Abstract
BackgroundThe molecular changes in vivo in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response.MethodsThe gene expression was examined by whole genome 44 k oligo microarrays and 12 k cDNA microarrays in peripheral blood leukocytes collected from seven leukemia patients before treatment, 2–4 h and 18–24 h after start of chemotherapy and validated by real-time quantitative PCR. Statistically significantly upregulated genes were classified using gene ontology (GO) terms. Parallel samples were examined by flow cytometry for apoptosis by annexin V-binding and the expression of selected proteins were confirmed by immunoblotting.ResultsSignificant differential modulation of 151 genes were found at 4 h after start of induction therapy with cytarabine and anthracycline, including significant overexpression of 31 genes associated with p53 regulation. Within 4 h of chemotherapy the BCL2/BAX and BCL2/PUMA ratio were attenuated in proapoptotic direction. FLT3 mutations indicated that non-responders (5/7 patients, 8 versus 49 months survival) are characterized by a unique gene response profile before and at 4 h. At 18–24 h after chemotherapy, the gene expression of p53 target genes was attenuated, while genes involved in chemoresistance, cytarabine detoxification, chemokine networks and T cell receptor were prominent. No signs of apoptosis were observed in the collected cells, suggesting the treated patients as a physiological source of pre-apoptotic cells.ConclusionPre-apoptotic gene expression can be monitored within hours after start of chemotherapy in patients with acute myeloid leukemia, and may be useful in future determination of therapy responders. The low number of patients and the heterogeneity of acute myeloid leukemia limited the identification of gene expression predictive of therapy response. Therapy-induced gene expression reflects the complex biological processes involved in clinical cancer cell eradication and should be explored for future enhancement of therapy.
Highlights
The molecular changes in vivo in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response
Pre-apoptotic AML cells in peripheral circulation were without signs of apoptosis during the first 24 hours of chemotherapy In this study we examined seven AML patients before (-30 min), at 2–4 h and at 18–24 h after start of first day standard induction therapy [26], consisting of a 30 min infusion of anthracycline and a 24 h continuous infusion of cytarabine started concomitantly
We investigated the BCL2/BAX and BCL2/PUMA ratios as apoptotic responders to treatment at the different time points for each of the patients, and found a significant increase in gene expression of the apoptosis facilitators PUMA and BAX (Additional File 2) and a decrease in the BCL2/BAX ratio as well as BCL2/ PUMA for most of the AML samples (0 versus 4 h p-values < 0.0004, 0 versus 18 h p-values < 0.01, 4 versus 18 h pvalues 0.5 and 0.2, respectively) (Figure 2A,B)
Summary
The molecular changes in vivo in acute myeloid leukemia cells early after start of conventional genotoxic chemotherapy are incompletely understood, and it is not known if early molecular modulations reflect clinical response. Clinical response to chemotherapy and karyotype analysis of AML cells provide prognostic information about risk for relapse [9]. Gene expression analysis may provide important prognostic information in the 50% of patients with standard risk for relapse due to normal karyotype [10,11]. Recent studies of mutations or signaling response in AML have indicated potential for risk stratification [12,13,14] All these studies are based on bulk cell analysis, and propose that analysis of patient cells under DNA damaging therapy may provide biological important information about the therapy response. We hypothesize that earlier sampling and analysis of gene expression could provide us with information about therapy responses and early resistance mechanisms against intensive chemotherapy
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