Saturday, December 4, 2004, 06:00 PM
[1067] Single Knock-In PML-RARa Expressing Murine APL Tumors Provide a Unique Model for Studying In Vivo Sensitivity and Resistance to ATRA and Arsenic Trioxide.
Session Type: Poster Session 221-I
Adam M. Greenbaum, Matthew S. Holt, Timothy K. Ley, John F. DiPersio. Division of Oncology, Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, USA
Although approximately 70% of patients with acute promyelocytic leukemia (APL) are cured after treatment with all-trans retinoic acid (ATRA) and anthracycline based chemotherapy, those who relapse cannot be cured even with chemotherapy or arsenic trioxide salvage therapy. In order to study the molecular mechanisms of resistance to ATRA, liposomal ATRA, and arsenic trioxide, we have utilized a murine model of APL generated by "knocking in" the PML/RARa gene into the murine cathepsin G locus (Westervelt et al. Blood. 102(5):1857). Of the 76 single knock-in (SKI) mice followed for a median time of 368 days, 30 (39%) suffered from rapid-onset leukocytosis, anemia, thrombocytopenia, and hepato-splenomegaly. Spleens from these leukemic animals were removed and the tumors cells (> 90% CD34+/Gr-1+ APL cells) were frozen in aliquots and banked for future passive transfer into genetically compatible recipients. The SKI mice exhibited the expected median time to death of 222 days. Attempts to expand these tumors in vitro failed, thus precluding the use of in vitro methods to generate ATRA and arsenic resistant SKI APL tumor cells. An alternate in vitro model of identifying de novo resistant SKI APL tumors to the drugs was established by incubating individual SKI APL tumors with ATRA, liposomal ATRA, and arsenic in vitro for only 3 days and using ELISA to measure upregulation of MMP-9 (gelatinase B), a surrogate marker for neutrophil differentiation. ATRA, liposomal ATRA, and arsenic induced 6.2 ± 4.4, 5.7 ± 3.5, 1.7 ± 1.0 fold increase in accumulated levels of MMP-9, respectively. These data suggest that ATRA and liposomal ATRA are more potent inducers of terminal differentiation than arsenic in these APL tumors. More importantly, the SKI APL tumors were also assessed for in vivo resistance to these agents by injecting 1 x 106 banked APL tumor cells into the peritoneal cavity of wild-type genetically compatible mice. The mice were then treated i.p. with either arsenic trioxide (0.4 mg q.d.), liposomal ATRA (0.4 mg q.d.), or diluent control. Doses were administered daily starting on day +5 until death. Tumors in the recipient mice were tracked by PCR for the transgene and FACS. The tumors proliferated in the peritoneal cavity (days 1-14) followed by migration to the bone marrow and spleen (days 14-28). By the eighth week post-injection, all mice suffered from leukocytosis and eventually died. Of the 22 banked tumors studied, only two tumors showed de novo resistance to ATRA both in vitro and in vivo. We will present data on preliminary RNA profiling and DNA sequencing of the PML-RARa transgenes in these APL tumors and attempt to correlate these results and the results of in vitro induction of MMP-9. The SKI APL tumors will provide us with unique reagents to study the biology of resistance to ATRA and arsenic trioxide.
Abstract #1067 appears in Blood, Volume 104, issue 11, November 16, 2004
Keywords: Acute promyelocytic leukemia|MMP|Transgenic mouse
Poster Session: Diagnostic and Prognostic Markers in AML (6:00 PM-7:30 PM)
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[1277] The Ferrohemoglobin Affects Blood Arsenic Concentration and Individual Arsenic Tolerance.
Session Type: Poster Session 431-I
Jin Zhou, Ran Meng, Baofeng Yang (Intr. by Dao-Pei Lu). hematology, the first hospital of harbin medical university, Herbin, Heilongjiang, China
Background and objective: arsenic trioxide is effective to acute promyelocytic leukemia (APL), however, there were different tolerance and manifestations individually during arsenic therapy and consolidation. As we know, more than 90% arsenic combined with ferrohemoglobin after infused into blood circulation, and anemia was accompanied by most of the leukemia patients. Whether or not the anemia was one of the important factors that correlated to the incidence of arsenic side effects and clinical therapeutic effect, and how to defined a more proper therapeutic quantity of arsenic trioxide individually than routine 10 mg/ kg ·d in adult and 5mg / kg ·d in child, in this paper , we observed the effects of anemia on arsenic concentration in blood serum and the incidence of cardiac arsenic side effect.
Method: the arsenic concentration in serum and in erythrocyte of 58 cases of APL, who received arsenic treatment, were dynamically and simultaneously monitored by atom fluorescence meter in different time points. QTc and ST-T of these patients were monitored by 12-leads electrocardiograph. Arsenic trioxide 0.16mg / kg was infused in general speed daily, every duration of arsenic infusion was about 2 hours. The APL patients were divided into three groups: the mild anemia group (Hb = 60 g/L or below ),the middle anemia group(Hb = 61~100 g/L) and non-anemia group(Hb above 100 g/L).
Results:
The peak of serum arsenic in three groups appeared at the 4th hour, while the peak of intra-erythrocyte arsenic appeared at the10th hour after arsenic infusion, at the 24th, the arsenic levels in both the serum and the erythrocyte were very low. The peak and average arsenic concentrations in serum in anemia group were higher than that in non-anemia group of APL patients in the same time points.
The relationships among arsenic peaks in blood seums, the degree of anemia and the incidence of arsenic related cardiac side effects when received intravenous As2O3 infusion in general speed with the dosage of 0.16mg /kg daily were showed on table 1.
Conclusions:
The quantity of individual ferrohemoglobin affects the arsenic peak in blood serum, which shows a negative correlation ( r = - 0.97). There are positive correlation between the serum arsenic concentrations and the incidences of clinical arsenic related cardiac side effects. The APL patients with moderate degree of anemia have poor tolerance to arsenic trioxide. The proper therapeutic quantity of arsenic trioxide should be defined according to the quantity of ferrohemoglobin individually.
Tab.1: relationships among arsenic peak concentration in blood circulation, the degree of anemia and the incidence of arsenic related cardiac side effects when patients received intravenous As2O3 with the dosage of 0.16mg /kg daily.
Group N Hb As peak level(µg / L) long-QT (%) ST-T (%)
1 20 5.5±1.0 1048±32.4* 20(4 /20)* 40(8/20)*
2 20 8.5±1.5 846±41.2 5(1 /20) 15(4 /20)
3 18 11.5±1.5 714±28.9 0(0 /18) 5.5(1/ 18)
* t = 4.56 , p < 0.01 ; * x2 = 3.92 , * x2 = 4.02 , p < 0.05
{NOTE: Theres a graph after this table which can't be pasted here.}
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