Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes: A Prospective Validation Study in Low-Risk Patients with Normal Karyotype

Abstract Findings of recent studies indicate that flow cytometry (FCM) may be valuable in the diagnosis and prognostication of myelodysplastic syndromes (MDS). This approach appears particularly promising in patients with low-risk MDS without ringed sideroblasts and excess of blasts (i.e., with refractory anemia tout court) who have normal karyotype. These patients lack in fact any specific morphological or cytogenetic marker. However, the analytical methods reported so far require considerable technical skill, and therefore FCM has not yet become a routine procedure in the work-up of MDS patients. In this work, we developed a simple, reproducible FCM protocol for MDS and tested its validity prospectively. This study has been approved by the Ethics Committee, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy and by the Institutional Review Board of Nippon Medical School. The cytological diagnosis of MDS was made according to the WHO criteria by two independent cytologists who were blinded to clinical data. Three-color FCM was conducted at two laboratories (Tokyo and Pavia), which had received the details of the analytical method beforehand. The FCM protocol was developed in Tokyo and a part of which was reported previously (Leuk Res, 2008 32(5):699–707). The mandatory FCM parameters were CD34+ myeloblasts (% in all nucleated cells), CD34+ B-cell progenitors (% in all CD34+ cells), CD45 expression of CD34+ myeloblasts, and side scatter of mature myeloid cells. The optional parameters were CD11b, CD15, and CD56 expressions on CD34+ myeloblasts. These seven parameters were quantitatively analyzed and their reference ranges (RR) were determined using data from the cohort reported previously (Blood. 2006; 108(3): 1037–44). Bone marrow samples from 80 MDS patients with refractory anemia and normal karyotype, and from 82 controls were analyzed. Controls are patients who underwent routine diagnostic procedures for cytopenia and were eventually found to have conditions other than MDS and other clonal diseases. Abnormal data (outside the RR) in 2 or more parameters were common in MDS and were observed in 7 of 24 (29%) Japanese patients and 37 of 56 (66%) Italian patients when the four mandatory parameters alone were analyzed, and in 16 of 24 (67%) Japanese patients and 40 of 46 (87%) Italian patients when all seven parameters were analyzed (56 of 70 [80%] in total). A decreased CD34+ B-cell progenitor was the most common abnormality. By contrast, the occurrence of abnormalities in 2 or more FCM parameters was rare in control patients and was observed in 5 of 82 (6%) patients when all seven parameters were analyzed (56/70 versus 5/82, P < .0001). Therefore, when bone marrow samples lacking ringed sideroblasts and blast excess, and having normal karyotype show 2 or more abnormal FCM parameters, the likelihood ratio of MDS is 13.1 (95% confidence interval [CI], 6.4 to 29.3): the diagnostic sensitivity and specificity were 80% (95% CI, 74 to 84%) and 94% (95% CI, 89 to 97%), respectively. In conclusion, the findings of this study strongly indicate that the adopted FCM protocol is feasible and useful for diagnosing MDS in patients who lack specific morphological or cytogenetic markers.

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Diagnostic Potential of Immunophenotyping CD34+ Cells in Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v108.11.4836.4836 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4836-4836

Author(s):

Marc De Waele ◽

Barbara Leus ◽

Fabienne Trullemans ◽

Inge Verschraegen ◽

Montse Urbino ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

Myelodysplastic Syndromes ◽

Scoring System ◽

Refractory Anemia ◽

Low Grade ◽

Ringed Sideroblasts ◽

Cd34 Cells ◽

Diagnosis And Classification

Abstract Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoeitic stem cell disorders. They are characterized by abnormal bone marrow differentiation, peripheral blood cytopenia and a risk of transformation into acute myeloid leukemia (AML). The diagnosis of MDS depends on cytomorphology and cytogenetics and may be difficult especially in cases with normal numbers of blasts and without ringed sideroblasts in the bone marrow. Cytomorphology is subjective and dysplastic features may be present in other disorders than MDS. In this study we examined the potential of immunophenotyping CD34+ hematopoietic precursors for the diagnosis and classification of MDS. Bone marrow samples of 31 patients with low grade MDS (21 without and 10 with ringed sideroblasts), of 17 patients with refractory anemia with excess of blasts (RAEB), of 25 patients with AML and of 39 patients with cytopenia not due to MDS (controls) were examined. CD34+ cells were enumerated and the expression of B cell antigens (CD19), of myeloid antigens (CD13, CD33, CD117) and of immature antigens (CD133) was determined by flow cytometry. Statistical analysis was done with a Mann-Whitney test. A high number of CD34+ cells was found in MDS and AML. This was accompanied by an increase of the number of myeloid precursors and a decrease of the B cell precursors. CD117 appeared to be the best marker of myeloid precursors followed by CD13. A wide range of CD34+CD133+ and of CD34+CD33+ cells was found in all types of samples. Forty percent of the patients with low grade MDS showed an increased expression of CD117 on their CD34+ cells. In 25% of the cases without ringed sideroblasts a high expression of CD133 was present. Similar changes were more frequently found in RAEB and AML together with an increased expression of CD13 and CD33 and a low positivity for CD19. With a scoring system based on the expression of these antigens 57% of low grade MDS samples (score 1/6 or 2/6) could be distinguished from the controls (score 0/6). An elevated score was also found in respectively 84% and 100% of the RAEB and AML samples. 85% of them even had a score between 3/6 and 6/6. In conclusion, immunophenotyping of CD34+ cells is able to differentiate 60% of low grade MDS samples from other causes of cytopenia. Increased expression of CD117, CD133 and CD34 are the main differences. Similar changes are even more frequently found in RAEB and AML. A scoring system based on the antigen expression on the CD34+ cells is a powerful tool for the diagnosis and classification of MDS.

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

Blood ◽

10.1182/blood.v120.21.4946.4946 ◽

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.

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Difference in clinical features between Japanese and German patients with refractory anemia in myelodysplastic syndromes

Blood ◽

10.1182/blood-2005-01-0040 ◽

2005 ◽

Vol 106 (8) ◽

pp. 2633-2640 ◽

Cited By ~ 72

Author(s):

Akira Matsuda ◽

Ulrich Germing ◽

Itsuro Jinnai ◽

Motohiro Misumi ◽

Andrea Kuendgen ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Features ◽

Cumulative Risk ◽

Japanese Patients ◽

World Health ◽

Refractory Anemia ◽

Ringed Sideroblasts ◽

Fab Classification ◽

Health Organization ◽

French American British

AbstractSeveral reports indicate that there might be differences in clinical features between Asian and Western myelodysplastic syndrome (MDS) cases. We analyzed refractory anemia (RA) in French-American-British (FAB) classification cases diagnosed in Japan and Germany to perform a more exact comparison between Asian and Western MDS types. In the first step, we analyzed agreement of morphologic diagnosis between Japanese and German hematologists. Blood and bone marrow slides of 129 patients diagnosed with FAB-RA, FAB-RA with ringed sideroblasts (RARS), or aplastic anemia were selected randomly and evaluated separately by each group. The agreements of diagnoses according to FAB and World Health Organization (WHO) classifications were 98.4% and 83.8%, respectively. Second, we compared clinical features between 131 Japanese and 597 German patients with FAB-RA. Japanese patients were significantly younger than German patients. Japanese patients had more severe cytopenias. However, prognosis of Japanese patients was significantly more favorable than that of German patients. Japanese patients had a significantly lower cumulative risk of acute leukemia evolution than did German patients. Frequency of WHO-RA in Japanese patients with FAB-RA was significantly higher than that in German patients. In conclusion, our results indicate that the clinical features of Japanese patients with FAB-RA differ from those of German patients.

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Mitochondrial Ferritin Expression and Clonality of Hematopoiesis in Patients with Refractory Anemia with Ringed Sideroblasts.

Blood ◽

10.1182/blood.v106.11.3444.3444 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3444-3444 ◽

Cited By ~ 7

Author(s):

Matteo G. Della Porta ◽

Luca Malcovati ◽

Anna Galli ◽

Sabrina Boggi ◽

Erica Travaglino ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Early Stage ◽

Refractory Anemia ◽

Specific Marker ◽

Erythroid Cells ◽

Prognostic Information ◽

Sideroblastic Anemia ◽

Erythroid Progenitor ◽

Ringed Sideroblasts

Abstract Sideroblastic anemias are a heterogeneous group of disorders that have in common the presence of erythroblasts with iron-loaded mitochondria defined as ringed sideroblasts. We have previously demonstrated that mitochondrial iron deposition in these disorders is in the form of mitochondrial ferritin (MtF), suggesting that this latter may be a specific marker of sideroblastic anemia (Cazzola et al, Blood2003;101:1996–2000). The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts, which is generally associated with a relatively benign clinical course. In the present work, we studied the relationship between MtF expression and clonality of hematopoiesis in 55 consecutive female patients with low-risk MDS, including 20 cases with ringed sideroblasts and 35 cases without ringed sideroblasts. The expression of MtF, as well as that of cytosolic ferritin (H and L subunits) and of transferrin receptor (CD71), was evaluated by flow cytometry in bone marrow erythroid cells; in selected cases, these immunophenotypic investigations were also performed on liquid cultures of purified CD34-positive cells. X-chromosome inactivation patterns (XCIPs) were assessed in peripheral blood granulocytes and in bone marrow CD34-positive cells by analysis of both DNA methylation at the HUMARA and PGK loci and of IDS gene expression. Within informative females, 11 out of 12 patients with ringed sideroblasts displayed clonal XCIPs in granulocytes; by contrast, only 9 out of 22 patients without ringed sideroblasts displayed clonal XCIPs. Purified CD34-positive cells showed clonal XCIPs in 6 out of 7 patients with ringed sideroblasts but had polyclonal XCIPs in 4 out of 5 individuals without ringed sideroblasts. Flow cytometry evaluation of bone marrow erythroid cells showed that MtF expression was restricted to MDS patients with ringed sideroblasts. A close positive relationship was found between MtF and CD71 expression (r=.49, P=.001); this association may reflect the cytosolic iron deprivation induced by MtF overexpression. Analysis of cultured erythroid progenitor cells showed that MtF was detectable at a very early stage of differentiation from CD34-positive cells. Addition of erythropoietin to the culture system sustained the appearance of a polyclonal erythroid population in 2 out of 4 patients with ringed sideroblasts and clonal CD34-positive cells. These observations suggest that refractory anemia with ringed sideroblasts is a truly clonal stem cell disorder, while more than 50% of patients with refractory anemia without ringed sideroblasts have evidence of polyclonal hematopoiesis. The clonal pattern of CD34-positive cells and the early appearance of MtF during erythroid differentiation suggest that - despite be benign natural history of refractory anemia with ringed sideroblasts - the initial pathogenetic event in this condition occurs in multipotent stem cells. Although the mechanisms responsible for overexpression of MtF are still unclear, flow cytometry evaluation of this protein is a useful diagnostic tool that also provides helpful prognostic information.

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Myelodysplastic Syndromes in Adults - Preliminary Results From the First, Retrospective, Multicenter Brazilian Registry.

Blood ◽

10.1182/blood.v114.22.4841.4841 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4841-4841

Author(s):

Silvia M.M. Magalhães ◽

Rosane Bittencourt ◽

Elvira Velloso ◽

Maria de Lourdes Chauffaille ◽

Alita Andrade Azevedo ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Clinical Features ◽

Myelodysplastic Syndromes ◽

Tertiary Care ◽

Incidence Rates ◽

Low Risk ◽

Refractory Anemia ◽

Survival Curves ◽

Fab Classification

Abstract Abstract 4841 Myelodysplastic syndromes (MDS) are a group of acquired clonal stem cell disorders that mainly affect the elderly population, characterized by ineffective hematopoiesis and high risk of leukemic transformation. MDS are heterogeneous in terms of morphology, clinical features and survival. An increasing body of work reveals that there might be differences in clinical features between Asian and Western cases. Japanese patients seem to be younger, have a lower frequency of refractory anemia (RA) with ringed sideroblast (RARS) and a higher frequency of RA, according to FAB classification, as well as different prognostic factors such as the frequency of cytogenetic abnormalities. Incidence rates for MDS in Brazil are unavailable. The purpose of the study was to obtain epidemiological data of MDS adult patients who presented from January 2003 to December 2007 in 10 Brazilian tertiary-care hematology centers from different regions of the country. Patient data collected by participating physicians were entered and stored with the use of an internet-based, data collection tool. Blood counts, bone marrow aspiration, trephine biopsy and chromosomal study were recorded. Survival was estimated through Kaplan-Meier method and the difference between survival curves was assessed by means of Log-Rank Test. Death incidence rates were estimated and compared. Statistical analyses of relevant variables were performed. Three hundred and forty three patients with diagnosis of MDS according to FAB/WHO classification were included in this retrospective analysis. The mean age at presentation was 68 years (range 17 to 98). Fifty percent of cases were male. Cigarette smoking, alcohol abuse and pesticide/herbicide exposure were reported in 33.5%, 13.4% and 14.3% respectively. Median hemoglobin was 8.7 g/dL, median neutrophils count was 1,575/mm3 and median platelets count was 97,000/mm3. There was no excess of blasts in 68.4% of cases. Bone marrow biopsy was performed in 78.5% of patients. Lymphoid nodules were seen in 11.3% and any degree of fibrosis in 28.6%. Cytogenetic analysis was performed in 67.8% of cases and showed chromosomal abnormalities in 50.5%. The del(5q) isolated or combined with other alterations were observed in 6.0%. Flow cytometry analysis for CD55 and CD59 was performed in 11,3% and was normal in 97,4%. Near 8% of cases were classified as secondary MDS. The distribution of disease subtypes according to FAB classification was: RA 42,3%, RARS 9,0%, RA with excess of blasts (RAEB) 20,7%, RAEB-t 4,2% and chronic myelomonocytic leukemia (CMML) 3,9%. According to IPSS patients were stratified as low-risk (low risk plus intermediate I) 55,9% and high risk (intermediate II and high risk) 13,1%. In 30,1% no stratification was possible. In 26,5% of cases iron overload was diagnosed although only 28,3% of cases had performed serum ferritin. The follow-up time ranged from 1 to 78 months (mean: 28 months). Thirty-six percent of patients died and the death was MDS-related in 68.3% of cases. The high and low risk survival curves were significantly different (p<0,001), and, the death incidence rate (per 1000 person.month) was 8,7 (95% CI: 6,6-11,4) and 29,1 (95% CI: 19,5-43,4) for the low and high-risk group respectively. This clinical registry of adult Brazilian MDS patients represents a unique opportunity to gain insight about these disorders and its demographic and clinical features and provide an important baseline for future studies. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v122.21.2769.2769 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2769-2769

Author(s):

Patricia Font ◽

Dolores Subirá ◽

Sergio Matarraz ◽

Celina Benavente ◽

Teresa Cedena ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Monoclonal Antibodies ◽

Cell Count ◽

Myelodysplastic Syndromes ◽

Blast Cell ◽

Low Risk ◽

Cd34 Cells ◽

Blast Cells ◽

Good Concordance

Abstract Introduction In 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%. Objectives 1-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. Methods FCI 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. Results Finally, 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). Conclusions In 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.

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Do the SLC25A38 or ALAS2 Genes Play a Role In the Phenotypic Expression of Ringed Sideroblasts In Acquired Myelodysplastic Syndrome?

Blood ◽

10.1182/blood.v116.21.4954.4954 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4954-4954

Author(s):

Conrad V Fernandez ◽

Stephen Couban ◽

Robert Liwski ◽

Makota P Matsuoka ◽

Barbara Morash ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Myelodysplastic Syndromes ◽

Phenotypic Expression ◽

Complete Loss ◽

Refractory Anemia ◽

Loss Of Function ◽

Ringed Sideroblasts ◽

The Absolute ◽

Homozygous Form

Abstract Abstract 4954 Background. The SLC25A38 gene has recently been identified to play a role in the pathogenesis of congenital sideroblastic anemia (CSA). The erythroid specific mitochondrial carrier family protein SLC25A38 is important for the biosynthesis of heme. The ALAS2 gene is also frequently mutated in CSA. Refractory anemia with ringed sideroblasts (RARS) is an acquired myelodysplastic condition characterized by lineage dysplasia with an excess number of ringed sideroblasts in the marrow. A genetic cause for the expression of ringed sideroblasts in RARS or other myleodysplasias has not been clearly determined. We examine whether loss of function mutations in SLC25A38 or ALAS2 genes are associated with acquired myelodysplastic syndromes (MDS) with ringed sideroblasts. Methods. Participants had to have adequate banked DNA or bone marrow from diagnosis and meet WHO 2001 criteria of MDS and a high percentage of ringed sideroblasts. Medical records were retrospectively examined for patient demographics and outcomes. All diagnostic bone marrows were reviewed to confirm the diagnosis and the percentage of ringed sideroblasts and blasts was recorded. Cytogenetic findings were also recorded. DNA was extracted as needed by standard techniques. Coding exons and exon/intron boundaries of SLC25A38/ALAS2 were PCR-amplified using primers designed with Primer3 (http://frodo.wi.mit.edu/) from each affected individual. These products were then sequenced using the ABI 3130 xl electrophoresis instrument (Applied Biosystems). Sequence chromatograms were interpreted using the MutationSurveyor program from SoftGenetics, Inc., with gene annotations from GenBank looking for mutations predicted to result in loss of function. Results. 12 samples were identified from patients diagnosed between 2003 and 2008. The diagnosis by WHO 2001 classification of myelodysplastic syndrome was refractory anemia with ringed sideroblasts [RARS] (n=7), refractory cytopenia with multilineage dysplasia (RCMD-RS] (n=4) and refractory anemia with ringed sideroblasts with thrombocytosis [RARS-T] (n=1). The median age at diagnosis was 82 years (range 58–94 years). Participants were males (n=9) and females (n=3). The clinical status was as follows: Remission (n = 1), Active Disease (n = 7), and Unknown (n = 4). Treatment provided to patients included transfusion supportive care only (n =2), erythropoietin (n= 3) and MDS directed drug therapy (n=1) and unknown (n=5). For those with blood counts available at diagnosis, the median white blood cell count was 8 × 109/L (range 4.7–10.9), the hemoglobin was 94 g/L (range 85–112) and the platelets were 327 × 109/L (range 3–951). The absolute neutrophil count was 5.3 × 109/L (range 3.1–7) and the absolute lymphocyte count was 1.9 × 109/L (range 0.7 – 2.2). The median percentage ringed sideroblast count in the diagnostic bone marrow was 51% (range 15–81%). Conventional G-banding cytogenetics showed a definitive abnormality in only one of the 12 patients. Mutations predicted to result in complete loss of function occurred in 0/12 in the SLC25A38 gene and 0/12 in the ALAS2 gene. One previously unreported variant of unknown significance at SLC25A38 E03 (cDNA position #239C>G; 80T>R) was identified in homozygous form in a patient with RARS and normal cytogenetics. Conclusions. Variations in the coding regions of SLC25A38 or ALAS2 genes were not obviously associated with mutations predicted to result in complete loss of function in this cohort of patients with acquired myelodysplastic syndromes with ringed sideroblasts. We did identify one unique variant in homozygous form but its significance is uncertain. We plan to expand this study to confirm these findings. Whilst loss of complete function of these genes does not appear to be associated with acquired ringed sideroblasts, we have not ruled out a contribution of functional mutations resulting in reduced expression of these genes. Further examination of this question may clarify the physiological understanding of ringed sideroblasts in acquired MDS, which in turn may represent a target for therapy. Disclosures: No relevant conflicts of interest to declare.

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The influence of a dosage regimen of dexamethasone on detection of normal B-cell precursors in the bone marrow of children with BCP-ALL at the end of induction therapy

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2020-19-1-53-57 ◽

2020 ◽

Vol 19 (1) ◽

pp. 53-57

Author(s):

E. V. Mikhailova ◽

T. Yu. Verzhbitskaya ◽

J. V. Roumiantseva ◽

O. I. Illarionova ◽

A. A. Semchenkova ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Induction Therapy ◽

Therapy Group ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Intermittent Therapy ◽

Continuous Therapy ◽

B Cell Precursors

Minimal residual disease (MRD) monitoring by flow cytometry at the end of induction therapy is one of the key ways of a prognosis assessment in patients with acute lymphoblastic leukemia (ALL). In B-cell precursor ALL (BCP–ALL), this method of MRD detection is complicated due to the immunophenotypic similarity between leukemic cells and normal B-cell precursors (BCPs). A decrease in intensity of induction therapy can lead to a more frequent appearance of normal BCPs in the bone marrow, which significantly complicates the MRD monitoring. Aim: to assess the incidence of normal BCPs in bone marrow on the 36th day of induction therapy with two different regimens of glucocorticoid (GC) administration according to ALL-MB 2015 protocol. This study was approved by the Independent Ethical Committee and the Academic Council of Dmitriy Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology Ministry of Healthcare of Russian Federation. The study included 220 patients with BCP-ALL who were randomized to two types of GC-based induction therapy: a continuous administration of dexamethasone (n = 139) and an intermittent regimen with a 1-week dexamethasone therapy stop (n = 81). On the 36th day of induction therapy, MRD and normal BCPs were quantified in bone marrow samples by flow cytometry. On the 36th day of treatment, 43.2% of BCP(+) samples were established in the intermittent-therapy group, and 27.3% in the continuous-therapy group (p = 0.016). Comparison of the BCP level in BCP(+) samples revealed the more equitable distribution of BCPs at different developmental stages in the intermittent-therapy group, meanwhile mainly the immature BCPs in a quantity of less than 0.01% were found in the continuous-therapy group. Reduced-intensity induction therapy for patients with BCP-ALL leads to a noticeable increase of normal BCPs in bone marrow at the end of this treatment stage. A higher rate of BCP(+) bone marrow samples hinder the MRD detection due to the immunophenotypic similarity of BCPs and leukemic cells.

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Treatment of myelodysplastic syndromes with all-trans retinoic acid. Leukemia Study Group of the Ministry of Health and Welfare

Blood ◽

10.1182/blood.v81.5.1152.bloodjournal8151152 ◽

1993 ◽

Vol 81 (5) ◽

pp. 1152-1154

Author(s):

R Ohno ◽

T Naoe ◽

M Hirano ◽

M Kobayashi ◽

H Hirai ◽

...

Keyword(s):

Bone Marrow ◽

Retinoic Acid ◽

Myelodysplastic Syndromes ◽

Liver Function Tests ◽

High Serum ◽

Refractory Anemia ◽

Prior Therapy ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Advanced Stages

We treated 23 patients with myelodysplastic syndromes (MDS); 2 refractory anemia (RA) with prior therapy, 11 RA with excess of blasts (RAEB), and 10 RAEB in transformation (RAEB-T), with daily oral 45 mg/m2 all-trans retinoic acid (ATRA) in a multiinstitutional prospective study. In two patients with RAEB and one with RAEB-T, a more than 1,000/microL increase of peripheral neutrophil counts was observed with some reduction of blast percentage in the bone marrow 2 to 9 weeks after the start of ATRA. However, the effect was transient and did not last for more than 5 weeks despite the continuation of ATRA therapy. In one other patient with RA, one patient with RAEB, and one patient with RAEB-T, slight increase of hemoglobin levels or reduction of blast percentage in bone marrow was noted. Toxicities attributable to ATRA were minimal and included cheilitis, xerosis, dermatitis, gastrointestinal disorders, abnormal liver function tests, and high serum triglyceridemia. Although ATRA works remarkably as a differentiation therapy in acute promyelocytic leukemia, its effect in MDS included in this study was modest. Further study of this agent alone or in combination may be warranted in less advanced stages of this disease.

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