Refractory Cytopenia With Multilineage Dysplasia And Ringed Sideroblasts Research Articles

Considering Bone Marrow Blasts from Nonerythroid Cells Improves the Prognostic Evaluation of Myelodysplastic Syndromes with Ring Sideroblasts

Introduction: As showed in a recent study of our group, considering bone marrow (BM) blasts from nonerythroid cellularity (NECs) improves the prognostic evaluation of MDS (Arenillas et al, J Clin Oncol 2016). By enumerating blasts from NECs, 12% of MDS patients diagnosed within WHO categories with less than 5% BM blasts were reclassified into higher-risk categories and showed a poorer overall survival than did those who remained in the initial categories. Refractory anemia with ring sideroblasts (RARS) and refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS) have shown an special good outcome in different studies. As MDS with ring sideroblasts (MDS-RS) usually present a high percentage of BM erythroblasts, considering BM blasts from NECs could imply a risk overestimation of this subset of patients.Aim: we evaluated the relevance of considering BM blasts from NECs or from total nucleated cells (TNCs) on classification and prognostication of the group of patients diagnosed with MDS-RS.Methods: We retrospectively analyzed 3,924 de novo MDS diagnosed according to WHO 2001 and 2008 classifications from the MDS Spanish registry. 1,045 patients presented less than 5% BM blasts from TNCs and equal or greater than 15% BM ring sideroblasts, fulfilling current definition for RARS (WHO 2001 and 2008) and RCMD-RS (WHO 2001). Moreover 1,233 patients with equal or greater than 5% BM ring sideroblasts and less than 5% BM blasts were analyzed in order to explore the future definition of WHO 2016, that considered as MDS-RS those patients with 5%-<15% BM ring sideroblasts if SF3B1 mutation is present. This was a tentative analysis since SF3B1 mutation information was not available in our series. Percentage of BM blasts from NECs was calculated as follows: [%BM blasts from TNCs/(100 - %BM erythroblasts) x 100]. Survival curves were constructed by using the Kaplan-Meier method and compared using the log-rank test. Univariable and multivariable Cox regression analysis were also implemented.Results: Median age at diagnosis of MDS-RS was 76y (25-101) and 59% were males. Estimated median follow-up, as calculated by reverse Kaplan-Meier method, was 50.1 months (95% CI, 45.5-54.7) and median OS was 96.5 months. By enumerating blasts from NECs, 10% of MDS-RS patients were reclassified into categories with equal or greater than 5% BM blasts and showed a poorer overall survival (OS) than did those who remained in initial categories (median OS, 68.1 vs 97.6 months, P=0.025; Hazard ratio (HR): 1.41; 95%CI: 1.04-1.91; p=0.026). After adjusting the survival analysis by IPSS cytogenetic risk groups, the prognostic impact of BM blasts considered from NECs maintained its significance (HR: 1.37; 95%CI: 1.01-1.85; p=0.045). Similar results were observed applying this method to the group of MDS patients with equal or greater than 5% BM ring sideroblasts and less than 5% BM blasts. By considering blasts from NECs, 10% of this subset of patients were reclassified into categories with equal or greater than 5% BM blasts and showed a poorer OS than did those who remained in initial categories (median OS, 60.2 vs 85.8 months, P=0.003; HR: 1.51; 95%CI: 1.15-1.97; p=0.003; HR adjusted for IPSS cytogenetics: 1.46; 95%CI: 1.12-1.92; p=0.006).Conclusion: considering bone marrow blasts from nonerythroid cells improves the prognostic evaluation of MDS with ring sideroblasts.Written on behalf of the Grupo Español de Síndromes Mielodisplasicos (GESMD). DisclosuresNo relevant conflicts of interest to declare.

SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts

AbstractRefractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated erythroid dysplasia and 15% or more bone marrow ring sideroblasts. Ring sideroblasts are found also in other MDS subtypes, such as refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS). A high prevalence of somatic mutations of SF3B1 was reported in these conditions. To identify mutation patterns that affect disease phenotype and clinical outcome, we performed a comprehensive mutation analysis in 293 patients with myeloid neoplasm and 1% or more ring sideroblasts. SF3B1 mutations were detected in 129 of 159 cases (81%) of RARS or RCMD-RS. Among other patients with ring sideroblasts, lower prevalence of SF3B1 mutations and higher prevalence of mutations in other splicing factor genes were observed (P < .001). In multivariable analyses, patients with SF3B1 mutations showed significantly better overall survival (hazard ratio [HR], .37; P = .003) and lower cumulative incidence of disease progression (HR = 0.31; P = .018) compared with SF3B1-unmutated cases. The independent prognostic value of SF3B1 mutation was retained in MDS without excess blasts, as well as in sideroblastic categories (RARS and RCMD-RS). Among SF3B1-mutated patients, coexisting mutations in DNA methylation genes were associated with multilineage dysplasia (P = .015) but had no effect on clinical outcome. TP53 mutations were frequently detected in patients without SF3B1 mutation, and were associated with poor outcome. Thus, SF3B1 mutation identifies a distinct MDS subtype that is unlikely to develop detrimental subclonal mutations and is characterized by indolent clinical course and favorable outcome.

Genetic Determinants of Disease Phenotype and Clinical Outcome in Myelodysplastic Syndromes with Ring Sideroblasts

Ring sideroblasts (RS) characterize a group of myelodysplastic syndromes (MDS) categorized in the WHO classification as refractory anemia with RS (RARS) or refractory cytopenia with multilineage dysplasia and RS (RCMD-RS), according to the presence of 15% or more bone marrow RS and dysplasia in one or more myeloid lineages. A high prevalence of somatic mutations in SF3B1 was reported in MDS with RS [N Engl J Med 2011;365:1384-95], and recent unsupervised analyses suggested that MDS with SF3B1 mutation represent a homogeneous subset [Blood 2014 Jun 26]. In this study, we performed a comprehensive mutation analysis of genes implicated in myeloid disorders in a large and well-characterized cohort of myeloid neoplasms with 1% or more RS with the aim to identify mutation patterns that affect disease phenotype and clinical outcome.The study population consisted of 309 patients (pts), including: a) 244 with MDS, of whom 160 assigned to sideroblastic categories (RARS, RCMD-RS) and 84 to other WHO categories [34 RA or RCMD, 7 MDS with isolated del(5q), 20 RAEB-1, 23 RAEB-2]; b) 51 with myelodysplastic/myeloproliferative neoplasms (MDS/MPN: 9 CMML, 42 RARS-T); c) 14 with AML-MDS.SF3B1 mutations were observed in 151/244 pts with MDS and RS (62%). Within sideroblastic categories, SF3B1 mutation was found in 81/91 cases of RARS (89%), and 48/69 RCMD-RS (70%). Among pts classified in other MDS categories, significantly lower rate of SF3B1 mutations (22/84, P<.001) and higher prevalence of mutations in other RNA splicing factors (SRSF2, U2AF1, ZRSR2) (P<.001) were observed. Pts with MDS carrying SF3B1 mutation showed a limited pattern of recurrently co-mutated genes including those involved in DNA methylation (39%), chromatin modification (10%), and RUNX1 (5%). Variant allele frequencies (VAFs) analysis showed that in most cases (91%) SF3B1 mutation was in the dominant clone. Within SF3B1-negative MDS with RS, a significantly higher prevalence of mutations in TP53 was found (9/93, P=.001), 6 of 9 cases showing disease phenotype with multilineage dysplasia and no excess blasts.In a multivariable analysis, pts with SF3B1 mutation showed significantly better overall survival (OS) (HR .39, P=.001) and lower risk of disease progression (HR=.40, P=.024) compared with SF3B1-unmutated cases. The independent prognostic value of SF3B1 mutations was retained when the analysis was limited to sideroblastic categories (OS: HR=.32, P=.005; risk of progression: HR=.27, P=.036).Then, we focused on MDS associated with SF3B1 mutation (defined by SF3B1 mutation, no excess blasts or del(5q): 81 RARS, 48 RCMD-RS, 4 RA or RCMD) with the aim to identify genetic determinants of disease phenotype. We found that mutations in DNA methylation genes (TET2, DNMT3A) were significantly associated with multilineage dysplasia (P=.015). The analysis of VAFs showed that in 90% of cases there was no statistical evidence of subclonality of these gene mutations. When comparing pts with uni- or multilineage dysplasia, no significant difference was observed in hematological parameters. In addition, no significant effect of multilineage dysplasia was found on OS (P=.5) and risk of progression (P=.92). Taken together, these results suggest that MDS associated with SF3B1 mutation is indeed a homogeneous subset, and should be recognized as a distinct disease entity within MDS, irrespective of current WHO criteria.We then used Cox regression models in SF3B1-mutant MDS with the aim to identify mutation patterns associated with relevant clinical outcomes, including RBC transfusion-dependency, risk of disease progression and survival. We found that mutations in chromatin modifiers (ASXL1, EZH2) were significantly associated with development of transfusion-dependency (HR=3.85, P=.006). In addition, mutations in RUNX1 were significantly associated with worse OS (HR=6.98, P=.012) and increased risk of progression (HR=5.63, P=.023). Unambiguous statistical evidence of subclonality of these gene mutations was obtained in 46% of cases.In conclusion, this study shows that SF3B1 mutations identify a distinct subset of MDS with homogeneous features and favorable prognosis irrespective of current WHO classification criteria. Within MDS associated with SF3B1 mutation, concurrent or subclonal mutations in genes involved in DNA methylation, chromatin modification and RUNX1 account for variability in disease phenotype and clinical outcomes. DisclosuresNo relevant conflicts of interest to declare.

Molecular Genetic Analysis of Myelodysplastic Syndromes (MDS) Patients with Ring Sideroblasts (RS); Independent Confirmation of Association of SF3B1 Mutations with Better Prognosis

Background:Ring sideroblasts (RS) are erythroblasts with iron-loaded mitochondria, appears as blue granules when stained with Prussian blue, and are characteristic of myelodysplastic syndromes (MDS) subgroups refractory anemia with ring sideroblasts (RARS), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), and RARS with thrombocytosis (RARS-T) but are also infrequently found in other MDS groups. The etiology of RS remains unclear. Molecular genetic analysis of MDS patients with RS (MDS-RS) using exome and targeted sequencing identified mutations in several genes including SF3B1. While the RS phenotype was shown to be strongly associated with SF3B1 mutations, the association of SF3B1 mutations with better prognosis remained controversial. Also, there remained several patients who do not show any mutations in SF3B1 suggesting there are additional genes associated with RS phenotype. In addition to SF3B1 mutations, RARS-T patients show mutations in JAK2 (V617F and Exon 12), MPL and CALR genes, frequently mutated in Essential Thrombocytopenia (ET). But there remained a high number of patients who do not show any mutation in JAK2, MPL and CALR genes suggesting additional genes are associated with thrombocytosis. In order to gain further insight into the molecular genetics of MDS-RS patients and to elucidate their clinical significance we screened a large cohort of patients with RS (RARS/RCMD-RS) and a subgroup with thrombocytosis (RARS-T).Methods:This study is approved by the Institutional Review Board of Columbia University and informed consent was obtained from all the individuals participated in the study. Bone marrow mono nuclear cells were isolated from bone marrow aspirate and DNA was extracted using DNeasy Blood and Tissue kit. To screen for most frequent mutations in SF3B1 (exon 14 and 15), MPL (exon 10), JAK2 (exon 12), and CALR (exon 9) genes, primers were designed to amplify the exons and the exon-intron junctions using PCR and the amplified and purified PCR products were sequenced using Sanger sequencing on both strands. To screen for mutations V617F in JAK2, primers were designed to carry out allele specific PCR. Demographic and clinical data is collected from patient’s charts/reports. Statistical analysis including survival curve (Kaplan–Meier method) was performed using GraphPad Prism Software.Results:A total of 209 patients with RS phenotype were screened for SF3B1 mutations. Mutations in SF3B1 gene were detected in 62% (130/209) of RS patients, including 85% (23/27) of RARS-T and 59% (107/182) of RARS/RCMD-RS patients. Among all the SF3B1 mutations, K700E was the most frequent mutation (60%) followed by mutations at H662. Clinical significance of SF3B1 mutations on overall survival, using Kaplan-Meier method, showed SF3B1 mutations were associated with better prognosis (Fig 1). The median survival of patients with SF3B1 mutation is 110 months compared to those without mutations, 70 months (p value < 0.034). Studies to screen SF3B1 negative patients for additional mutations are being carried out using exome sequencing to identify genes associated with RS phenotype.In addition to SF3B1, RARS-T patients were also screened for JAK2 V617F and exon 12 mutations. JAK2 V617F mutations were detected in 11% of RARS-T patients and no mutations were found in JAK2 exon 12. Patients negative for JAK2 were screened for mutations in MPL and CALR genes. No mutations were found in MPL but 8% (2/23) of those negative for JAK2 were found to have CALR mutations. Both CALR mutations were frameshift mutations that alter the C-terminus thus abolishing the KDEL sequence required for CALR function. There are still 80% RARS-T patients in our cohort who do not show any mutations in JAK2, MPL, CALR genes.Conclusions:The association of SF3B1 mutation with prognosis is controversial with some studies suggesting a better prognosis while other reports no effect. Our study, using a large cohort of well-characterized RS patients provides an Independent verification of the observation that SF3B1 mutations are associated with better overall survival. There remains a large group of patients (40% in RARS/RCMD-RS groups) without SF3B1 mutation but with a RS phenotype suggesting yet other gene(s) is associated with RS phenotype. Similarly, there remain patients who do not show any mutations in JAK2, MPL and CALR suggesting other genes are involved in the etiology of thrombocytopenia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pathway Mutations in the Splicing Machinery in Myeloid Neoplasms

AbstractAbstract SCI-14During the past decade, significant progress has been made in our understanding of the molecular pathogenesis of myelodysplastic syndromes (MDS) and related myeloid neoplasms, in which one of the major findings was frequent mutations of genes in epigenetic regulation, such as DNA methylation (DNMT3A, TET2, and IDH1/2) and chromatin modifications (ASXL1, EZH2, EED, and SUZ12). They are also found in comparable or even higher fractions of other myeloid neoplasms, underscoring the common impact of deregulated epigenetic regulation on myeloid leukemogenesis. On the other hand, a new class of pathway mutations has been uncovered recently that commonly involve the RNA splicing machinery (1, 2, 3). Thus, at least eight different components of the machinery, invariably engaged in the 3' splice site recognition, have been reported to be mutated in as high as 45 percent to 85 percent of cases with different subtypes of MDS and related myeloid neoplasms mostly in a mutually exclusive manner. This indicates that the 3' splice site recognition is the functional target of these mutations. There exist discrete mutational hotspots in three out of four major targets, including SF3B1, SRSF2, U2AF35, and ZRSR2, and these mutant alleles can induce abnormal RNA splicing, indicating the gain-of-function nature of the mutations (2). Splicing factor mutations are largely specific to myelodysplasia phenotypes but relatively rare in acute myeloid leukemia and myeloproliferative neoplasms (2), suggesting their primary roles in the pathogenesis of myelodysplasia. The genotype-phenotype association is especially prominent in the case of SF3B1 mutations, which were found in 76 percent to 83 percent of cases of refractory anemia with ringed sideroblasts (RARS), refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), and refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) (1, 2). In this session, the updated findings on the spliceosome mutations found in myelodysplasia, including their clinical and functional aspects, will be discussed. Disclosures:No relevant conflicts of interest to declare.

Clinical importance of SF3B1 mutations in Chinese with myelodysplastic syndromes with ring sideroblasts

Recent studies report SF3B1 mutations in about 20% of persons of European descent with myelodysplastic syndromes (MDS). Mutations are especially common in persons with ring sideroblasts (RS). SF3B1 mutation state was determined in 104 Chinese with MDS-RS. SF3B1 mutations were found in 55 subjects (53%) including 25 of 39 with refractory anemia and RS (RARS), 26 of 45 (58%) of those with refractory cytopenia with multi-lineage dysplasia and RS (RCMD-RS), 3 of 6 with refractory anemia with excess blasts-1-RS (RAEB1-RS) and 1 of 14 with RAEB2-RS. There were significant correlations between SF3B1 mutation state and platelet levels (P=0.007), mean RBC corpuscular volume (MCV; (P<0.001), proportion of RS (P<0.001) and percent bone marrow erythroblasts (P=0.012) and myeloblasts (P=0.044). Multivariate analyses using a Cox proportional hazards regression model including sex, age, SF3B1 mutation state, hemoglobin concentration, absolute neutrophil level, platelet level, MCV, international prognostic scoring system (IPSS) cytogenetics category, WHO morphologic category and treatment showed SF3B1 mutation state to independently predict survival. These data increase our knowledge of the impact of SF3B1 mutations in persons with MDS. They indicate a similar favorable impact of SF3B1 mutation on survival in Chinese with MDS as reported for persons of European descent.

Possible association of postmeiotic segregation increased 2 (PMS 2) gene deletion and myelodysplastic syndrome.

e17021 Background: Postmeiotic segregation Increased 2 (PMS2) gene is one of the gene family members found in clusters on chromosome 7 involved in DNA mismatch repair. Mutations in this gene are associated with hereditary nonpolyposis colorectal cancer and Turcot syndrome. PMS-2 defects are not extensively studied and hence there is still considerable potential to detect new hemato-oncological associations with same. Chromosome 7 defects are the most commonly associated genetic abnormalities associated with myelodysplastic syndrome (MDS). Here, we describe a case of a patient with known PMS 2 gene deletion presenting with early onset MDS. Methods: A 52 year old caucasian female came in to our oncology clinic for evaluation after being diagnosed with DCIS of right breast. She underwent surgical resection followed by tamoxifen and surviellance mamograms. She developed gradual onset of fatigue, prompting follow-up laboratory tests which revealed WBC of 8,600, Hgb of 8.5, MCV of 101, and Plt 383,000. B12, folate, and iron studies were normal. Bone marrow biopsy revealed cellularity 80% and cytogenetics with trisomy 8.This lead to diagnosis of myelodysplastic syndrome, refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS). Patient is currently treated with supportive transfusions after lack of benefit of darbopoietin (baseline epo level 67). Family history is pertinent for a son who died at age 21 from colon cancer and a daughter diagnosed at age 22 with colon cancer, who survives. Her daughter subsequently tested positive for G750 PMS-2 from her father and complete deletion of PMS2 from her mother (our patient). Results: PMS2 gene deletion might predispose patients to MDS. Conclusions: Given the common link with chromosome 7 abnormalities, it is therefore possible that there is an associated increased risk of MDS in PMS-2 patients. While our patient did not have any general abnormalities by karyotype, current assays and general karyotyping are of potentially limited value unless specific mutation points are identified. Therefore, additional evaluations may be necessary to better identify at-risk patients.

123 Hypocellular RAEB and hypocellular AML: Cytogenetic, immunophenotypic and morphological review of a six-year patient cohort from a regional diagnostic service

Allogeneic stem cell transplantation is the only known curative therapy for myelodsyplastic syndromes (MDS). We present the transplant outcomes for 84 adult MDS patients, median age 50 (18-69 years), undergoing allogeneic hematopoietic stem cell transplantation (HSCT) at the University of Minnesota between 1995 and 2007. By WHO criteria 35 (42%) had refractory anemia with excess blasts (RAEB-1 or 2), 23 (27%) had refractory cytopenia with multilineage dysplasia (RCMD) or RCMD and ringed sideroblasts (RCMD-RS), and the remaining 26 (31%) had refractory anemia (RA), myelodysplastic syndrome-unclassifiable (MDS-U), chronic myelomonocytic leukemia (CMML), myelodysplastic/myeloproliferative disease (MDS/MPD), or myelodysplastic syndrome-not otherwise specified (MDS-NOS). Graft source was related in 47 (56%), unrelated donor (URD) marrow in 11 (13%), and unrelated cord blood (UCB) in 26 (31%). The conditioning regimen included total body irradiation (TBI) in 94% of transplantations; 52 (62%) myeloablative (MA) and 32 (38%) nonmyeloablative (NMA) regimens. Cumulative incidence of neutrophil engraftment by day +42, acute graft-versus-host disease (aGVHD) by day +100, and chronic GVHD (cGVHD) by 1 year were 88% (80%-96%, 95% confidence interval [CI]), 43% (36%-50%, 95% CI), and 15% (10%-20%, 95% CI), respectively. One-year treatment-related mortality (TRM), relapse, disease-free survival (DFS), and overall survival (OS) were 39% (28%-50%, 95% CI), 23% (12%-32%, 95% CI), 38% (28%-48%, 95% CI), and 48% (38%-58%, 95% CI) respectively. Cumulative incidence of relapse at 1 year in patients with pre-HCT complete remission (CR) or <5% blasts was improved at 18% (8%-28%, 95% CI) compared to 35% (16%-54%, 95% CI) in patients with 5%-20% blasts (P = .07). Additionally, with MA conditioning, the incidence of relapse at 1 year trended lower at 16% (6%-26%, 95% CI) versus 35% (18%-52%, 95% CI) in NMA (P = .06), and a statistically significant decrease in relapse was noted in patients entering HCT with CR or <5% blasts with an incidence of 9% (0%-18%, 95% CI) (MA) versus 31% (11%-51%, 95% CI) (NMA) (P = 0.04). For those patients with ≥5% blasts, MA conditioning did not significantly decrease relapse rates. One-year TRM was similar between MA and NMA conditioning. For patients entering transplant in CR or with <5% blasts, prior treatment to reach this level did not impact rates of relapse or transplant-related mortality when all patients were analyzed; however, when broken down by conditioning intensity, there was a trend toward improved DFS in those NMA patients who were pretreated. Finally, 1-year DFS was similar using related donor peripheral blood stem cell (PBSC)/marrow, URD marrow, or UCB grafts. These data suggest that (1) blast percentage <5% at HSCT is the major predictor of improved DFS and relapse and prior treatment to reach this disease status may have value in leading to improved DFS; (2) MA conditioning is associated with lower relapse risk, particularly in patients with CR or <5% blasts, but is not able to overcome increased disease burden; (3) NMA conditioning yields equivalent TRM, DFS, and OS, and is reasonable in patients unsuited for MA conditioning; (4) the donor sources tested (PBSC, bone marrow [BM], or UCB) yielded similar outcomes.

Novel TET2 Mutations Associated With UPD4q24 in Myelodysplastic Syndrome

Cryptic chromosomal aberrations, such as regions of uniparental disomy (UPD), have been shown to harbor homozygous mutations and are a common feature in myelodysplastic syndrome (MDS). We investigated the sequence integrity of 4q24 candidate tumor suppressor gene TET2 in MDS patients with UPD on chromosome 4. The coding exons of TET2 were analyzed by 454 deep sequencing and Sanger sequencing in nine patients with UPD on 4q. Four patients had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) and UPD4q24, and five patients (refractory anemia with excess blasts-II, n = 1; 5q- syndrome, n = 1; RCMD-RS, n = 1; refractory anemia, n = 1; refractory cytopenia with multilineage dysplasia, n = 1) had no UPD4q24. Mutations on TET2 were identified in all four patients with UPD4q24. These were localized to exons 3, 6, and 9 and resulted in two premature stop codons, one frameshift mutation, and one cysteine to glycine amino acid change. Mutant clone size varied between 30% and 85%. One patient with UPD outside of q24 (UPD4q28.3) displayed additional TET2 mutations, but these were at low clonal levels (13%, 4%, and 4% for a silent mutation, a 180-base pair deletion in exon 3, and a lysine to phenylalanine substitution in exon 11, respectively). The other patients who did not have UPD4q24 did not have verifiable TET2 mutations. Our data identify novel TET2 mutations in a dominant clone in patients with UPD4q24. The presence of UPD4q24 and mutations in RCMD-RS patients may suggest specificity to this subtype. Our preliminary results need to be confirmed in a large cohort of all MDS subtypes.

Allogeneic Stem Cell Transplantation for Adults with Myelodysplastic Syndromes: Importance of Pretransplant Disease Burden

Allogeneic stem cell transplantation is the only known curative therapy for myelodsyplastic syndromes (MDS). We present the transplant outcomes for 84 adult MDS patients, median age 50 (18-69 years), undergoing allogeneic hematopoietic stem cell transplantation (HSCT) at the University of Minnesota between 1995 and 2007. By WHO criteria 35 (42%) had refractory anemia with excess blasts (RAEB-1 or 2), 23 (27%) had refractory cytopenia with multilineage dysplasia (RCMD) or RCMD and ringed sideroblasts (RCMD-RS), and the remaining 26 (31%) had refractory anemia (RA), myelodysplastic syndrome-unclassifiable (MDS-U), chronic myelomonocytic leukemia (CMML), myelodysplastic/myeloproliferative disease (MDS/MPD), or myelodysplastic syndrome-not otherwise specified (MDS-NOS). Graft source was related in 47 (56%), unrelated donor (URD) marrow in 11 (13%), and unrelated cord blood (UCB) in 26 (31%). The conditioning regimen included total body irradiation (TBI) in 94% of transplantations; 52 (62%) myeloablative (MA) and 32 (38%) nonmyeloablative (NMA) regimens. Cumulative incidence of neutrophil engraftment by day +42, acute graft-versus-host disease (aGVHD) by day +100, and chronic GVHD (cGVHD) by 1 year were 88% (80%-96%, 95% confidence interval [CI]), 43% (36%-50%, 95% CI), and 15% (10%-20%, 95% CI), respectively. One-year treatment-related mortality (TRM), relapse, disease-free survival (DFS), and overall survival (OS) were 39% (28%-50%, 95% CI), 23% (12%-32%, 95% CI), 38% (28%-48%, 95% CI), and 48% (38%-58%, 95% CI) respectively. Cumulative incidence of relapse at 1 year in patients with pre-HCT complete remission (CR) or <5% blasts was improved at 18% (8%-28%, 95% CI) compared to 35% (16%-54%, 95% CI) in patients with 5%-20% blasts ( P = .07). Additionally, with MA conditioning, the incidence of relapse at 1 year trended lower at 16% (6%-26%, 95% CI) versus 35% (18%-52%, 95% CI) in NMA ( P = .06), and a statistically significant decrease in relapse was noted in patients entering HCT with CR or <5% blasts with an incidence of 9% (0%-18%, 95% CI) (MA) versus 31% (11%-51%, 95% CI) (NMA) ( P = 0.04). For those patients with ≥5% blasts, MA conditioning did not significantly decrease relapse rates. One-year TRM was similar between MA and NMA conditioning. For patients entering transplant in CR or with <5% blasts, prior treatment to reach this level did not impact rates of relapse or transplant-related mortality when all patients were analyzed; however, when broken down by conditioning intensity, there was a trend toward improved DFS in those NMA patients who were pretreated. Finally, 1-year DFS was similar using related donor peripheral blood stem cell (PBSC)/marrow, URD marrow, or UCB grafts. These data suggest that (1) blast percentage <5% at HSCT is the major predictor of improved DFS and relapse and prior treatment to reach this disease status may have value in leading to improved DFS; (2) MA conditioning is associated with lower relapse risk, particularly in patients with CR or <5% blasts, but is not able to overcome increased disease burden; (3) NMA conditioning yields equivalent TRM, DFS, and OS, and is reasonable in patients unsuited for MA conditioning; (4) the donor sources tested (PBSC, bone marrow [BM], or UCB) yielded similar outcomes.

SNP Arrays in Patients with Myelodysplastic Syndromes and Ringed Sideroblasts: A Karyotypic and Disease Association Analysis

Introduction: The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders. Ringed sideroblasts (RS) are found in the following subclasses of MDS: refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), and refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T). The objective of this study was to evaluate the use of single nucleotide polymorphism (SNP) arrays (SNP-A) in patients with MDS and RS and specificallyto compare chromosomal abnormalities detected by metaphase karyotyping (MC) with those detected using high-resolution SNP based karyotyping (which can detect unbalanced genomic lesions in addition to copy-neutral loss of heterozygozity) andto conduct a disease association analysis using the SNP-A.Methods: We reviewed the electronic records of patients with MDS and RS seen at our institution between 2002 and 2008. DNA was extracted using the Puregene DNA Purification Kit. Gene Chip Mapping 250K Assay Kit (Affymetrix) was used. Signal intensity and genotype calls were analyzed using CNAG v.3.0. For the disease association analysis, the Fisher's p-value was used to compare SNPs found in patients with MDS and RS versus 150 normal controls.Results: 83 patients with MDS who have RS were identified. 40 (48%) had RARS, 25 (30%) had RCMD-RS, and 18 (22%) had RARS-T. The mean age of these patients was 70.7 years, 53 patients (64%) were males, and 70 (84%) were Caucasian. Of those 83 patients, 45 had available DNA for SNP analysis, 23 (51%) of whom had RARS, 11 (24%) had RCMD-RS, and 11 (24%) had RARS-T. The mean age of these 45 patients was 69.9 years, 29 (64%) were males, and 39 (87%) were Caucasian. By MC, 20/45 (44.5%) patients had abnormal karyotypes and 25/45 (55.5%) patients had normal karyotypes. Using SNP-A, chromosomal abnormalities including UPD were identified in 29/45 (64.5%) of patients. Of the 25 pts who had normal karyotypes by MC, 11 (44%) had abnormal karyotypes by SNP-A. The chromosomal distributions of the lesions detected by MC were as follows: chromosome 5 (18.4%), chromosome 7 (15.8%), chromosome 8 (13.1%), chromosome 17, 18, 19, 20, 21 (5.2% in each), and others (26.3 % in total). The distribution of chromosomal lesions detected by SNP-array analysis (excluding UPD) was as follows: chromosome 8 (18.7 %), chromosome 5 (14.6%), chromosome 7 (12.5%), chromosome 17 (10.4%), chromosome 20 (8.3%), chromosome 4 (6.2%), chromosomes 2, 3, 13, 22 (4.1% each), and others (12.5% in total). UPD was found in 12/45 (26.7%) patients mostly affecting chromosome 1 (27.8%). A large number of SNPs were found to be significantly more prevalent in patients with MDS with RS than in controls (with p-value < 0.0001). Genes within 50 kb from these SNPs were scrutinized. At least 11 of those genes (RP1, LIMD1, CHL1, ATP6V1F, TEAD2, SPTLC2, CDH13, DIAPH2, DLEU2, FAM10A4, TRPM8) are known to be related to cancer in the literature. Given that karyotypic abnormalities were more prevalent in chromosomes 8, 5, and 7, we looked specifically at the SNPs in those chromosomes which were significantly associated with disease (rs 409429, rs 446153, rs 453186 and rs 509273 in chromosome 8; rs6891109 in chromosome 5; and rs6970371 in chromosome 7). The genes within 50 kb of these SNPs that are known to be associated with cancer are: RP1 in chromosome 8 (colon cancer), and ATP6V1F in chromosome 7 (prostate cancer).Conclusion: This study shows that SNP-A based karyotyping is a useful tool for karyotyping and can detect more chromosomal abnormalities than MC (64.5 versus 44.5%, odds ratio 1.45). We also found that about half of the patients who had normal karyotypes by MC were found to have karyotypic abnormalities by SNP-A. In addition, we show multiple candidate genes that could be important in the pathogenesis of MDS with RS.

Granulocyte JAK2 (V617F) Mutation Status in Myeloid Neoplasms with Ringed Sideroblasts.

The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts (RARS). We have previously demonstrated that mitochondrial iron accumulation in this condition is in the form of mitochondrial ferritin (MtF). A gain-of-function mutation of JAK2 is found in most patients with chronic myeloproliferative disorders. A high frequency of this mutation has been also reported in RARS associated with marked thrombocytosis (RARS-T), a provisional entity characterized by marked increase in platelet count, hypercellular marrow with increased megakaryocytes, and ringed sideroblasts. In this study, we investigated the granulocyte JAK2 (V617F) mutation status in 73 patients receiving a diagnosis of myeloid malignancy with ringed sideroblasts at the Division of Hematology, University of Pavia Medical School & IRCCS Policlinico San Matteo Pavia, Italy between 2001 and 2006. According to the WHO classification of the myeloid neoplasms, 23 patients had RARS, 17 had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), 16 had refractory anemia with blasts excess, four had MDS with isolated del(5q), and 13 fulfilled the criteria for RARS-T. JAK2 (V617F) mutation status was analyzed on peripheral blood granulocytes through a quantitative real time PCR-based allelic discrimination assay. We compared clinical and biological features of patients with RARS-T with those of patients with refractory anemia or cytopenia with ringed sideroblasts, and found that RARS-T patients had higher neutrophil and platelet counts, lower frequency of cytogenetic abnormalities and higher incidence of the JAK2 (V617F) mutation (P values ranging from <.001 to .02). JAK2 (V617F) was detected in six out of 63 evaluable cases (9.5%), one being diagnosed as MDS with isolated del(5q) and five as RARS-T, resulting in an incidence of mutation in the latter group of 45%. The proportion of mutant alleles ranged between 2.8% and 18.4%, values commonly observed by us in essential thrombocythemia [Blood. 2006 May 1;107(9):3676–82]. Focusing the analysis on RARS-T, a significantly higher hemoglobin level at diagnosis was found in mutated (median value 11.2 g/dL, range 10.1–15.4) compared with non mutated patients (median value 9 g/dL, range 6–9.9) (P=.009). JAK2-positive patients also showed a significantly lower percentage of ringed sideroblasts in the bone marrow (P=.01), and an increased marrow reticulin fibrosis (P=.03). We then evaluated the clonality of hematopoiesis in female patients through analysis of X-chromosome inactivation patterns (XCIPs) in circulating granulocytes and bone marrow CD34-positive cells. Twenty-one out of 23 informative female patients with ringed sideroblasts (91%), as well as 5/6 RARS-T (83%) displayed a clonal pattern of X-chromosome inactivation. These observations suggest that refractory anemia with ringed sideroblasts with marked thrombocytosis is a clonal stem cell disorder significantly associated with the JAK2 (V617F) mutation. This disorder shows both dysplastic and proliferative features, the presence of the mutation being associated with a predominant myeloproliferative phenotype. The recognition of a heterogeneous genetic background in myeloid neoplasms with ringed sideroblasts suggests that different mechanisms might be involved in the induction of mitochondrial ferritin expression in these disorders.

Usefulness and prognostic impact on survival of WHO reclassification in FAB low risk myelodyplastic syndromes

In 1999, WHO proposed a revised classification for myelodysplastic syndromes (MDS). According to this system, FAB low risk MDS (RA and RARS) were defined as such when the presence of dysplastic features was only restricted to the erythroid lineage, and new categories, refractory cytopenia with multilineage dysplasia (RCMD) and refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), were added. In a retrospective analysis of 240 consecutive patients diagnosed at our institution as having FAB RA and RARS, we reclassified the disease following the WHO criteria and we found that 179/214 patients (84%) still remained in the RA category, while 35/214 (16%) moved to RCMD. Moreover, 17/26 patients (65%) maintained the RARS diagnosis, whereas 9/26 (35%) were re-classified as RCMD-RS. We detected differences among the WHO subgroups as to age and sex distribution as well as to median survival observed by stratifying patients according to different prognostic scoring systems. Furthermore we confirmed the usefulness of WHO segregation with regard to its predictive value for evolution into acute leukaemia. Our study provides evidence that WHO classification may have prognostic impact on MDS subgroups which are usually categorized by FAB as having a favourable outcome.