Milena Fava

Real-time quantitative polymerase chain reaction detection of minimal residual disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study

PURPOSE Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial. PATIENTS AND METHODS Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls. RESULTS Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate > or = 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004). CONCLUSION Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.

Significant Correlation Between the Degree of WT1 Expression and the International Prognostic Scoring System Score in Patients With Myelodysplastic Syndromes

PURPOSE To determine whether pattern of WT1 gene expression is a useful marker for establishing prognosis and tracking disease progression in patients with myelodysplastic syndromes (MDS). PATIENTS AND METHODS We performed a quantitative assessment of the WT1 transcript amount by real-time quantitative polymerase chain reaction (RQ-PCR) in 173 samples (131 bone marrow samples and 42 peripheral-blood samples) from 131 patients with MDS (79 patients with refractory anemia [RA], 31 with RA with excess blasts [RAEB], 18 with secondary acute myeloid leukemia [s-AML] evolved from MDS, and three with deletion of 5q as the sole cytogenetic abnormality). Values obtained were correlated with the blast percentage and International Prognostic Scoring System (IPSS) score. RESULTS Sixty-five percent of BM and 78% of PB samples for RA and 100% of BM and PB samples of RAEB and s-AML expressed WT1 transcript amounts greater than the level observed in healthy volunteers. The degree of WT1 expression was highly correlated with the type of MDS, was much higher in RAEB and s-AML compared with RA, and increased during disease progression. Moreover, a significant correlation was found between WT1 expression levels, blast cell percentage, and the presence of cytogenetic abnormalities. Therefore, we found a significant correlation between the amount of WT1 transcripts and the IPSS score, which currently represents the most reliable risk index of disease progression available for MDS patients. CONCLUSION WT1 is a useful molecular marker for risk assessment in MDS patients.

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.

Sensitivity to imatinib therapy may be predicted by testing Wilms tumor gene expression and colony growth after a short in vitro incubation

The objective of the current study was to verify the ability to predict response to imatinib therapy using in vitro assays to evaluate the inhibition of Wilms tumor gene (WT1) expression and colony growth after samples obtained from patients with chronic myelogenous leukemia (CML) before the start of treatment were subjected to short‐term incubation with imatinib.

Alternative BCR/ABL Splice Variants in Philadelphia Chromosome–Positive Leukemias Result in Novel Tumor-Specific Fusion Proteins that May Represent Potential Targets for Immunotherapy Approaches

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in approximately 80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.

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-PDGRFα 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.

Molecular monitoring in patients with chronic myelogenous leukemia

Imatinib has revolutionized the treatment of chronic myelogenous leukemia (CML). Given the high rates of complete cytogenetic remission achieved with imatinib therapy, molecular monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction has become the method of choice to assess the amount of residual disease below the cytogenetic threshold. BCR-ABL transcript levels measured at specific times during therapy may predict durable cytogenetic remission and prolonged progression-free survival or, on the contrary, failure and suboptimal response, thus directing clinical decisions. Recently, recommendations have been established for harmonizing the methodologies used to measure BCR-ABL transcripts in patients with CML, allowing results to be expressed on a standardized comparable international scale. Rising levels of BCR-ABL transcripts indicate the need for an analysis of kinase mutations, the major mechanism of imatinib resistance. The early detection and the characterization of these mutations may allow timely and appropriate treatment to overcome resistance.