With the ALL-REZ BFM 87 protocol, more than one-third of patients may be regarded as cured from recurrent ALL with second complete remissions lasting more than 10 years. Immunophenotype and time point of relapse are important prognostic factors that allow us to adapt more precisely treatment intensity to individual prognosis in future trials.
Hypoxia-inducible factor 1 (HIF-1) controls angiogenesis and glycolysis, two leading characteristics of solid tumor invasion, metastasis, and lethality. Increased angiogenesis is also found in the bone marrow (BM) of leukemias. Less is known in leukemia about the role of HIF-1 and vascular endothelial growth factor (VEGF), the most important proangiogenic target gene of HIF-1. We show by immunohistochemistry that the oxygen-regulated component of HIF-1 (HIF-1a) is overexpressed in clusters of leukemic cells in BM specimens of childhood acute lymphoblastic leukemia (ALL) and absent in biopsies of normal BM. Half the HIF-1a-positive ALL biopsies exhibited VEGF coexpression. Among 96 children with relapsed ALL, diagnostic BM aspirates with high VEGF mRNA levels were associated with a significantly lower probability of eventfree survival at 3 years (0.3170.08 vs 0.6570.07, P ¼ 0.003). Those with poor molecular response to therapy (evaluated by MRD assessment) had 2.2-fold higher VEGF levels than those responding well to chemotherapy (P ¼ 0.005). In conclusion, the data demonstrate activation of the HIF pathway in the BM of ALL patients and indicate that the expression of HIF target genes, such as VEGF, play an important role in leukemia progression, therapy response, and outcome.
Background: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors, and hematologic malignancies. The expression of isoforms of VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formation.
Methods: We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to β2-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.
Results: Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF121 and VEGF165 constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF189 constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF121 and VEGF165 accounted for 94% (SD = 7.6%) and VEGF189 only 6% (SD = 3.7%).
Conclusions: Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.
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