A Feasibility Study of Rapamycin with Hyper-CVAD Chemotherapy in Adults with Acute Lymphoblastic Leukemia (ALL) and Other Aggressive Lymphoid Malignancies and Evaluation of mTOR Signaling Using Phosphoflow

Therapies for adults with acute lymphocytic leukemia (ALL) fail to cure the majority of patients. The mTOR inhibitor, rapamycin, is a potent chemosensitizer in ALL cells and combination rapamycin- methotrexate is curative in an ALL xenotransplantation model. We therefore explored the feasibility of adding rapamycin to a multi-drug ALL regimen in subjects with de novo or relapsed Philadelphia chromosome negative ALL or other aggressive lymphoid malignancies. Additionally we performed pharmacodynamic analysis of peripheral blood blasts to estimate mTOR activation at baseline and with rapamycin.

Subjects were treated with rapamycin 12 mg on day 1, followed by 4 mg daily on days 2–7. To allow steady state inhibition of mTOR, systemic therapy with hyper-CVAD (A cycles) alternating with high dose methotrexate and high dose cytarabine (B cycles) was given following the day 3 rapamycin dose of each cycle, with rituximab if CD20 positive and intrathecal prophylaxis if indicated.

Peripheral blood specimens were collected serially during the first cycle of chemotherapy and were aliquoted, incubated with signal modulators as controls for 30 minutes then fixed with 4% formaldehyde and permeabilized. No ficoll separation was performed. Thereafter, cell samples from all time points for a subject were thawed, denatured with ice-cold methanol, and stained for cytometer analysis using a uniform antibody cocktail including alexa fluor-488 conjugated to phosphorylated S6 kinase.

We report results on the first 7 subjects, median age 45, (range 26–65) of which 3 had relapsed B-precursor ALL and the remainder were newly diagnosed T lymphoblastic lymphoma, adult T- cell lymphoma (ATLL), mantle cell (MCL) and Burkitt’s lymphoma (BL).

Feasibility. Thus far 16 ‘A’ and 15 ‘B’ cycles have been administered. The median time to next rapamycin was 23 days (range 19–57) and to chemotherapy was 25 days (range 21–59). Median time to recovery of absolute neutrophil count of > 500 and platelet count of >50 was 16 (range 0–27) and 17 (range 0–46) days, respectively. Two subjects never had count recovery in the setting of persistent disease. The following > grade 3 non- hematologic toxicities were observed: 5 neutropenic fevers, 5 infections, 4 non-neutropenic fevers, 1 ataxia (cytarabine related), psychosis, and hypophosphatemia. No fungal infections were noted. No treatment-related mortality has been observed.

Responses. Of 7 subjects, 4 achieved complete responses (CR) after cycle 2B (MCL, BL, ATLL and T- lymphoblastic lymphoma). The ATLL and MCL patients completed 5 and 4 cycles respectively and remain in CR after allogeneic BMT. The T-lymphoblastic lymphoma and BL patients completed 8 and 7 cycles respectively and remain in CR. All three subjects with relapsed ALL were taken off study with persistent disease after 1 to 4 cycles, and have subsequently died.

Pharmacokinetics. Rapamycin levels fell within the range typically targeted for transplant immunosuppression -mean (SD) 8.57 (3.33), range 4.7–14.7.

Pharmacodynamics. Two of 3 subjects with pre B- ALL had sufficient peripheral blast count of >200/μ l to perform intracellular phosphoflow. Both subjects’ blasts displayed constitutive phosphorylation of the ribosomal S6 protein (25% & 31%) at baseline with maximal inhibition at 72 hours of in vivo rapamycin in the 1^st sample and submaximal inhibition in the 2^nd sample (2.9% & 12.4%). Rapamycin trough levels were 9.5 and 8.7 respectively. The addition of 1000nM ex vivo rapamycin produced further reduction (0.68% & 2.07%). Despite exhibiting varying degrees of rapamycin sensitivity, both subjects had persistent disease after only 1 and 2 cycles of therapy respectively.

We show that the addition of rapamycin to Hyper-CVAD in adults with ALL and other aggressive lymphoid malignancies is feasible and results in similar toxicities to Hyper-CVAD alone without increased myelosuppression or treatment related mortality. At basal state, pS6, measured by flow cytometry, is heterogeneous in primary ALL samples and likely only demonstrable in a subset of blasts. Whole blood intracellular flow cytometry is a novel, feasible and potentially powerful technique to monitor pharmacodynamic response to novel therapeutics that inhibit mTOR signaling in ALL. Expansion of this trial to better characterize toxicity, response rates and the feasibility of performing PD/PK correlation is planned.

Off Label Use: Rapamycin. This is FDA approved for rejection prevention in solid organ transplant. It has been used investigationaly in this study for treating ALL. Carroll:Glaxo Smith Kline, Inc.: Research Funding; Sanofi Aventis Corporation: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.