82 newly diagnosed MM patients uniformly treated with a dexamethasone based induction regimen and frontline ASCT. Using the HistoRx PM-2000 quantitative immunohistochemistry platform, coupled with the AQUA analysis software, we have examined the expression of the following proteins: FGFR3 which is associated with t(4;14), cyclin B2 and Ki-67 which are associated with cellular proliferation, TACI which is associated with maf deregulation, and phospho-Y705 STAT3 and p65NF-κB, which are associated with myeloma cell growth and survival. For FGFR3, patients were divided into FGFR3 positive and negative groups based on hierarchical clustering of their AQUA score. For all other proteins examined, based on AQUA scores, the top quartiles or quintiles of patients were classified as high expression groups. Based on the univariate analysis, patients were further classified as “High Risk” MM if they had been identified as high expressers of either TACI, p65NF-κB or FGFR3. The Kaplan-Meier method was used to estimate time to progression and overall survival. Multivariate analysis was performed using the Cox regression method. Results: 82 patients were included in this study. In univariate analysis, FGFR3 and p65NF-κB expression were associated with significantly shorter TTP (p=0.018 and p=0.009) but not OS (p=0.365 and p=0.104). TACI expression levels predicted for worse OS (p=0.039) but not TTP (p=0.384). High expression of Ki67 or phospho-Y705 STAT3 did not affect survival. Of the 82 cases, 67 were included in the multivariate analysis since they had AQUA scores available for all markers: 26 (38.8%) were considered as High Risk by their AQUA scores and had significantly shorter TTP (p=0.014) and OS (p=0.006) compared to the Low Risk group. The median TTP for the Low and High Risk groups was 2.9 years and 1.9 years, respectively. The 5-years estimates for OS were 60.6% for the High Risk group versus 83.5% for the Low Risk group. Multivariate analysis was performed using del13q and our risk group classification as variables. Both our risk group classification and del13q were independent predictors for TTP, having 2.4 and 2.3 greater risk of relapse, respectively. Our risk group classification was the only independent predictor of OS with the High Risk group having a 5.9 fold greater risk of death. Conclusions: We have found that the expression of FGFR3, TACI, and p65NF-κB, in an automated and fully quantitative tissue-based array, is a powerful predictor of survival post-ASCT in MM and eliminates the “observer-dependent” bias of scoring TMAs. A validation of this “High Risk” TMA based signature is currently underway in larger and independent cohorts. Figure Figure

Platelet Functions and Risk Prognosis in Myelodysplastic Syndromes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3806-3806
Author(s):  
Nora V. Butta ◽  
Mónica Martín Salces ◽  
Raquel de Paz ◽  
Elena G. Arias Salgado ◽  
Ihosvany Fernández Bello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Risk ◽  
Myelodysplastic Syndromes ◽  
Surface Expression ◽  
Low Risk ◽  
Control Group ◽  
Platelet Surface ◽  
High Risk Patients ◽  
Fibrinogen Receptor ◽  
Risk Patients

Abstract Abstract 3806 The myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders with peripheral cytopenias and increased incidence of leukemic transformation. The prognosis of MDS is determined by several factors, including the presence of specific cytogenetic abnormalities, the percentage of blastoid cells in bone marrow and peripheral blood, the number of affected cell lineages, and transfusion dependency. The most commonly used risk stratification system is the International Prognostic Scoring System (IPSS). This score divides patients into a lower risk subset (low and intermediate-1) and a higher risk subset (intermediate-2 and high). Patients with MDS may have hemorrhagic complications with serious outcomes that are among the major causes of death in this population. These bleeding episodes that are often related to thrombocytopenia also occur in MDS patients with normal platelet count. The aim of this work was to study functional characteristics of platelets in MDS patients and their relationship to risk evaluated as indicated by IPSS. Eighty diagnosed MDS patients risk-stratified according to IPSS were included: 40 with low-risk, 29 with intermediate-1-risk (I-1), 8 with intermediate-2-risk (I-2) and 3 with high-risk. Eighty healthy donors were included as control group. Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-μm aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time, CT), which is expressed in seconds. Platelet activation was determined through FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 μM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (αIIb and β3 subunits) was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane phosphatidylserine (PS) exposed in basal conditions. I-2 and high-risk patients were gathered together in a high-risk group in order to analyze experimental results. Statistical analysis was performed with one-way ANOVA and Tukey test. CTs obtained with COL-EPI and COL-ADP cartridges in controls and low risk patients were similar and significantly shorter than CTs observed in I-1-risk and high-risk MDS patients (p<0.05). Platelets from all MDS patients showed a reduced capability for being activated by 100 μM TRAP. This impairment was more evident in I-1-risk and high-risk patients: PAC-1 binding, in arbitrary units (AU), was 11368±1017 in controls; 7849±789 in low-risk MDS (p<0.05); 4161±591 in I-1-risk MDS (p<0.01 versus control and p<0.05 versus low-risk) and 492±184 in high-risk MDS (p<0.01 versus control and p<0.05 versus low-risk). The platelet surface expression of P-selectin induced by 100 μM TRAP was also reduced: 5102±340 AU in controls, 3318±400 AU in low-risk MDS (p<0.05); 1880 ±197 AU in I-1-risk MDS (p<0.05 versus control and versus low-risk), and 1211±130 AU in high-risk MDS (p<0.05 versus control and versus low-risk). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients. Platelets from MDS patients expressed more PS than controls under basal conditions. Mean fluorescence values for FITC-annexin binding were: 383±16 in controls; 444±21 in low-risk (p<0.05); 575±52 in I-1-risk MDS (p<0.05 versus control and versus low-risk); 611±17 in high-risk MDS (p<0.05 versus control and versus low-risk). Our results indicated that platelets from MDS patients had less ability to be activated and were more apoptotic than control ones. These dysfunctions were more pronounced when the risk of the disease was higher according to IPSS. Disclosures: No relevant conflicts of interest to declare.

Identification Of Low Risk Group In Infants With Acute Lymphoblastic Leukemia By Flow Cytometric Minimal Residual Disease Measurement At Day 15 Of Interfant-99 and Interfant-06 Protocols Treatment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1333-1333
Author(s):  
Alexander Popov ◽  
Barbara Buldini ◽  
Paola de Lorenzo ◽  
Emanuella Giarin ◽  
Annamaria Di Meglio ◽  
...  
Keyword(s):  
Residual Disease ◽  
Risk Group ◽  
Cox Model ◽  
Lymphoblastic Leukemia ◽  
Low Risk ◽  
Older Children ◽  
Flow Cytometric ◽  
Minimal Residual ◽  
Risk Of Relapse

Abstract Background Acute lymphoblastic leukemia (ALL) in infants is a relatively rare disease with peculiar biological features and worse outcome in comparison to ALL in older children. Infant ALL is characterized by a high frequency of MLL gene rearrangements, mainly CD10-negative B-cell precursor ALL (BCP-ALL) immunophenotype and high tumor burden at diagnosis. Even with new therapeutic approaches event-free survival (EFS) in this subgroup of patients does not exceed 50%. Although flow cytometric (FCM) minimal residual disease (MRD) detection at day 15 of remission induction is well established for patients' stratification in older children treated with the AIEOP-BFM-2009 protocol, the prognostic value of FCM MRD in infant ALL is not fully known yet. Aim of the present study was to evaluate the prognostic significance of FCM MRD measurement in infants with ALL treated with Interfant-99 and Interfant-06 protocols in AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) centers in Italy. Patients and methods Between May 1999 and December 2011, 120 consecutive infants aged 0 to 365 days with newly diagnosed ALL were treated in AIEOP centers with the Interfant99 and the on-going Interfant-06 protocols. Among these patients, 51 (42.5%) with available day 15 follow-up bone marrow samples were included in this study on FCM MRD. In 39 (76.5%) cases, different types of MLL gene rearrangements were identified by fluorescence in situ hybridization (FISH), while 12 (23.5%) patients had germline MLL. MRD detection was performed by 4-6-color FCM. Median follow-up time was 3.5 years (range: 1 month – 7.5 years). Outcome was estimated by evaluating the probability of EFS and the cumulative incidence of relapse (CIR). Analysis of prognostic relevance of FCM MRD in combination with other criteria used for stratifying patients enrolled in the Interfant-06 protocol was performed with the Cox model on the cause-specific hazard of relapse. Results and discussion We classified infants according to the AIEOP-BFM day 15 stratification into three risk groups: 14 patients (27.5%) were considered at standard risk (SR: MRD less than 0.1%), 9 patients (15.7%) at high risk (HR: MRD 10% or more), and the majority of infants (29, 56.9%) at intermediate risk (IR: MRD 0.1% to 10%). As the 14 SR patients had 3-year EFS and CIR significantly better than other patients, we considered two major groups of patients with different outcome: SR group (MRD<0.1%) with 3-year EFS 77.9% (standard error, SE, 11.3) and CIR 14.9% (SE 10.2), and non-SR group with 3-year EFS 32.0% (SE 8.5) and CIR 58.0% (SE 8.8, p=0.0104 and p=0.0085, respectively). Half of SR group (7 of 14 cases) had germline MLL. 4 out of 7 MLL-positive SR-patients were in continuous complete remission (CCR) In contrast, the majority of infants in the non-SR group carried various types of MLL rearrangements. Only 5 cases in the non-SR group were MLL germline and only two of them are still in CCR. We evaluated the prognostic impact of day 15 MRD in MLL-positive cases (n=39). In this cohort of patients, we also observed a difference, although not statistically significant, between SR and non-SR groups both in 3-year EFS (57.1%, SE 18.7 and 30.9%, SE 9.2, respectively; p=0.3630) and in 3-year CIR (28.6%, SE 18.9 and 60.9%, SE 9.5, respectively; p=0.1733). We evaluated the suitability of MLL negativity and of day 15 FCM MRD <0.1% as single criterion for the identification of low-risk patients. Each factor, when separately analyzed in a Cox model, was significantly correlated with a reduction in the risk of relapse, as shown in Table 1, left panel. Nevertheless, as day 15 FCM MRD levels are strictly related to MLL status, the Cox model which analyzes jointly the two factors, is unable to identify the one independently impacting on the risk of relapse (Table 1, right panel). Thus, although being a strong prognostic factor by itself, day 15 FCM MRD stratification did not confer an advantage in relapse prediction when considered in combination with MLL status, which is the only low-risk group criterion in the Interfant-06 stratification. Conclusion Day 15 FCM MRD proved to be a suitable variable predicting treatment failure and can be used as an alternative or in combination with Interfant-06 stratification criteria to identify SR patients. Disclosures: Popov: Alexion: Research Funding.

Venous thromboembolism in psychogeriatric in-patients – A study of risk assessment, incidence, and current prophylaxis prescribing

2013 ◽  
Vol 25 (6) ◽  
pp. 913-917 ◽  
Author(s):  
Xinsheng Liu ◽  
Fintan O'Rourke ◽  
Huong Van Nguyen
Keyword(s):  
Risk Assessment ◽  
Venous Thromboembolism ◽  
Scoring System ◽  
Risk Group ◽  
Low Risk ◽  
Vte Prophylaxis ◽  
Retrospective Audit ◽  
Medium Risk ◽  
Risk Scoring ◽  
Risk Scoring System

ABSTRACTBackground: While venous thromboembolism (VTE) risk assessment and prophylaxis is well established for medical and surgical in-patients, there is a paucity of evidence, and therefore guidelines, in this area for psychogeriatric in-patients. We wished to determine VTE incidence, risk, and use of prophylaxis, in a psychogeriatric in-patient population.Methods: Retrospective audit of consecutive psychogeriatric patients aged 65 years and over admitted to Bankstown Hospital over a 3-year period, 2007–2009. Using an adapted VTE risk scoring system, patients were assigned as low, medium, or high VTE risk.Results: A total of 192 patients were included in the study. Mean age was 79.1 ± 7.0 years. Out of the total, 55.2% of patients had diagnosis of dementia, and 33.3% had depression. Overall, 81.8% (157/192) were assessed as low risk, and 18.2% (35/192) as medium risk. Also, 16.7% (32/192) received VTE prophylaxis.Four new VTE events occurred in medium-risk group, and one in low-risk group (p = 0.004). Overall VTE incidence was 10.5/10,000 patient-days, but 44.2 per 10,000 in medium-risk group. VTE risk score was predictive of VTE events – IRR 6.02 (95% Confidence Intervals (CI) = 1.76–20.7, p = 0.004) for every one-point increment in risk. Depression was associated with significantly higher VTE occurrence (6.3% in those with diagnosis vs. 0.8% without, p = 0.043).Conclusion: Using a VTE risk scoring system adapted for psychogeriatric in-patients, those assessed to be at medium risk had a significantly increased rate of VTE. On this basis, we would recommend VTE prophylaxis be prescribed for psychogeriatric in-patients assessed to be at medium and high level of risk.

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