WT1 mRNA Research Articles

Two distinct effector memory cell populations of WT1 (Wilms' tumor gene 1)-specific cytotoxic T lymphocytes in acute myeloid leukemia patients.

Wilms' tumor gene 1 (WT1) protein is a promising tumor-associated antigen for cancer immunotherapy. We have been performing WT1 peptide vaccination with good clinical responses in over 750 patients with leukemia or solid cancers. In this study, we generated single-cell gene-expression profiles of the effector memory (EM) subset of WT1-specific cytotoxic T lymphocytes (CTLs) in peripheral blood of nine acute myeloid leukemia patients treated with WT1 peptide vaccine, in order to discriminate responders (WT1 mRNA levels in peripheral blood decreased to undetectable levels, decreased but stayed at abnormal levels, were stable at undetectable levels, or remained unchanged from the initial abnormal levels more than 6 months after WT1 vaccination) from non-responders (leukemic blast cells and/or WT1 mRNA levels increased relative to the initial state within 6 months of WT1 vaccination) prior to WT1 vaccination. Cluster and principal component analyses performed using 83 genes did not discriminate between responders and non-responders prior to WT1 vaccination. However, these analyses revealed that EM subset of WT1-specific CTLs could be divided into two groups: the "activated" and "quiescent" states; in responders, EM subset of the CTLs shifted to the "quiescent" state, whereas in non-responders, those shifted to the "activated" state following WT1 vaccination. These results demonstrate for the first time the existence of two distinct EM states, each of which was characteristic of responders or non-responders, of WT1-specific CTLs in AML patients, and raises the possibility of using advanced gene-expression profile analysis to clearly discriminate between responders and non-responders prior to WT1 vaccination.

Hypoxia-Sensitive Epigenetic Regulation of an Antisense-Oriented lncRNA Controls WT1 Expression in Myeloid Leukemia Cells

WT1 is a transcription factor expressed in hematopoietic stem cells and in most cases of myeloid leukemia. We investigated the roles of hypoxia and epigenetics in the regulation of WT1 expression in myeloid leukemia cells. WT1 expression correlates with hypomethylation of the CpG island in Intron 1, and pharmacologic demethylation of this CpG island induces WT1 mRNA expression. Hypoxia causes decreases in DNMT expression and activity and increased expression and activity of TET2 and TET3, resulting in demethylation of this CpG island and expression of WT1 mRNA. Demethylation of the CpG island, either from pharmacologic treatment or induction of hypoxia, results in transcription of an antisense-oriented lncRNA, and inhibiting lncRNA expression with shRNA blocks WT1 mRNA expression. These results reveal a novel model of hypoxia-mediated epigenetic gene regulation. In addition, this is the first report that TET2 and TET3, increasingly recognized as important epigenetic regulators of gene expression in stem cells and in cancer cells, can be regulated by hypoxia, providing a solid mechanistic link between hypoxia and epigenetic regulation of gene expression with important implications for the role of hypoxia in stem cell function.

Wilms Tumor 1 rs16754 predicts favorable clinical outcomes for acute myeloid leukemia patients in South Chinese population

The single nucleotide polymorphism (SNP) rs16754 in WT1 shows a clinical implication in Caucasus population. However, the results were not reproducible in different population cohorts. We evaluated the clinical significance of rs16754 for 205 de novo acute myeloid leukemia (AML) patients in South Chinese population, 188 healthy volunteers were recruited as healthy controls. WT1 mRNA expression was investigated in 81 pretreatment bone marrow specimens. WT1GA/AA patients showed better overall survival (OS, P=0.006) and relapse-free survival (RFS, P=0.025) as compared with WT1GG patients, and the favorable clinical outcomes were most prominent in older patients with superior OS (P=0.001) and RFS (P=0.003). In multivariable analysis, rs16754 was still associated with favorable OS (HR=1.533, P=0.042). The WT1GG patients showed significantly higher WT1 mRNA expression than the WT1GA/AA patients (P=0.01). In summary, WT1 rs16754 may serve as an independent biomarker in AML patients from South Chinese.

Sodium butyrate-induced histone hyperacetylation up-regulating WT1 expression in porcine kidney fibroblasts.

Wilms' tumor 1 gene (WT1) is essential for the development of kidney and histone acetylation and is involved in its expression regulation in mice. However, whether WT1 expression is associated with histone acetylation in porcine kidney cells is unclear. Here, the effect of histone deacetylase inhibitor sodium butyrate (NaBu)-induced hyperacetylation on WT1 expression in porcine kidney fibroblasts (PKF) was examined. Treatments of NaBu (1, 3, 6 mM) for 24 h increased PKF viability, and 24, 48 h-treatments of 1 mM NaBu enhanced PKF proliferation. WT1 mRNA levels were significantly elevated in NaBu-treated (1, 3 mM for 24, 48 h, respectively) PKF samples. Consistently, strengthened expression of WT1 protein and histone acetylation level were detected in NaBu-treated PKF cells. Together, NaBu-induced hyperacetylation up-regulates WT1 expression in PKF, suggesting the involvement of histone acetylation in the transcriptional modulation of WT1 in porcine kidney cells.

Review of the Results of WT1 Peptide Vaccination Strategies for Myelodysplastic Syndromes and Acute Myeloid Leukemia from Nine Different Studies.

We performed a systematic review of data from nine clinical trials of WT1 peptide vaccination in patients with myelodysplastic syndromes and/or acute myeloid leukemia (MDS/AML), published between 2004 and 2012. A total of 51 patients were eligible for analysis. Vaccination with WT1 peptides proved safe and feasible in patients with MDS/AML, in studies from different institutions. Additionally, clinical responses and clinical benefit were observed, with some patients achieving and maintaining remission long-term (more than 8 years). A significant correlation between induction of WT1-specific T cells and normalization/reduction of WT1 mRNA levels and progression-free survival was noted in a number of studies. However, larger studies are warranted to confirm these results. Interestingly, the majority of trials reported the presence of WT1-specific T cells with limited or absent functionality prior to vaccination, which increased in frequency and function after vaccination. In conclusion, WT1 peptide vaccination strategies were safe in this heterogeneous group of patient with MDS/AML. Larger and more homogeneous studies or randomized clinical trials are needed to quantify the contribution of WT1 peptide vaccines to clinical responses and long-term survival.

Molecular and morphological differentiation of testes and ovaries in relation to the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina

Ambient temperatures during embryonic development determine gonadal sex in many reptiles. The temperature sensitive period for sex determination has been defined by shifting eggs between female- and male-producing temperatures in a few species. This phase spans 20–35% of embryogenesis in most species, which makes it difficult to define the mechanisms that transduce temperature into a signal for ovarian versus testicular development. We present an extensive set of studies that define a brief period when high temperature specifies, and then determines, ovarian fate in a northern population of snapping turtles, Chelydra serpentina. We shifted embryos from male to female temperatures, or vice versa, at various stages of development. Gonads in embryos incubated at female temperatures commit to ovarian fate earlier (by stage 18) than gonads in embryos incubated at male temperatures commit to testicular fate (by stages 19–21). In double shift studies, embryos were incubated at a female temperature, exposed to a male temperature for set times, and shifted back to the original temperature, or vice versa. The time required to induce ovarian development (≤6 days at female temperatures) was much shorter than the time required to induce testicular formation (>20 days at male temperatures). Differentiation of the gonads at the histological level occurred after the sex-determining period. Nevertheless, we found that a change in temperature rapidly (within 24h) influenced expression and splicing of WT1 mRNA: the absolute abundance of WT1 mRNA, the relative abundance of +KTS versus –KTS isoforms, as well as the ratio of +KTS:–KTS isoforms was higher in gonads at a male versus a female temperature. In conclusion, ovarian fate is more readily determined than testicular fate in snapping turtle embryos. The short sex-determining period in this species (6–8% of embryogenesis) will facilitate studies of molecular mechanisms for specification and determination of gonad fate by temperature.

Clinical Phase II Study of WT1 Peptide Vaccination in Pediatric Leukemia Patients after Stem Cell Transplantation and Analysis of Immunological Monitoring

IntroductionAllogeneic stem cell transplantation (allo-SCT) is a radical treatment for pediatric patients with relapsed and/or chemotherapy-resistant leukemia. Because these leukemia cells seem refractory to chemotherapy and irradiation, immunotherapy are needed. WT1 is highly expressed in pediatric leukemia and essential for leukemia cell growth, leading to the hypothesis that the WT1 protein-based immunization after allo-SCT may improve the clinical outcome of the SCT therapy. We investigated the clinical efficacy and immunological effect of immunotherapy targeting WT1 protein for pediatric patients after allo-SCT. MethodMajor inclusion criteria were as follows: Patients with HLA-A*2402 and/or HLA-*A0201 aged 20 years or younger and WT1 mRNA expression in leukemic cells; donors with HLA-A*2402 and/or HLA-*A0201. The HLA A*2402 restricted 9mer modified WT1 peptide or HLA A*0201 restricted natural WT1 peptide emulsified in Montanide ISA 51 adjuvant was injected intradermally. The dose of WT1 peptide depended on patient weight. The vaccinations were scheduled to be given weekly for 12 consecutive weeks, and if no recurrence and severe adverse effect were observed, vaccination was continued. Absolute number and frequency of WT1 specific cytotoxic T lymphocytes were analyzed by WT1 tetramer assay.ResultsA total of 18 patients with 9 AML/MDS, 8 ALL and 1 NHL were enrolled. Seven patients were those with high risk (HR) of relapse who had received SCT at 3rd CR or on disease. The other 11 patients were at standard risk (SR) but had either relapsed after SCT or shown induction failure. 16 had HLA-A*2402 and the other 2 had HLA-A*0201. 12 of the 18 patients received WT1 peptide vaccination monthly after the first 12-time consecutive vaccinations with one-week interval and are still in complete remission for 16-72 months (median 31 mo.). One patient discontinued the vaccination because of pancreatitis due to treatment with steroid for GVHD. One year probabilities of PFS is 66.7% in all, and it is notable that 4 of 7 HR patients are maintained in long-term CR. Although, of these 12 patients with persistence of CR, seven patients had high WT1 mRNA level before vaccination in peripheral blood (PB) or bone marrow (BM), WT1 mRNA levels decreased to within normal level after vaccination. WT1-specific CTLs in PB were detected and increased after vaccination in all cases and absolute numbers of these cells were significantly increased after vaccination in all cases (p< 0.05). The fold increase of WT1-specific CTL numbers after the vaccination was significantly higher in cases which maintained CR (p< 0.05). The WT1-specific CTLs with high tetramer staining were detected during the first 12-time vaccinations in 14 patients, and of these patients, 12 patients are still in CR. In the other 4 patients, only very few WT1 specific CTLs were detected, and three of the four patients showed recurrence. Five patients (1AMKL, 4 ALL) had recurrence. There was no significant difference in the fold increase of WT1-specific CTL numbers after the vaccination between cases with immunosuppressive agents for the prevention of GVHD and those without the agents.DiscussionWe reported the interim result of the phase II clinical study of WT1 peptide immunotherapy after SCT for pediatric patients, in which promising clinical efficacy of the therapy was suggested. In addition, we found that an increase in WT1-specific CTLs was associated with a decrease in WT1 mRNA, suggesting occurrence of WT1-driven graft versus leukemia (GVL) effect, which is consistent with strategies to enhance leukemia antigen-specific GVL response without deterioration of GVHD. The following three mechanisms are suggested. 1) After SCT, many kinds of cytokines are produced and proliferation of T lymphocytes occur; 2) lymphodepletion after SCT allows development of T cells specific to WT1 peptide; and resultantly, 3) efficient homeostatic expansion of WT1-specific CTLs is induced. Post-SCT vaccination with WT1 peptide can be a safe and effective strategy to prevent the recurrence of the disease without deterioration of GVHD in pediatric hematological malignancy. For avoiding late side effect due to preconditioning regimen and GVHD, effective post SCT immunotherapy is a promising strategy for pediatric patient. Larger studies are warranted on application of WT1 targeted immunotherapy to prevent recurrence after pediatric SCT DisclosuresNo relevant conflicts of interest to declare.

Vaccination with WT1 mRNA-Electroporated Dendritic Cells: Report of Clinical Outcome in 66 Cancer Patients

Tumor recurrence and lack of tumor control are major problems in cancer treatment. In order to control malignant disease, we performed phase I/II dendritic cell (DC) vaccination studies in an adjuvant setting in 30 patients with acute myeloid leukemia (AML) and in 36 patients with solid tumors. Following chemotherapy, the patients underwent leukapheresis; CD14+ monocytes were isolated, cultured into clinical-grade mature DC, electroporated with mRNA encoding the Wilms’ tumor protein WT1 and injected intradermally (Van Tendeloo et al. PNAS 2010;107:13824-9). No major DC-related systemic toxicity was observed.DC vaccination as a post-remission treatment was evaluated in 30 AML patients following chemotherapy; 27 patients were in complete remission (CR) but at very high risk of relapse and 3 in partial remission (PR). WT1 mRNA levels in blood and marrow were followed as a measure of residual disease. Clinical and molecular response, as determined by normalization of WT1 transcript levels in blood and/or marrow, occurred in 8/23 patients who had increased levels of that marker at the start of DC vaccination. Of these 8 responding patients, 5 are still in complete and molecular remission, all of them now more than 5 years after diagnosis and most probably cured; 1 of those 5 patients was in PR following chemotherapy and was brought into complete and molecular remission by the DC vaccination only. There was a possible effect of DC vaccination in 6 additional patients: 3 with stable disease, some of it late; and 3 at high risk of relapse but without increased WT1 mRNA levels before DC vaccination: 1 patient with erythroleukemia and 2 patients with initial leucocytosis >20,000/µL have remained in CR, now at respectively 57, 52 and 45 months post-diagnosis. Overall 8/30 patients have not relapsed yet, with a median follow-up from diagnosis and start of DC vaccination of respectively 70 months (range 45 - 92 months) and 63 months (range 39-90 months). Delayed type hypersensitivity (DTH) testing showed immunoreactivity to the DC vaccine in all patients tested. WT1 epitope tetramer+ CD8+ T-cells were evaluated in 13 HLA-A2+ patients: an increase following DC vaccination in tetramer+ T-cells for at least 2/4 epitopes tested was only observed in patients with long-standing CR.Ten patients with unresectable, epithelial-type malignant pleural mesothelioma and non-progressive disease after platinum/pemetrexed-based chemotherapy received DC vaccination. Evaluation of response according to RECIST criteria showed 7 patients with stable disease and 3 with progressive disease. DTH testing showed vaccine-elicited immunity in 9/10 patients. Median overall survival (OS) from start of chemotherapy was 32 months; this compares with an OS of 22 months reported in the literature for a similar subgroup of patients treated with chemotherapy only (Hillerdal et al. J Thorac Oncol 2008).Twenty-six patients with other advanced and pre-treated cancers also underwent DC vaccination (13 with breast cancer (12 with metastatic disease), 5 with glioblastoma multiforme (GBM), 1 with brain stem astrocytoma and 1 each with metastatic melanoma, Ewing sarcoma, esophageal, colon, pancreatic, renal cell and ovarian cancer). Significant DTH responses were recorded in all patients. At a median follow-up from start of DC vaccination of 23.3 months, 8/26 patients (31%) are still alive and median OS was 23.5 months. Evaluation of response showed 3 patients with PR (1 brain stem astrocytoma, 1 GBM and 1 breast cancer) and 1 patient with CR (1 GBM). In the breast cancer patient subgroup, 5/13 patients are still alive (38%) and median OS was 33.5 months after start of DC vaccination; this compares with OS data from the literature of 21.7 months after diagnosis (Kiely et al. J Clin Oncol 2011;29:456-63). In the GBM (n=5) patient subgroup, 1/5 patients is alive in CR 26 months and median OS was 14.7 months after start of DC vaccination; this compares with OS data from the literature of 14.6 months after diagnosis (Stupp et al. N Engl J Med 2005;352:987-96).In conclusion, WT1-targeted DC vaccination is feasible, safe and immunogenic in cancer patients. In AML, metastatic breast cancer and malignant glioma, there is evidence of objective response. In addition, OS data in solid tumors compare favorably with the best data reported so far for similar cohorts of patients, suggesting that adjuvant WT1/DC-based immunotherapy provides a clinical benefit to these patients. DisclosuresOff Label Use: Dendritic cells as immunotherapy.

WT1 Protects Leukemic Cells from Cytotoxic Replicative Stress

BACKGROUND:The transcription factor WT1 is a “pan-leukemic marker”. WT1 is overexpressed in a significant number of AML, CML, ALL, and MDS cells. Moreover, the persistence of WT1 expression after chemotherapy indicates residual leukemic stem cells. Hydroxyurea can decrease the white blood cell count in leukemic patients. Hydroxyurea inhibits ribonucleotide reductase and consequently depletes the cellular dNTP pool. This process induces replicative stress and activates the intra-S phase checkpoint. The prolonged arrest of DNA replication leads to double strand DNA breaks and apoptosis. The aim of our study was to investigate whether WT1 has an impact on the survival of AML and CML cells treated with hydroxyurea. Furthermore, we analyzed whether replicative stress affects WT1 expression. We also addressed whether clinically relevant drugs modulating WT1 influence apoptosis, autophagy, and DNA damage signaling in leukemic cells.METHODS:We used human AML and CML cells (MV4-11, RS4-11, NB4, HEL, K562) and primary AML cells with various genetic lesions (FLT3-ITD, MLL-AF4, PML-RARα, JAK2V617F, BCR-ABL; p53 mutant or null) or a complex karyotype. Cells were exposed to 0,5 mM hydroxyurea and analyzed by FACS, Western Blot and mRNA expression analyses. RNAi with morpholinos targeting the WT1 mRNA as well as the tyrosine kinase inhibitor imatinib (0,1-1 µM) and epigenetic modifiers of the histone deacetylase inhibitor family (HDACi: 1,5-5 µM entinostat and 10-100 nM panobinostat) were used to decrease WT1 levels.RESULTS:1.The stability of WT1 is causally linked to the survival of leukemic cells undergoing replicative stress. Hydroxyurea-treated leukemic cells retaining WT1 (K562 and RS4-11 cells) accumulate in S phase and survive. However, cells that lose WT1 (NB4, MV4-11, HEL, and primary AML cells) succumb to apoptosis (57-70% apoptosis after 24 h; p<0,001).2.The specific elimination of WT1 in K562 cells with morpholino antisense nucleotides (10 nM) significantly sensitizes the cells to hydroxyurea-induced apoptosis (26-30% increased apoptosis rates after 24 h; p<0,005).3.The hydroxyurea-induced loss of WT1 in sensitive cells is caspase-dependent but does not require the p53 tumor suppressor. The pan-caspase inhibitor ZVAD-FMK (20-50 µM) and the ectopic overexpression of mitochondrial anti-apoptotic proteins (BCL-XL/BCL2) prevents the hydroxyurea-induced depletion of WT1 and apoptosis in NB4 cells (from 62-69% to 9-14%; p<0,001).4.Moreover, our data reveal that hydroxyurea promotes the occurrence of previously unrecognized WT1 cleavage products. These are different from the known HTRA2-induced WT1 fragments, as they contain the WT1 zinc fingers required for DNA binding. These novel cleavage products also contain the domain for transcriptional activation, but lack the domain for transcriptional repression. We demonstrate that caspases cleave the full-length WT1 protein into such fragments, which accumulate in the nucleus of hydroxyurea-treated NB4 cells.5.Resistance of BCR-ABL-positive K562 CML cells to pro-apoptotic effects of hydroxyurea can be overcome with the tyrosine kinase inhibitor imatinib. This drug inhibits the WT1 promoter and sensitizes the cells to hydroxyurea (22-48% increased apoptosis rate after 24 h; p<0,005).6.The HDACi entinostat and panobinostat suppress the WT1 gene and accelerate the proteasomal degradation of WT1 by an increased expression of the E2 ubiquitin conjugase UBCH8. The depletion of WT1 by HDACi renders K562 cells sensitive towards hydroxyurea-induced killing and causes a synergistic activation of cell death (apoptosis increased by 43-55% after 24 h; p<0,001).7.In K562 cells hydroxyurea/entinostat combinations lead to a caspase-dependent cleavage of the DNA repair enzyme PARP-1 and to the accumulation of double-strand DNA breaks.8.We further show that entinostat significantly shifts the balance from a hydroxyurea-induced autophagy program to apoptosis (from 60% to 16% autophagy; p<0,001) in K562 cells. We reveal that entinostat depletes proteins critical for autophagy and autophagosome-lysosome fusion (Beclin-1 and HDAC6) in these cells.CONCLUSIONS:Our data illustrate that WT1 is a novel biological target of hydroxyurea, independent of a specific genetic lesion and irrespective of a functional p53 pathway. The inhibition of WT1 might be therapeutically exploitable for targeting approaches against leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Wilms' tumor gene (WT1) expression correlates with vascular epithelial growth factor (VEGF) in newly acute leukemia patients undergoing chemotherapy.

Today, leukemia is one of the biggest problems worldwide. The Wilms' tumor gene (WT1) and the vascular endothelial growth factor (VEGF) gene are highly expressed in patients with various cancers. This study concerned the relationship between expression of WT1 and VEGF in patients with acute leukemia. We evaluated expression of WT1 mRNA and VEGF mRNA using real-time quantitative RT-PCR in the peripheral blood (PB) of 8 newly diagnosed AML and 4 newly diagnosed ALL patients, serially monitored for 2 months. A further 12 normal PB samples served as controls. In the patient group, in comparison with the normal ranges, WT1 and VEGF gene expression was increased, the average values for the expression of these two genes being 0.2852±0.11 and 0.2029±0.018, respectively. While was no significant relevance between the two genes pre-treatment, a positive link between the two genes in 75% of patients with AML was noted during the procedure of chemotherapy, whereas in 75% of patients with ALL an antiparallel association was observed. Leukemia is associated with production of WT1, which may affect the expression of VEGF.

Real-time assessment of relapse risk based on the WT1 marker in acute leukemia and myelodysplastic syndrome patients after hematopoietic cell transplantation

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (alloHCT) for acute leukemia and myelodysplastic syndrome (MDS). Wilms' tumor antigen (WT1) is overexpressed in the majority of acute leukemia and MDS patients and has been proposed as a universal diagnostic marker for detection of impending relapse. Comprehensive studies have shown that WT1 transcript levels have predictive value in acute leukemia patients in complete remission after chemotherapy. However, the focus of this study is the period after alloHCT for predicting relapse onset. We analyzed the accumulation of WT1 mRNA transcripts in peripheral blood of 82 leukemia and MDS patients and defined specific molecular ratios for relapse prediction. The extensively validated WT1/c-ABL ratio was used to normalize increases in WT1 transcript levels. The observed lead time of crossing or exceeding set WT1 levels is presented along with linear interpolation to estimate the calculated day the WT1 thresholds were crossed. The WT1/c-ABL transcript ratio of 50 or above yielded 100% specificity and 75% sensitivity reliably predicting future relapse with an observed average of 29.4 days (SD=19.8) and a calculated average of 63 days (SD=29.3) lead time before morphologic confirmation. A lower ratio of 20 or above gave lower specificity but higher sensitivity (84.8 and 87.5%, respectively) identified more patients that relapsed, at earlier times, providing an earlier warning with actual average lead time of 49.1 days (SD=30.8) and calculated average of 78 days (SD=28.8). WT1 transcript levels serve as a diagnostic relapse test with greater sensitivity than the morphologic approach used in the clinic as a readout.

Abnormal WT1 gene expression in paroxysmal nocturnal hemoglobinuria

To explore the pathogenesis of abnormal WT1 expression in paroxysmal nocturnal hemoglobinuria (PNH). The expression of WT1 mRNA in CD59⁻ and CD59⁺ bone marrow mononuclear cells (BMMNC) were measured by semi-quantitative reverse transcription PCR. After WT1 gene silence by RNA interference (RNAi) technology, biological characteristics of BMMNC were investigated by flow cytometry. The relative expression of WT1 mRNA in PNH CD59⁻ BMMNC (1.06 ± 0.12) was significantly higher than that in PNH CD59⁺ BMMNC (0.90 ± 0.12) and normal BMMNC (0.86 ± 0.05, P<0.05), but there was no significant difference between PNH CD59⁺ BMMNC and normal BMMNC (P>0.05). WT1 mRNA expression in PNH was positively correlated with the proportion of CD59⁻ cells (r²=0.490, P=0.016), but had no relationship with the proportion of CD59⁺ cells. After WT1 gene silence by siRNA in PNH CD59⁻ BMMNC, WT1 mRNA expression was decreased. The proportions of G0/G1 phase in PNH CD59⁻ cell blank control group and siRNA-scr transfected group were (92.73 ± 3.71)% and (93.06 ± 4.14)%, and the proportions of S phase were (6.99 ± 3.61)% and (6.73 ± 4.08)%, respectively. The proportions of G0/G1 and S phase in siRNA-WT1 transfected group was (94.46 ± 3.71)% and (5.40 ± 3.55)%, respectively. There were significant differences in the proportions of G0/G1 phase and S phase among the controls, siRNA-WT1 transfected group and siRNA-scr transfected group (P<0.05). The rate of apoptosis in siRNA-WT1 transfected group [(35.91 ± 22.36)%] was significantly higher than those in controls [(26.12 ± 17.10)%] and siRNA-scr transfected group [(27.39 ± 18.99)%] (P<0.05). siRNA-WT1 could effectively suppress the WT1 gene expression of CD59⁻ clone in PNH patients, inhibit its proliferation, and promote its apoptosis. WT1 gene expression might contribute to PNH clone proliferation.

Nestin and WT1 expression in atheromathous plaque neovessels: association with vulnerability.

Neoangiogenesis is crucial for the progression and vulnerability of atheromasic lesions. Since adult vasa vasorum, which represent the neoangiogenetic burden of healthy arteries, constitutively express Nestin and Wilms Tumor (WT1), the aims of the present study are: i) to describe and quantify Nestin and WT1 in plaque neovessels; ii) to investigate the relationship between neovessel phenotype and plaque instability. We prospectively evaluated 49 consecutive carotid endarterectomy specimens. Histopathological characteristics were separately collected, particularly the intraplaque histological complications. Immunohistochemistry was carried out for CD34, Nestin and WT1; the density of positivity was evaluated for each marker. RT-PCR was performed to assess Nestin and WT1 mRNA levels on the first 10 plaques and on 10 control arteries. Six (12.2%) plaques showed no neoangiogenesis. In the others, the mean immunohistochemical densities of CD34, Nestin, and WT1-positive structures were 41.88, 28.84 and 17.68/mm2. Among the CD34+ neovessels, 68% and 42% expressed Nestin and WT1 respectively, i.e., nearly 36% of the neovessels resulted to be Nestin+/WT1-. Furthermore, complicated plaques (n=30) showed significantly more CD34 and Nestin-positive vessels than uncomplicated plaques (n=13; P=0.045 and P=0.009), while WT1 was not increased (P=0.139). RT-PCR confirmed that WT1 gene expression was 3-fold lower than Nestin gene in plaques (p=0.001). Plaque neoangiogenesis shows both a Nestin+/WT1- and a Nestin+/WT1+ phenotype. The Nestin+/WT1- neovessels are significantly more abundant in complicated (vulnerable) plaques. The identification of new transcription factors in plaque neoangiogenesis, and their possible regulation, can open new perspectives in the therapy of vulnerable plaques.

Expression of WT1 and PRAME gene in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome

This study was aimed to explore the transcription level of WT1 and PRAME two genes in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome(MDS) and their relationship with bone marrow dysplasia and karyotype. The quantitative expression of WT1 and PRAME transcripts detected by RQ-PCR in the bone marrow samples of 203 MDS patients and 19 aplastic anemia(AA), 6 other benign anemia(BA), 4 paroxysmal nocturnal hemoglobinuria(PNH) patients from July 2009 to June 2012 and 14 healthy donors, and in 92 peripheral blood samples. The results showed that WT1 and PRAME expression levels in both BM and PB samples of MDS group were higher than those in normal controls, AA, and BA patients (BM: WT1:P = 0.000, 0.000, 0.000, PRAME: P = 0.048, 0.000, 0.064; PB: WT1:P = 0.012, 0.000, 0.011, PRAME: P = 0.020, 0.004, 0.003). What is more, this expression in high risk MDS group (RAEB1, RAEB2, MDS-AML) were higher than those in low risk group (RCUD, RCMD, MDS-U) and AA and BA. The WT1 and PRAME mRNA expression levels in PB and BM were well correlated (WT1:r = 0.6028, P = 0.001; PRAME: r = 0.7628, P = 0.000), as well as the WT1 expression levels in BM samples with the Karyotype (P = 0.049). In addition, the same positive rate of WT1 or PRAME expression existed in BM and PB samples of MDS patients. It is concluded that the WT1 and PRAME gene expression levels in both BM and PB samples of MDS patients are higher than those in healthy controls, AA and other benign anemia patients, and increase with the progression of the disease. The WT1 and PRAME transcripts constitute good molecular markers for the clinical diagnosis and prognosis and monitoring minimal residual disease after treatment of MDS. What is more, when bone marrow is not so convenient to get, the transcript levels of PB samples can be detected.

Simultaneous induction of Tri-antigen specific cytotoxic T lymphocytes(CTLs) using DCs transferred with WT1, survivin and TERT mRNA for adoptive T cell therapy

T cell to tumor cell ratios, increasing anti-tumor activity and improving patient survival. We therefore incorporated the OX40 and CD28 co-stimulatory endodomains into our GD2-CAR construct in the hope they would enhance in vivo expansion and persistence of CAR T-cells in patients with relapsed/refractory neuroblastoma. Methods: This is a Phase I, dose-escalating, safety trial administering 3 generation GD2-CAR T cells to patients with relapsed/refractory neuroblastoma. Results: We have manufactured 4 autologous 3 generation GD2-CAR T cell products (patient ages: 4 e 23 years). Starting with a median of 20x10 PBMCs, we obtained a median of 450x10 CAR T cells (range 240x10 to 480x10, 18-fold expansion) after 10 3 days of culture. The transduction efficiency was 79 5% by FACS. Phenotypically, the T cell lines were 27 11% CD4 and 67 10% CD8, with 8 2% central memory (CD45ROCD62LCCR7), and 12 10% naive (CD45RACCR7) cells in the final products. Cr release assays showed the T cell lines produced 70 7% and 55 8% lysis of GD2 targets (LAN-1 and CHLA255, respectively) at a 20:1 effector:target ratio. One patient has been treated, so far without significant toxicity. Clinical and immunological results are pending. Conclusion: Generation of autolgous 3 generation CAR T cells for the treatment of high-risk neuroblastoma appears feasible and the safety and antitumor activity of the approach are now being assessed.

Successful repeated treatment of acute myeloid leukemia in early relapse with gemtuzumab ozogamicin alone

A 68-year-old female was diagnosed with acute myeloid leukemia (AML-M2 without 8/21 translocation) in December 2006. Although a complete remission (CR) was obtained after induction chemotherapy, the first post-remission therapy was discontinued because of severe cardiovascular complications. She had a relapse of AML with CD33-positive myeloblasts which comprised 38.4% of the bone marrow cells in November 2007. She received two courses of low-dose chemotherapy because of the previous complications. The amount of Wilm's tumor 1 (WT1) mRNA in the peripheral blood was 13,000copies/μg RNA after the first course of the chemotherapy, and 4.8% myeloblasts remained in the bone marrow after the second course. She was treated with a single course of gemtuzumab ozogamicin (GO), with a subsequent CR with 0.9% marrow myeloblasts and fewer than 50 copies of WT-1 mRNA (normal level). Thereafter, she received five courses of GO monotherapy at each occasion of early AML relapse. Hematological remission has been sustained over a period of about 24months with the GO monotherapy alone. This case suggests that GO monotherapy is a useful salvage therapy for early relapse of CD33-positive AML in situations in which standard chemotherapy is not indicated.

WT-1 Expression Level In BM Is The Great Prognostic Marker In Three Of Classification IPSS, WPSS, and Latest Revised IPSS(IPSS-R)

Objective/material/methodIt is known new classification named revised IPSS(IPSS-R) as a prognosis in MDS patients in 2012. To investigate the clinical utility of WT1 mRNA expression level including IPSS-R, we studied of MDS patients who provided consent at 17 medical institutions nationwide from Dec. 2008 to Sep. 2009 were enrolled in Japan. A total of 172 subjects including 115 MDS patients by IPSS (RA:69, RARS:9, RAEB:24, RAEB-t :13 ). 13 patients who developed AML from MDS, and 44 patients with non malignant hematological disorders who provided consent. This study was designed to follow up 82 patients, who gave secondary consent, among 115 MDS patients registered in study ODK-0801 for 5 years up to 2014 to analyze in detail the relationship between the WT1 mRNA expression level and prognosis. WT1 mRNA level in PB and BM were measured using the WT1 mRNA assay kit “OTSUKA” (OTSUKA PHARMACEUTICAL CO., LTD). WT1 mRNA expression levels in peripheral blood (and in bone marrow, if possible) were measured periodically to evaluate the usefulness of the WT1 mRNA expression level as a monitoring marker of MDS and analyze changes in pathology in individual patients by central review and changes in WT1 mRNA expression levels. In this study, value of 50 copies/μgRNA was set as the cut off value for WT-1mRNA expression. The differences in clinical and demographic data were assessed in the chi-square test and logistic regression analysis. The survival data was analyzed using Kaplan-Meier method and compared by the log-rank test. This study presents the results of interim analysis 3 years after the start of the investigation. ResultsThe comparison study between the patients categorized into three groups by the WT1 mRNA level in BM ,GroupI:less than 102 copies/μgRNA(n=35), Group II: 102 to 104 copies/μgRNA(n=30), and GroupIII: more than 104 copies/μgRNA(n=17) resulted that the survival rates decreased significantly as the WT1 mRNA level increased(GI vs GII P<0.01, GI vs GIII P<0.01, GII vs GIII p<0.067), respectively. In multivariate Cox proportional hazard regression analysis of IPSS, five out of fifteen parameters, which are WBC count (P=0.0001), IPSS score (P=0.0003), blast in PB(P=0.0011), WT1 mRNA level in BM(P=0.0055), sex(P=0.093) were independently associated with survival time, respectively. And we studied same analysis used WPSS. WBC count (P=0.0001), WPSS socre(P=0.0001), blast in PB(P=0.0037), WT1 mRNA level in BM(P=0.0029), sex(P=0.012) were independently associated with survival time, respectively. Also we analyzed using IPSS-R. In multivariate Cox proportional hazard regression analysis, five out of fifteen parameters, WBC count (P=0.0001), IPSS-R score(P=0.0001), blast in PB(P=0.0012), WT1 mRNA level in BM(P=0.01), sex(P=0.01) were independently associated with survival time, but not ANC(both score and absolute number), Hb, platelet, karyotype or other parameters. Using these selected five valiables, we provided the level of WT-1mRNA with 4 risk groups which classified :100, 50100, 100,100, 150,100copies/μRNA). According to increasing of WT-1mRNA level, survival duration shortened the increase with the increase in each risk group. DiscussionThe WT-1 mRNA level in BM was positively correlated with the prognosis of MDS stage and tended to be correlated with advanced risk of IPSS, WPSS, and IPSS-R. Therefore it was considered to be a useful prognostic marker for MDS. ConclusionWe found that the survival time was shortened significantly with an increase in WT1 mRNA expression levels in both peripheral blood and bone marrow, demonstrating that WT1 mRNA expression levels are a highly useful prognostic factor in MDS even if we use any of classification of IPSS, WPSS, IPSS-R. This assay has great possibility to contribute to more appropriate therapeutic decisions in MDS patients as well as diagnosis and to evaluate the timing of allogeneic transplantation. Disclosures:Kurokawa: Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding. Usuki:Alexion Pharmaceuticals, Inc.: Speakers Bureau. Ohyashiki:Novartis: Honoraria, Research Funding.

Pre-Treatment WT1 mRNA Expression Level In Peripheral Blood Predicts Response and Overall Survival Of Myelodysplastic Syndrome Patients In The Azacitidine Era

BackgroundThe Wilms' tumor gene (WT1), originally discovered as a tumor suppressor has been proven to have an oncogenic role in leukemia and several other cancers. WT1 mRNA expression levels in peripheral blood (PBWT1) has been reported as a useful marker for the risk evaluation of myelodysplastic syndrome (MDS). In the era of hypomethylating agents, the significance of PBWT1 on MDS prognosis is still unknown. This study aimed to clarify the impact of pre-treatment PBWT1 levels on overall response (OR) and overall survival (OS) in MDS patients treated with azacitidine (AZA). Patients and MethodsWe retrospectively analyzed all patients from March 2011 to March 2013 with World Health Organization 2008 defined MDS, CMML or AML with 20–30% bone marrow blasts who received AZA treatment in our department for at least one cycle (37.5–75.0 mg/m2/day during 7 days, every 28 days). Patients' peripheral blood specimens were collected before AZA initiation, mRNA was extracted from leukocytes using the RNeasy Mini-Kit (Qiagen, Valencia, CA), and the amount containing WT1 mRNA was measured using a WT1 mRNA Assay Kit (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). Hematologic response was evaluated according to International Working Group 2006. OR was defined as a best overall response of complete remission (CR), partial remission, marrow CR, or hematologic improvement. Univariate analyses for OR were carried out using Fisher's exact test. Factors associated with at least borderline significance (p < 0.10) were subjected to a multivariate analysis, using logistic regression model. OS was estimated according to the Kaplan-Meier method. Multivariate analysis was performed with proportional hazard Cox model, including all variables with p < 0.10 in univariate analyses. ResultsOf 55 patients enrolled, pre-treatment PBWT1 levels were available in 41 patients and the median level was 790 copies/µg RNA (range, less than 50–310000). Baseline characteristics according to PBWT1 levels (≤ 790 [lower group] [n = 21] and > 790 [higher group] [n = 20]) are summarized in Table 1. Median number of AZA treatment cycles was 4 (range, 1–18). Four patients (2 in higher group, and 2 in lower group) received allogeneic stem cell transplantation (alloSCT) after AZA treatment. OR rates were significantly lower in PBWT1 higher group than lower group (30.0 vs 71.4%, p = 0.03). In univariate analysis, other significant risk factors or with borderline significance for OR were higher serum ferritin levels (> 1000 ng/ml) and RBC transfusion dependency ≥ 4 units/8 weeks. In multivariate analysis, higher PBWT1 levels independently predicted reduced likelihood of OR (odds ratio = 0.212, 95% CI 0.01-0.95, p = 0.02). OS was significantly inferior in PBWT1 higher group as shown in Figure 1. In univariate analysis, other significant factor was Revised International Prognostic Scoring System (IPSS-R) risk groups (high risk defined as IPSS-R high or higher, and low risk defined as IPSS-R intermediate or lower). In multivariate analysis, higher PBWT1 levels (hazard ratio [HR] = 9.75, 95% CI 1.22-77.58, p = 0.03) and IPSS-R high risk (HR=7.04, 95% CI 1.43-34.48, p = 0.02) were independent predictors for OS.Table 1Patient baseline characteristics according to pre-treatment PBWT1 levelsPBWT1(copies/µg RNA)≤ 790 (n=21)>790 (n=20)p valueMedian age, years71.070.5WHO 2008 classification, %0.202RARS4.80.0RCMD19.010.0RAEB-123.840.0RAEB-238.140.0CMML-114.30.0AML0.010.0IPSS-R risk group, %0.003Very low0.00.0Low28.60.0Intermediate33.330.0High33.325.0Very high4.845.0Absolute neutrophil count, mean ± SD, 103/µl2.0 ± 2.61.4 ± 1.20.392Platelets, mean ± SD, 104/µl15.1 ± 25.610.8 ± 18.10.545Hb level, mean ± SD, g/dl9.5 ± 2.37.7 ± 1.50.008Ferritin level, mean ± SD, ng/ml531.1 ± 902.1741.0 ± 1008.10.503Marrow blast, %0.111< 5 %52.325.0≥ 5 %47.675.0RBC transfusion dependency, %0.326Yes23.840.0No76.260.0 [Display omitted] ConclusionOur results suggest that PBWT1 can predict both response and survival of MDS patients treated with AZA. Although salvage therapy including alloSCT can affect the survival, poor survival might result from inferior response rates in PBWT1 high patients. In MDS with high PBWT1, restoration of epigenetically silenced tumor suppressor genes with AZA might not induce apoptosis. We propose that alternative therapeutic strategies should be sought in MDS patients with high PBWT1 levels. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow WT1 Levels In Long-Term Survivors Of Core-Binding Factor AML and Acute Promyelocytic Leukemia

BackgroundQuantitation of WT1 mRNA levels may be used in minimal residual disease studies in AML cases lacking a suitable molecular target. The normal thresholds of bone marrow WT1 levels at different time–points have not been clearly stated. Difficulties arise as a consequence of the relatively high WT1 expression in normal bone marrow and the unknown influence of chemotherapy. ObjectiveTo establish the reference range value of bone marrow WT1 levels in treated CBF-AML and APL in complete remission. Patients and MethodsPatients with bone marrow samples obtained from ≥2 years (2-11 y) after initial diagnosis of CBF-AML (AML1-ETO n:21 patients CBFb-MYH11 n:21 patients) or APL(n:22 patients) were included in the study (120 samples). Ages at diagnosis ranged from 1 to 76 years. Mean age was: AML1-ETO 36.8(2-76y),CBFb-MYH11 32.4(1-55y), PML-RARa 47.3(21-76y). Peripheral blood, reticulocyte count and a bone marrow aspirate were also available in 86 cases. WT1 was analyzed following the primers and conditions of the ELN group. Specific chimeric real time PCR was simultaneously performed in each sample in accordance with the BIOMED protocol. Each WT1 or chimeric PCR was analyzed in triplicate whereas the control gene (abl) was analyzed in duplicate. ResultsAll patients with an available BM study were in morphologic complete remission. Two samples corresponded to an anemic patient (2/86). Six cases had a reticulocyte count above 2%. Platelet counts were below 120x109/l in 3 cases (3/86). Leukocyte counts below 3.5x109/l were observed in 6 samples (6/86). The calculated mean WT1 copy number from the 120 bone marrow samples was 47.23 (AML1-ETO:61.33, CBFb-MYH11:43.51, PML-RARa:36.4 p:ns). The mean chimeric copy number was 0.75 (AML1-ETO:0.82, CBFb-MYH11:0.72, PML-RARa:0.69 p:ns). Only 5 samples had a specific copy number of chimeric transcripts above 5 (3 AML1-ETO,1 CBFb-MYH11 and one PMAL-RARa patient who had a molecular relapse one month later and was succesfully treated). In 6 samples, the specific chimeric copy number was between 1 and 5 copies ( 4 AML1-ETO and 2 CBFb-MYH11). In the remaining 109 BM samples, the specific copy number was <1. In 9 BM samples, the WT1 levels were in the range of 100-300 copies (7 AML1-ETO and 2 CBFb-MYH11). All these 9 samples had specific chimeric gene number below 1.5 copies. In three samples (1 AML1-ETO, 1 CBFb-MYH11 and 1 PML-RARa) peaks of WT1 levels of ≥300 copies were detected. In these three samples the specific chimeric gene was undetectable (<0.01). ConclusionsBone marrow WT1 copy number in treated CBF-AML and APL was 47.23. In ten percent of cases, the WT1 copy number was greater than 100 copies. Occasionally, WT1 peaks (≥300 ) were detected. The meaning of the differential bone marrow WT1 transcriptional activity in long-term survivors of AML remains to be investigated. Disclosures:No relevant conflicts of interest to declare.

Prevention Of Relapse In Acute Myeloid Leukemia By Dendritic Cell Vaccination: Report on a Phase II Study With 29 Patients

Relapse remains a major problem in acute myeloid leukemia (AML) and has an important impact on survival, especially in the majority of older patients who cannot undergo allogeneic hematopoietic stem cell transplantation. In a phase II study, we investigated the impact on relapse of dendritic cell (DC) vaccination as a post-remission treatment in 29 AML patients; 26 patients were in complete remission and 3 in partial remission following chemotherapy. DC were derived from blood monocytes, electroporated with mRNA encoding the Wilms' tumor 1 protein (WT1) and administered via the intradermal route. Three different WT1 constructs were used to generate mRNA by in vitro transcription: 'construct 1' encoding full-length WT1, 'construct 2' with a Sig-DC-LAMP major histocompatibility complex (MHC) class II-skewing signal and a deletion of the WT1 nuclear localization signal and 'construct 3', a codon-optimized version of construct 2. WT1 mRNA levels in blood and marrow were followed as a measure of minimal residual disease.DC electroporated with mRNA derived from constructs 1, 2 and 3 were used to vaccinate respectively 13, 6 and 10 AML patients at very high risk of relapse. In those 3 groups, clinical and molecular response, as determined by normalization of WT1 transcript levels in blood and/or marrow, occurred in respectively 7/13, 1/4 and 0/6 patients who had increased levels of that marker at the start of DC vaccination. Of these 8 responding patients, 3 relapsed and died; the other 5 are still in complete remission, 4 of them now more than 5 years after diagnosis and most probably cured; 1 of those 4 patients was in partial remission following chemotherapy and was brought into complete and molecular remission by the DC vaccination only. All 15 patients who did not normalize WT1 mRNA levels following DC vaccination relapsed and/or died. There was a possible effect of DC vaccination in 6 additional patients: 3 with stable disease, some of it late; and 3 at high risk of relapse but without increased WT1 mRNA levels before DC vaccination: 1 patient with erythroleukemia and 2 patients with initial leucocytosis > 20,000/µL have remained in complete remission, now at respectively 46, 42 and 35 months post-diagnosis. Overall 8/29 patients have not relapsed yet, with a median follow-up from diagnosis and start of DC vaccination of respectively 60 months (range 35 - 84 months) and 53 months (range 27-78 months).Delayed type hypersensitivity (DTH) testing showed immunoreactivity to the DC vaccine components in all patients tested. There was also evidence of natural killer (NK) cell activation following DC vaccination. WT1 epitope tetramer+ CD8+ T-cells were evaluated in 13 HLA-A2+ patients: an increase following DC vaccination in tetramer+ T-cells for at least 2/4 epitopes tested was only observed in patients with long-standing complete remission.In conclusion, DC vaccination had a demonstrable antileukemic effect in 8/29 AML patients and a possible effect in another 6. Contrary to expectations, the WT1 constructs with MHC class II-skewing signal did not seem to have superior activity over the full-length WT1 construct without that signal. Vaccination with WT1 mRNA-transfected DC is emerging as a non-toxic strategy to prevent or delay relapse in AML. Disclosures:Off Label Use: Dendritic cell vaccination.