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Pathophysiology and treatment of congenital neutropenials
$3-B1-1-03 PATHOPHYSIOLOGY AND TREATMENT OF CONGENITAL NEUTROPENIAS K. Welte, C. Zeidler, E. Yakisan, A. Reiter, H. Riehm Kinderklinik, Medical School Hannover, D-30625 Hannover, Germany Severe congenital neutropenia (SCN) is a heterogenous group of disorders characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (Kostmann-Syndrom) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (ANC) of less than 500/#1 (Cyclic neutropenia). Patients with SCN suffer from severe recurrent bacterial infections beginning in early infancy. The pathophysiology of both diseases is still unclear. In SCN patients we were able to detect normal or elevated G-CSF serum levels. Binding of G-CSF to its receptor was normal and the number of receptors on myeloid cells from SCN patients were rather elevated as judged by Scatchard analysis. Reverse transcriptase PCR products of GCSF receptor messenger RNA did not reveal any mutation in SCN patients. However, recent data from one of our patients with SCN who developed myelodysplasia/leukemia after 2 years on G-CSF therapy showed a point mutation in the G-CSF receptor RNA resulting in a cytoplasmatic truncation of the receptor protein. In phase I/II clinical studies in SCN patients, treatment with recombinant human G-CSF (r-metHuG-CSF; Filgrastim) resulted in a rise in the ANC associated with a reduction of infections. We have treated 32 patients with SCN and 4 patients with cyclic neutropenia for 4-6 years. 30 of 32 patients with SCN and all patients with cyclic neutropenia responded to r-met-HuG-CSF treatment at dosages between I and 120 ug/kg/d, subcutaneously, with an increase in the median ANC to above 1000/#1 and maintained their ANC stable during long-term treatment up to 6 years. Clinical responses included significant reduction of severe bacterial infections, reduction of intravenous antibiotic use and improvement of the quality of life. Significant adverse events noted included osteopenia (n=15), splenomegaly (n=12), and myeloid leukemia (n=2). These findings demonstrate that r-metHuG-CSF is the most promising treatment of patients with SCN.
$3-B1-1-04 CYTOKINE/ CHEMOTHERAPY INTERACTIONS IN MYELOID LEUKEMIA: CLINICAL RESULTS AND LABORATORY CORRELATES M. Andreeff, J. Drach, C. Koller, S. O'Brian, H. Kantarjian, L.E. Robertson, S. Kornblau, S. Escudier, S. Zhao, E. Estey, A. Deisseroth The University of Texas, M.D. Anderson Cancer Center, Houston, Texas Cytokinetic resistance, i.e. diminished response of non-cycling cells to chemotherapy, has been identified as a factor contributing to poor response to induction therapy and short remission durations in AML (Proc. NY Acad Sci 468, 368, 1986). In vitro studies have demonstrated recruitment of quiescent AML cells and increased cell kill by combinations of G-CSF or IL-3 and Ara-C (Leukemia 4, 826, 1990). We treated 112 patients (pts) with newly diagnosed AML or poor prognosis MDS (69 AML, 43 MDS) with G-CSF (400 ttg/m2s.c, daily) 24 hours before, during, and after chemotherapy (fludarabine [30mg/m 2] and Ara-C [2g/m"q given days 1-5 [FLAG]). Sixty-three percent 71/112 of pts achieved complete remission (CR), 45/69 (65%) AML and 26/43 (60%) MDS. In pts with chromosome 5 and 7 abnormalities the CR rate was 64%, a significant increase from pts treated with FA only (36%). Remission duration and survival (p=0.036) were also longer for these pts. In 22 pts, we investigated the in vivo cytokinetic effects of G-CSF. Bone marrow biopsies and aspirates were studied prior to and 24 hrs after the first dose of G-CSF. Cell kinetic assays included DNA/RNA, PCNA, and Ki-67 flow cytometry (FCM), and the fluorescent in situ hybridizaUon (FISH)/BRdU technique. In 13/22 = 59% pts, significant recruitment (depletion of G 0, increase in S, increase in PCNA/Ki-67 expression) was observed at 24 hrs. The median increase in S-phase was 2.0 fold (range 1.3 to 9.7). We conclude that FLAG induces CR in a high proportion of poor prognosisAML and MDS pts and exerts biological effects within the first 24 hrs. Inpts with cytogenetic abnormalities detectable by FISH, a combined method of FACS sorUng, BRdU and FISH was developed that detects as few as 1 leukemic in 10,000 normal cells. Minimal Residual Disease (MRD) was found in 5/13 pts in morphological CR; 4/5 pts with MRD relapsed shortly afterward (median 10 wks); 1 pt died in CR at 4 wks. Only 1/8 pts without detectable MRD relapsed. Twenty-one pts with relapsed/refractory AML were treated with IL-3 (0.5 - 1.0/~g/kg bid x 7d) and Idarubicin (12 m g / m 2q.d. x 3d) + Ara-C (1.5 g/m2daily x 4) starting on day 4, 17 are evaluable. IL-3 receptors (IL-3R+) were found in 7/8 pts studied. Recruitment was observed in 6/10 pts (4/6 IL-3R+) with significant increases in PCNA (27.4% 44.5%) and Ki-67 (29.4% - 39.9%). Marrow aplasia was observed in 8/14 evaluable pts and CR in 3/17. Results are not consistent with the suggested protective effect on IL-3 on leukemia cells. The MTD was 0.5 ttg/kg/d of IL-3. These studies demonstrate in vivo effects of G-CSF and IL-3 on leukemic cells and may result in more effective combination therapies for poor prognosis rnyeloid leukemias.
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$3-B1-1-04 CYTOKINE/ CHEMOTHERAPY INTERACTIONS IN MYELOID LEUKEMIA: CLINICAL RESULTS AND LABORATORY CORRELATES M. Andreeff, J. Drach, C. Koller, S. O'Brian, H. Kantarjian, L.E. Robertson, S. Kornblau, S. Escudier, S. Zhao, E. Estey, A. Deisseroth The University of Texas, M.D. Anderson Cancer Center, Houston, Texas Cytokinetic resistance, i.e. diminished response of non-cycling cells to chemotherapy, has been identified as a factor contributing to poor response to induction therapy and short remission durations in AML (Proc. NY Acad Sci 468, 368, 1986). In vitro studies have demonstrated recruitment of quiescent AML cells and increased cell kill by combinations of G-CSF or IL-3 and Ara-C (Leukemia 4, 826, 1990). We treated 112 patients (pts) with newly diagnosed AML or poor prognosis MDS (69 AML, 43 MDS) with G-CSF (400 ttg/m2s.c, daily) 24 hours before, during, and after chemotherapy (fludarabine [30mg/m 2] and Ara-C [2g/m"q given days 1-5 [FLAG]). Sixty-three percent 71/112 of pts achieved complete remission (CR), 45/69 (65%) AML and 26/43 (60%) MDS. In pts with chromosome 5 and 7 abnormalities the CR rate was 64%, a significant increase from pts treated with FA only (36%). Remission duration and survival (p=0.036) were also longer for these pts. In 22 pts, we investigated the in vivo cytokinetic effects of G-CSF. Bone marrow biopsies and aspirates were studied prior to and 24 hrs after the first dose of G-CSF. Cell kinetic assays included DNA/RNA, PCNA, and Ki-67 flow cytometry (FCM), and the fluorescent in situ hybridizaUon (FISH)/BRdU technique. In 13/22 = 59% pts, significant recruitment (depletion of G 0, increase in S, increase in PCNA/Ki-67 expression) was observed at 24 hrs. The median increase in S-phase was 2.0 fold (range 1.3 to 9.7). We conclude that FLAG induces CR in a high proportion of poor prognosisAML and MDS pts and exerts biological effects within the first 24 hrs. Inpts with cytogenetic abnormalities detectable by FISH, a combined method of FACS sorUng, BRdU and FISH was developed that detects as few as 1 leukemic in 10,000 normal cells. Minimal Residual Disease (MRD) was found in 5/13 pts in morphological CR; 4/5 pts with MRD relapsed shortly afterward (median 10 wks); 1 pt died in CR at 4 wks. Only 1/8 pts without detectable MRD relapsed. Twenty-one pts with relapsed/refractory AML were treated with IL-3 (0.5 - 1.0/~g/kg bid x 7d) and Idarubicin (12 m g / m 2q.d. x 3d) + Ara-C (1.5 g/m2daily x 4) starting on day 4, 17 are evaluable. IL-3 receptors (IL-3R+) were found in 7/8 pts studied. Recruitment was observed in 6/10 pts (4/6 IL-3R+) with significant increases in PCNA (27.4% 44.5%) and Ki-67 (29.4% - 39.9%). Marrow aplasia was observed in 8/14 evaluable pts and CR in 3/17. Results are not consistent with the suggested protective effect on IL-3 on leukemia cells. The MTD was 0.5 ttg/kg/d of IL-3. These studies demonstrate in vivo effects of G-CSF and IL-3 on leukemic cells and may result in more effective combination therapies for poor prognosis rnyeloid leukemias.
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