Engraftment capacity of mesenchymal cells following hematopoietic stem cell transplantation in patients receiving reduced-intensity conditioning regimen.

The engraftment ability of mesenchymal cells was investigated in 26 patients receiving allogeneic transplantation from HLA-identical siblings with reduced-intensity conditioning (RIC). The stem cell source was bone marrow (BM) in eight patients and G-CSF-mobilized peripheral blood hematopoietic cells in 18 cases. A total of 32 patients engrafted very quickly and the chimerism evaluation (both on myeloid and on lymphoid subsets) showed that they were full donor by day 60. At the time of the study they were in complete hematological remission and displayed a full donor hematopoiesis. Two patients showed early disease progression while one did not engraft. Forty-eight out-marrow samples harvested from the 26 patients generated a marrow stromal layer adequate for the chimerism evaluation. Monocyte-macrophage contamination of marrow stromal layers was always reduced below 2% by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. The chimerism evaluation was performed by PCR analysis of STRs microsatellites and the amelogenin locus, by using capillary electrophoresis (CE) and by FISH analysis in case of the sex mismatch. In eight patients, a partial donor origin of stromal cells was shown (7-86% cells of donor). The source of hematopoietic cells was BM in three patients and mobilized peripheral blood in the other five.

Ex vivo pharmacological purging of leukapheresis collections with nitrogen mustard: amifostine pretreatment improves both early and late peripheral blood progenitor cell recovery.

Ex vivo pharmacological purging of bone marrow has been used to eliminate clonogenic tumor cells contaminating the autograft and potentially responsible of relapse. A considerable improvement of pharmacological purging would be achieved only if normal marrow progenitor cells could be selectively protected by the cytotoxicity of these agents. Amifostine (WR-2721; Ethyol), a phosphorylated aminothiol compound, has been shown to have this property both in vivo and in vitro. We describe here, an experimental model for ex vivo purging of peripheral blood progenitor cell (PBPC) collections based on the combination of 3 mg/ml of amifostine and the alkylating agent nitrogen mustard. Amifostine pretreatment resulted in a statistically significant protection of normal late and early progenitor cells. Under the same experimental conditions, we observed a 4-6 log reduction of contaminating leukemic cells (i.e., K-562 and CEM) and in contrast to the protection of normal peripheral blood progenitor cells, preincubation of contaminating K-562 or CEM with amifostine did not significantly alter the LD95 nitrogen mustard concentration. Moreover, when we tested fresh human leukemia progenitor cells, amifostine pretreatment sensitized the leukemic cells to the cytotoxic effects of NM.

Optimization of the yield of PBSC for autotransplantation mobilized by high-dose chemotherapy and G-CSF: proposal for a mathematical model.

We analyzed the results of 71 leukapheresis procedures performed in 21 patients to identify the best predictive factors affecting the yield of peripheral blood progenitors after high-dose chemotherapy followed by G-CSF administration. An average of 1 +/- 1 x 10(8) MNC/kg, 5 +/- 6 x 10(4) CFU-GM/kg and 4 +/- 6 x 10(6) CD34+ cells/kg was collected for each leukapheresis. When we defined > or = 5 x 10(4)/kg as the minimum number of CFU-GM per procedure for a 'satisfactory' collection, multiparameter analysis of clinical features and laboratory findings showed that the only factors that predicted the numbers of CFU-GM collected were prior treatment with the MOPP regimen and the number of mononuclear cells identified in the basophil channel of the H*1 = Technicon. A logistic regression analysis performed to generate a mathematical model revealed four predictive factors: the number of previous cycles of chemotherapy, previous MOPP chemotherapy, the interval from latest chemotherapy and the number of mononuclear cells/microliter. This model was valuable in defining the optimal time for the first leukapheresis procedure. In contrast, the number of circulating CD34+ cells did not correlate with CFU-GM numbers collected whereas the numbers of mononuclear cells did provide a simple and reliable index. Thus the principal factor affecting the efficiency of peripheral blood stem cell collection was prior therapy with MOPP.

DHAP regimen plus G-CSF as salvage therapy and priming for blood progenitor cell collection in patients with poor prognosis lymphoma.

We studied 14 patients affected by lymphoma to assess the toxicity, efficacy and mobilization capability of salvage DHAP regimen followed by G-CSF 5 micrograms/kg/day. Ten patients were affected by intermediate-grade NHL and 4 by HD; all of them were in relapse or in PR. We administered a total of 34 courses of DHAP plus G-CSF (median 2 per patient; range 1-5) and did not observe either life-threatening extrahematologic toxicity or severe infections during the short neutropenic period. A significant tumor burden reduction was observed in 86% of patients (50% CR, 36% PR). A total of 35 aphereses were performed (median 3 per patient; range 1-5). The hemopoietic progenitors showed a very rapid increase from day +11 with a synchronous and impressive peak on day +13. We collected a median of 2.6 x 10(6)/kg CD34+ cells, 10 x 10(4)/kg CFU-GM, 5 x 10(4)/kg BFU-E and 0.5 x 10(4)/kg CFU-GEMM per apheresis. All patients transplanted with PBPC had a rapid and sustained hematological recovery. The DHAP regimen followed by G-CSF proved to be a very effective and well-tolerated schedule for debulking disease before transplantation and for enhancing progenitor cell mobilization.

Addition of erythropoietin to granulocyte colony-stimulating factor after priming chemotherapy enhances hemopoietic progenitor mobilization.

Hemopoietic progenitor cell mobilization intended for autotransplantation is now feasible in many patients, following the administration of single cytokines (G-CSF, GM-CSF, IL-3) or their combination. Erythropoietin (EPO) is a cytokine which showed an interesting activity also on non-erythroid progenitors, however the clinical relevance of this activity has not been sufficiently investigated yet. This retrospective study has attempted to assess the effectiveness of the combination of EPO plus G-CSF after priming chemotherapy to increase the number of blood progenitor cells, as compared to the results obtained by G-CSF alone. Thirty-four patients underwent priming chemotherapy followed by cytokine administration: 18 patients received G-CSF 5 micrograms/kg/day and 16 patients G-CSF plus EPO 50 U/kg/day. The two groups were homogeneous as regards the main clinical characteristics which are thought to affect BPC mobilization. As for hemopoietic progenitor cell mobilization, we observed that the combination of EPO and G-CSF was more effective in comparison with G-CSF alone, with a median of 1.9-fold for circulating MNC, 4.0-fold for CFU-GM, 4.7-fold for BFU-E and 2.8-fold increase for CD34+ cells. The results of apheresis collections revealed that the same group of patients showed better results for total blood progenitor cells/kg. The difference was statistically significant both for BPC mobilization and collection. Our findings suggest that EPO has a synergistic activity with G-CSF in mobilizing hemopoietic progenitors; the good results obtained, despite our pretreated patients, suggest that this cytokine combination has both biologic and clinical relevance.

Very low toxicity and good quality of life in 48 elderly patients autotransplanted for hematological malignancies: a single center experience.

Between May 1994 and May 2000, we autotransplanted 48 consecutive patients, 21 females and 27 males aged over 60 years (range: 60-78, median: 63). Sixteen patients had multiple myeloma (MM), 14 high-grade non-Hodgkin's lymphoma (HGNHL), six low-grade non-Hodgkin's lymphoma (LGNHL), nine acute myeloid leukemia (AML), one chronic lymphocytic leukemia (CLL), one Hodgkin's disease (HD) and one breast cancer; the performance status (WHO) was 0-1. Seventeen patients were in 1st CR (35.4%) and one in 2nd CR (2.1%), 25 in PR (52.1%), while five patients had been transplanted with progressive disease (10.4%); seven patients with MM received a double transplant. Patients received high-dose therapy including melphalan alone (13) or associated with other drugs (26), busulfan-cyclophosphamide (three), BEAM (11) and TBI (two). All patients took a median of 11 (range: 8-25) days to reach neutrophils >500/microl, 13 (range: 9-83) days to reach platelets > 20,000/microl and 17 (range: 11-83) days to reach platelets > 50,000/microl. Hematological toxicity, hospital stay and supportive care did not differ from those of a cohort of younger patients. At present, 31 patients are alive (14 in CR, five in PR, five in PD and seven in relapse) and 16 died from PD at a median follow-up of 37 months (1-67). Only one patient died from transplant-related toxicity. Quality of life, evaluated using a QLQ-C30 questionnaire in 25 patients at day +90, was good. In our experience PBPC mobilization and transplantation is feasible in patients aged > or = 60 years and the toxicity of this procedure is acceptable, with an early transplant-related mortality of 1.8%; therefore patients with hematological malignancies potentially curable with high-dose therapy (HDT) should also be candidates for HDT.

Combined administration of alpha-erythropoietin and filgrastim can improve the outcome and cost balance of autologous stem cell transplantation in patients with lymphoproliferative disorders.

We compared the use of G-CSF plus EPO in a group of 32 multiple myeloma and lymphoma patients with historical controls receiving G-CSF alone. Haemopoietic reconstitution was significantly faster in patients receiving G-CSF+EPO (group B), with a median time of 10 days to achieve an ANC count >0.5 x 10(9)/l, compared to 11 days in the historical group (A). The median duration of severe neutropenia (ANC count <100/ml) was significantly shorter in group B compared to group A; platelet counts >20 x 10(9) and >50 x 10(9)/l were achieved at days + 13 and + 17, respectively in group B, compared to days + 14 and + 24, respectively, in group A (P = 0.015, 0.002) patients. The transfusion requirement was reduced in group B, with 0 (0-6) RBC units and 1 (0-5) platelet unit transfused in group B vs 2 RBC (0-9) and 2 platelet units (0-8) in group A. Median days of fever, antibiotic therapy and hospital stay were reduced in group B (9.5 days vs 22). The mean cost of autotransplantation per group A patient was 23,988 Euro, compared with 18,394 Euro for a group B patient. Our study suggests that the EPO + G-CSF combination not only accelerates engraftment kinetics, but can also improve the clinical course of ASCT.

Direct demonstration of cytokeratin filaments by electron microscopy in K562 cell lines in liquid culture.

Our previous study suggested the presence of cytokeratins in the supernatant of human K562 erythroleukemic cell line. In this study we confirm, by using an electron microscopy technique, that K562 cells contain typical intermediate tonofilaments with the characteristics of cytokeratins. After variable intervals of liquid culture, K562 cells have been examined for their clonogenic ability by immunostaining and ultrastructural study. K562 cells showed variable amounts of medium-sized filaments (intermediate filaments) of 10 nm mean size, having the submicroscopic pattern consistent with tonofilaments, arranged as electron-dense curve-shaped bundles, in perinuclear position independently from the different growth phases. A pool of monoclonal antibodies against cytokeratins confirmed the presence of cytokeratins in immunostaining, while the cytofluorimetric analysis showed an unexpected positivity of the CD34 antigen, associated with the typical adhesion molecule VLA5. In conclusion, the immunostaining with monoclonal antibodies and the ultrastructural findings suggest the expression of epithelial features in human K562 leukemic cells.

Pharmacologic bone marrow purging: is there any place for etoposide? In vitro comparison with mafosfamide.

Residual leukemic cells in a bone marrow graft may increase the risk of relapse after autotransplantation. We have compared the efficacy of etoposide with that of mafosfamide, which has been used mainly for purging in acute leukemias. First, we examined the effects of VP-16 and ASTA-Z on the normal hematopoietic progenitors and on the erythroleukemic cell K562. Subsequently, we evaluated purging activity in cocultures using two different ratios of leukemic contamination. The most effective drug concentrations in inhibiting 100% of K562 growth were 50 micrograms/ml of ASTA-Z and 70 micrograms of VP-16. Residual growth of normal colony-forming units-granulocyte-macrophage (CFU-GM) was 4.8% with VP-16 and 32.5% with ASTA-Z. In treated cocultures, ASTA-Z produced a higher inhibition of the K562 line than VP-16 at both levels of leukemic cell contamination. At 0.5% contamination, VP-16 showed higher toxicity toward normal hematopoietic progenitors than ASTA-Z. At the 5% contamination level, VP-16 completely inhibited colony formation, whereas ASTA-Z spared some normal progenitor cells (21.2%). In conclusion, in our experimental model, VP-16 did not show improved efficacy over ASTA-Z in killing leukemic cells and in sparing normal progenitors.