Onur Ek

Prognostic Significance of T-Lineage Leukemic Cell Growth in SCID Mice: A Children's Cancer Group Study

Contemporary intensive therapies are effective for the majority of pediatric T-lineage acute lymphoblastic leukemia (ALL) patients, thus current challenge is to identify patients who may benefit from alternative treatment modalities. Previously, we demonstrated that human leukemic cell growth in the severe combined immunodeficiency (SCID) mouse was a significant prognostic factor for very high risk B-lineage ALL patients. In the current report we show that primary leukemic cells from 24 of 88 (27%) T-lineage ALL patients (SCID+) caused histopathologically detectable leukemia in SCID mice. These SCID+ patients were similar to SCID- (n = 64) patients with respect to virtually all presenting features, including age, sex, race, and leukocyte count. Growth of primary leukemic cells in SCID mice was not a significant predictor of outcome for the aggregate population of T-lineage ALL patients. Two-year event-free survival (EFS) outcomes for SCID+ patient and SCID- patients were 76.2% (SD = 5.6%) and a 64.0% (SD = 10.4%; p = 0.20). Overall survival also was similar between the two groups (p = 0.36). Among the subset of patients with M1 or M2 marrow status by day 7 of induction chemotherapy (rapid early responders), those who were SCID+ had poorer outcomes than those who were SCID-, with a 2-year EFS of 68.4% (SD = 11.9%) vs. 85.7% (SD = 6.0%) and relative hazard rate of 3.06 (p = 0.06). These data suggest that leukemic cell growth in SCID mice may identify a subset of T-lineage ALL patients who are at higher risk for relapse despite achieving a rapid early response to induction chemotherapy.

In Vivo., Toxicity and Pharmacokinetic Features of B43(Anti-CD19)-Genistein Immunoconjugate

B43(anti-CD19)-Genistein immunoconjugate targets genistein, a naturally occurring protein tyrosine kinase inhibitory isoflavone to the membrane-associated anti-apoptotic CD19-LYN complexes and triggers apoptotic cell death. In this preclinical study, the toxicity profiles of B43-Genistein as well as unconjugated genistein were evaluated in mice. B43-Genistein and genistein were administered either as single bolus injections or daily injections for 10 consecutive days via the intraperitoneal route to mice. Genistein was not toxic to mice at the highest dose of 40 mg/kg and no test article-related histopathological lesions were found in any of the 64 genistein-treated mice. B43-Genistein had a significantly longer elimination half-life and slower plasma and tissue clearance than unconjugated genistein. B43-Genistein was not toxic to mice at the highest single dose of 40 mg/kg or highest cumulative dose of 100 mg/kg and no test article-related histopathological lesions were found in any of the 108 mice treated with B43-genistein. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a tyrosine kinase inhibitor-containing immunoconjugate.

Combined Therapeutic Efficacy of the Thymidylate Synthase Inhibitor ZD1694 (TOMUDEX) and the Immunotoxin B43(Anti-CD19)-PAP in a SCID Mouse Model of Human B-Lineage Acute Lymphoblastic Leukemia

The quinazoline antifolate N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ZD1694; Tomudex) is a potent inhibitor of thymidylate synthase and causes cell death through disruption of DNA synthesis and repair by blocking the obligatory thymidine nucleotide synthesis. B43(anti-CD19)-PAP immunotoxin is a potent inhibitor of protein synthesis in CD19+ B-lineage acute lymphoblastic leukemia (ALL) cells and causes apoptosis. In this model, 100% of SCID mice challenged with 1 x 10(6) human NALM-6 B-lineage ALL cells develop overt and invariably fatal leukemia. All of the 22 control SCID mice treated with phosphate-buffered saline died of disseminated human leukemia between 31 and 61 days with a median survival of 41.2 days. Treatment with ZD 1694 resulted in improved leukemia-free survival with a median survival of 69.2 days (P < 0.001, log-rank test). B43-PAP treatment was more effective than ZD1694 (P=0.026) and resulted in 51.0% long-term leukemia-free survival with a median survival of 187.5 days (P < 0.0001. log-rank test). The combination of ZD1694 and B43-PAP was more effective than either agent alone and resulted in 100% long-term leukemia-free survival. To our knowledge, this preclinical study is the first to demonstrate the feasibility and therapeutic advantage of combining an anti-leukemia immunotoxin with a thymidylate synthase inhibitor.

Evaluation of Temozolomide in a SCID Mouse Model of Human B-Cell Precursor Leukemia

We used a SCID mouse model of human B-lineage acute lymphoblastic leukemia to examine the antileukemic activity of temozolomide in comparison to as well as in combination with B43-PAP anti-CD19 immunotoxin. One hundred percent of the 20 PBS-treated control mice died of disseminated human B-lineage ALL at 32 to 64 days after the inoculation of 1x10(6) NALM-6 cells, with a median event free survival time of 43 +/- 1 days. Temozolomide, when administered i.p. for 5 consecutive days at a dose level of 411 mg/m2 or as a single 750 mg/m2 bolus dose, elicited significant antileukemic activity and improved survival in this SCID mouse model of human B-lineage ALL. The median survival times were 43 +/- 1 days for PBS-treated mice, 56 +/- 16 days for mice injected with the 5-day temozolomide program, and 64 +/- 15 days for mice treated with a single bolus dose of temozolomide. However, temozolomide was not as effective as B43-PAP. Whereas only 40 +/- 21% of mice treated with temozolomide survived beyond 120 days, B43-PAP treatment resulted in 74 +/- 7% survival in the same model system. The combination of temozolomide with B43-PAP was well tolerated by mice but it was not significantly more effective than B43-PAP alone. Temozolomide may have very limited potential as an antileukemic agent for treatment of B-lineage ALL.