Expression of WT-1 mRNA in Peripheral Blood from Myelodysplastic Syndromes

Abstract (INTRODUCTION) The Wilms’ tumor gene WT-1 is overexpressed in various types of solid tumor as well as in hematologic malignancies, i.e., acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myeloid leukemia, and myelodysplastic syndromes (MDS). It was reported that WT-1 overexpression in peripheral blood (PB) and bone marrow (BM) is useful for the monitoring of minimal residual disease and early detection of relapse in AML. MDS are clonal hematologic stem cell disorders characterized by cytopenias and a risk of progression to acute leukemia. It is important for decisions on treatment strategy to assess disease progression and predict the prognosis in MDS. The aim of this study was to investigate the clinical significance of WT-1 mRNA expression in PB obtained from patients with MDS. (METHODS AND RESULTS) PB was obtained from 92 patients with MDS or leukemia transformed from MDS (AL-MDS): 40 refractory anemia (RA), 5 RA with ringed sideroblasts (RARS), 27 RA with excess blasts (RAEB), 5 RAEB in transformation (RAEB-t), and 15 AL-MDS cases in the FAB classification (RA 27, RARS 5, refractory cytopenia with multilineage dysplasia 12, RAEB-1 13, RAEB-2 14, 5q- 1, and AL-MDS 20 cases in the WHO classification). RNA was isolated from PB mononuclear cells (PBMCs) and converted into cDNA. The levels of WT-1 mRNA expression were assessed using the real-time quantitative polymerase chain reaction. We analyzed whether the level of WT-1 mRNA expression in PBMCs was associated with MDS subtype in the FAB classification, clinical characteristics (hemoglobin value, white blood cell counts, neutrophil counts, lymphocyte counts, chromosomal abnormality, number of cytopenias, blast percentages in BM, lactate dehydrogenase values, C-reactive protein values), International Prognostic Scoring System (IPSS) score, survival, and time to leukemia transformation. High expression of WT-1 mRNA, which was defined as more than 50 copies/μg mRNA according to the results of normals, was observed in 42.5%, 40.0%, 85.2%, 80.0%, and 100% of RA, RARS, RAEB, RAEB-t, and AL-MDS patients, respectively. The WT-1 mRNA levels increased with the aggressiveness of disease subtype and IPSS. FAB subtypes included RARS (mean ± SD, 129 ± 111 copies/μg), RA (220 ± 134), RAEB (5554 ± 2593), RAEB-t (14284 ± 9056), and AL-MDS (51591 ± 30309). IPSS score were divided into low/intermediate-1 risk (316 ± 136) and intermediate-2/high risk (7901 ± 3035) (P < 0.05 for RA vs RAEB, RAEB-t or AL-MDS; RAEB vs AL-MDS; and low/intermediate-1 vs intermediate-2/high risk). Furthermore, the WT-1 mRNA level was inversely correlated with the neutrophil percentage in PB and positively correlated with the blast percentage in both PB and BM. Among RA patients, those with favorable cytogenetics had lower WT-1 mRNA values compared with other patients (P < 0.05). When patients were divided into three groups based on the log value of WT-1 mRNA (<2, 2–4, and >=4), the survival times of those groups were 73.1, 55.6, and 15.3 months, respectively (P < 0.005). (CONCLUSIONS) Higher quantitative expression of WT-1 mRNA in PBMCs is associated with aggressive disease behavior in MDS patients. This may justify anti-WT-1 immunotherapy under investigation for MDS treatment (PNAS2004;101:13885–13890).

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

Blood ◽

10.1182/blood.v114.22.3821.3821 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3821-3821

Author(s):

Hideto Tamura ◽

Kazuo Dan ◽

Norio Yokose ◽

Rika Iwakiri ◽

Masatsugu Ohta ◽

...

Keyword(s):

Immune Response ◽

Mrna Expression ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Quantitative Polymerase Chain Reaction ◽

Mrna Level ◽

Prognostic Significance ◽

Refractory Anemia ◽

Mrna Levels ◽

Disease Subtype

Abstract Abstract 3821 Poster 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.

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Thrombocytosis as a Presenting Feature in Myelodysplastic Syndromes (MDS) in Adults - 40 Year Experience from a Single Institution in the USA.

Blood ◽

10.1182/blood.v108.11.4831.4831 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4831-4831

Author(s):

Dhatri Kodali ◽

Hector Mesa ◽

Ajay Rawal ◽

Pankaj Gupta

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Who Classification ◽

Clinical Course ◽

Refractory Anemia ◽

Risk Category ◽

Platelet Counts ◽

Hydroxyurea Treatment

Abstract Thrombocytosis at presentation is uncommon in the myelodysplastic syndromes (MDS), and its influence on the clinical course of the disease and on prognosis is uncertain. To determine the clinical course and long-term outcomes of patients (pts) with thrombocytosis at initial diagnosis of MDS, we conducted a retrospective analysis of 503 pts diagnosed with MDS between Jan 1966 and July 2006 at the Minneapolis VA Medical Center. Original bone marrow and peripheral blood slides and reports were reviewed with a hematopathologist (H.M.) in all pts with high platelet counts (> 400 × 103/μL) and evidence of dysplasia. Clinico-pathological correlation was obtained by chart review. Patients with inadequate data, secondary causes of thrombocytosis, transient thrombocytosis, and those without evidence of dysplasia were excluded. Of 503 pts, 41 (8.2%) were found to have thrombocytosis at presentation. Their median age was 74 years. The spleen was enlarged (by imaging) in 6 pts. Peripheral blood counts (mean; range) at diagnosis were: hemoglobin (10.9 g/dL; 7.4 – 17.1), absolute neutrophils (7.9 × 103/μL; 0.8 – 30.7) and platelets (627 × 103/μL; 402 – 1231). The cases were re-classified according to the WHO classification of myeloid disorders as follows: chronic myelomonocytic leukemia-1 (CMML-1) = 17 (41%), refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) = 13 (32%) and others = 11 (27%; including 2 pts each with RA, MDS/myeloproliferative disorder-unclassified [MDS/MPD-U] and 5q- syndrome, and 1 pt each with RA with excess blasts [RAEB-1], therapy related MDS [tMDS], refractory cytopenia with multilineage dysplasia [RCMD], MDS-U and atypical chronic myeloid leukemia [Aty CML]). Bone marrow fibrosis was increased in 3 pts with CMML-1 and 1 with RARS-T, and was normal or only focally increased in all other pts. The IPSS risk category was Low in 15 pts, Int-1 in 3 pts and Int-2 in 2 pts. Cytogenetic data was not available in the other pts. Jak-2 mutation analysis was positive in 2 pts with RARS-T, negative in 1 pt each with RARS-T and MDS/MPD-U, and is pending in others. On follow-up, 2 pts with CMML-1 and 1 pt with Aty CML transformed to acute myeloid leukemia (AML) and both pts with 5q- syndrome transformed to CMML-2. Two pts with RARS-T progressed to RAEB-2 and 1 pt with CMML-1 progressed to CMML-2. One pt with CMML-1 developed marked myelofibrosis. Marked thrombocytosis required hydroxyurea treatment in 5 pts. One MDS-U pt received 5-azacytidine. Four of 41 (10%) pts experienced major bleeding events; 3 were receiving aspirin. Five pts required ongoing red cell transfusions. The median survival (MS) was 36 months in RARS-T, 60 months in CMML-1 and 27 months in others; the MS of all 41 pts was 48 months. Causes of death were AML in 3 pts, cytopenias due to MDS in 6 pts and unrelated/unknown in 21 pts. Eleven pts are currently alive. In conclusion, the majority of pts presenting with myelodysplasia and thrombocytosis fall in the MDS/MPD category of the new WHO classification (most commonly CMML or RARS-T), be older, and have low-risk features (IPSS Low or Int-1). The risks of spontaneous bleeding, transformation to AML, progression of disease or myelofibrosis are low, and the overall prognosis is relatively favorable. Platelet counts may reach levels requiring cytoreductive therapy. This study helps better understand the natural history and prognosis of this varied spectrum of MDS and overlap syndromes.

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

Blood ◽

10.1182/blood.v114.22.4841.4841 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4841-4841

Author(s):

Silvia M.M. Magalhães ◽

Rosane Bittencourt ◽

Elvira Velloso ◽

Maria de Lourdes Chauffaille ◽

Alita Andrade Azevedo ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Clinical Features ◽

Myelodysplastic Syndromes ◽

Tertiary Care ◽

Incidence Rates ◽

Low Risk ◽

Refractory Anemia ◽

Survival Curves ◽

Fab Classification

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

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Decreased levels of miR-28-5p and miR-361-3p and increased levels of insulin-like growth factor 1 mRNA in mononuclear cells from patients with hereditary hemorrhagic telangiectasia

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2018-0508 ◽

2019 ◽

Vol 97 (6) ◽

pp. 562-569 ◽

Cited By ~ 4

Author(s):

Anthony Cannavicci ◽

Qiuwang Zhang ◽

Si-Cheng Dai ◽

Marie E. Faughnan ◽

Michael J.B. Kutryk

Keyword(s):

Growth Factor ◽

Peripheral Blood ◽

Hereditary Hemorrhagic Telangiectasia ◽

Mononuclear Cells ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Manifestations ◽

Mrna Levels ◽

Insulin Like Growth Factor ◽

Peripheral Blood Mononuclear ◽

Micro Rnas

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder inherited in an autosomal dominant manner. Patients with HHT can develop vascular dysplasias called telangiectasias and arteriovenous malformations (AVMs). Our objective was to profile and characterize micro-RNAs (miRNAs), short noncoding RNAs that regulate gene expression posttranscriptionally, in HHT patient-derived peripheral blood mononuclear cells (PBMCs). PBMCs, comprised mostly of lymphocytes and monocytes, have been reported to be dysfunctional in HHT. A total of 40 clinically confirmed HHT patients and 22 controls were enrolled in this study. PBMCs were isolated from 16 mL of peripheral blood and purified for total RNA. MiRNA expression profiling was conducted with a human miRNA array analysis. Select dysregulated miRNAs and miRNA targets were validated with reverse transcription–quantitative polymerase chain reaction. Of the 377 miRNAs screened, 41 dysregulated miRNAs were identified. Both miR-28-5p and miR-361-3p, known to target insulin-like growth factor 1 (IGF1), a potent angiogenic growth factor, were found to be significantly downregulated in HHT patients. Consequently, IGF1 mRNA levels were found to be significantly elevated. Our research successfully identified miRNA dysregulation and elevated IGF1 mRNA levels in PBMCs from HHT patients. This novel discovery represents a potential pathogenic mechanism that could be targeted to alleviate clinical manifestations of HHT.

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Relative survival following response to 7 + 3 versus azacytidine is similar in acute myeloid leukemia and high-risk myelodysplastic syndromes: an analysis of four SWOG studies

Leukemia ◽

10.1038/s41375-018-0275-x ◽

2018 ◽

Vol 33 (2) ◽

pp. 371-378 ◽

Cited By ~ 2

Author(s):

Megan Othus ◽

Mikkael A. Sekeres ◽

Sucha Nand ◽

Guillermo Garcia-Manero ◽

Frederick R. Appelbaum ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Myeloid Leukemia ◽

Relative Survival ◽

Acute Myeloid

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Dose-dependent immune response in milk cells and mammary tissue after intramammary administration of lipopolysaccharide in dairy cows

Veterinární Medicína ◽

10.17221/1877-vetmed ◽

2008 ◽

Vol 52 (No. 6) ◽

pp. 231-244 ◽

Cited By ~ 9

Author(s):

C. Werner-Misof ◽

M.W. Pfaffl ◽

R.M. Bruckmaier

Keyword(s):

Immune Response ◽

Mrna Expression ◽

Real Time ◽

Polymorphonuclear Neutrophils ◽

Mammary Tissue ◽

Mrna Levels ◽

Rt Pcr ◽

Lps Challenge ◽

Cell Counts ◽

Cox 2

The immune response in milk cells and the status of mammary tight junctions (TJ) in response to intramammary (IM) infusion of different doses of Escherichia coli lipopolysaccharide (LPS) was investigated. Experiment I: Seven German Braunvieh cows were IM infused into one quarter with 1 μg (LPS-1) and 3 μg (LPS-3) of LPS, respectively, and the contralateral control quarter with saline (9 g/l; C). Milk samples were taken immediately before and 12, 24, 36, 48, 60, 84 and 108 h after infusion and analysed for somatic cell counts (SCC), lactose, sodium (Na) and chloride (Cl) ions, and electrical conductivity (EC). Milk cell mRNA expression of various inflammatory factors was quantified by real-time RT-PCR. Blood samples were taken immediately after milking for the analysis of leukocytes (WBC), polymorphonuclear neutrophils (PMN), Na and Cl. Milk SCC, lactose, Na, Cl and EC did not differ significantly between LPS-1 and C quarters after the challenge. In LPS-3 quarters SCC levels increased within the first 12 h, reached peak levels between 12 and 36 h (P ≤ 0.001) and decreased (P ≤ 0.05) thereafter to reach baseline at 108 hours. Lactose in LPS-3 quarters decreased (P ≤ 0.05) to a minimum at 24 h and increased slightly thereafter while EC, Na, and Cl increased transiently in response to LPS-3. WBC and PMN levels in both groups decreased numerically within 24 h after LPS administration. In LPS-1, WBC at 24, 48 and 108 h were significantly lower whereas in LPS-3 they were significantly higher than at time 0. TNFα-mRNA expression in both groups did not change in response to IM LPS-challenge. IL-1β-mRNA expression at 12, 24 and 36 h in LPS-1 quarters increased significantly as compared to time 0. In LPS-3 quarters the mRNA expression values of all tested ILs increased significantly as compared to time 0 within 12 h after LPS-challenge. IL-1β-mRNA expression decreased (P ≤ 0.05) at 48 and 84 h in LPS quarters. IL-8 mRNA was significantly decreased at 84 h after challenge in LPS-3 quarters. COX-2-mRNA expression in LPS-1 quarters decreased significantly as compared to time 0 at 48, 84 and 108 h, with a minimum at 84 h (P ≤ 0.05). In LPS-3 quarters COX-2-mRNA levels increased (P ≤ 0.05) within 48 h after the LPS-challenge. Experiment II: Six cows (5 German Braunvieh, 1 Brown Swiss) were injected in one quarter with 100 μg LPS and in the contralateral quarter with saline (9 g/l; C). Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of TJ proteins occludin (OCLN) and zonula occludens (ZO-) 1, 2 and 3 were quantified by real-time RT-PCR. OCLN-mRNA expression did not change in response to the IM infusion while that of ZO-1, ZO-2 and ZO-3 decreased significantly within six hours. In conclusion, a dose of 1 μg LPS did not initiate a immune response in the mammary gland. Furthermore the dose of 100 μg of LPS enhanced TJ permeability by reducing TJ plaque proteins density.

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Polyclonal primitive hematopoietic progenitors can be detected in mobilized peripheral blood from patients with high-risk myelodysplastic syndromes

Blood ◽

10.1182/blood.v86.10.3660.bloodjournal86103660 ◽

1995 ◽

Vol 86 (10) ◽

pp. 3660-3667 ◽

Cited By ~ 54

Author(s):

M Delforge ◽

H Demuynck ◽

P Vandenberghe ◽

G Verhoef ◽

P Zachee ◽

...

Keyword(s):

Stem Cell ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Allogeneic Bone Marrow Transplantation ◽

High Dose ◽

Allogeneic Bone ◽

Hematopoietic Progenitors ◽

Daughter Cells ◽

Mobilized Peripheral Blood

Myelodysplastic syndromes (MDS) form a heterogeneous group of clonal hematopoietic disorders with unfavourable prognosis. Allogeneic bone marrow transplantation is the only potentially curative treatment, but remains limited to a small subgroup of younger patients with HLA- compatible donors. As autologous stem cell transplantation is currently being explored as an alternative treatment strategy for MDS, more information needs to be acquired regarding the clonal nature of the progenitor cells in these autografts. Therefore, we have analyzed the clonal patterns of highly purified hematopoietic progenitors and their mature daughter cells in mobilized peripheral blood collections produced from five female patients with high-risk MDS in complete hematologic remission. X-chromosome activation patterns of flow-sorted immature (CD34 + 38low, CD34 + 33low) and committed (CD34 + 38high, CD34 + 33high) progenitors were studied with the polymerase chain reaction-based HUMARA assay. In four patients, a polyclonal remission was shown in all stem cell subpopulations and their mature daughter cells whereas one patient was found to remain skewed in all fractions, except T lymphocytes. This study provides strong evidence that polyclonal immature hematopoietic progenitors can be mobilized and harvested in patients high-risk MDS after treatment with high-dose chemotherapy.

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