Hemochromatosis-associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts

Abstract Complex interaction between a multitude of genetic variants may be responsible for differential susceptibility to specific diseases, and be responsible for phenotypic variability and heterogeneity of clinical presentations. Such a variability in clinical features confounded for many years investigations into the pathogenesis of myelodysplastic syndromes (MDS). We made a curious observation of increased ferritin levels in some newly diagnosed patients with MDS RARS (refractory anemia with ringed sideroblasts) in whom transfusional iron-overload was unlikely due to very low transfusion burden. Hence, we hypothesized that RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this particular subset of MDS. We studied a cohort of 109 MDS patients; 42 with RARS, and 67 with other forms of MDS (18 RA, 12 RAEB, 7 RAEB-T, 1 CMML, and 29 MDS/MPD overlap). All patients were genotyped using restriction fragment length polymorphism (RFLP) method, designed to detect presence of C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygozity for the C282Y mutation in 21% of RARS patients (vs 9% in control population, n=2016, p= 0.017) while H63D genotype was not increased. The possible pathogenic role of this finding in RARS was supported by the normal distribution of mutant HFE alleles in patients with other forms of MDS (5% vs. 9%, p =0.35). Interestingly, 3/7 patients with RA not fulfilling the RARS criteria, but having increased numbers of ringed sideroblasts (<15%) also showed heterozygozity for either C282Y or H63D allele. To correlate the presence of C282Y allele with clinical features of RARS patients, we have performed a subset analysis. Within this group we have included patients with a rather nebulous and rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt); 7 of these patients (n=10) were found to have either C282Y or H63D allele resulting in a frequency of 30% and 40% of C282Y or H63D allele, respectively. The combined prevalence of either of these alleles in the control population is 33% (vs. 70% in RARSt, p=.01). Previously, we have demonstrated that RARSt patients are characterized by a high prevalence of the V617F JAK2 mutation (Szpurka et al, Blood 2006) suggestive of the pathophysiologic derivation of this syndrome from MPD rather than MDS. Consequently, we have tested the frequency of HFE gene variants associated with hemochromatosis in patients with MPD and Jak2 mutations. Of note is that patients with RARS harbored more C282Y alleles than those with other forms of MDS or MPD with Jak2 mutation (except for those with RARSt; (21% vs 5% and 3%, p =0.036 and .012, respectively). We conclude that hemochromatosis associated mutations may contribute to the pathogenesis of RARS. In patients with MPD and Jak2 mutation, concomitant presence of hemachromatosis-predisposing HFE variants may result in the unusual presentation associated with ringed sideroblasts.

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Immature and mature monocyte-derived dendritic cells in myelodysplastic syndromes of subtypes refractory anemia or refractory anemia with ringed sideroblasts display an altered cytokine profile

Leukemia Research ◽

10.1016/j.leukres.2006.11.007 ◽

2007 ◽

Vol 31 (10) ◽

pp. 1373-1382 ◽

Cited By ~ 19

Author(s):

Ling Ma ◽

Jan Ceuppens ◽

Ahmad Kasran ◽

Michel Delforge ◽

Marc Boogaerts ◽

...

Keyword(s):

Dendritic Cells ◽

Myelodysplastic Syndromes ◽

Cytokine Profile ◽

Refractory Anemia ◽

Ringed Sideroblasts

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Validation Of a Multi-Diagnostic Approach Including Flow Cytometry To Identify Specific Risk Categories Within Low and Intermediate-1 Risk Myelodysplastic Syndromes Within a Prospective Clinical Trial: A Study On Behalf Of The HOVON89 Study Group

Blood ◽

10.1182/blood.v122.21.1521.1521 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1521-1521

Author(s):

Eline M.P. Cremers ◽

Theresia M. Westers ◽

Canan Alhan ◽

Claudia Cali ◽

Heleen A. Visser-Wisselaar ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Scoring Systems ◽

Diagnostic Approach ◽

Refractory Anemia ◽

Genomic Profiling ◽

Intermediate Risk ◽

Ringed Sideroblasts ◽

Multilineage Dysplasia ◽

Risk Categories ◽

Good Risk

Abstract Myelodysplastic syndromes (MDS) constitute a heterogeneous group of hematopoietic stem cell disorders, characterized by ineffective hematopoiesis resulting in cytopenias and variable risk of acute myeloid leukemia (AML). To make an accurate distinction between specific risk categories in MDS, especially in low and intermediate risk MDS, a multi-diagnostic approach is recommended. To verify the efficacy of multiple diagnostic tools in MDS we used the HOVON89 study-cohort (a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low-intermediate-1 risk MDS; trial registered at www.trialregister.nl as NTR1825; EudraCT nr.: 2008-002195-10. Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). We collect data on cytomorphology (CM), conventional cytogenetics (CCG), fluorescence in situ hybridization (FISH) and microarray-based genomic profiling. In addition, we performed flow cytometric (FC) analysis according to European LeukemiaNet guidelines (Van de Loosdrecht et al., Haematologica 2009 and Leukemia 2012). Current CM results (N=98) identified: 8 refractory anemia (RA); 16 refractory anemia with ringed sideroblasts (RARS); 43 refractory cytopenia with multilineage dysplasia with/without ringed sideroblasts (RCMD/RCMD-RS); 16 refractory anemia with excess blast-1 (RAEB-1), 5 chronic myelomonocytic leukemia-1 (CMML-1) and 10 patients with isolated del(5q). CCG analysis (N=101) indicated 2 very good risk, 84 good risk, 13 intermediate risk and 2 poor risk patients according to the IPSS-R risk categories (Greenberg et al., Blood 2012). In addition, interphase FISH analysis (N=72) was normal in 15 patients, in 6 patients the del(5q) was confirmed. From 68 MDS patients data from both CCG and microarray were available. Microarray-based genomic profiling identified genomic abnormalities such as copy neutral loss of heterozygosity and small (<5 Mb) copy number alterations coinciding with a cancer gene in 13 patients with normal CCG. As expected in one patient the balanced translocation t(3;3)(q21;q26) was not identified by microarray-based genomic profiling. FC analysis (N=82) evaluated aberrancies with regard to count, marker expression level and lineage infidelity marker expression on myeloid progenitors, B cell progenitors, maturing myeloid/monocytic and erythroid cells. Current, validated FC-scoring systems identified 60/81 (Della Porta et al., Haematologica 2012) and 61/81 (Wells et al., Blood 2003) patients with MDS. Both scoring systems do not evaluate dyserytropoiesis and dysmegakaropoiesis, thereby possibly not recognizing RA, RARS or RCMD patients with only dyserytropoiesis and dysmegakaropoiesis. We integrated both scoring systems into one score (Van de Loosdrecht et al., Leukemia 2012, and JNCCN 2013). Patients were divided into 3 categories: not likely MDS (A), signs of dysmyelopoiesis (B) and fitting MDS (C). This score diagnosed 69/78 patients as probably or likely MDS by FC (B or C, figure 1). Remarkably, patients scored as ‘category A’ only displayed dyserytropoiesis and/or dysmegakaryopoiesis by CM. FC identified dyserytropoiesis in these patients, however, a consensus erytroid flow score is not yet validated. In addition, FC identified different risk categories within the patient group with no genetic abnormalities (based on CCG, FISH and microarray-based genomic profiling; data not shown). In conclusion, this is the first prospective study in low/int-1 risk MDS that validates FC as a valuable diagnostic tool in MDS (sensitivity of FC in this cohort: 88%). FC only failed to recognize some patients with only dyserytropoiesis and dysmegakaryopoiesis by CM, not evaluated by the current scoring system. Thirteen patients with unilineage dysplasia by CM had multilineage dysplasia by FC. We postulate that RA/RARS patients with multi-lineage dysplasia by FC may have clinical features of RCMD patients and therefore a higher risk on transformation to AML. Clinical follow-up data are expected within 1.5 year. In near future, a multi-diagnostic approach may i) identify risk categories within well defined IPSS-R subgroups, ii) predict risk on transformation and iii) select patients who might benefit from new emerging drugs for low-int-1 risk MDS. Disclosures: No relevant conflicts of interest to declare.

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Reclassifying myelodysplastic syndromes: what's where in the new WHO and why

Hematology ◽

10.1182/asheducation-2015.1.294 ◽

2015 ◽

Vol 2015 (1) ◽

pp. 294-298 ◽

Cited By ~ 27

Author(s):

Daniel A. Arber ◽

Robert P. Hasserjian

Keyword(s):

Advisory Committee ◽

Diagnostic Criteria ◽

Myelodysplastic Syndromes ◽

Who Classification ◽

Prognostic Significance ◽

Gene Mutations ◽

Refractory Anemia ◽

Ring Sideroblasts ◽

Actual Publication

Abstract A revision to the 4th edition of the WHO Classification of myelodysplastic syndromes (MDSs), originally published in 2008, is expected in mid-2016. Based on recommendations of a Clinical Advisory Committee, the revision will aim to incorporate new discoveries in MDS that impact existing disease categories. Although the basic diagnostic principles of the WHO classification remain unchanged, several changes to the classification are proposed. All revisions are considered preliminary until the actual publication of the monograph and online document. Proposals for change include abandoning the routine use of “refractory anemia/cytopenia” in the various disease names, including the prognostic significance of gene mutations in MDS, revising the diagnostic criteria for MDS entities with ring sideroblasts based on the detection of SF3B1 mutations, modifying the cytogenetic criteria for MDS with isolated del(5q), reclassifying most cases of the erythroid/myeloid type of acute erythroleukemia, and recognizing the familial link in some cases of MDS. This review will provide details of the major proposed changes as well as rationale for the revisions.

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Myelodysplastic syndromes: prognostic significance of multilineage dysplasia in patients with refractory anemia or refractory anemia with ringed sideroblasts

Blood ◽

10.1182/blood-2001-11-0120 ◽

2002 ◽

Vol 99 (10) ◽

pp. 3870-3872 ◽

Cited By ~ 14

Author(s):

Scott M. Dunkley ◽

Arumugam Manoharan ◽

Yiu Lam Kwan

Keyword(s):

Myelodysplastic Syndromes ◽

Prognostic Significance ◽

Refractory Anemia ◽

Ringed Sideroblasts ◽

Multilineage Dysplasia

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

Blood ◽

10.1182/blood.v112.11.3634.3634 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3634-3634

Author(s):

Kiyoyuki Ogata ◽

Matteo G. Della Porta ◽

Luca Malcovati ◽

Cristina Picone ◽

Norio Yokose ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Myelodysplastic Syndromes ◽

Normal Karyotype ◽

Japanese Patients ◽

Low Risk ◽

Refractory Anemia ◽

Nippon Medical School ◽

Ringed Sideroblasts

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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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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