Gene expression profile of slowly responding subclones responsive for relapse in childhood acute lymphoblastic leukemia
01 / 2005 - 12 / 2009
Treatment of pediatric acute lymphoblastic leukemia (ALL) is based on the concept of tailoring the intensity of therapy to a patients relapse risk. Recently, clinical studies were started in which patients are stratified according to the levels of minimal residual disease after induction therapy and early during further treatment, since it has been shown that MRD level is the best predictive factor for disease outcome. More recently, it has been shown that gene expression profiles of leukemic cells at diagnosis might be correlated with prognosis. Based on results in our previous studies we reported that slow responding subclones represent the clones causative for a leukemic relapse in oligoclonal disease. Based on these results, we hypothesize that gene expression profiles of the slow responding subclones present after the first weeks of chemotherapy might be more predictive than the profiles of all leukemic cells at diagnosis. Using Ig-gene rearrangements as surrogate markers in order to follow the biological behavior of subclones, we have demonstrated that relapse-causing subclones are already present in the very beginning of the disease and show the slowest rate of reduction even during induction therapy. At diagnosis, they are in vivo present submerged among therapy-sensitive subclones. So far, the analysis of gene expression profiles has been focused on the recognition of the prognostically different subgroups only at the time of diagnosis. During and at the end of induction therapy, the limited number of leukemic cells that can be obtained hampers the application of micro-array technology, which needs at least 20ug RNA from 20 x 10e6 cells. - PURPOSE. The aim of our project is to investigate whether the expression profiles of leukemic cells during and after induction treatment are prognostically better than the profiles of the leukemic cells at diagnosis. - PLAN OF INVESTIGATION. In the present project we will apply a new technique, i.e. multiplex ligation amplification (MLPA) assay, which enables us to quantify up to 45 genes in one single PCR reaction per sample, reducing the number of leukemic cells needed to 100,000. Initially, we will select 20-40 genes based on the presently available data on prognostic cDNA microarray studies of cytogenetically defined subgroups of childhood ALL (e.g.TEL-AML1, hyperdiploid ALL). For the different cytogenetic subgroups, MLPA-assays will be developed. The MLPA-assay will be tested on immunologically purified leukemic cells sampled at the time of diagnosis, after 2 weeks of therapy, at the end of therapy and at eventual relapse. - POSSIBLE RESULTS/RELEVANCE FOR CANCER. In this way it can be investigated whether the expression profile of leukemic cells that respond slowly and cause relapse differs from the profile at diagnosis. By testing a cohort of well defined patients, including patients who have been included in prognostic microarray expression profile analysis, it will be studied whether the MPLA profile of leukemic cells early during treatment has a better prognostic value than the expression profile of the total leukemic cell pool present at diagnosis. Furthermore, this multiplex assay might be an attractive second-generation profiling test for routine prognostic testing at diagnosis and end of induction, once the laborious and expensive micro-array approach has identified the most relevant target genes that should be applied.