Prognosis Of Myelodysplastic Syndrome Patients Research Articles

TP53 Mutation Status Divides MDS Patients with Complex Karyotypes into Distinct Prognostic Risk Groups: Analysis of Combined Datasets from the International Working Group for MDS-Molecular Prognosis Committee

Background:Complex karyotypes in patients with myelodysplastic syndromes (MDS) are defined by the presence of 3 or more cytogenetic abnormalities and are considered indicators of a poor prognosis by nearly all models in clinical use. There is evidence that the prognostic significance of complex karyotypes in MDS can be refined by considering the number of cytogenetic abnormalities or the presence of specific lesions, the presence of monosomy, and the mutation status of the TP53 gene. Mutations of TP53 are strongly associated with adverse clinical features including complex karyotypes, yet still have independent prognostic significance. Whether these mutations or other elements associated with complex karyotypes carry the greatest prognostic significance is unknown and has been the subject of recent contradictory findings. Here we examine the relationship between TP53 mutations, the number and type of chromosomal abnormalities, and clinical features including overall survival in a combined analysis of MDS patients with complex karyotypes.Methods:Data on 258 complex karyotype MDS patients from the US and Europe gathered by members of the International Working Group for Prognosis in MDS-Molecular Committee were combined for this analysis. Patients gave informed consent to have their data and clinical samples collected at their respective institutions in accordance with the Declaration of Helsinki. Tumor samples were examined for TP53 mutations primarily with next generation sequencing techniques. Categorical variables were compared using a Fisher exact test or Kruskal-Wallis test as appropriate, while continuous variables were compared using a Wilcoxon rank-sum test. All p-values reported are two-sided, unadjusted for multiple testing, and considered significant at the 0.05 level.Results:Of the 258 complex karyotype MDS samples examined, TP53 mutation status was determined in 223, of which 133 (60%) carried a TP53 mutation. Patients with TP53 mutations were slightly older than unmutated patients (median age 70.8 vs. 65.5 years, p=0.055), were more thrombocytopenic (median platelet count 42 vs. 78 x109/L, p=0.002), and had a greater percentage of bone marrow blasts (7.75% vs 4%, p=0.03). Mutated patients were also more likely to have a monosomal karyotype (85% vs. 59%, p<0.0001), have 5 or more chromosomal abnormalities (85% vs. 51%, p<0.0001), and have abnormalities of chromosome 17 (38% vs. 23%, p=0.03).Patients with TP53 mutation had a significantly shorter median overall survival of 7.7 months compared to 23.4 months for patients with unmutated TP53 (p<0.0001, see figure). Multivariate analysis that considered potential confounders such as monosomal karyotypes, number of abnormalities, blast proportions and platelet count identified TP53 mutation status as the most significant prognostic marker for overall survival (hazard ration [HR] 2.12, p<0.0001). The presence of only 3 or only 4 karyotype abnormalities was protective compared with 5 or more such lesions (HR 0.385, p=0.0005 for 3 lesions and HR 0.505, p=0.0083 for 4 lesions).Discussion:Despite strong associations with adverse clinical and cytogenetic abnormalities incorporated into existing prognostic scoring systems, TP53 mutations carry significant independent prognostic value for patients with MDS. In our multivariable analyses, TP53 mutation appears to dominate over monosomal karyotype as an adverse prognostic marker in MDS patients with complex cytogenetics. Many of the TP53 mutations identified had a small variant allele fraction suggesting that they were present in a disease subclone. The small number of mutant cells in these cases may be insufficient to alter clinical measures, but could still indicate a poor prognosis if they represent a clone capable of treatment resistance and rapid disease progression.This combined analysis is a work in progress that will include data from additional groups and explore mutation status of other MDS-related genes, TP53 variant allele fraction, clinical risk scores, and data from patients without complex karyotypes.Conclusions:Somatic TP53 mutations are highly enriched in MDS patients with complex karyotypes and identify a group with significantly shortened overall survival. Sequencing of TP53 can improve the prediction of prognosis in MDS patients with 3 or more chromosomal abnormalities. [Display omitted] DisclosuresBejar:Genoptix Medical Laboratory: Consultancy, Honoraria, Licensed IP, no royalties Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Other. Garcia-Manero:Celgene: Research Funding. Sekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen Corp: Membership on an entity’s Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity’s Board of Directors or advisory committees. Campbell:14M Genomics Limited: Consultancy, Equity Ownership.

SP-007 MYELODYSPLASTIC SYNDROMES: OLD AND NEW CLASSIFICATION AND PROGNOSTIC SYSTEMS

The myelodysplastic syndromes (MDS) consist of a heterogeneous spectrum of myeloid clonal hemopathies. This presentation will focus on the recently defined prognostic clinical and molecular abnormalities intrinsic to the aberrant marrow hematopoiesis in MDS as they relate to clinical features and potential for disease progression. The Revised International Prognostic Scoring System (IPSS-R) provides a recently refined and improved method for clinically evaluating the prognosis of MDS patients. Novel components of this scoring method compared to the IPSS include: 5 rather than 3 cytogenetic prognostic subgroups with specific and new classifications of a number of less common cytogenetic subsets, splitting the low marrow blast percentage value, and depth of cytopenias. Other differentiating clinical features in the IPSS-R were additive to the five major parameters for predicting survival although not AML evolution: age, performance status, serum ferritin and LDH levels. Recent investigations from a number of centers have validated the clinical utility of this IPSS-R system for risk analysis of both untreated and treated MDS patients. Molecular profiling has recently generated extensive data describing critical hematopoietic molecular and biologic derangements contributing to the patients’ clinical phenotypes. These studies have demonstrated the roles of specific somatic gene mutations of bone marrow cells for the development and clinical outcomes of MDS, including their propensity to progress to more aggressive stages such as AML. These mutations have been identified in more than 80% of MDS patients, including most cases with a normal karyotype. The reported abnormalities involve genes engaged in molecular signaling and differentiation, regulation of cell cycle progression, apoptosis, transcription, translation and epigenetics. Genetic alterations have included oncogenic mutations, amplifications or deletions, transcriptional RNA splicing abnormalities, epigenetic changes and/or altered telomere dynamics. Molecular features in del(5q) MDS, such as ribosomal biogenesis dysfunction, altered micro RNA (miRNA) levels and p53 mutations also contribute to the phenotype and prognosis of these patients. These specific molecular alterations in marrow cells from MDS patients explain much of the clinical heterogeneity demonstrated by this spectrum of diseases. Sequential combinations of cooperative mutations appear to be required for the disease and its progression, possibly together with epigenetic changes of specific gene expression. These molecular abnormalities are being combined with the known clinical features of the IPSS-R to develop a more precise prognostic classification schema. Further, these findings have the potential to help discover critical targets for biospecific therapy for this group of diseases.

Expression Profiles of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinases (TIMPs) in Myelodysplastic Syndromes (MDS): Level of MMP-9 Is Associated with Improved Prognosis in MDS Patients

AbstractAbstract 3845 Introduction:Matrix metalloproteinases (MMPs) belong to a unique family of zinc dependent endopeptidases, they are strictly regulated by their endogenous inhibitors called tissue inhibitor of MMPs (TIMPs). MMPs have been associated with tumorigenesis. In addition to their role in extracellular matrix turnover and cancer cell migration, MMPs regulate signaling pathways that control cell growth, inflammation, or angiogenesis and may even work in nonproteolytic manner. MMPs play a key role during invasion and metastasizing of cancer cells and they have been shown to be associated to invasive phenotype and poor prognosis in several solid tumors. Little is known about their role in MDS. A total of 23 different human MMPs and 4 TIMPs have been identified. To better understand the role of MMPs in MDS, we performed an expression profile screen study by QPCR to detect the expression level of all MMP and TIMP family members, except MMP23, in CD34+ cells from a cohort (N=10) of newly diagnosed, untreated patients with MDS, and compared the expression level with CD34+ cells from 5 normal donors. Of these 26 genes, we identified MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 as aberrantly up-regulated genes in MDS and expanded the study to a larger cohort of patients to correlate with clinical features and clinical outcomes. Methods:CD34+ cells from 98 newly diagnosed patients with MDS and 5 normal donors were evaluated for MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 expression profiling. CD34+ cells were sorted from patients and normal donor bone marrow. Total cellular RNA was isolated using Trizol, cDNA was obtained by using TaqMan reverse transcription reagent (Applied Biosystems). For real-time PCR, all assays were purchased from Applied Biosystems and analyzed with an Applied Biosystems Prism 7500 Sequencing detection system. GAPDH was used as internal control. Immunohistochemistry was used to detect MMP9 protein level in cytospin from CD34+ cells. MMP9 abtibody was obtained from R&D systems, MMP9 ELISA kit (R&D systems) was used to detect the MMP9 protein level in bone marrow plasma. Results:For the 98 MDS patients studied, 78% were older than 60 years; by IPSS score, 17 (17.3%) low risk, 35 (35.7%) INT-1, 24 (24.5%) INT-2, 10 (10.2%) high risk, 12 (12.2%) not available. By Cytogenetics, diploid 44 (45%), 20q- 7(7%), −5/5q- 7 (7%), −7/7q- 7 (7%), −5/5q- and −7/7q- 6 (6%), 8+ 6 (6%), IM 6 (6%), MISC 14 (14%). By QPCR and comparing MDS CD34+ cells with normal CD34+ cells, aberrant up-regulation (fold>2) of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3 was detected in 40%, 93.8%, 94%, 84%, 15.6%, 45%, 21% and 27% of patients respectively. Up-regulation of MMP8, MMP9 and MMP25 were significant with a mean fold value 350.7 (0–3363.7), 1112.4 (0–15641) and 143.8 (0–1017.9) respectively. We performed MMP9 immunohistochemistry of MDS CD34+ cytospins from 16 pts with higher and lower MMP9 RNA relative expression level and found consistent protein expression level in cytoplasm. No elevated MMP9 protein levels were detected in bone marrow plasma. We then performed an analysis of associations with clinical variables and observed that relative expression value of MMP9 was associated with lower bone marrow blast (p=0.001) and longer survival (p=0.02) (figure 1). We did not found association between survival and the other 7 up-regulated genes. Conclusions:Bone marrow CD34+ cells from patients with MDS have abnormal up-regulation of MMP2, MMP8, MMP9, MMP25, MMP28, TIMP1, TIMP2 and TIMP3. MMP9 up-regulation is associated with longer survival. Our results suggest that MMP-9 could be a useful prognostic indicator for MDS and that this family of proteins needs further study in MDS. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clinicopathologic Characterization of Acute Myeloid Leukemia and Myelodysplastic Syndrome with Inv(3)(q21q26.2)/t(3;3)(q21;q26.2) Reveals That Complex Karyotype but Not Blast Percentage Is Associated with Poor Survival; A Bone Marrow Pathology Group Study

AbstractAbstract ▪3847▪This icon denotes a clinically relevant abstractAcute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2); RPN1-EVI1 [inv3/t3] is a distinct type of AML with recurrent genetic abnormalities (RGA) in the 2008 WHO classification, with poor response to therapy and poor prognosis. The resulting dysregulation of EVI1 plays an important role in stem cell self-renewal and leukemogenesis. Although myelodysplastic syndrome (MDS) with inv3/t3 has a high risk of progression to AML, inv3/t3 is not among the genetic abnormalities sufficient for diagnosis of AML, irrespective of blast percentage (%) in the WHO classification. The revised International Prognostic Scoring System (IPSS-R) includes comprehensive cytogenetic subgrouping to better define prognosis in MDS patients. In this system, inv3/t3 is included in a poor risk karyotype group. The objective of this multicenter study was to evaluate a series of patients with MDS/AML and inv3/t3 in order to characterize their clinicopathologic features and outcome, and to apply the IPSS-R to inv3/t3 MDS patients.111 patients (40 MDS and 71 AML with inv3/t3) were gathered from 8 medical centers. The median age at diagnosis was 56.5 years and was significantly older in MDS than AML with inv3/t3 patients (65 vs 54.5, p=0.03). Patients typically presented with normocytic anemia, thrombocytopenia and mild leukopenia (median Hb 9.1 g/dL, platelet 91 x109/L, WBC 3.6 x109/L). MDS with inv3/t3 patients had lower WBC than AML with inv3/t3 (median 3.1 vs 5.5, p<0.001). 19% of patients had hepatosplenomegaly. The median bone marrow (BM) blast% was 4% in MDS and 50% in AML with inv3/t3 and BM cellularity was higher in AML (70%) than MDS (40%) with inv3/t3 (both p<0.001). 88% of patients showed dysmegakaryopoiesis with characteristic small uni/bilobated forms. Dysgranulopoiesis (46%) and dyserythropoiesis (56%) were common and 59% of patients displayed multilineage dysplasia. The cytogenetics showed isolated inv3/t3 in 41% of patients, one additional abnormality in 33% and complex karyotype in 26%. −7/del7q (37%) was a frequent additional abnormality. Philadelphia chromosome (Ph) was noted in 10% of AML with inv3/t3. Overall 83% of patients (75% of MDS and 87% of AML with inv3/t3) expired (median follow up of 7.9 months (mo)). Most patients received therapy including 54% with chemotherapy (CTX; topoisomerase II inhibitors and/or antimetabolites) alone, 27% with CTX and allogeneic stem cell transplant (SCT) and 19% with supportive care. 16% of patients (10 MDS and 8 AML with inv3/t3) were associated with prior therapy for solid tumors and lymphomas. 57% of MDS with inv3/t3 patients subsequently evolved to AML.There was no significant difference in overall survival (OS) between MDS and AML with inv3/t3 (12.9 vs 8.0 mo, Cox PH p=0.11, Figure 1). There was no OS difference between MDS and AML after excluding Ph+ cases (Cox PH p=0.17) nor between de novo and therapy related MDS/AML with inv3/t3 (Cox PH p=0.89). Patients with isolated inv3/t3, one additional cytogenetic abnormality, and a complex karyotype showed progressively shorter OS (12.9, 10.0 and 4.3 mo, Cox PH p<0.001, Figure 2). The patients who received CTX and SCT showed superior OS to patients receiving CTX alone or supportive care only (15, 7 and 5 mo, respectively, Cox PH p=0.02). In multivariate analysis, choice of therapy and complex karyotype retained independent prognostic significance (Cox PH p= 0.02 and p<0.001, respectively).MDS with inv3/t3 patients were classified into IPSS Intermediate (Int)-1 (21), Int-2 (13), and high (6) risk groups. IPSS-R categorized MDS patients into low (3), Int (6), high (14) and very high (17) risk groups. 57% of IPSS Int-1 risk group patients (expected OS 3.5 year) were reclassified to high or very high risk group in IPSS-R (expected OS <1.6 year). Thus, the IPSS-R scores were higher relative to IPSS score (signed rank test, P<0.001). However, 72.5% and 77.5% of MDS with inv3/t3 patients had shorter OS than expected OS by IPSS-R and IPSS scores.The IPSS-R better reflects the OS of inv3/t3 than IPSS but may not fully reflect the generally dismal prognosis. Patients with MDS and AML with inv3/t3 follow a similarly aggressive clinical course, supporting classification of MDS with inv3/t3 as an AML with RGA irrespective of blast%. Additional cytogenetic abnormalities are associated with shorter OS in AML/MDS with inv3/t3 and our data suggest that aggressive therapy with SCT should be considered in these patients. [Display omitted] [Display omitted] Disclosures:Vardiman:Celgene Corporation: review of slides for clinical trials not relevant to this abstract Other. Foucar:e. Honoraria–Scientific Symposium Pathology Education: ASCP Press; ARP, Amirsys, ASCP Press; ARP, Amirsys Patents & Royalties, Honoraria, Not relevant to this abstract Other.

Simultaneous analysis of the expression of 14 genes with individual prognostic value in myelodysplastic syndrome patients at diagnosis: WT1 detection in peripheral blood adversely affects survival

Several studies have evaluated the prognostic value of the individual expression of certain genes in patients with myelodysplastic syndromes (MDS). However, none of them includes their simultaneous analysis by quantitative polymerase chain reaction (PCR). We evaluated relative expression levels of 14 molecular markers in 193 peripheral blood samples from untreated MDS patients using real-time PCR. Detectable WT1 expression levels, low TET2, and low IER3 gene expression were the only markers showing in univariate analysis a poor prognostic value for all treatment-free (TFS), progression-free (PFS), and overall survival (OS). In multivariate analysis, molecular parameters associated with a shorter TFS were: WT1 detection (p = 0.014), low TET2 (p = 0.002), and low IER3 expression (p = 0.025). WT1 detection (p = 0.006) and low TET2 (p = 0.006) expression were associated with a shorter PFS when multivariate analysis was carried out by including only molecular markers. Molecular values with an independent value in OS were: WT1 detection (p = 0.003), high EVI1 expression (p = 0.001), and undetectatable p15-CDKN2B (p = 0.037). WT1 expressers were associated with adverse clinical-biological features, high IPSS and WPSS scoring, and unfavorable molecular expression profile. In summary, detectable WT1 expression levels, and low TET2 and low IER3 expression in peripheral blood showed a strong association with adverse prognosis in MDS patients at diagnosis. However, WT1 was the only molecular marker displaying an independent prognostic value in both OS and TFS.

Methylation of the CpG Island Near SOX7 Gene Promoter Is Correlated with the Poor Prognosis of Patients with Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS), characterized by the decreased production of blood cells, often progresses to acute myeloid leukemia (AML), a sign of poor prognosis of MDS. In AML, the Wnt/β-catenin pathway is aberrantly activated, suggesting that the increased pathway activity may be correlated with the development and prognosis of MDS. SOX7 protein, encoded by the sex-determining region Y-box 7 (SOX7) gene, inhibits the activity of the Wnt/β-catenin pathway. Because the DNA methylation can regulate the transcription of SOX7 gene, we used the methylation-specific PCR to investigate the methylation status of the CpG island in MDS patients to determine the potential correlation of the SOX7 methylation with the development and prognosis of MDS. We found that the CpG island of the SOX7 gene was methylated in 58.1% (97/167) of MDS patients, but not in any healthy control. Furthermore, the percentage of patients with the methylated CpG island of the SOX7 gene was significantly higher in patients at advanced stages of MDS than in the patients at early stages. The increased percentages of this SOX7 methylation were also correlated with age, marrow blast levels, and International Prognostic Scoring System (IPSS) risk. After prognostic analysis, we found that patients with the methylated CpG island of the SOX7 gene had shorter overall survival and cumulative survival than patients with unmethylated CpG island. Our findings suggest that the methylation of the CpG island of the SOX7 gene can be used as a predictive factor for the development and prognosis of MDS patients.

Methylation Analysis of the Wnt Antagonist in Myelodysplastic Syndrome

Abstract 5048 Background and Objectives:Myelodysplastic syndrome (MDS) is one of the most threatening hematological malignancies. Recently, the epigenetic changes have been recognized in the MDS, and some research found that the aberrant DNA methylation had close relationship with the MDS. Meanwhile, some studies showed that the activation of the Wnt signaling pathway and abnormal methylation of the Wnt antagonists had close relationship with hematological malignancies, such as AML AALL Aand CLL, but less is known in MDS. In our research, we studied the methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in the patients with MDS and evaluated the role of them in the pathogenesis and progression of MDS, with providing a new theory support for clarifying the complicated pathogenesis and progression of MDS. Methods:The methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in pretreatment bone marrow samples from 53 patients with MDS was measured by methylation-specific polymerase chain reaction (MSP). On the other hand, we collected the clinical materials of the patients with MDS and follow up the patients. Then the correlation between methylation and clinical features as well as prognosis of MDS patients was analyzed byχ2 test and Kaplan-Meier method. Results: In 53 bone marrow samples, the methylation frequencies of the Wnt antagonists were as follows: SFRP4 for 62.3 % (33/53), DKK1 for 45.3 % (24/53), HDPR1 for 34.0 % (18/53), SFRP1 for 15.1 % (8/53), DKK3 for 9.4 % (5/53), and WIF-1 for 5.7 % (3/53). After analyzing individual tumor suppressor gene, clinical parameters and prognostic information, it was found that the patients with the percentage of BM blast above 5% had higher methylation frequency of HDPR1 than the patients with the percentage of BM blast bellow 5% (44.4%∼a13.3%, P=0.034); besides, the methylation frequency of HDPR1 exhibited significant differences in the MDS subtypes (P=0.019), and was significantly correlated with the WPSS (P=0.037); In addition, Kaplan-Meier survival curve indicated that the mean overall survival of patients with methylation was significantly shorter than that of patients without HDPR1 methylation (457.3 days vs. 919.6 days, P=0.037) (Fig.1). Conclusion:The methylation of the Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) were observed in patients with MDS and their methylation frequencies were different. The aberrant methylation of HDPR1 may play an important role in the progression and prognosis of MDS. Disclosures:No relevant conflicts of interest to declare.

A Novel Missense Mutation In An MDS Patient and Effects on TET2 mRNA Expression and Clinical Outcomes

AbstractAbstract 1889 IntroductionThe ten-eleven translocation 2 (TET2) gene has recently been recognized to be mutated at a relatively high frequency in all myeloid malignancies, representing a compelling unifying feature of these related but phenotypically distinct conditions. The TET2 paralog TET1 catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine; TET2 shares a homologous domain thought to catalyze this conversion, and is hypothesized to act as a tumor suppressor gene (TSG) by regulating DNA methylation and epigenetic control of gene expression at critical loci important for myelopoiesis and leukemogenesis. The prognostic impact of TET2 mutations is largely unknown. The hypomethylating agents 5-azacytidine and decitabine, approved agents with activity in myelodysplastic syndrome (MDS), are incorporated into DNA and/or RNA, inhibiting DNA methyltransferase and preventing promoter methylation and TSG silencing. Given the role of TET2 in epigenetic regulation, the aim of this study was to determine whether TET2 mutations were associated with prognosis in MDS patients (pts) treated with hypomethylating therapies. MethodsBone marrow aspirates from 12 MDS pts who received hypomethylating agents were obtained, and PCR and bidrectional sequencing of TET2 coding exons was performed. RT-PCR was performed in triplicate and analyzed by the comparative Ct method. ResultsThe median age of the 12 pts was 68 (49-80), the median international prognostic scoring system (IPSS) score was 1.75 (0-3) and 7/12 (58%) had normal cytogenetics. Two of 12 (17%) pts had exonic TET2 mutations. Pt 7 exhibited a previously reported heterozygous V1718L missense mutation, while pt 3 had 2 previously unreported heterozygous mutations: a nonsense mutation in exon 3, Q1068X, and a missense mutation in exon 6, R1214V. TET2 mRNA expression from all pt samples that expressed GAPDH was quantified. Pt 3 had decreased TET2 mRNA expression compared to pt 7, MDS patients with wild type TET2 and non-diseased control samples (p=.01). Pts with wild-type TET2 had an overall response rate (defined as complete responses plus partial responses) of 20%, while neither of the patients with TET2 mutations responded to hypomethylating agents. The post-treatment median survival was significantly different between patients harboring TET2 mutations and those with wild type TET2 (log rank test p<.003). Both pts with mutated TET2 ultimately died, 4 months and 6 months after treatment, while 7/10 (70%) pts with wild type TET2 died, with a median time to death or last follow-up of 26 months (95% CI 6.1–47.6) ConclusionsWe report the existence of two novel mutations in TET2, one of which is a heterozygous nonsense mutation that is predicted to result in a truncated protein lacking the conserved domains. The reduced expression of TET2 observed in this pt may be due to nonsense-mediated decay, a post-transcriptional process that recognizes and degrades mRNA that contains a premature translation termination codon. The adverse association seen in this series between TET2 mutations and clinical outcomes in MDS pts who received hypomethylating drugs merits replication in a larger sample. Disclosures:No relevant conflicts of interest to declare.

Expression of Differentiation Antigens on Bone Marrow Myeloid Cells of the Patients with Myelodysplastic Syndromes and it's Clinical Significances

AbstractAbstract 4967 ObjectiveTo study the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients. MethodsQuantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality of these antigen expression and the prognosis of MDS cases were analyzed. ResultsThe granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The “right hook”, “sickle” and “retroflex 7” shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+(0.39±0.34)and CD16-/CD16+(1.33 ±0.77)of high-risk MDS group were significantly higher than those of control group (0.07±0.05 and 0.39 ±0.31 respectively) (P<0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while their MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ (3.88%±3.07%), CD11b- HLA-DR- (16.23%±15.59%), CD16-HLA-DR- (41.12%±24.53%), CD11b+CD16- (33.53%±17.26%) and CD13+CD16- (44.51%±21.99%) granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P<0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double antigen positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double antigen positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases correlated directly with their IPSS (international prognostic scoring system) (r=0.690, P=0.000) and WPSS (WHO adapted prognostic scoring system) (r=0.651, P=0.000) scores. ConclusionThere were abnormal expressions of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity of these abnormal expressions was correlated with their prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS. Disclosures:No relevant conflicts of interest to declare.

Expression of differentiation antigens on bone marrow myeloid cells of the patients with myelodysplastic syndromes and its clinical significances

To detect the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients. Quantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality and the prognosis of MDS cases were analyzed. The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The "right hook", "sickle" and "retroflex 7" shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+ (0.39 +/- 0.34) and CD16-/CD16+ (1.33 +/- 0.77) were significantly higher in high-risk MDS group than those of control group (0.07 +/- 0.05 and 0.39 +/- 0.31 respectively) (P < 0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ 3.88% +/- 3.07%, CD11b-HLA-DR- 16.23% +/- 15.59%, CD16-HLA-DR- 41.12% +/- 24.53%, CD11b+CD16- 33.53% +/- 17.26% and CD13+CD16- 44.51% +/- 21.99% granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P < 0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases per case correlated directly with their IPSS (international prognostic scoring system) (r = 0.690, P = 0.000) and WPSS (WHO adapted prognostic scoring system) (r = 0.651, P = 0.000) scores. There is an abnormal expression of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity is correlated with the prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.

Prognostic Significance of WT1 mRNA and Anti-WT1 Antibody Levels in Peripheral Blood in Patients with Myelodysplastic Syndromes.

Abstract 3821Poster Board III-757(INTRODUCTION) The Wilms tumor gene (WT1) message is overexpressed in tumor cells from various solid cancers as well as hematologic malignancies including myelodysplastic syndromes (MDS). We reported previously that WT1 mRNA expression in peripheral blood mononuclear cells (PBMCs) as well as bone marrow (BM) cells increased with the aggressiveness of MDS disease subtype as defined by the French-American-British (FAB) classification and that a humoral immune response, IgG- or IgM-type anti-WT1 antibody (Ab) expression, was detected in sera from most MDS patients. In this study, we investigated whether WT1 mRNA expression and anti-WT1 Ab titers in PB were associated with prognosis in MDS patients by examining their long-term follow-up data. (METHODS AND RESULTS) (1) WT1 mRNA expression in PBMCs was examined in 80 patients: 35 with refractory anemia (RA); 5 with RA with ringed sideroblasts (RARS); 24 with RA with excess blasts (RAEB); 5 with RAEB in transformation (RAEB-t); and 11 with acute myeloid leukemia transformed from MDS (AML-MDS). Levels of WT1 mRNA expression were assessed using the real-time quantitative polymerase chain reaction [Tamaki H, et al, Leukemia 1999]. WT1 mRNA levels increased with the aggressiveness of disease subtype (mean: RA, 220.9; RARS, 129.4; RAEB, 5,554.3; RAEB-t, 14,284.0; AML-MDS, 56,272.7 copies/μg) and with the aggressiveness of the International Prognostic Scoring System (IPSS) category (mean: low, 114.5; intermediate-1, 360.8; intermediate-2, 12,041.6; high, 7,357.9 copies/μg) in these patients. (2) IgG- and IgM-type anti-WT1 Ab titers were determined using the dot-blot assay [Elisseeva OA, Blood 2002] in sera from 45 of the 80 patients: 15 RA; 3 RARS; 18 RAEB; 3 RAEB-t; and 6 AML-MDS. IgM and IgG WT1 Abs were detected in 31 (79.5%) and 34 (87.2%) MDS patients, and 5 (83.3%) and 6 (100%) AML-MDS patients, respectively. WT1 Abs levels were not correlated with FAB subtype, IPSS, or WT1 mRNA expression in PBMCs. (3) When patients were divided into three groups based on the WT1 mRNA level (fewer than 100 copies/μg, 100 to 10,000 copies/μg, and more than 10,000 copies/μg), their survival rates differed significantly (P = 0.0186): survival was worse in those with increased WT1 mRNA levels. Specifically, a high WT1 mRNA level was a strong predictor of rapid AML transformation even if adjusted by the IPSS (P = 0.0005). Furthermore, patients with high levels of either IgM or IgG WT1 Abs had significantly better survival compared with those whose IgM and IgG WT1 Abs values were both low (P = 0.0007) even when adjusted by the IPSS (P = 0.0019). (CONCLUSIONS) This study showed for the first time that high WT1 mRNA expression and high WT1 Ab titers in PB affected the prognosis of MDS patients negatively and positively, respectively, suggesting that an optimal immune response against WT1 may beneficial. Recently, clinical trials of WT1 peptide-based immunotherapy have been conducted for various malignancies including MDS. Our data presented here may provide a rationale for anti-WT1 immunotherapy in MDS. Disclosures:No relevant conflicts of interest to declare.

Analysis of the Immunophenotypic Features of Blast Cells, Lymphocytes and Granulocytes by Multiparametric Flow Cytometry in Bone Marrow (BM) Cells of Myelodysplastic Syndromes.

Abstract 4843 BackgroundThe myelodysplastic syndromes (MDS) are a group of clonal heterogeneous bone marrow disorders characterized by peripheral cytopenias, ineffective hematopoiesis, and unilineage or multilineage dysplasia. Multiparametric flow cytometry is increasingly being used as an adjunct to the establishing of MDS. While many antigens have been described to be aberrantly expressed in MDS the findings are generally heterogeneous and there is no consistent finding that would be present in all cases with MDS. AimTo investigate the immunophenotypic features of MDS and non-MDS patients and the characteristic of subtypes of MDS. MethodsBM samples were collected from 22 MDS patients including 3 RA (2 male, 1 female, median age 57), 3 RAS (2 male, 1 female, median age 72), 12 RAEB (6 male, 6 female, median age 67.5), 4 MDS-AML (2 male, 2 female, median age 69.5) and 20 non-MDS (11 male, 9 female, median age 32.5, 7 AA, 5 PNH, 3 IDA, 1 ALL, 2 CML, 2 MM). The multiparametric flow cytometric analysis was performed using an extensive panel of monoclonal antibodies and using the conventional and secondary gating strategies to analysis the immunophenotypic features of BM cells. ResultsThis study showed that the proportion of blast cells increased significantly than non-MDS group (P=0.002). As the disease progressing, the percentage of blast cells became higher and significantly difference compared to the non-MDS group (P=0.226, P=0.464, P=0.001 and P=0.000, respectively). The expressions of CD34+ and CD7+ on blast cells were significantly difference between MDS and non-MDS groups (P=0.005, and P=0.002, respectively). Compared with the subtypes of MDS and non-MDS group, the expressions of CD34+ and CD7+ on blast cells became high gradually (P=0.534, P=0.487, P=0.009, P=0.004 and P=0.294, P=0.166, P=0.002, P=0.001) and the high percentage of blast cells and high expression levels of CD34+ and CD7+ might indicate poor prognosis. The expression of CD7+ on lymphocytes was similar with CD34+ and CD7+ on blast cells, but the expressions of CD19+ and CD56+ on lymphocytes were no significantly difference (P=0.076, P=0.252, respectively). The expressions of antigens on granulocytes showed that the expressions of CD15+CD11b+, CD10+ and HLA-DR were significantly difference between MDS and non-MDS groups(P=0.000, P=0.009 and P=0.007, respectively), meanwhile, as the disease progressing, the expression rates of CD15+CD11b+, CD10+ and HLA-DR in subtypes of MDS increased gradually and the survival time of these patients who had over-expression of these antigens was shorter than control group(P=0.002). However, the expressions of CD33+, CD13+, CD56+ and CD15+CD11b- were significantly difference between subtypes of MDS and non-MDS group (P=0.059, P=0.588, P=0.063 and P=0.207, respectively). ConclusionsOur results showed that using multiparametric flow cytometry to analyze the immunophenotypic features of BM cells could provide clinically useful information for the diagnosis, classification and prognosis of MDS patients. Particularly, the percentage of blast cells, the expression of CD34+ and CD7+ on blast cells, the expression of CD7+ on lymphocytes and the expression of CD15+CD11b+, CD10+ and HLA-DR on granulocytes may provide the much more useful information. However, further studies including larger number of patients with a longer follow-up are necessary to confirm these results. DisclosuresNo relevant conflicts of interest to declare.

Changes in Gene Expression Profiles during Treatment with Erythropoetic Growth Factors in Myelodysplastic Syndromes.

Background: Gene expression profiles have been associated with prognosis in myelodysplastic syndromes. WT1 is a tumor-suppressor gene coding for a transcription factor located on chromosome 11p13, which was originally identified for its involvement in the pathogenesis of the Wilms' tumor. In normal bone marrow, WT1 expression is low or undetectable, whereas it is aberrantly expressed in hematological malignancies. Evidence indicates that WT1 is important in the lineage-specific differentiation of hematopoietic cells and leukemogenesis. In myelodysplastic syndromes (MDS), WT1 expression has prognostic significance: it is directly correlated with the type of MDS, with IPSS score and with disease progression. Recent data demonstrate that WT1 is a potent activator of the EPO gene under normoxia and it is suggested that WT1 may regulate paracrine EPO synthesis in a tissue-specific manner. Bmi-1 is a transcriptional repressor gene which may be expressed restrictedly in stem cells and progenitors and is required to regulate the adult self-renewing hematopoietic and leukemic stem cells. It appears that it also plays an important role in providing cells the potential for proliferation. A number of reports on Bmi-1 provide perspectives on the close association of its expression with the progression of hematopoietic malignancies. Furthermore, flow cytometry has shown that Bmi-1 positivity in CD34+ cells is positively correlated with IPSS score.Introduction: We have designed a study to evaluate changes in gene expression profiles of bone marrow mononuclear cells of primary low and intermediate-1 IPSS risk MDS patients receiving erythropoetic growth factors (darbepoetin or high-dose rHuEpo alpha). Associations with response, changes in Hb, in percentage of CD34+ and apoptotic cells are evaluated. We present preliminary results in 6 patients.Methods: Bone marrow samples were obtained before and after 12 weeks of treatment. Mononucleated cells were cryopreserved and later thawed for total RNA extraction and cDNA synthesis. Gene expression profiling and the expressions of WT1 and Bmi-1 by RT-PCR were evaluated.Results: Baseline median Hb was 9,5 g/dL (interquartile range 8,7 10,1). Baseline mean Bmi-1 was 14011 (± SD 3927). All patients had a major erythroid response to treatment. Preliminary results demonstrate a significant decrease in WT1 from baseline median 1635 (interquartile range 1251–2150) to 1192 (interquartile range 1005–1365, P=0.046). [Display omitted] Discussion: Though few patients have yet been studied, it is suggested that WT1 decreases in patients responding to erythropoetic growth factors. Further evaluation with stratification for erythropoetic response in the extended study may furnish novel associations between gene expression, erythropoetic growth factors and prognosis in MDS patients.

Clinical Features and Prognosis of MDS Patients with Chromosome 5 Abnormalities Other Than ‘5q-Syndrome': Results of Multi-Center Analysis in Korea.

Introduction: The myelodysplastic syndrome (MDS) is frequently associated with various chromosomal abnormalities. ‘5q− syndrome' is low-risk MDS known as good responder of lenalidomide recently. However, the patients with other abnormalities in chromosome 5 showed quite different clinical features from those with ‘5q− syndrome'. The aim of this study was a retrospective evaluation for Korean MDS patients with abnormalities in chromosome 5 other than ‘5q− syndrome'.Materials and Methods: Among 456 patients with MDS diagnosed at 16 hospitals in Korea between 1996 and 2006, 370 with available cytogenetic data entered the study. Univariate and multivariate analysis were performed.Results: Ninety three patients (25.1%) showed abnormalities in chromosome 5 and the ‘5q− syndorme' was only 10 patients (2.7%). Among the rest, 39 patients (10.5%) had various abnormalities other than 5q deletion such as translocation or 5 monosomy, 38 (10.3%) had complex abnormalities with 5q−, and 2 had mosaic pattern with normal chromosome. Four patients had isolated 5q− but blasts in marrow were over 5%. The deletion of 5q was interstitial but with a predominance for 5q13-33 deletions (34.8%). MDS patients with chromosome 5 abnormalities other than ‘5q− syndrome' didn't share the clinical features with ‘5q− syndrome'. There was no leukemic transformation in ‘5q− syndrome' group, but 18 (21.7%) with other abnormalities in chromosome 5 finally transformed to acute leukemia. Five year overall survival was significantly inferior in non-'5q− syndrome' patients than ‘5q− syndrome' (14.3% vs. 79.6%, P=0.0115).Conclusions: Patients with isolated 5q− and excess blast (>5%), other abnormalities than isolated 5q−, or mosaic chromosome with isolated 5q− and normal chromosome didn't share the clinical features such as lower rate of leukemic transformation and long survival.

Place de la cytogénétique dans la prise en charge des syndromes myélodysplasiques primaires

Cytogenetic abnormalities in myelodysplastic syndromes (MDS) are complex and heterogeneous. The most frequent rearrangements (gains or losses of genetic material) vary from patient to patient, and within the same patient. The prognostic value of these rearrangements has been extensively studied. They allowed the definition of a risk based classification system for MDS (the International Scoring System for evaluating Prognosis, IPSS), proven to be a highly useful method for evaluating prognosis in MDS patients. Despite recent progress in mapping and definition of minimally deleted chromosomal regions, the primary critical genetic events remain to be determined. The recurrent cytogenetic abnormalities associated with MDS are likely to be secondary events contributing to but not initiating the neoplastic phenotype of the disease.

Trisomy of chromosome 8 in myelodysplastic syndrome: Significance of the fluctuating trisomy 8 population

Chromosome analyses were performed in five patients with myelodysplastic syndrome (MDS) who showed trisomy of chromosome 8 during the course of their disease. Four of these patients showed trisomy 8 at the diagnosis of MDS, and the remaining one had trisomy 8 when the leukemia phase developed. The proportion of bone marrow (BM) cells with trisomy 8 in the four patients who showed trisomy 8 at MDS diagnosis fluctuated, and this fluctuation was not related to the percentage of blasts in the BM or to progression of the disease. However, in two patients, metaphase cells with trisomy 8 disappeared when their anemic state improved, although leukothrombocytopenia was still present, suggesting that the decrease in the number of BM cells with trisomy 8 reflects hematologic features in some MDS patients. These findings indicate that trisomy 8 in our MDS patients was possibly not the primary event in the genesis of the disease, and that there may have been competition between a normal karyotype clone and a trisomy-8-positive clone. Our results further suggest that the presence of a clone with trisomy 8 is not always a sign of disease progression or of poor prognosis in MDS patients.