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In this report, we describe the homing of hematopoietic stem cells (HSCs) to non-hematopoietic tissues in lethally irradiated (9Gy) hybrid mice transplanted intravenously with lin(-)/CD117(+) bone marrow cells from ROSA26 mice. The numbers of CFU-GM in spleen of irradiated transplanted mice were well above the levels found in non-irradiated group as early as day 8 after transplant. On 12th day regeneration of lymphocytes was observed, an increase in granulocytes was detected as late as on 33rd day. Transplanted cells containing lacZ gene were detected in recipient mice by histochemistry and their location in the thymus, liver, stomach and ileum was followed during 33days post-transplantation. On 8 and 33days post-transplantation, we found massive presence of donor (lacZ(+)) cells in the thymic cortex. Hematopoietic stem cell transplantation led not only to recovery of hematopoietic and lymphoid tissues but also facilitated recovery of the small intestinal mucosa, which was significantly damaged by ionizing radiation.

Group of 152 patients (investigated before autologous transplantation) and 35 healthy donors for allogeneic transplantation was examined for the risk of infection transmission that can be associated with the infusion of cryopreserved peripheral blood progenitor cells to the patient and/or cross-contamination of stored grafts. No laboratory signs of active infection were found in 22 donors (63 %) and in 91 patients (60%). The most common was active infection by herpes viruses--50 cases in patients, 21 cases in donors; hepatitis B was found in only two cases. The rate of clinically unsuspected (but dangerous) infections in donors and patients thus remains relatively high in spite of the fact that the system of donor search and the whole transplantation procedure have improved in the last years. The system of safety assurance is extremely important and the whole palette of preventive tests according to EBMT (European Blood and Marrow Transplantation Group) and ISHAGE (International Society for Hemotherapy and Graft Engineering) is fully justified.

In a group of 71 patients and 22 donors the danger of infection transmission by infusion of cryopreserved peripheral blood progenitor cells to the patient and/or cross contamination of stored grafts was evaluated. No laboratory signs of active infection were found in 15 donors (13 related, 2 unrelated; 68%) and in 55 patients (77%). Active infection by herpesviruses was the most common (in 13 patients and 7 donors), hepatitis B being found in only one case. The cytomegalovirus IgG test was the most common marker of previous infection; it was found in 14 donors and 55 patients. The rate of clinically unsuspected infections in donors and patients including cases requiring immediate treatment among the patients group is relatively high and fully justifies the practice of prophylactic serological testing in the whole range of tests according to the European Blood and Marrow Transplantation Group and International Society for Hematotherapy and Graft Engineering in both autologous and allogeneic transplantations of hematopoietic stem cells.

Hemopoietic growth factors (HGF) and leukapheresed peripheral progenitor cells (PBPC) are increasingly used for supportive care in high-dose chemotherapy (HDC) of solid tumors. Presently, therapeutic protocols with cyclic HDC plus PBPC support are successfuly used in breast cancer patients. Administration of PBPC significantly influences hemopoietic recovery in terms of shortening the pancytopenia period which reduces the risk of dangerous complications, especially the risk of infection. As a certain controversy exists about efficacy of this therapy, large randomized studies are conducted to find more accurate conclusions. In 1998 National Cancer Institute (NCI) gave top priority to four randomized studies of HDC with PBPC support. In recent years, rising yields of PBPC are obtained. The use of new combinations and dosages of hemopoietic growth factors leads to a significant increase of progenitor cells circulating in peripheral blood. Effective mobilization regimens combinations of chemotherapy and cytokines - enable to increase the numbers of circulating progenitors as much as 100-fold. Another aspect, how to minimize the risks is to reduce the transplant volume and so reduce the amount of cryoprotective agent DMSO (dimethyl sulfoxide) and hemolysed erythrocytes. This led to the idea to use only whole blood enriched for PBPC. At present it has been used also in our patients. The results show that enriched whole blood can be used as sufficient substitution for support in intensive cyclic chemotherapy in breast cancer patients.

The aim of our project was to compare the efficacy of mobilised whole blood versus cryopreserved PBPC (peripheral blood progenitor cells) obtained by leukapheresis in the support of hematopoietic recovery in cyclic intensive chemotherapy. Twenty-nine women with breast carcinoma were treated. The mean age was 46 years. In stage III were 23, in stage IV were 6. They received 6 cycles of epirubicin 150 mg/m2 and cyclophosphamide 1250 mg/m2. In the first cycle, 24 hours after chemotherapy, application of G-CSF 5 microg/kg/day was started, and discontinued when leukaphereses and whole blood collections were done. Leukapheresed progenitors were then divided into 3 aliquots, cryopreserved and reinfused after the 4th, 5th and 6th chemotherapy cycles. Mobilised whole blood was collected on day 14 of the 1st and 2nd cycles and reinfused 24 hours after chemotherapy. The occurrence of grade IV leukopenia was 1.82 times higher with whole blood support and grade IV thrombocytopenia 2.64 times higher than in cycles with cryopreserved PBPC support. This resulted from the fact that in one application the numbers of CD34+ cells and CFU-GM were nearly double in cryoconcentrates. The yields of CD34+ cells in 450 ml of whole blood were 1.8 x 10(6)/kg, which is not sufficient for optimal hemopoietic recovery.

The efficacy of autologous peripheral stem cells given as mobilized whole blood or leukapheresis product for hematopoietic rescue after intensive chemotherapy was studied in 34 consecutive female patients with high-risk breast cancer. All patients received six cycles of chemotherapy regimen EC (epirubicin 150 mg/m2 and cyclophosphamide 1250 mg/m2) at 14-day intervals. In the first cycle, chemotherapy was given on day 1, and 24 h later mobilization of PBPC was started with G-CSF at a dose of 5 microg/kg/day for 13 days. In all other cycles, G-CSF was given at the same dose from day 7. On days 11, 12, and 13, leukaphereses were performed, and whole blood was collected on day 14 (the peak incidence of colony-forming units-granulocyte-macrophage [CFU-GM] burst-forming units-erythrocyte [BFU-E], and colony-forming unit-granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]). The second cycle of chemotherapy was started on day 15, and 24 h later, whole blood (collected in the first cycle) was reinfused, and the same was done in the third cycle. In the fourth to sixth chemotherapy cycles, leukapheresis product was used for hematopoietic rescue. The median increment of absolute values in both whole blood and leukapheresis product was as follows: CD34+ cells over baseline was approximately 17.4-fold, CFU-GM was 85.3-fold, BFU-E was 95.9-fold, and CFU-GEMM was 44.2-fold. In the cycles with whole blood support, the mean values of applied progenitors per cycle were CD34+ cells 1.52 x 10(6)/kg, CFU-GM, 1.18 x 10(5)/kg, BFU-E 2.54 x 10(5)/kg, CFU-GEMM 0.31 x 10(5)/kg. In the courses with PBPC support, the mean values of progenitors were CD34+ 2.04 x 10(6)/kg, CFU-GM 1.59 x 10(5)/kg, BFU-E 2.87 x 10(5)/kg, and CFU-GEMM 0.34 x 10(5)/kg. Leukopenia in patients supported with whole blood versus leukapheresed PBPC was as follows: grade 4, 13/6 (38.2%/17.6%), grade 3, 19/23 (55.9%/70.6%), and grade 2, 1/4 (2.9%/11.8%), respectively. Thrombocytopenia was grade 4, 11/6 (32.4%/17.6%), grade 3, 10/7 (29.4%/20.6%), grade 2, 7/13 (20.6%/38.2%), and grade 1, 6/6 (17.6%/17.6%), respectively. The median follow-up analysis was at 24.6 (7-36) months. High-risk patients previously treated with surgery and adjuvant chemotherapy (n = 5) were not evaluated for response. In 21 patients with locally advanced or inflammatory breast carcinoma the response rate (RR) was 94%, CR was 90%, and PR was 15%. No response to therapy was observed in 1 patient. In 8 patients with metastatic disease, RR was 75%, there was no CR, and PR was 75%. Two patients died during therapy. Relapse-free survival (RFS) in the adjuvant group was 23.7 (range 12-36) months and in the group with locally advanced disease was 18.2 (range 7-27) months. In the group with metastatic disease, time to tumor progression (TTP) was 12.1 (range 1-16) months. Mean duration of hospital stay for whole blood reinfusion in the second and third chemotherapy cycles was 6.7 (range 5-8) days and for PBPC in the fourth to sixth cycles was 6.2 (range 4-8) days, which at p < 0.001 was not statistically significant.

The role of erythropoietin (EPO) plus granulocyte-colony stimulating factor (G-CSF) combination in hemopoietic recovery was studied in patients with high-risk breast carcinoma and compared to a control group of previously treated identical patients who were not given EPO plus G-CSF. Eleven consecutive patients admitted to this study had Stage III or IV breast cancer. They received 6 cycles of intensive chemotherapy (epirubicin 150 mg/m2 and cyclophosphamide 1300 mg/m2). The 1st cycle served for mobilization of peripheral blood progenitor cells (PBPC). At its end leukaphereses collections of PBPC were performed to be used as hematologic support (PBPCT) in the 5 remaining cycles. The administration of EPO plus G-CSF was started when leukocyte (WBC) count in peripheral blood dropped below 1 x 10(9)/l and hemoglobin (Hb) level fell below 100 g/l. The treatment was stopped when leukocyte count rose to 5 x 10(9)/l and Hb to 130 g/l. EPO plus G-CSF combination after PBPCT produced significant effects in terms of hemopoietic recovery, clinical benefit and supportive care requirements when compared with 12 historic control patients: Periods of leukopenia were shorter which resulted in reduced risk of infectious complications. The grades of leukopenia in the study and control groups were as follows: grade 4 (36 vs. 18%), grade 3 (57 vs. 30%), grade 2 (7 vs. 13%) respectively. Significantly shorter was the time of PLT recovery < 50 x 10(9)/l (p < 0.001). The grades of thrombocytopenia were: grade 4 (29 vs. 11%), grade 3 (21 vs. 12%), grade 2 (25 vs. 36%) respectively. The number of necessary transfusions was significantly reduced as well as the length of hospital stay (p < 0.001). In conclusion, our results obtained in this study confirm that combination of EPO plus G-CSF not only increases the rate of hemopoietic recovery, reduces the number of necessary red blood cell and platelet transfusions but, at the same time, simplifies the clinical management and is more tolerable for the patients.

To support multicyclic, dose-intensive chemotherapy in breast cancer, we assessed the effects of reinfusing hematopoietic progenitors either as a leukapheresis product or as mobilized unprocessed whole blood. In this clinical study, 16 consecutive female breast cancer patients were given six cycles of chemotherapy regimen EC (epirubicin (150 mg/m2) and cyclophosphamide (1250 mg/m2) on day 1). In the first cycle, 24 h after chemotherapy, mobilization of the peripheral blood progenitor cells (PBPC) was started with growth factor G-CSF (Neupogen; Amgen-Roche) at a dose of 5 microg/kg/day for 13 days. In all other cycles G-CSF had been given at the same dose from day 7. On days 11, 12 and 13 the leukaphereses were performed and their products cryopreserved. On day 14 whole blood was collected. The median peak incidence of CFU-GM (granulocyte-macrophage colony-forming unit) in peripheral blood was approximately 50 times the baseline level. The leukapheresed PBPC were divided into portions and reinfused after the fourth, fifth and sixth chemotherapy courses. The support with mobilized whole blood was given after the second and third cycles. The effects of the support of whole blood vs leukapheresed PBPC on hematopoietic recovery were compared. The best yields of leukaphereses were achieved on day 13 after initiation of the chemotherapy. The mean number of CD34+ cells was 4.93 x 10(6)/kg (s.d. 2.7; range 0.36-10.54 x 10(6)/kg) the amount of CFU-GM was 2.18 x 10(5)/kg (s.d. 1.3; range 0.07-4.2 x 10(5)/kg). The yields of CFU-GM in 450 ml whole blood collected on day 14 reached 0.51 x 10(5)/kg (s.d. 0.28; range 0.05-1.5 x 10(5)/kg) and of CD34+ cells were 1.3 x 10(6)/kg (s.d. 0.8, range 0.18-2.58 x 10(6)/kg). PBPC yields in 450 ml of unprocessed whole blood were in some cases not sufficient for good hematopoietic recovery after the EC cycles. Grade 4 leukopenias and thrombocytopenias were two times higher in cycles with whole blood support than in cycles with cryopreserved PBPC support. An increase of PBPC harvest can be simply achieved by collecting larger amounts of unprocessed blood, as used by some authors.