WT1 Transcript Research Articles

Wilms' tumor gene 1 expression analysis by real-time quantitative polymerase chain reaction for monitoring of minimal residual disease in acute leukemia

Wilms' tumor gene 1 (WT1) gene expression was analyzed in 32 patient with acute myeloid leukemia (AML) and 18 with acute lymphoblastic leukemia (ALL) to investigate whether it could serve as a MRD marker. Ninety-four percent of patients with acute leukemia showed high WT1 expression at presentation. WT1 expression as a MRD marker was evaluated in 36 patients. The rise of WT1 expression preceded the hematological relapse by ∼4 months (mean time 129 days; range 6–298). The prognostic significance of WT1 expression was analyzed in 30 patients with AML. WT1 expression higher than 20 WT1 copies /104ABL copies after induction and consolidation chemotherapy was associated with shorter OS. WT1 expression analysis could be a useful tool for MRD monitoring in acute leukemia.

Predictive Value of Minimal Residual Disease (MRD) Monitoring by RQ-PCR in WT1 Positive Patients Entered in the UK MRC AML-15 Trial

Relapse remains the main cause of treatment failure in Acute Myeloid Leukaemia (AML). Some 65% of AML patients lack a specific fusion transcript which can serve as a molecular target for detecting residual disease. However, in over 75% of AML cases, the Wilm's Tumor (WT1) gene is over-expressed and can therefore be used for MRD monitoring by RQ-PCR. The clinical value of serial MRD monitoring in WT1 positive patients was prospectively assessed in the AML-15 Trial which opened in 2002. The trial compared 3 induction regimens (DA v/s ADE v/s FLAG Ida), followed by randomisation in consolidation (courses 3 and 4) to either MACE + MidAC or 2 doses of Ara-C (3g/m2 or 1.5g/m2) and to stop or have a 5th course (Ara-C 1.5g/m2). Patients were also randomised to receive Gemtuzumab Ozogamicin (3mg/m2) at induction and/or consolidation. Adults in the standard and poor risk groups were scheduled to receive an allograft if a matched donor was available. An “in house” WT1 assay used primers/probes covering exons 7 and 8 in the 3′ region of the WT1 gene. WT1 transcripts from BM and PB were measured by real-time quantitative PCR (RQ-PCR) on the 7900 HT ABI machine, at diagnosis, after each course of chemotherapy, stem cell transplant, and during remission for up to 3 years. WT1 copies were normalised to ABL gene and expressed per 105ABL copies. WT1 was over-expressed in >80% of AML cases screened, when compared to normal BM copies (median 544, range 31–1173), PB (median 10, r 0–58), PBSC (median 71, r 0–483). In this study, 700 patients (excluding Core Binding Factor positive AML) with diagnostic BM WT1 levels >104 copies have been recruited and data on 416 are presented. Follow up is complete to 1/1/08 with 244 relapses after a median follow-up of 40 months. At complete remission (CR) following induction, a greater log reduction in WT1 transcripts was associated with a significantly reduced relapse risk even after adjustment for age, WBC, cytogenetics, performance status and de Novo/Secondary AML (hazard ratio (HR) 0.57 (0.46–0.71) per log reduction p<0.0001). The 5yr relapse rates (RR) for patients with a log reduction of <2, 2–3, and 3 or more were 77%, 50% and 26% respectively (p <0.00001). Furthermore, presence or absence of a 2 log reduction adds to the MRC risk index (developed to identify patients who might benefit from transplant or be candidates for experimental therapies, Burnett et al. ASH 2006, Abstract 18) for survival from CR (HR 0.59 (0.37–0.91), p=0.02). With respect to post induction WT1 transcripts, there were naturally three groups for both BM and PB levels for relapse risk: <1000, 1000–4999, 5000+ copies for BM (5 yr RR 45%, 74%, 82%, p<0.0001 ) and <1000, 1000–2999 and 3000+ copies for PB (5yr RR 53%, 82%, 91%, p<0.0001). After consolidation/SCT, BM and PB transcript levels were also highly predictive of relapse: BM <5000 copies RR 37%, BM 5000+ copies RR 84% (p<0.0001); PB <1000 copies RR 32%, PB 1000+ copies RR 90% (p<0.0001). Looking at all data together, in a multivariate analysis adjusted for MRC risk group, the most important prognostic variables for relapse, relapse free survival and overall survival were post induction log reduction and post chemotherapy PB transcript level >500 copies. In all cases PB transcript levels were more prognostic than levels from BM. We conclude that MRD monitoring in WT1 positive AML patients allows risk stratification based on treatment response, and in this group of patients, MRD monitoring adds to already known risk factors. Whether patients identified as being at high risk of relapse benefit from further treatment, including allogeneic stem cell transplantation, remains to be determined in future studies.

Leukemia Relapse after Allogeneic Transplants in AML: Predictive Role of WT1 Expression and Protective Effect of DLI.

Background: relapse remains a significant problem in patients with acute leukemia undergoing an allogeneic hemopoietic transplant (HSCT) and a predictive molecular marker of disease would be useful.Aim of the study. Assess the role of WT1 expression in AML patients undergoing an allogeneic HSCT, topredict relapse anddrive immune interventionMethods. We studied 81 patients with AML who received an allogeneic HSCT between 30/1/04 and 11/2/08 in our Unit: 52 patients were in first remission, and 29 patients had more advanced disease. Median age 45 (16–66), prepared with CY-TBI (n=52) or RIC regimen (n=29). WT1 mRNA levels of expression were normalized with respect to the number of ABL transcripts and expressed as WT1 copy numbers every 104 copies of ABL. Patients bone marrow cells were monitored for WT1 expression, and cytology, pretransplant, at day +30 and every month untill day +180, then at scheduled follow up. Patients were considered WT1 positive, when WT1 copy numbers were greater than 180. Escalating doses of donor lymphocyte infusions (DLI) were given, when possible, in WT1+ patients.WT1 pre-transplant. WT1 pre-HSCT was available in 63 patients, 39 were WT1- and 24 WT1+: their relapse risk was 10% vs 62% (p=0.00002) and their crude survival 79% vs 29% (p=0.0001). When CR1 patients were selected, relapse was 10% vs 50% for WT1- vs WT1+ patients (p=0.02), and survival 81% vs 50% (p=0.9).WT1 post-transplant. Fifty patients were always WT1- after HSCT, and 31 were found WT1+ at a median interval from HSCT of 80 days (range 24–696): relapse was 8% vs 68% for WT1- vs WT1+ patients (p=<0.0001), and survival 78% vs 26% (p=0.0001).Cox analysis: in multivariate analysis on relapse WT1 pre-transplant (p<0.0001) and post-transplant (p<0.0001) were independent predictors, whereas disease phase (CR 1/>1) was not (p=0.39). Survival was predicted by all 3 variables, WT1+ pre (p=0.001), WT1+ post (p=0.03) and phase of disease (p=0.002).Immune intervention. Of the 31 WT1+ patients, 13 had BM blasts on cytology (6– 80%) at the time of first WT1 positivity, and were treated with chemotherapy. 18 patients were WT1+ and had marrow in CR on cytology: 9 of these received DLI and 9 did not; hematologic relapses were seen in 2/9 and 7/9 respectively (p=0.02) and crude survival is 6/9 vs 2/9 (p=0.07). The median interval between WT1+ and relapse was 96 days (39– 246).Conclusions. This study suggests that WT1 positivity before and after HSCT are independent predictors of relapse and survival in patients with AML. WT1 expression predicts relapse better than disease phase. The interval between WT1 positivity and relapse is approximately 3 months: the use of DLI in this time interval appears to protect against hematologic relapse.

Two Novel WT-1 Derived Peptides Induce CD4+ peptide–specific T Cell Proliferation in Patients with Myelodysplastic Syndrome (MDS)

The Wilms tumor gene WT1 is overexpressed in hematopoietic malignancies such as Myelodysplastic syndromes and leukemias and the WT1 protein was demonstrated to be an attractive target antigen for an immunotherapeutic approach to these diseases. Most of the efforts have been focused to the search for immunogenic peptides suitable for inducing cytotoxic T lymphocytes (CTLs) and to less extent for CD4+ T lymphocytes with potential cytotoxic activity. On this matter, in our previous experience with a p210-derived peptide vaccine developed for chronic myeloid leukemia patients with minimal residual disease, the main immune and therapeutic effect observed after vaccinations appeared to be mediated by peptide-specific CD4+ T cells induced by the longest peptide (25 mer) included in our vaccine. CML-peptide specific T cells were found to be either CD4+/perforin+ or CD4+/CD25+/Foxp3+ and we recently showed their direct cytotoxicity against a CML cell line. Thus to pursue a vaccine strategy mainly devoted to a similar CD4+ T cell immune response, we screened WT1 protein through Syfpeithi database to identify original peptides with a suitable length (23–25 amino acids) to be processed by several HLA class II molecules and to induce a strong CD4+ T cell stimulation. Additionally, in order to maximize the immunogenic potential of the novel peptides, we focused our attention on areas of the protein with known CTLs/CD4 T cells immunogenic epitopes. We identified two peptides that fulfilled these requirements: SEPQQMGSDVRDLNALLPAVPSLGG (WT1-iso5 64–88) which includes 5 amino acid from the alternative splicing derived isoform 5 of WT1 and the first 20 aa of “canonical” WT1 sequence and RPFMCAYPGCNKRYFKLSHLQMHSR (WT1321–345). Both 25mer peptides showed strong HLA binding properties for HLA-DRB1*0101, HLADRB1* 0401, HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1501 and HLADRB1* 0301( DR17). We first tested them in vitro for their capability to induce peptide-specific CD4+ T cells. Briefly, CD4+ T cells freshly isolated from PBMC were cultured for 21 days in 5% AB human serum media while undergoing to 3 rounds of stimulation with autologous CD14+ cells and both WT1-iso5 64–88 and WT1 321–345 peptides at 20μg/ml in the presence of IL-15. This in vitro stimulation was performed in 3 normal subjects and in 3 MDS patients with high levels of bone marrow WT1 transcript (2 patients presenting a low-International Prognostic Scoring System (IPSS) refractory anemia (RA) and 1 with intermediate IPSS RA). In all 3 healthy donors tested, both peptides were able to induce peptide specific CD4+ T cell proliferation as measured by standard 3HThymidine assay, with a stimulation index (SI) ranging from 2.0 to 2.5 regardless of their HLA-DR phenotype ( SI= cpm CD4+ T cells plus test peptides/CD4+ T cell alone or CD4+ T cells plus control peptides; peptide-specific T cell proliferation was considered positive for SI≥2). Similar results were obtained in all 3 MDS patients in which WT1-iso5 64–88 and WT1 321–345 induced peptide-specific CD4+ T cell proliferation with a SI value of 2.5, 2.9 and 3.0 respectively. In conclusion the present study identified 2 novel WT1-derived 25 mer peptides which were able to easily induce in vitro a peptide-specific CD4+ T cell response in MDS patients. WT1-specific CD4+ T cells proliferated with similar SI values in normal donors and in WT1 positive MDS patients, the latter being highly exposed to this antigen and thus potentially tolerant to it. A possible cytotoxic activity of these WT1-specific CD4+ T cells is under evaluation and in vivo vaccinations of low-intermediate IPSS MDS patients with these peptides are planned.

BAALC and WT1 Transcript Amount Discriminates Secondary or Reactive Eosinophilia from Idiopathic Hypereosinophilic Syndrome or Chronic Eosinophilic Leukemia

Idiopathic hypereosinophilic syndromes (HES) or chronic eosinophilic leukemia (CEL) comprise a spectrum of indolent to aggressive diseases characterized by persistent hypereosinophilia. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to eosinophilia, it can present in many myeloproliferative disorders or alternatively it may be a reactive form, secondary to many clinical conditions. The fusion gene FIP1L1-PDGFR alpha was identified in a subset of patients presenting with HES/CEL. In spite of this, the majority of HES/CEL patients do not present detectable molecular lesions and for many of them the diagnosis is based on exclusion criteria and sometimes it remains doubt. CD34-positive progenitor cells from bone marrow (BM) express BAALC and WT1. Overexpression of BAALC and WT1 were seen in patients with AML and ALL. In a subset of AML it marked poor prognosis, suggesting a role for BAALC or WT1 overexpression in acute leukemia. To explored the possibility to distinguish between HES/CEL and reactive hypereosinophilia based on the measurement of BAALC and WT1 transcript amount. Twenty-two patients with hypereosinophilia were characterized at the molecular level and analyzed for BAALC and WT1 expression. The transcription of FIP1L1-PDGFRalpha fusion gene was detected by nested RT-PCR. The relative transcript amount of BAALC and WT1 were determined by real time PCR analyses. The FIP1L1-PDGFRalpha fusion gene expressed has been identified in bone marrow mononuclear cells of 4 cases. The relative expression level of BAALC and WT1 in these 4 cases with positive FIP1L1-PDGFRalpha fusion gene expression were 2.27(0.27–6.8) and 0.39(0.002–0.90), respectively. Whereas the relative amount of transcripts of BAALC and WT1 in 18 patients with negative FIP1L1-PDGFRalpha fusion gene were 0.069(0.015–0.11) and 0.054(0–0.34) respectively. The relative amount of transcripts of BAALC and WT1 in patients with HES/CEL were 32 times and 7 times than that in those with negative FIP1L1-PDGFRalpha fusion gene, respectively. These results clearly demonstrates that BAALC and WT1 quantitative assessment allows to discriminate between HES/CEL and reactive eosinophilia and represents a useful tool for disease monitoring especially in the patients lacking a marker of clonality.

Expression patterns of WT1 and PRAME in acute myeloid leukemia patients and their usefulness for monitoring minimal residual disease

Both WT1 and PRAME are highly expressed in acute myeloid leukemia (AML) patients. To assess the efficacy of their simultaneous detection for the purpose of monitoring minimal residual disease (MRD), we used real-time quantitative RT-PCR to quantify both WT1 and PRAME transcript levels in the bone marrow of 204 newly diagnosed AML patients, and 21 patients were serially monitored for a median of 11 months. The 22 normal bone marrow samples which served as controls collectively expressed low levels of WT1 and PRAME. An increase of >1-log over the upper limit of normal bone marrow was defined as positive. The positive rates of WT1 and PRAME for all patients were 79.2% and 55.4%, respectively. For the 108 patients lacking a specific fusion gene, the positive rate of WT1 was significantly higher than that of PRAME (76.9% vs. 35.2%, P < 0.001). PRAME was positive in 9/25 WT1 (−) patients and the log increase of PRAME was higher than that of WT1 in 12/83 WT1 (+) patients. Dynamic patterns of WT1 and PRAME during follow-up showed that a consistent elevation or a rise over time to exceed the normal range predicted clinical relapse. The exception was that one patient’s WT1 significantly decreased at relapse compared to diagnosis. Therefore, a simultaneous detection of WT1 and PRAME might not only provide AML patients with either a positive or a more sensitive molecular marker for MRD monitoring. Moreover, it might also avoid false negativity in the case of expression alteration.

Pilot trial of a Wilms tumor-1 (WT1) peptide vaccine in patients with thoracic and myeloid neoplasms

3051 Background: The transcription factor, WT1, is overexpressed in several solid tumors and leukemias and has been targeted by investigators for immunotherapy. Using computer prediction analysis we designed analog peptides derived from WT-1 sequences by substituting amino acids at key HLA-A0201 binding positions. One peptide (WT-A1) was capable of stimulating CD8 T-cells for killing of HLA matched tumor cells. A modified version of the protein (WT1–122A1) created by adding flanking amino acids to the core sequence, generated CD4 responses as well. Two additional peptides were generated (WT1–427 & WT1–331) which were capable of recruiting CD4 T-cell response across multiple HLA-DR subtypes. These four peptides administered together with adjuvant Montanide and GM-CSF comprise the vaccine. Methods: The primary objective of the study is to test the safety, activity and immunogenicity of WT1 vaccine in 10 patients with mesothelioma (meso) or non-small cell lung cancer (NSCLC) expressing WT1 protein by immunohistochemistry, and 10 patients with acute myeloid leukemia (AML) or myelodysplastic syndrome with WT1 transcript measured by RQ-PCR. Patients receive up to 12 vaccinations over 9 months. Immune responses are evaluated by delayed-type hypersensitivity, CD4 T-cell proliferation, CD4 and CD8 T-cell interferon release, and WT1 peptide tetramer staining. Results: 12 patients (5 AML, 5 meso, 2 NSCLC) have been enrolled and 2 are currently active. 4 meso patients discontinued the vaccines after 3 or 6 doses because of progression. 1 meso patient has stable disease after 12 vaccinations. 3 AML patients relapsed on the vaccine (after 3rd, 6th, 8th dose) and had evidence of increasing WT-1 transcript. 3 of 10 patients tested developed a DTH skin reaction. 6 of 7 patients evaluated had a CD8 or CD4 immune response to the vaccinating peptides. One AML patient had grade 2 urticaria after the 5th vaccination and was removed from study. No other significant adverse events occurred. Conclusions: This analog WT1 peptide vaccine is well-tolerated and immune responses can be elicited. Future studies will incorporate this vaccine as adjuvant therapy in meso and AML. Supported by NIH PO1 23766, Innovive Pharmaceuticals, MARF, The Experimental Therapeutics Center and the Baker Street Foundation. No significant financial relationships to disclose.

Early prediction of treatment outcome in acute myeloid leukemia by measurement of WT1 transcript levels in peripheral blood samples collected after chemotherapy

The Wilms' tumor gene WT1 is a reliable marker for minimal residual disease assessment in acute leukemia patients. The study was designed to demonstrate the potential use of WT1 to establish quality of remission in acute leukemia patients for early identification of patients at high risk of relapse. A prospective study based on a quantitative Real-Time PCR (TaqMan) assay in 562 peripheral blood samples collected from 82 acute leukemia patients at diagnosis and during follow-up was established. The evaluation of WT1 in peripheral blood samples after induction chemotherapy can distinguish the continuous complete remission patients from those who obtain only an "apparent" complete remission and who could relapse within a few months. WT1 helps identify patients at high risk of relapse soon after induction chemotherapy allowing post-induction therapy in high risk patients to be intensified.

Leukemia-associated antigen-specific T-cell responses following combined PR1 and WT1 peptide vaccination in patients with myeloid malignancies

We describe the safety and immunogenicity of a combined vaccine of 2 leukemia-associated antigenic peptides, PR1 and WT1. Eight patients with myeloid malignancies received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor. Patients were reviewed weekly for 4 weeks to monitor toxicity and immunologic responses. Toxicity was limited to grades 1 to 2. Using peptide/HLA-A 0201 tetramers and intracellular interferon-gamma staining, CD8(+) T cells against PR1 or WT1 were detected in 8 of 8 patients after a single vaccination. To monitor the kinetics of vaccine-induced CD8(+) T-cell responses and disease regression after vaccination, absolute PR1 and WT1(+)CD8(+) T-cell numbers and WT1 expression were studied weekly after vaccination. Responses occurred as early as 1 week after vaccination. After vaccination, the emergence of PR1 or WT1(+)CD8(+) T cells was associated with a decrease in WT1 mRNA expression as a marker of minimal residual disease, suggesting a vaccine-driven antileukemia effect. Conversely, loss of response was associated with reappearance of WT1 transcripts (P < .01). This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. These results support further studies of combination immunization strategies in leukemia patients.

WT1 protein expression in childhood acute leukemia

In patients with acute leukemia, Wilms' tumor gene 1 (WT1) has been used as a target for the detection of minimal residual disease (MRD) by PCR techniques. The expression of WT1 protein, however, has not been extensively studied. To determine the relation between expression of WT1 transcripts and of the encoded protein, we examined leukemic cell lines and primary childhood leukemia samples using both real-time quantitative PCR (RQ-PCR) and flow cytometry. WT1 protein was highly expressed in the leukemic cell lines K562, HL-60, PLB 985, KG-1a and CEM. By contrast, 40 primary samples of acute lymphoblastic leukemia (ALL; B-ALL, n = 15 and T-ALL, n = 10) and acute myeloid leukemia (n = 15) expressed low levels of WT1 protein. RQ-PCR detected WT1 transcript levels in the same range as reported in earlier studies in childhood acute leukemia. The results of this study indicate the following: (i) there are considerable discrepancies between WT1 transcripts and protein expression; (ii) WT1 is not a suitable marker for flow cytometric MRD detection in childhood acute leukemia.

Pilot Study of a Wilms Tumor Protein (WT1) Heteroclitic Peptide Vaccine in Patients with Myeloid and Thoracic Neoplasms.

WT1 is a transcription factor over-expressed in several types of leukemia and solid tumors which has the potential to serve as a target for immune based therapies. Previous studies have demonstrated the ability of WT1 peptides to induce specific HLA restricted T cell cytotoxic responses. In an effort to overcome immune tolerance associated with this self-antigen, we used computer prediction analysis to design heteroclitic peptides derived from WT1 protein sequences by substituting various amino acids at key HLA A0201 binding positions. A selected peptide (WT1-A1) was found to stabilize MHC class I molecules better than native sequences and was also able to cross react with native WT1 sequences to kill HLA matched tumor cells. A modified version of the heteroclitic peptide was also created by adding flanking amino acids (WT1-122A1) to the core sequence in an effort to extend reactivity of this peptide to also include a broad range of HLA class II molecules. These 2 peptides were then combined with 2 native WT1 sequences (WT1-427 & WT1-331) capable of recruiting CD4 T cell response, to form a polyvalent vaccine with the potential to react across a number of HLA-DR subtypes. A clinical pilot study was designed to test the safety, activity and immunogenicity of this vaccine in patients with Acute Myelogenous Leukemia (AML) in complete remission, but with molecular evidence of WT1 transcript as measured by quantitative polymerase chain reaction (RQ-PCR) or with advanced mesothelioma or non-small cell lung cancer (expressing WT-1 protein by immunohistochemistry). Each patient receives 6 vaccinations with the 4 WT1 peptides (200mcg each) plus the immune adjuvant Montanide over 12 weeks. In addition, GM-CSF injections are administered to local sites of inoculation 2 days before and on the day of vaccination. Study design called for 10 patients in each group (solid tumor; hematologic malignancy). Immune responses are evaluated by delayed-type hypersensitivity, CD4 T-cell proliferation, CD4 and CD8 T-cell interferon-gamma release, and WT1 peptide tetramer staining. In the AML patients, minimal residual disease was assessed by sequential RQ-PCR at baseline and after the 3rd and 6th vaccines. To date, 8 patients (4 AML, 3 mesothelioma, 1 non-small lung cancer) have been enrolled on study. Two of the mesothelioma patients discontinued the vaccines after 3 and 6 doses because of progression of disease. One other mesothelioma patient has stable disease following 10 vaccinations. Two of the 4 AML patients have relapsed while receiving the vaccine (one after 3rd dose, the other after the 6th). Each had evidence of increasing WT-1 transcript at the time of relapse. One other remains in CR and has a transcript level 50% of baseline. No significant adverse events have been observed to date in the patients undergoing vaccination. Three of 4 evaluable patients showed evidence of either a CD8 or CD4 immune response to the vaccinating peptides and native peptides in vitro. In summary, preliminary data suggest that this heteroclitic WT1 peptide vaccine can be administered safely to patients and that immune responses can be elicited.

CD16+CD56− NK Cells in the Peripheral Blood of Cord Blood Transplant Recipients: A Unique Subset of Immature NK Cells Possibly Associated with Graft-Versus-Leukemia Effect.

NK cells play a major role in the activity of graft-versus-host (GVL) effect after an HLA-mismatched stem cell transplantation. In unrelated cord blood transplantation (CBT) where there is often an HLA mismatch between the donor and recipient, NK cells may also play a vital role, though their roles have not been extensively studied. Cord blood (CB) is known to have a unique subset of NK cells characterized by a CD16+CD56− phenotype. CD16+CD56− NK cells in CB are thought to be progenitors of CD16+CD56+ NK cells because CD16+CD56− NK cells acquires CD56 expression after in vitro culture in the presence of IL-2. However, the function of this immature NK cell subset after CBT remains unknown. A marked increase in the number of CD16+CD56- NK cells in the peripheral blood of an HLA-mismatched CBT recipient with acute myeloid leukemia (AML) was recently observed. A 56-year old male, who received a reduced intensity CBT following a full relapse after allogeneic stem cell transplantation from an HLA-matched sibling donor, showed an increase in the copy number of WT-1 mRNA in the peripheral blood around day 80 after the CBT, but the WT-1 copy number decreased from 1500/microliter RNA to 230/microliter RNA in association with the increase in the number of CD16+CD56- NK cells, and his molecular remission lasted more than 1.5 years thereafter. This case prompted an investigation of CD16+CD56− NK cells in the peripheral blood after allogeneic stem cell transplantation. A similar increase in the proportion of CD16+CD56− NK cells (20% or more) in the peripheral blood CD16+ NK cells was observed in 64% (7/11) of CBT recipients, all of whom maintained remission, but in none of the 11 bone marrow and 8 peripheral blood stem cell transplant recipients examined (Figure 1). CD16+CD56− NK cells in CBT recipients expressed receptors specific to NK cells such as NKp30 and NKp46 same level as CD16+CD56− NK cells of fresh CB cells. CD16+CD56− NK cells isolated from CBT recipients became CD56+ when they were cultured in the presence of IL-2 with or without K562-mb15-4-1BBL. When cultured NK cells derived from the CD16+CD56− NK cells were separated into CD158b+ and CD158b− cells, CD158b+ cells failed to kill 721–221 cells transfected with HLA-C*0301 while they killed untransfected or HLA-C*0401-transfected 221 cells. Despite the presence of the corresponding KIR ligand (C*0304), cultured CD16+CD56− NK cells showed cytotoxicity against the patient's leukemic cells. These findings suggest that an increase in the proportion of CD16+CD56− NK cells is unique to recipients of CBT and that this immature NK-cell subset in CBT recipients may undergo differentiation into mature NK cells in vivo capable of killing residual leukemic cells, thereby contributing to the GVL effect regardless of the presence of the KIR ligand. [Display omitted]

Real-Time Quantitative PCR (RQ-PCR) Detection of Minimal Residual Disease (MRD) by Optimized WT1 Assay To Enhance Risk Stratification in Acute Myeloid Leukemia (AML): A European LeukemiaNet Study.

Relapse remains the major cause of treatment failure in AML; however, RQ-PCR assays to detect leukemic fusion transcripts have been shown to identify reliably those patients at highest risk of relapse, allowing development of a more individualized treatment approach. In cases lacking a leukemia-specific MRD marker, quantification of genes over-expressed in AML e.g. Wilms' Tumor gene (WT1) could provide more a precise measurement of disease response and quality of remission, potentially enhancing risk scores such as the one developed by the MRC used to identify those patients most and least likely to benefit from allogeneic transplant in first CR. WT1 is over-expressed in at least 75% of AML cases. Within the European LeukemiaNet we systematically evaluated 9 published and “in house” WT1 assays. Assays were excluded due to demonstrated lack of RNA-specificity or location within the 3′ region of the gene which has been shown to be subject to deletion or mutation in AML. An assay located within the 5′ region associated with superior sensitivity was ultimately selected following parallel testing in 11 labs and evaluated in 238 diagnostic peripheral blood (PB) and 386 bone marrow (BM) samples. WT1 was over-expressed in the majority, with comparable levels in PB and BM (PB - median 4637 WT1 copies/104ABL copies, range 0–1132709; BM - median 7212, 0–750571), as compared to normal BM (median 19.8, 0–213), PB (median 0.01, 0.01–47.6) and PBSCs (median 6.1, 0–39). In cases over-expressing WT1, kinetics of transcript reduction were evaluated following induction. A greater response was associated with a significantly reduced risk of relapse (hazard ratio 0.65 per log reduction (95% CI 0.43–0.96), p=0.03), although this failed to remain significant when adjusted for age, presenting WBC and cytogenetic risk group, which are key variables in the MRC risk index. Indeed, there was a highly significant correlation between larger log reduction in normalized WT1 transcript level and better risk score (p=0.0001). Sequential analysis of PB and BM samples from 15 AML cases with low WT1 expression (<250 copies) showed no significant modulation in transcript level on regeneration after chemotherapy, indicating that in WT1+ AML, transcript levels detected in follow-up samples reliably reflect disease status. This study provides evidence that recognized pre-treatment risk factors for relapse correlate closely with kinetics of response to induction therapy and lend support to the evaluation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification and identify those patients most suited to proceed rapidly to allogeneic transplant.

Graft-versus-leukemia effects associated with detectable Wilms tumor-1–specific T lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia

AbstractTo determine whether the leukemia-associated Wilms tumor antigen (WT1) contributes to a graft-versus-leukemia (GVL) effect after allogeneic stem-cell transplantation (SCT) for acute lymphoblastic leukemia (ALL), we studied CD8+ T-cell responses to WT1 in 10 human lymphocyte antigen (HLA)–A*0201–positive ALL patients during the early phase of immune recovery after SCT (days 30-120). Seven of 10 patients had detectable WT1 expression in their peripheral blood (PB) before SCT by quantitative reverse-transcription polymerase chain reaction. Using WT1/HLA-A*0201 tetramers and intracellular interferon-γ (IFN-γ) staining, WT1+ CD8+ T-cell responses after SCT were found only in patients with detectable WT1 expression before SCT (5 of 7 vs. 0 of 3; P < .05). To monitor the kinetics of WT1+ CD8+ T-cell responses and disease regression after SCT, absolute WT1+ CD8+ T-cell numbers and WT1 expression were studied for each time point. The emergence of WT1+ CD8+ T cells was associated with a decrease in WT1 expression, suggesting a WT1-driven GVL effect. Loss of WT1+ CD8+ T-cell responses was associated with reappearance of WT1 transcripts, consistent with a molecular relapse (P < .001). WT1+ CD8+ T cells had a predominantly effector–memory phenotype (CD45RO+ CD27−CD57+) and produced IFN-γ. Our results support the immunogenicity of WT1 after SCT for ALL and highlight the potential for WT1 vaccines to boost GVL after SCT for ALL.

6021 POSTER Clinical features and treatment outcome of patients with myeloid antigen coexpression in acute lymphoblastic leukemia: a study of 214 Peruvian patients

Acute myeloid leukemia with mutated nucleophosmin (NPM1m+AML) is a heterogeneous entity. We investigated whether NPM1m+AML with monocytic or myeloid differentiation have distinct immunophenotype. The study included 160 NPM1m+AMLpatients and 178 AML patients without NPM1 mutation and recurrent cytogenetic abnormality (NPM1wt−AML). We analyzed the immunophenotype by flow cytometry. NPM1 mutation was detected by PCR. Compared with NPM1wt−AML patients, NPM1m+AML patients showed higher positive rates of CD33 and CD9 and lower positive rates of CD34, HLA-DR, CD7, CD15 and CD117 (all P < 0.05). HLA-DR, CD64, CD14, CD11b, CD15, CD4, CD9 and CD10 were higher (P < 0.001) and CD117 was lower (P < 0.01) in monocytic NPM1m+AML compared with myeloid NPM1m+AML. Similar rates of lymphoid antigen (CD19, CD2, and CD7) and myeloid antigen (CD13, CD33) positivity were detected in monocytic and myeloid NPM1m+AML. Compared with NPM1wt−AML, CD34 expression was lower both in myeloid and monocytic NPM1m+AML subgroups, although HLA-DR was lower in NPM1m+AML compared with NPM1wt−AML only in myeloid subgroup. Comparisons of NPM1m+AML and NPM1wt−AML showed no differences in monocyte-associated markers such as CD14 and CD11b in myeloid and monocytic subgroup. Myeloid NPM1m+AML correlated with the female gender (P = 0.001), lower WBC counts (P = 0.04) and higher WT1 transcripts (P = 0.006) compared with monocytic NPM1m+AML.These results suggested monocytic and myeloid-derived NPM1m+AML exhibit distinct immunophenotypes.

Prognostic significance of WT1 gene expression in pediatric acute myeloid leukemia

The Wilms Tumor gene (WT1) encodes a transcription factor involved in kidney development and malignancy. WT1 expression in a subpopulation of early CD34+ cells has suggested its involvement in hematopoiesis. WT1 is aberrantly expressed in leukemias. High expression of WT1 at diagnosis has been associated with unfavorable prognosis in adult acute myeloid leukemia (AML). The prognostic relevance of WT1 expression in pediatric AML was evaluated in only one study, including 47 patients, which showed that very low levels of WT1 at presentation were associated with an excellent outcome. To test the validity of these findings we measured levels of WT1 in 41 newly diagnosed pediatric AML of the non-M3 FAB subtype. Patients were treated according to an AML-BFM 83-based protocol in a single institution. Mononucleated cells obtained from presentation BM aspirates were cryopreserved and later thawed and used for total RNA extraction and cDNA synthesis. The quantitative assessment of WT1 transcripts was made by real-time PCR (RQ-PCR). WT1 transcripts values were normalized with respect to the number of ABL transcripts. WT1 levels were significantly higher in patients bearing favorable chromosome abnormalities, t(8;21) and inv(16) (P = 0.002). Higher levels of WT1 expression were unexpectedly associated with a higher probability of overall survival by Cox regression analysis (P = 0.002). Multivariate regression analysis could not discriminate between the effects of WT1 and cytogenetics on survival. Higher WT1 expression was associated with favorable cytogenetics subtypes and accordingly with better outcome in children with AML in this study.

WT1 transcript amount discriminates secondary or reactive eosinophilia from idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia

Idiopathic hypereosinophilic syndromes (HES) comprise a spectrum of indolent to aggressive diseases characterized by persistent hypereosinophilia. Hypereosinophilia can result from the presence of a defect in the hematopoietic stem cell giving rise to eosinophilia, it can be present in many myeloproliferative disorders or alternatively it may be a reactive form, secondary to many clinical conditions. The hybrid gene FIP1L1-PDGRFalpha was identified in a subset of patients presenting with HES or chronic eosinophilic leukemia (CEL). In spite of this, the majority of HES patients do not present detectable molecular lesions and for many of them the diagnosis is based on exclusion criteria and sometimes it remains doubt. In this study we explored the possibility to distinguish between HES/CEL and reactive hypereosinophilia based on WT1 transcript amount. For this purpose, 312 patients with hypereosinophilia were characterized at the molecular and cytogenetic level and analyzed for WT1 expression at diagnosis and during follow-up. This study clearly demonstrates that WT1 quantitative assessment allows to discriminate between HES/CEL and reactive eosinophilia and represents a useful tool for disease monitoring especially in the patients lacking a marker of clonality.

Clinical and Immunological Activity of WT1 Peptide Vaccination in Patients with Acute Myeloid Leukemia and Myelodysplasia: Results of a Phase II Trial.

The transcription factor Wilms tumor protein (WT) 1 belongs to a new generation of tumor antigens, which are essential for tumor cell proliferation. WT1 is highly expressed in AML and in MDS upon appearance of blasts. A phase II trial of vaccination with the HLA-A2-restricted WT1.126–134 peptide was performed in patients with AML and MDS and overexpression of WT1 to determine immunogenicity and clinical activity. Patients received vaccinations with 0.2 mg WT1.126–134 peptide (day 3), 62.5 mcg dendritic cell-stimulating adjuvant GM-CSF (days 1–4) and 1 mg T helper protein keyhole limpet hemocyanin (day 3). The initial 13 patients were to receive 4 biweekly and subsequent 4-weekly vaccinations, the subsequent 13 patients were continuously vaccinated biweekly. Vaccination was continued in absence of overt disease progression. WT1 levels were assessed by quantitative RT-PCR and WT1-specific T cell responses by tetramer analyses and cytokine flow cytometry. Response assessment following IWG-MDS criteria was used, capturing stable disease and hematologic improvement. A duration of 8 weeks was required for stable disease. Enrolment was completed in June 2006 with 24 patients with AML and 2 with MDS (RAEB). Of the 24 AML patients, 16 had > 5% marrow blasts at study onset (8 without prior chemotherapy, 4 with disease persistence following chemotherapy, 4 with PR), and 8 were in CR at high risk for relapse. A median of 10 (range 4 – 23) vaccinations was administered with 8 patients currently still under treatment. No significant toxicity occurred. To date, 22 patients are evaluable for clinical response. Overall, 8/16 patients with > 5% marrow blasts at study onset displayed clinical efficacy of vaccine treatment (SD or better). One AML patient achieved CR for 12 months after brief initial progression, and 7 patients had disease stabilization (2, 2+, 3, 3, 6, 10+, 14 months). One of these patients with RAEBII had a major response of neutrophils and platelets, and one AML patient had initial progression and subsequent transient complete clearance of peripheral blasts. WT1 transcripts as molecular disease marker decreased at least 3-fold (range 3-fold - >50-fold, median >10-fold) in 12 of 20 currently evaluated patients, including all 8 patients with evidence of clinical efficacy and 4 of 5 AML patients vaccinated in CR. The generation of a WT1-specific T cell response in peripheral blood and bone marrow was detected in 12 of 16 evaluated patients including all 6 of these 16 patients with evidence for clinical efficacy. This study shows that WT1 vaccination has promising antileukemia activity. A multicenter comparative WT1 vaccination study in CR patients at high risk of relapse is currently initiated.

CD34+ Donor Chimerism and Wilms Tumor Gene 1 (WT1) Expression Provide an Early Indication of Relapse in Patients with Acute Leukemias and MDS after Hematopoietic Cell Transplantation (HCT) with Reduced-Intensity Conditioning.

HCT following reduced intensity conditioning (RIC) relies mainly on immunological effects for disease control. The early detection and quantification of minimal residual disease and the timely adjustment of immune suppression are therefore particularly important in this setting. Since appropriate disease-specific gene markers are available only in a minority of patients with acute leukemias or MDS, the potential of both donor chimerism and Wilms Tumor gene 1 (WT1) expression to provide quantification of MRD was investigated.Patients and Methods: Ninety-five consecutive patients with AML (n=68), ALL (n=7) and intermediate/high-risk MDS (n=20) were analyzed. Patients were 60 (median; range 21–74) years old and in CR1 (n=45), CR2 (n=25), or more advanced disease status (n=25). Grafts were obtained either from related (n=24) or unrelated (n=71) donors. Conditioning regimens consisted of fludarabine 30mg/kg BW day -4 to -2 (n=90) and total body irradiation with 2 Gy at day 0 (n=95), and post-transplant immunosuppression employed cyclosporin A and mycophenolate mofetil. Total donor chimerism (TDC, n=93 patients, 236 samples), CD34+− chimerism (n=89, 219 samples) and disease-specific molecular markers detected by FISH (DSM, n=39, 77 samples) were all determined prospectively from bone marrow (BM) samples at baseline and on days +28, +56 and +84 post-transplant. WT1 expression was analyzed retrospectively by RT-PCR from stored peripheral blood (PB) samples (n=95, 321) from the same time points.Results: With a median follow-up of 11.7 (range 2–61) months, 34 (36%) patients relapsed (defined by BM blasts >5%). Since complete results from all techniques were available up to day 84, we analyzed the diagnostic power of all methods to predict hematological relapse one month in advance up until the fourth month after HCT (n=21 patients, 22%). First, we estimated the value of the three different prospective MRD techniques (DSM, TDC and CD34+ chimerism) using Receiver Operating Curves (ROC). Relapse was predicted 1 month in advance by CD34+−chimerism [p= 0.001, area under the curve (AUC) = 0.875], but not by TDC or DSM, (n=30). The cut-off value of 5% decrease in CD34+ chimerism in a one month period achieved a sensitivity of 71% and specificity of 91%. In comparison, WT1 expression was similarly associated with a pending relapse (p< 0.0001, AUC = 0.861). The optimal cut-off value of 24 WT-1 copies per 10000 ABL copies was assessed by ROC and resulted in a sensitivity of 79% and specificity of 91%. In a logistic regression model, we estimated the ratios of the odds of relapsing within the next month. WT1 achieved a higher odds ratio (36/1) than CD34+ chimerism (25/1). Combining both techniques yielded a specificity of 98% and an odds ratio of 72/1Conclusion: WT1 expression in PB and CD34+ chimerism in BM are superior to full donor chimerism and disease-specific markers determined by FISH in predicting relapse in all patients with acute leukemia and MDS. Both markers are suitable to develop effective post-transplant treatment strategies to further decrease the relapse rate using immunological or cytotoxic approaches.

Phase II Study of Liposome Encapsulated Doxorubicin Citrate Complex, NPLD (Myocet™) in Myelofibrosis with Myeloid Metaplasia (MMM).

MMM is a chronic myeloproliferative disorder accompanied by an intense bone marrow stromal reaction that includes collagen fibrosis, osteosclerosis, and angiogenesis.The molecular pathogenesis of MMM has not been fully characterized despite the recent discovery of an activating JAK2 mutation (JAK2V617F) in approximately half of the patients. Conventional drug therapy in MMM not improves survival and is used for palliative purposes only.Myocet™ is a proprietary liposomal formulation of doxorubicin designed to enhance delivery of the drug to tumor cells and reduce side effects while maintaining equivalent anti-tumor efficacy.Moreover liposomes are avidly removed from circulation by the macrophages of the RES or MPS and are taken up in the spleen, liver and bone marrow were show both a cytotoxic and anti-angiogenic activityWe thus designed a phase II study for patients with primary MMM. The treatment program consisted of 25 mg total dose of Myocet™ given I.V. every two weeks for six months. Study eligibility criteria included a histologically confirmed diagnosis of MMM, platelet count ≥ 100 × 109/L, and neutrophil count ≥ 1 × 109/L, PS ≤ 2, LVEF > 50%, normal liver and renal function.8 patients (median age, 66 years; range, 60–73) were enrolled to the trial. Median duration of disease prior to protocol treatment was 45 months (range, 24–144). All pts (100%) had received prior therapy for MMM, including thalidomide 3 (37%), hydroxyurea 8 (100%), interferon 2 (25%)), anagrelide 2 (25%), and erythropoietin 8 (100%). 3 patients (37%) were RBC transfusion-dependent and 1 had severe constitutional symptoms (night sweats or disease related fever).Baseline median (range) values for palpable spleen size, leukocyte count, platelet count, CD34 cell count, and serum lactate dehydrogenase (LDH) were 10 cm BCM (median, range 6 to 30), 8.5 × 109/L (3–36.2), 250 × 109/L (160–950), 151.1 × 106/L (1.9–356), and 680 U/L (316–868), respectively. 8/8 pts had JAK2 V617F mutation. In addition, pre-treatment and post-treatment quantitative assessment by real-time quantitative polymerase chain reaction (RQ-PCR) of WT1 transcripts in the peripheral blood were performed and the values were expressed as WT1 copies every 104 copies of ABL. 7/8 pts are evaluable for response and toxicity (i.e., received six months of therapy) 1 pt discontinued because of progression.Clinico-hematologic responses have been observed in 7/7 (100%) pts and were evaluated according to European Myelofibrosis Network criteria These include 1 CR, 5 Major Response,1 Minor Response1 of 3 transfusion-dependent pts have become transfusion independent. The median time to response was 12 weeks (range, 4 to 18). Therapy has been well tolerated. Only a Grade 3 thrombocytopenia was seen.Striking WT1 gene expression levels in samples that were obtained after treatment were found to be significantly lower (median 480 range 210–520) than those before (median 1000, range 560–1230) (P < .0001, Mann-Whitney U test) The median post-treatment follow up at the time of this writing was 10 months (range 8–13)We conclude that Myocet has clinical activity in a subset of patients with myelofibrosis with an acceptable toxicity profile.