Endogenous VEGF-C mRNA Expression Increases In Vitro Drug Resistance of Pediatric AML Cells and Is an Independent Prognostic Factor for the Time To Reach Complete Remission in AML.
Authors: de Jonge Hendrik J.M. , ter Elst Arja , Boezen H. Marike , Weidenaar Alida C. , Scherpen Frank J.G. , Steege Jessica C.A. Bouma-ter , Kaspers Gertjan J.L. , Goemans Bianca F. , Creutzig Ursula , Kamps Willem A. , de Bont Evelina S.J.M. 
Published: April 2020

Abstract
VEGF-C, has been characterized as a lymphangiogenic and angiogenic growth factor and mediates Acute Myeloid Leukemic (AML) cell proliferation and survival by FLT-4 signaling. Moreover, exogenously added VEGF-C protected 2 AML cell lines and 5 primary AML samples from in vitro chemotherapy-induced apoptosis for 3 drugs via an increased bcl-2/bax ratio (Dias, S. et al. Blood, Mar 15;99(6):2179–84, 2002). We hypothesized that in vitro as well as in vivo cellular drug resistance of AML cells are increased as a result of downstream effects of endogenous VEGF-C signaling. In primary AML cells from 40 pediatric patients VEGF-C mRNA expression with a quantitative PCR as well as in vitro cellular resistance to 9 drugs with a total cell-kill MTT assay were studied. LC50 values (drug concentration needed to kill 50% of the leukemic cells) were used as a measure of resistance. The best available in vivo equivalent for in vitro drug resistance is the time in days from diagnosis to complete remission (CR). CR is defined as less than 5% leukemic cells in a regenerated bone marrow aspirate. Correlations were calculated using the Spearman rank correlation coefficient. P-values ≤0.05 were considered to be statistically significant. VEGF-C mRNA expression levels varied largely in the samples (median: 0.02, given as arbitrary units, range: 0.004–0.33, n=40). Increased endogenous VEGF-C mRNA expression levels correlated significantly with increased in vitro resistance for 6 of 9 drugs (for cytarabine such a trend was seen), these drugs were all typical AML drugs (Table 1a and 1b). For time to reach CR, cox regression analysis was used to study the effect of VEGF-C, age at diagnosis, sex, treatment protocol, FAB type, cytogenetic risk profile and white blood cell counts on time to reach CR. Interestingly, VEGF-C was in vivo an independent prognostic factor for the time to reach CR (p=0.006, Odds ratio:0.224, 95% confidence interval: 0.077–0.655). LC50 values of the various drugs did not correlate to the time to CR (all p-values were above 0.1). So, high VEGF-C mRNA levels are correlated to a slower decrease of the total number of leukemic blasts in vitro as well as in vivo upon drug exposure. In conclusion, these results suggest for the first time that increased endogenous VEGF-C mRNA expression, probably resulting in increased anti/pro-apoptotic ratios, are correlated with in vitro as well as in vivo drug resistance of pediatric AML cells resulting in a longer time to reach CR.
Table 1a. Correlations endogenous VEGF-C mRNA expression and LC50 values. Drugs Cytarabine Gemcitabine Fludarabine 6-thioguanine Daunorubicin Etoposide VEGF-C rho 0.28 0.33 0.31 0.40 0.46 0.43 p 0.08 0.04 0.05 0.01 0.005 0.008 Table 1b. Drugs Cladibrine Vincristine L-asparaginase Rho indicates spearman rank correlation coefficient. p ≤ 0.05 was considered statistically significant. VEGF-C rho 0.45 0.28 0.22 p 0.003 0.12 0.21