Andries R. Jonkhoff

Extensive early apoptosis in frozen–thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation

Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze–thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen–thawed grafts, threshold numbers for adequate haematological recovery of 2.8–3.0 × 106 CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2–1.3 × 106 CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3–0.4 × 106CD34+ cells/kg). In contrast to freeze–thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4°C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen–thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery.Bone Marrow Transplantation (2002) 29, 249–255. doi:10.1038/sj.bmt.1703357

Peripheral blood stem cell transplantation as an alternative to autologous marrow transplantation in the treatment of acute myeloid leukemia

The clinical use of autologous marrow transplantation in acute myeloid leukemia (AML) has been hampered by the inability to collect adequate numbers of cells after remission induction chemotherapy and the notably delayed hematopoietic regeneration following autograft reinfusion. Here we present a study in which the feasibility of mobilizing stem cells was investigated in newly diagnosed AML. Among 96 AML patients, 76 patients (79%) entered complete remission. Mobilization was undertaken with low dose and high dose schedules of G-CSF in 63 patients, and 54 patients (87%) were leukapheresed. A median of 2.0 × 106 CD34+cells/kg (range 0.1–72.0) was obtained in a median of three leukaphereses following a low dose G-CSF schedule (150 μg/m2) during an average of 20 days. Higher dose regimens of G-CSF (450 μg/m2 and 600 μg/m2) given during an average of 11 days resulted in 28 patients in a yield of 3.6 × 106 CD34+ cells/kg (range 0–60.3) also obtained following three leukaphereses. The low dose and high dose schedules of G-CSF permitted the collection of 2 × 106 CD34-positive cells in 46% and 79% of cases respectively (P = 0.01). Twenty-eight patients were transplanted with a peripheral blood stem cell (PBSC) graft and hemopoietic repopulation was compared with the results of a previous study with autologous bone marrow. Recovery of granulocytes (>0.5 × 109/l, 17 vs 37 days) and platelets (>20 × 109/l; 26 vs 96 days) was significantly faster after peripheral stem cell transplantation compared to autologous bone marrow transplantation. These results demonstrate the feasibility of PBSCT in the majority of cases with AML and the potential advantage of this approach with respect to hemopoietic recovery.

Hypophyseal Non-Hodgkin's Lymphoma presenting with clinical panhypopituitarism successfully treated with chemotherapy

SummaryA patient is described with a testicular Non-Hodgkins Lymphoma (NHL) presenting with panhypopituitarism caused by a hypophyseal localization. A67Gallium scintigraphy showed avid uptake in the hypophyseal region. Obviously67Gallium could reach the tumor, by the intravenous route, which was the reason to treat the patient with intravenous chemotherapy. A complete remission was induced, which seems to be lasting (+ 25 months). As far as we know this is the first report of panhypopituitarism caused by a hypophyseal NHL in the hypophysis and successfully treated by intravenous chemotherapy.

Gallium-67 radiotoxicity in human U937 lymphoma cells

Promising clinical results have been obtained with radiolabeled antibodies in lymphoma patients. The higher uptake by lymphomas of 67Gallium (67Ga) compared with monoclonal antibodies makes selective radiotherapy by the widely available 67Ga appealing. However, the gamma radiation of 67Ga used in scintigraphy is considered to be almost non-toxic to lymphoma cells. However, in addition to photon radiation 67Ga emits low energy Auger electrons and 80-90 keV conversion electrons which could be cytotoxic. The objective of the present study was the assessment of radiotoxicity of 67Ga on a lymphoid cell line: U937. Proliferation (MTT-assay) and clonogenic capacity (CFU-assay) were measured after 3 and 6 days incubation with 10, 20 and 40 microCi ml-1 67Ga. Growth inhibition was 36% after 3 days incubation and 63% after 6 days incubation with 40 microCi 67Ga ml-1. Clonogenic capacity was reduced by 51% after 3 days and 72% after 6 days incubation with 40 microCi ml-1 67Ga. A survival curve showed an initial shoulder and became steeper beyond 200-250 pCi cell-1 (low linear energy transfer type). Iso-effect doses of 67Ga and 90Yttrium (90Y) were determined. The iso-effect dose of 40 microCi 67Ga ml-1 (cumulative dose of conversion electrons 306 cGy) was 2.5 microCi 90Y ml-1 (cumulative dose 494 cGy) and the iso-effect dose of 80 microCi 67Ga ml-1 was 5.0 microCi 90Y/ml. The main cytotoxic effect of 67Ga seems to be induced by the 80 keV conversion electrons. We conclude that the conversion electrons of 67Ga have a cytotoxic effect on U937 cells and that in our experiments a 16-fold higher microCi-dose of 67Ga than of 90Y was needed for the same cytotoxic effect. We believe that 67Ga holds promise for therapeutic use.

A single-step colony-forming unit assay for unseparated mobilized peripheral blood, cord blood, and bone marrow.

The colony-forming unit (CFU) assay is exposed to a lot of variation, part of which is introduced by several enrichment strategies that are routinely performed before assessment of clonogenic capacity in mobilized peripheral blood (PB), bone marrow (BM), or cord blood (CB). We investigated the possibility to perform a single-step CFU assay by direct plating of PB, BM, or CB into CFU culture medium to obtain more reproducible results than after a standard Ficoll or lysis procedure. Direct plating implies the presence of red blood cells (RBC), white blood cells (WBC), and plasma in the CFU assay, which could possibly influence the outcome of the assay. Of all components, only the RBC was found to negatively influence CFU-GM growth if a concentration of > 0.02 x 10(9)/ml was present in the CFU culture medium. Subsequently, depending on the RBC concentration PB, BM, and CB samples were prediluted in triplicate or quadruplicate and plated into CFU medium. Lysis and/or Ficoll procedures were also performed in triplicate or quadruplicate on the same samples, and the mean colony number and coefficient of variation (CV) of the three techniques were compared. Significantly smaller CV values were found using the direct plating technique (all assays, mean 7.5%, range 1.6-15.6%) than after Ficoll separation (mean 18.0%, range 2.2-62.5%). Intermediate results were obtained with the lysis method (mean CV 11.6%, range 3.3-29%). In most samples, and especially in those with a very low number of clonogenic cells per milliliter, more colonies were detected with the direct plating method than with either the lysis or Ficoll method. In conclusion, the single-step direct plating method significantly enhances reproducibility of the CFU assay for PB, BM, and CB samples in comparison with standard techniques by circumvention of loss of colony formation and by decreasing variability. Furthermore, the direct plating technique is a timesaving assay.

Storage of unprocessed G-CSF-mobilized whole blood in a modified Leibovitz's L15 medium preserves clonogenic capacity for at least 7 days.

Autologous stem cell transplantation using unprocessed, G-CSF-mobilized whole blood (WB) is a simple, cost-reducing procedure and supports high-dose chemotherapy regimens not exceeding 72 h. Thereafter, clonogenic capacity rapidly decreases if routine anticoagulants are used for storage. In order to increase clinical applicability, we investigated the requirements for optimal preservation of unprocessed WB for 7 days. During storage at 22°C in CPDA-1, a decrease in pH was noted, which was at least partially responsible for the low recovery of clonogenic cells. Subsequently, WB cells were stored in various cell culture media (RPMI 1640, α-MEM, X-VIVO15, CellGro SCGM and Leibovitzs L15 medium) containing either serum, serum-free substitutes or no additives. Leibovitzs L15 showed significantly better CFU-GM recoveries than the other media. Using a calcium-free modification of L15 medium (added 3:10 to WB), 94 ± 24% of CD34+ cells, 41 ± 14% of BFU-E, 56 ± 17% CFU-GM and 90 ± 14% of LTC-IC were preserved during storage for 7 days at 22°C. Storage at 4°C was also feasible, but showed less optimal recoveries of 52 ± 29% (CD34), 32 ± 10% (BFU-E), 13 ± 7% (CFU-GM) and 58 ± 9% (LTC-IC). The expression of CD38, Thy-1, c-kit, AC133, L-selectin and CXCR4 on CD34-positive cells remained unchanged. In conclusion, a modified Leibovitzs L15 medium better meets the metabolic requirements of a high-density cell culture and allows safe storage of G-CSF mobilized WB for at least 7 days. The results encourage further exploration of WB transplants stored for 7 days for clinical use. Bone Marrow Transplantation (2001) 28, 145–155.

CELL CYCLE DEPENDENCY OF 67GALLIUM UPTAKE AND CYTOTOXICITY IN HUMAN CELL LINES OF HEMATOLOGICAL MALIGNANCIES

67Gallium (67Ga) is a radionuclide which accumulates in hematological malignancies and is used for diagnostic imaging. We investigated in this in vitro study the cell cycle dependency of cellular uptake and cytotoxicity of 67Ga. Cell cycle synchronization of cells was achieved by counterflow centrifugal elutriation and the use of cytostatic drugs. The human lymphoma cell lines U-937 and U-715 were used and in elutriation experiments we also used the leukemic cell line HL-60. The transferrin receptor (CD71) expression, 67Ga uptake and cell proliferation inhibition were the parameters measured. We also studied cytotoxicity in various schedules for combination of 67Ga and drugs and the residual proliferative capacity was measured. The CD71 expression in the three cell lines increased from 106-177% on S phase cells and from 118-233% on G2M cells, as compared to the G0/G1 cell fraction. The 67Ga uptake varied from 108-127% for S cells and 128-139% for G2M cells. The drugs chosen induced cell cycle phase accumulation in S and/or G2M phase during preincubation. 67Ga preincubation induced accumulation in the G2M phase. Almost all combinations of 67Ga and drugs resulted in a non-interactive effect, except for methotrexate which resulted in an antagonistic effect. No preferential effect of any of the incubation schemes was seen. CD71 expression and 67Ga uptake were increased in S and G2M cells. Combination of 67Ga with drugs which arrest cells in these cell cycle phases did not result in a change in cytotoxicity. However, these results implicate that 67Ga and the cytostatic drugs tested except for methotrexate might be used together or sequentially in therapy.

Biological effectiveness of 67-gallium decay in HL60 cells compared with external low dose rate gamma irradiation : effects on proliferation, G2 arrest, and clonogenic capacity

PURPOSE Estimation of the relative biological effectiveness of electron emissions of 67Gallium for cell growth delay and inactivation. METHODS AND MATERIALS Human myeloid HL60 cells were incubated in vitro with 0.74 MBq/mL, 1.48 MBq/mL, or 2.96 MBq/mL of 67Gallium for 4 days. Proliferation (vital cell counts and colorimetric tetrazolium assay), clonogenic survival, and cell-cycle effects were compared with responses of HL60 cells externally irradiated with 0.78, 10.37, and 13.22 Gy by an external 67Gallium source. Dosimetric calculations were performed by Monte Carlo simulations (10,000 events). RESULTS Proliferation of cells was equally inhibited after 67Gallium incubation with 1.48 and 2.96 MBq/mL compared with 10.37 and 13.22 Gy external irradiation. Irradiation with 10.37 and 13.22 Gy caused a 81% and 89% reduction of Colony Forming Units, compared with 34%, 66%, and 80% reduction after 67Gallium incubation with 0.74, 1.48, and 2.96 MBq/mL, respectively. Peak values for G2/M accumulation were reached on day 4 for the cells externally irradiated with 10.37 and 13.22 Gy (47.5% and 56.7%) and on day 5 after 67Gallium incubation with 0.74 MBq/mL, 1.48 MBq/mL, and 2.96 MBq/mL (26.7%, 43.4%, and 58.2%). CONCLUSIONS 67Gallium incubation exerts a significant cytotoxic effect on human HL60 cells, which, on the basis of dosimetric studies, may be mainly ascribed to conversion electrons (80 KeV) and 8 KeV Auger electrons. Low energy (< 1 keV) Auger electrons do not contribute significantly. The relative biological effectiveness of 67Gallium compared with external low dose rate gamma irradiation is about 1.0 for clonogenic survival and approximately 1.8 and 1.5 for proliferation inhibition and G2 arrest, respectively. For in vivo therapy, this might implicate that higher doses of 67Gallium than 131Iodine or 90Yttrium are necessary for the same biological effect.

Radiotoxicity of 67-Gallium on myeloid leukemic blasts

Promising clinical results are obtained with radiolabeled antibodies in leukemia patients. 67Gallium (67Ga) is a radionuclide that accumulates in many malignant tissues without need for a monoclonal antibody. For this reason, the use of 67Ga as a therapeutic agent is appealing. In the present we study, we report data about the radiotoxicity of 67Ga on peripheral blast cells of 23 patients with acute myelogenous leukemia (AML) in vitro. Isolated blast cells were incubated for 4 days with 0.74 MBq/ml (20 microCi/ml), 1.48 MBq/ml (40 microCi/ml) or 2.96 MBq/ml (80 microCi/ml) 67Ga. Compared with non-irradiated control cells proliferation during incubation was almost abolished. Clonogenic survival was measured by a colony forming unit assay (CFU-assay). In 13 of the 23 patients (56%) sufficient colony growth was observed for evaluation. The mean clonogenic survival of blasts after incubation with 0.74 MBq/ml and 2.96 MBq/ml 67Ga was 22.5, 11.3 and 3.5%, respectively. In some cases colony growth was completely abolished after incubation with only 0.74 MBq/ml 67Ga. No correlation was found between cellular 67Ga-uptake, (micro)dosimetry and transferrin receptor density (CD-71) via which 67Ga enters the cell. In vitro the blasts received a dose of > 10 Gy in 9 of the 2.96 MBq/ml, in 3 of the 1.48 MBq/ml and in 2 of the 0.74 MBq/ml incubations. In one patient, even a radiation dose > 40 Gy was reached. Low dose rate irradiation is known to arrest cells in G2/M-phase of the cell cycle, but no such arrest was observed during incubation with 67Ga. Thus, 67Ga induces clonogenic cell death in leukemic blasts. Cellular uptake of 67Ga in vitro varies between patients and can be very high in some patients. The easy availability, low costs and absence of immunological problems warrant further investigation of the therapeutic potential of 67Ga in refractory or relapsed AML patients.

Granulocyte colony-stimulating factor mobilized whole blood containing over 0.3 x 106/kg CD34+ cells is a sufficient graft in autologous transplantation for relapsed non-Hodgkin's lymphoma

Summary. The feasibility of unprocessed, granulocyte colony‐stimulating factor (G‐CSF)‐mobilized whole blood (WB) as an alternative stem cell source for autologous stem cell transplantation was studied. Forty‐seven relapsed non‐Hodgkins lymphoma (NHL) patients entered the study. After two or three ifosfamide, methotrexate and etoposide (IMVP) courses, 1 l of G‐CSF‐mobilized WB was collected and stored refrigerated for 72 h. Meanwhile, BAM conditioning was given: BCNU (carmustine) 300 mg/m2, high‐dose cytarabine 6000 mg/m2 and melphalan 140 mg/m2. Toxicity, haematological recovery and survival were assessed and compared with peripheral blood stem cell transplantation (PBSCT) and bone marrow transplantation (BMT) reference groups. High‐dose G‐CSF (2 × 12 µg/kg/d) gave the best mobilization results. Haematological recovery was related to the WB CD34+ content. A CD34+ threshold of ≥ 0·3 106/kg, obtained in 90% of patients using high‐dose G‐CSF, correlated with adequate recovery: absolute neutrophil count (ANC) > 0·5 × 109/l: median 12 d (range 9–19). Platelet recovery > 20 and > 50 × 109/l was 19 (11–59) and 30 d (14 not reached) respectively. Overall survival of patients < 60 years was 57% at 4 years and event‐free survival was 32%. Survival was comparable with PBSCT and BMT after BEAM (BCNU, etoposide, cytarabine, melphalan). Remarkably, haematological recovery after BAM + WB was rapid and comparable (ANC) or slightly prolonged (platelets) in comparison with BEAM + PBSCT, despite a 10–20 times lower CD34+ cell dose in the WB graft. In conclusion, transplantation of WB containing ≥ 0·3 × 106/kg CD34+ cells after BAM conditioning is a safe procedure, and offers a fully equivalent and less costly alternative for PBSC.