Daily G-CSF Research Articles

Phenotypic and Functional Analysis of T-Cells Mobilized in HLA-Matched Sibling Donors Following Treatment with the Chemokine Antagonist AMD3100.

We are currently evaluating a novel method for the procurement of peripheral blood stem cells from HLA matched sibling donors using a direct antagonist of the CXCR4/SDF-1 interaction called AMD3100 (A). Donors receive a single subcutaneous injection of A and then undergo a 20 liter leukapheresis (LP) four hours later. The LP product is then cryopreserved and subsequently transplanted following ablative conditioning. To date, we have performed 15 transplants with allografts collected following A alone. In comparison to allografts collected following five days of G-CSF, A mobilized allografts contain approximately 50% less CD34+ cells but 2–3 times more CD3+ cells. Nevertheless, the kinetics of neutrophil and platelet engraftment have been virtually identical to that observed following G-mobilized allografts and grades 2–4 acute GVHD has been observed in only 20% of recipients. We sought to analyze the functional and phenotypic properties of T cells collected following A alone to understand the relatively low rates of acute GVHD despite the transplantation of higher T-cell doses. In 3 donors, extensive T cell phenotyping was performed on donor peripheral blood prior to A, 6 hours following A, and also on the LP product collected after A. Specifically, we were seeking to determine whether any alteration in CD4+ or CD8+ subsets had occurred. We analyzed T-cell subsets using well described markers for central memory, effector memory, naïve, and effector memory RA phenotypes. We also assessed expression of CD62L, CD127, CCR7, and SLAM family members (CD48, CD150, and CD244) on both CD4+ and CD8+ cells. The activation status on CD4 and CD8 cells was assessed using markers for CD25, CD30, and CD69. Finally we assessed for quantitative changes in the mobilization of regulatory T cells by assaying the proportion of CD4+CD25+FoxP3+ cells mobilized following A. In none of these analyses could we detect any significant alteration in the relative ratios of CD4 or CD8 subsets mobilized by A. Finally, the functional capacity of purified CD3+ cells collected following A was assessed using a NOD/SCID xenogeneic GVHD model we have recently developed. In that model, survival of mice transplanted with A mobilized T-cells was similar to that observed with untreated T cells, suggesting that A mobilized T cells retain their GVHD-inducing capacity. In summary, these preliminary data suggest that AMD3100 induces a “pan-mobilization” of T cell subsets without any apparent skewing toward a particular subset. These studies are in contrast to others suggesting subtle phenotypic and functional changes in donor T cells after mobilization with G-CSF. Further studies evaluating A mobilized allografts are ongoing.

AMD3100 with G-CSF for Autologous Peripheral Blood Progenitor Cell (PBPC) Mobilization in Non-Hodgkin's Lymphoma (NHL) and Multiple Myeloma (MM): Mobilization, Engraftment, and Pharmacokinetics (PK).

Background: AMD3100 potentiates the effect of G-CSF (Filgrastim) to mobilize hematopoietic PBPCs from the bone marrow into peripheral blood (PB) and may decrease the number of aphereses required to collect sufficient PBPCs for transplantation.Methods: Mobilization treatment consisted of subcutaneous G-CSF 10 ug/kg given in the morning on 5 consecutive days and a single dose of AMD3100 240 ug/kg in the evening of day 4, 10 – 11 hours prior to leukapheresis. These doses of G-CSF and AMD3100 were continued for up to 4 additional days in order to reach the target of ≥ 5 × 10^6 CD34+ cells/kg. Blood samples for FACS analysis of CD34+ cells were obtained immediately prior to dosing with AMD3100 and pre-apheresis the next morning. PK parameters were determined from samples obtained immediately prior to dosing, 0.25, 0.5, 1, 2, 4, 6 and 8 hours after the injection of AMD3100, and just prior to apheresis.Clinical Results: Nineteen pts have thus far been enrolled, 11 MM and 8 relapsed NHL. AMD3100 was well tolerated. Mobilization characteristics are summarized in table 1; 100% of MM pts and 63% of NHL pts reached the target of 5 × 10^6 CD34+ cells/kg; all collected > 2 ×10^6 CD34+ cells/kg. For apheresis day 1, AMD3100 increased absolute CD34+ counts ≥ 2-fold in 100% of pts; the median increase for MM pts was 3-fold (range: 2–7.7 fold) or an absolute increase of 108 CD34+ cells/uL (range: 40–166), whereas the median increase for NHL pts was 2.7-fold (range: 2.2–3.7 fold) or an absolute increase of 35.5 CD34+ cells/uL (range: 11–99). Among the relapsed NHL pts, who were characterized by lower pre-AMD3100 CD34+ counts, 4 had increase in PB CD34+ cells/ul from < 20 to > 20 and one other increased from 6 to 17. Of the 17 pts who have subsequently been transplanted; median time to engraftment of neutrophils >0.5 ×10^9/L and platelets >20 ×10^9/L was 11 and 18 days, respectively.PK Results: PK determinations for AMD3100 were completed in a subset of patients (4 MM; 3 NHL) using plasma samples obtained after the first dose. AMD3100 was rapidly absorbed following subcutaneous injection with a median tmax of 30 (range, 15–60) minutes and Cmax of 0.84 (range, 0.77–1.03) ug/ml. Plasma concentrations declined in a bi-exponential manner, with a median elimination half-life of 4.6 (range, 2.71–5.32) hours. The median AUC 0-infinity was 4410 (range, 2682–5165) ug-hr/ml. AMD3100 pharmacokinetics were consistent with results previously obtained in studies conducted using healthy volunteers in the absence of G-CSF.Conclusions:AMD3100 was generally safe and well tolerated in both groups.PK in the cancer patients was similar to that in previously studied healthy volunteers.AMD3100 increased PBPC mobilization following 4 days of G-CSF, and resulted in collecting large numbers of PBPCs in MM patients and facilitated adequate PBPC collections for poor mobilizers within the relapsed NHL group.Table 1:Mobilization characteristicsNo. of ptsNo. apheresis days (range)Median CD34+ cells collected (×10^6/kg)>2×10^6/kg collected on day 1>5×10^6/kg collected on day 1MM111 (1–2)13.1 (5.8–19.7)100%82%NHL82 (1–5)5.6 (2.8–10.6)88%50%All191 (1–5)9.0 (2.8–19.7)95%68%

Prospective randomised comparison of the COBE Spectra version 6 and Haemonetics MCS+ cell separators for hematopoietic progenitor cells leucapheresis in patients with multiple myeloma

A randomised crossover trial of two separators was undertaken to compare the mononuclear cell, CD34(+) cell and CFU-GM yield, in patients (<61 years) with previously untreated symptomatic multiple myeloma. After first-line therapy, all patients received mobilising chemotherapy (cyclophosphamide 4 g/m(2)) and daily G-CSF. The first leucapheresis was performed on the first day the peripheral blood absolute CD34(+) cell count was > 20 cells/microl. All patients underwent 2 leucaphereses on consecutive days. The patients were randomised to undergo either the first or second leucapheresis using the COBE Spectra. The target duration of the procedure on the COBE Spectra was 2 total blood volumes, and for the Haemonetics MCS(+) it was 20 cycles with four recirculations. Between September 2003 and March 2005, 60 patients were entered in the study. COBE Spectra version 6 processed significantly larger volumes of blood than the Haemonetics MCS(+) (8,845 and 5,680 ml, respectively, P < 0.01). The absolute yield of mononuclear cells (2.1 vs. 1.5 x 10(8)/kg, P = 0.04), CFU-GM (11 vs. 3 x 10(4)/kg, P = 0.01) and CD34(+) cells (3 vs. 1.7 x 10(6)/kg, P = 0.02) were all significantly higher with the COBE Spectra version 6, as were the yields per unit volume of blood processed. In conclusion, our study shows that COBE Spectra Version 6 is faster and has a better yield than the Haemonetics MCS(+), in patients with multiple myeloma.

High Dose Cyclophosphamide (CY) for Severe Aplastic Anemia (SAA): Safety and Long Term Follow-Up.

Background: Acquired SAA is a potentially life-threatening bone marrow failure disorder that is usually immune-mediated. SAA can be effectively treated with bone marrow transplantation (BMT), immunosuppressive therapy (IST), or CY. BMT can cure the disease but ideally requires a matched sibling donor. IST has a high response rate, but up to 50% of patients relapse, become dependent on cyclosporine, or develop secondary clonal disease such as paroxysmal nocturnal hemoglobinuria (PNH) or myelodysplastic syndromes (MDS). We previously showed that CY induces durable remissions in the majority of SAA patients, but concern has been raised about the safety of this approach.Methods: Since 1996, we treated 38 previously untreated, and 17 IST failed, SAA patients with CY (50 mg/kg/d x 4) followed by daily G-CSF (5 ug/kg/day) until the neutrophil count (ANC) reached 1000/dl. Response was defined as ANC > 1000/dl and transfusion independence without growth factor support for > 3 months. Relapse was defined as no longer meeting criteria for response. PNH was monitored by flow cytometry.Results: The median age of the newly diagnosed patients was 40 (range 2–68) years. With a median follow-up of 41 (range, 6 – 111) months, 33/38 patients survive (actuarial survival of 86%, 95% CI 72–95%) with 28 (74%, 95% CI 58–85%) achieving remission, most being complete. Median time to ANC of 500, last platelet and red cell transfusion was 50, 99, and 181 days, respectively. Before treatment, 15 patients met criteria for very (v) SAA (ANC < 200). Mortality within 6 months after CY occurred in 4 (10.5%) patients, all with vSAA; 1 additional patient died from bacterial sepsis 18 months after CY. 22/23 (96%) SAA patients survive (20 in remission) compared to 11/15 (73%) with vSAA (10 in remission). Eight patients had a severe infection at the time of beginning treatment and 5 survive in remission. PNH screening revealed a PNH population ranging from 0.5–40% of granulocytes in 12 patients, and all 12 achieved a durable remission (p = 0.039). No patient in this series has progressed to PNH or MDS, and the PNH clone is regressing in all 12 patients. Two patients have relapsed. One patient, whose first remission lasted 5 years, was retreated with CY into a persisting second complete remission 3 years ago; another patient recently relapsed 3 years after achieving remission. Ten of the 17 patients who failed IST (median age of 31, range 6–58) are alive and nine are in remission.Conclusions: Cy is safe and highly effective therapy for both untreated and relapsed SAA. Relapses after CY are rare and progression to PNH or MDS in previously untreated patients has not been observed in this series with now 15 patients out beyond 5 years. The presence of a PNH population may be a favorable risk factor, perhaps by excluding non-immune mediated forms of SAA. [Display omitted]

Distinct Gene Expression Pattern of CD34+ Stem and Progenitor Cells Mobilized by Pegfilgrastim after Cytotoxic Therapy in Multiple Myeloma in Comparison to G-CSF.

Background: Current regimens for peripheral blood stem cell (PBSC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of G-CSF starting shortly after cytotoxic therapy. Recently a polyethylenglycole (PEG)-conjugated G-CSF (pegfilgrastim) has been introduced which has a substantially longer half-life than the original formula. Here, we compared the molecular phenotypes of CD34+ stem and progenitor cells mobilized by G-CSF with those mobilized by pegfilgrastim.Study design and Methods: We examined immunomagnetically enriched CD34+ cells from leukapheresis products of 8 patients who received G-CSF and of 8 patients who were given pegfilgrastim using Affymetrix HG Focus GeneChips covering 8793 genes. The statistical scripting language ‘R' was used for data analysis. Significantly differentially expressed genes were identified with the Significance Analysis of Microarrays (SAM) algorithm.Results: Comparing CD34+ cells mobilized by G-CSF with pegfilgrastim-mobilized CD34+ cells 108 genes were differentially expressed (fold change 1.25 – 14.0, q- value 2.45–14.44%). 38 genes had a higher and 70 genes had a lower expression in CD34+ cells mobilized by G-CSF.We found upregulation of genes characteristic for erythropoietic differentiation including haemoglobin chains and Erythroid Kruppel-like factor in G-CSF-mobilized CD34+ cells. Utilizing clonogenic assays we were able to functionally corroborate this finding as G-CSF-mobilized cells gave rise to a significantly higher number of burst-forming units erythroid (BFU-E) as compared to colony forming units granulocyte-macrophage (CFU-GM) (p=0.016). Cell cycle regulating genes were differentially expressed as well. Genes encoding for proteins that cause cell cycle arrest including human HTm4 were upregulated in G-CSF-mobilized cells, as opposed to an upregulation of cell cycle-promoting genes including Cyclin D2 and Hepatocyte Leukemia Factor (HLF) in pegfilgrastim-mobilized cells.Moreover in pegfilgrastim-mobilized CD34+ cells we saw an upregulation of multiple genes involved in cellular immunogenicity like MHC class I and II antigens and genes encoding for proteins playing a role in antigen presentation.Conclusion: Unconjugated G-CSF seems to be associated with an increased mobilisation of erythroid progenitors or an induction of erythropoiesis. Pegfilgrastim might result in mobilization of more immunogenic CD34+ cells. Unconjugated G-CSF and pegfilgrastim both seem to have an effect on cell cycle. Unconjugated G-CSF might rather induce cell cycle arrest and pegfilgrastim seems to lead to an increase of the cell cycle activity. This may be due to potentially different effects of continuously high serum levels of G-CSF maintained by pegfilgrastim and the pulsatile daily G-CSF injections on CD34+ cells.

A Detailed Phenotypic Analysis Using Six Color Flow Cytometry of Lymphocyte Subsets Mobilized with AMD3100 Compared to G-CSF.

AMD3100 (AMD) is a bicyclam compound that rapidly mobilizes hematopoietic progenitor cells into circulation by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. Preliminary data in healthy donors and cancer patients show large numbers of CD34+ cells are mobilized following a single injection of AMD3100. To determine whether AMD3100 mobilized cells would be suitable for allografting, we performed a detailed phenotypic analysis using 6 color flow cytometry (CYAN Cytometer MLE) of lymphocyte subsets mobilized following the administration of AMD3100, given as a single 240mcg/kg injection either alone (n=4) or in combination with G-CSF (n=2: G-CSF 10 mcg/kg/day x 5: AMD3100 given on day 4). Baseline peripheral blood (PB) was obtained immediately prior to mobilization; in recipients who received both agents, blood was analyzed 4 days following G-CSF administration as well as 12 hours following administration of AMD3100 and a 5th dose of G-CSF. AMD3100 alone significantly increased from baseline the PB WBC count (2.8 fold), Absolute lymphocyte count (ALC: 2.5 fold), absolute monocyte count (AMC: 3.4 fold), and absolute neutrophil count (ANC: 2.8 fold). Subset analysis showed AMD3100 preferentially increased from baseline PB CD34+ progenitor counts (5.8 fold), followed by CD19+ B-cells (3.7 fold), CD14+ monocytes (3.4 fold), CD8+ T-cells (2.5 fold), CD4+ T-cells (1.8 fold), with a smaller increase in CD3−/CD16+ or CD56+ NK cell counts (1.6 fold). There was no change from baseline in the % of CD4+ or CD8+ T-cell expressing CD45RA, CD45RO, or CD56, CD57, CD27, CD71 or HLA-DR. In contrast, there was a decline compared to baseline in the mean percentage of CD3+/CD4+ T-cells expressing CD25 (5.5% vs 14.8%), CD62L (12.1% vs 41.1%), CCR7 (2.1% vs 10.5%) and CXCR4 (0.5% vs 40.9%) after AMD3100 administration; similar declines in expression of the same 4 surface markers were also observed in CD3+/CD8+ T-cells. A synergistic effect on the mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF (Figure). In those receiving both AMD3100 and G-CSF, a fall in the % of T-cells expressing CCR7 and CXCR4 occurred 12 hours after the administration of AMD3100 compared to PB collected after 4 days of G-CSF; no other differences in the expression of a variety activation and/or adhesion molecules on T-cell subsets were observed. Whether differences in lymphocyte subsets mobilized with AMD3100 alone or in combination with G-CSF will impact immune reconstitution or other either immune sequela (i.e. GVHD, graft-vs-tumor) associated with allogeneic HCT is currently being assessed in an animal model of allogeneic transplantation. [Display omitted]

The Kinetics of Etoposide (VP-16) Peripheral Blood Progenitor Cell (PBPC) Mobilization.

The kinetics of G-CSF PBPC mobilization are well described. CD34+cells in peripheral blood rise four days after G-CSF administration, and decline after the eighth day. VP-16 is increasingly used with G-CSF as a mobilizing regimen; however, the kinetics of mobilization are sparsely described. We retrospectively reviewed 275 patients (pts) with NHL or HD who received VP-16 plus G-CSF as a primary PBPC mobilizing regimen. 214 (78%) had NHL; 31% received prior radiation therapy; 63% were male; 85% had responsive disease. Pts received VP-16 (2 gm/m2) followed by daily G-CSF (10 mcg/kg). All pts experienced a significant WBC nadir. Pts began pheresis when their WBC recovered to 5,000. Pts were pheresed for at least 2 days or until 7.0 x 106 CD34+ cells/kg were collected; pheresis continued until a minimum of 2.0 x 106 CD34+ cells were collected. WBC nadir occurred day +6 after VP-16 and WBC recovery to 5,000 occurred day +13 (median) after VP-16. Pts were pheresed for a median of 3 days which yielded a median of 9.7 x 106 CD34+ cells/kg (range, 2.0–100.1 x 106). 72% of pts began collection on day +12, day +13, or day +14. The average CD34+ cell collection was maximal on the first 3 days of pheresis, but reasonable yields continued to be obtained for approximately 10 days of pheresis, as shown below: [Display omitted] 81% collected ≥ 5 x 106 CD34+ cells/kg, and 72% collected ≥ 7 x 106 CD34+ cells/kg. The platelet count on the first day of pheresis correlated with the ability to collect 5 and 7 x 106 CD34+ cells/kg (p<0.001). 50 of 275 (18%) pts did not begin pheresis until day +15 or longer after VP-16 administration. The variables that correlated with these problematic pts was the platelet count at day 1 of pheresis and refractory disease at the time of mobilization; of 24 pts with refractory disease, 11 (46%) had poor WBC recovery after VP-16 and initiated mobilization on a median of day +16 (p<0.001). We conclude that VP-16 + G-CSF is a very effective mobilizing regimen. The majority of pts mobilize large numbers of CD34+ cells requiring only 3 days of leukopheresis for excellent yields. Unlike the kinetics of G-CSF for PBPC mobilization in which yields of CD34+ cells are optimal for only 3–4 days, CD34+ cell collection continues for approximately 10 days from the initiation of pheresis. A minority of pts have poor WBC recovery after VP-16 administration, and these pts tend to be those with refractory disease.

Computational Optimisation of Chemotherapy Regimens Accounting for Heterogeneity of Myelotoxicity - a Worked Example for Lymphoma.

In the last years, the development of dose and time intensified chemotherapeutic regimens led to considerable improvement of treatment outcome in several diseases such as Hodgkin's lymphoma (Diehl et al, NEJM 2003), aggressive Non-Hodgkin's lymphoma (Pfreundschuh et al, Blood 2004) or breast cancer. Although the granulopoietic growth factor G-CSF is widely used, leukopenia is still a major limiting factor for such therapy intensifications. The degree of leukopenia is very inhomogeneously distributed in the population of patients even if similar dose levels are applied. Thus, further intensification is usually limited by a small subgroup of patients who respond with particularly severe toxicity.It was our objective to develop a method for estimating the potential for a differential dose-escalation with regard to expected myelotoxicity. For this purpose we used a biomathematical model of human granulopoiesis, which allows simulation of the dynamics of peripheral neutrophil concentration under a variety of chemotherapy regimens with or without G-CSF support (Engel et al, Blood 2004). This model is adapted to describe the observed heterogeneity of myelotoxic drug response.We analyzed clinical data obtained from several large phase III-trials of the German Study Group for Aggressive Non-Hodgkin's Lymphoma using CHOP-like chemotherapies. Patients were stratified into 3 risk groups (low, medium, high myelotoxicity) of approximately equal size. Stratification was performed using individual prognostic factors such as age, gender, ECOG performance status and LDH serum concentration.We were able to estimate the drug induced bone marrow damages as functions of the given dose and the related stratum. These estimates could be used to modify drug dosing and scheduling of chemotherapy in order to achieve a homogenization of myelotoxicity between strata. The aim was to intensify therapy for the low and medium risk group without exceeding the expected toxicity of the high risk group.The model simulations suggest that CHOP treatment intervals (G-CSF day 4-13) can be shortened in elderly patients from 14 to 11 and 12 days in the low and medium risk group, respectively. Alternatively, one may increase the dose of cyclophosphamide by 280mg/m2 and 100mg/m2 or apply additional etoposide (300mg/m2 and 150mg/m2) in these groups. Similar results can be obtained by a different approach using the observed degree of leukopenia during the first therapy cycle as a predictor for leukopenia in subsequent cycles. Using this risk factor for stratification into two groups, our model simulations show that cyclophosphamide doses can be increased by 360mg/m2 in the low risk group to yield a similar degree of leukopenia expected for the high risk group.We conclude that our computational model can help to design more effective chemotherapy regimens for future dose intensified clinical trials by increasing the total dose delivered in the population. Our approach may be also applicable for myelotoxic chemotherapies of other malignancies.

Phase II trial of docetaxel plus doxorubicin and cyclophosphamide in locally advanced breast cancer (LABC)

855 Introduction: It has been show that patients achieving a pathological complete response to primary chemotherapy benefit in terms of prolonged overall survival (NSABP B18). Docetaxel (D) plus doxorubicin (d) is a highly active combination in metastatic breast cancer (Mackey JR, Proc ASCO 2002) as well as in the neo-adyuvant setting (Ian C, JCO 2002). A three-drug combination could improve these results. Methods: In this study, females diagnosed of stage II-III LABC between May 2001 to October 2003 with 18–75 years, PS 0–1, and adequate renal, liver and hematological functions were included. Chemotherapy schema (TAC): D 75 mg/m2, Cyclophosphamide 500 mg/m2 and d 50 mg/m2 on day 1, every 21 days plus G-CSF days 5th to 11th. Four to six courses (c) were scheduled and assessment for clinical response (CR) was made after 2nd, 4th and 6th c. Results: 33 patients (p) were enrolled and pathological response (PR) was already available in 26p. Patient characteristics: median age 53 years (39–73); 25p had PS=0 (75,7%); 36,4% presented with stage IIA, 30,3% stage IIB, 30,3% stage IIIA, and 3,0% stage IIIB; 27p had ductal invasive histology and 6p had a lobular invasive cancer; ER were positive in 22p (66,6%) and negative in 11 p (33,4%); c-erb-B2 was +++/+++ in 7p (21,2%), ++/+++ in 9p (27,3%) and negative in 17p (51,5%). Chemotherapy: 137c were given (median 4 c/p). Surgery had been performed in 78,8% of p. Toxicity: all p had grade 3 alopecia and 4p had grade 3 asthenia; 9p had a neutropenic fever episode but none resulted in septic death. No other severe toxicity was recorded (including clinical cardiotoxicity). CR rate was assessed in 30p (90%) and it was complete in 9p and partial in 21p (overall 100%). 6p achieved a complete pathological response (PR) (18%) and 8 p a partial PR (24,2%). 15 p had a mastectomy performed (45,4%). Conclusions: This phase II study of TAC as induction chemotherapy in patients with LABC showed a high (both clinical and pathological) response rate. The main significant toxicity was the high incidence of neutropenic fever despite the routine use of stimulating factors. Final results will be presented. No significant financial relationships to disclose.

Phase II trial of docetaxel plus doxorubicin and cyclophosphamide in locally advanced breast cancer (LABC)

855 Introduction: It has been show that patients achieving a pathological complete response to primary chemotherapy benefit in terms of prolonged overall survival (NSABP B18). Docetaxel (D) plus doxorubicin (d) is a highly active combination in metastatic breast cancer (Mackey JR, Proc ASCO 2002) as well as in the neo-adyuvant setting (Ian C, JCO 2002). A three-drug combination could improve these results. Methods: In this study, females diagnosed of stage II-III LABC between May 2001 to October 2003 with 18–75 years, PS 0–1, and adequate renal, liver and hematological functions were included. Chemotherapy schema (TAC): D 75 mg/m2, Cyclophosphamide 500 mg/m2 and d 50 mg/m2 on day 1, every 21 days plus G-CSF days 5th to 11th. Four to six courses (c) were scheduled and assessment for clinical response (CR) was made after 2nd, 4th and 6th c. Results: 33 patients (p) were enrolled and pathological response (PR) was already available in 26p. Patient characteristics: median age 53 years (39–73); 25p had PS=0 (75,7%); 36,4% presented with stage IIA, 30,3% stage IIB, 30,3% stage IIIA, and 3,0% stage IIIB; 27p had ductal invasive histology and 6p had a lobular invasive cancer; ER were positive in 22p (66,6%) and negative in 11 p (33,4%); c-erb-B2 was +++/+++ in 7p (21,2%), ++/+++ in 9p (27,3%) and negative in 17p (51,5%). Chemotherapy: 137c were given (median 4 c/p). Surgery had been performed in 78,8% of p. Toxicity: all p had grade 3 alopecia and 4p had grade 3 asthenia; 9p had a neutropenic fever episode but none resulted in septic death. No other severe toxicity was recorded (including clinical cardiotoxicity). CR rate was assessed in 30p (90%) and it was complete in 9p and partial in 21p (overall 100%). 6p achieved a complete pathological response (PR) (18%) and 8 p a partial PR (24,2%). 15 p had a mastectomy performed (45,4%). Conclusions: This phase II study of TAC as induction chemotherapy in patients with LABC showed a high (both clinical and pathological) response rate. The main significant toxicity was the high incidence of neutropenic fever despite the routine use of stimulating factors. Final results will be presented. No significant financial relationships to disclose.

Hematopoietic stem cell (HSC) redistribution for cardiac repair following acute myocardial infarction (AMI)

Controversy surrounds the role of HSC in repairing cardiac damage. In some animal studies, intracoronary or intramyocardial injection of autologous HSC contributed to new myocardial tissue following acute ischemic insult. The role of removing and reinjecting HSC is uncertain. HSC mobilized from the bone marrow circulate widely. We hypothesize that circulating HSC can localize to the area of revascularized cardiac damage following myocardial infarction and that HSC engraftment may have a beneficial effect on cardiac function. A pilot trial was conducted to study the feasibility and safety of HSC mobilization following AMI. Five patients that experienced large transmural anterior AMIs received 4 daily subcutaneous injections of 10 ug/kg G-CSF. At presentation, all patients had high-grade occlusions of a coronary artery that were successfully revascularized by percutaneous procedures. Baseline cardiac bloodflow (rubidium-82 PET) and myocardial metabolism (FDG-PET) studies confirmed transmural infarction. All patients started G-CSF within 7 days of AMI. Peripheral venous blood contained a median of 1.7 CD34(+)cells per ul (range 0.7 to 7.5) prior to G-CSF and a median of 75.6 CD34(+)cells per ul (range 72 to 91) following 4 days of G-CSF. There was no toxicity associated with G-CSF administration. The median baseline left ventricular ejection fraction (LVEj) was 30% (range 23–40%). Four weeks following HSC mobilization, the median LVEj increased to 40% (range 30 to 48%). This increase was clinically and statistically significant. Improvement in FDG uptake and a trend for increased perfusion were also observed. We conclude that administration of G-CSF following AMI is safe and mobilizes HSC into the circulation. G-CSF is associated with improved left ventricular function in a group of patients with large anterior wall AMI, a greater improvement than would be expected from historical controls. These findings suggest that G-CSF acts directly upon the heart to trigger recovery or G-CSF mobilized HSC migrate and engraft in the heart aiding recovery of cardiac function. A larger, randomized trial is planned to explore these issues.

Granulocyte colony stimulating factor does not induce long-term DNA instability in healthy peripheral blood stem cell donors.

To establish the safety of using G-CSF in healthy PBSC donors, we prospectively determined the degree of DNA destabilization in peripheral blood WBC. Donors were treated with SC G-CSF for 5 days. A baseline sample of peripheral blood was collected before G-CSF treatment and consecutive samples were collected on day 5, 1 month, and 2 months after treatment. The extent of double-stranded DNA relaxation and de novo synthesis of DNA was determined in each sample. We found that both parameters of DNA destabilization were significantly increased after 5 days of G-CSF and returned to baseline level at 1 and 2 months. We assume that these findings suggest that rhG-CSF administration to healthy PBSCT donors for 5 days may be a safe procedure.

A prospective randomised concurrent comparison of the COBE Spectra Version 4.7, COBE Spectra Version 6 (auto PBSC™) and Haemonetics MCS+ cell separators for leucapheresis in patients with haematological and non-haematological malignancies

A prospective study of the CD34+ cell collection efficiency of three cell separators was undertaken comparing the mononuclear cell, CD34+ cell and CFU-GM yield. Twenty patients were entered in the study, all had received mobilising chemotherapy and daily G-CSF ( 5 μg/ kg subcutaneously). The first leucapheresis was performed when the peripheral blood absolute CD34+ cell count was ⩾20 cells/ μl . All patients underwent two leucaphereses on consecutive days. The patients were randomised to undergo either the first or second leucapheresis using the COBE Spectra Version 4.7 and then randomised to either the COBE Spectra Version 6 or Haemonetics MCS+ for the other leucapheresis. The target durations of the procedure on the COBE Spectra Version 4.7 and Version 6 were 180 min or two total blood volumes (TBV), and for the Haemonetics MCS+ was 20 cycles with four recirculations. All machines were operated on the 1997 software supplied by the respective manufacturers. The time taken for the procedure was significantly longer with both the Haemonetics MCS+ and the COBE Spectra Version 6 than the COBE Spectra Version 4.7. Both COBE Spectra versions processed significantly larger volumes of blood than the Haemonetics MCS+. The absolute yield of mononuclear cells, CFU-GM and CD34+ cells were all significantly lower with the Haemonetics MCS+ compared with both COBE Spectra Versions, as were the yields per unit volume of blood processed. There was no significant difference in the reduction in the platelet count following leucapheresis with any of the machines.

Limited toxicity in IIIA/B lung cancer patients treated with neoadjuvant concomitant chemo-twice daily radiation and G-CSF support

Limited toxicity in IIIA/B lung cancer patients treated with neoadjuvant concomitant chemo-twice daily radiation and G-CSF support

Reduction of marrow hematopoietic progenitor and stem cell content is not sufficient for enhanced syngeneic engraftment.

The mechanisms regulating long-term engraftment of primitive stem cells are largely unknown. Most conditioning strategies use myeloablative agents for experimental or clinical hematopoietic stem cell transplantation. Host conditioning regimens, in part, have been designed on the assumption that transplanted cells home to specific marrow sites and if these sites are occupied by host stem cells, engraftment will not take place. However, there is now evidence that stable long-term syngeneic engraftment may occur in the absence of host marrow stem cell depletion. To further study the association of engraftment with stem cell depletion, we investigated whether the marked egress of hematopoietic progenitor and stem cells from the marrow into the peripheral blood in C57BL6 mice following a single dose of cyclophosphamide (day 1) and four days of G-CSF (days 3-6) afforded an increased opportunity for long-term syngeneic donor engraftment. During and after mobilization, glucose phosphate isomerase (GPI)-1(b) mice received 30 x 10(6) GPI-1(a) marrow cells without further myeloablation. The level of donor/recipient chimerism was assessed in cell lysates after six months. Increased long-term syngeneic donor engraftment was observed prior to mobilization (before day 6), during a period of active hematopoietic regeneration following the administration of cyclophosphamide. Hematopoietic regeneration was evidenced by a reduced but rapidly increasing marrow cellularity and an increased proportion of hematopoietic progenitors in S-phase. In contrast, long-term syngeneic donor engraftment was not increased over controls during the period of maximum progenitor and stem cell mobilization (after day 5). At this time there were minimal numbers of progenitor and stem cells in the marrow. These data suggest that in the absence of host stem cell ablation, maximal engraftment does not occur during marrow progenitor or stem cell depletion, suggesting that the presence of "open" marrow sites is not a prerequisite for engraftment. The mechanisms for increased engraftment during progenitor cell regeneration following cyclophosphamide need further investigation. Understanding the mechanisms for engraftment without host stem cell ablation may allow strategies for improved long-term engraftment of syngeneic or autologous stem cells with reduced post-transplant toxicity.

Effects of short-term administration of G-CSF (filgrastim) on bone marrow progenitor cells: analysis of serial marrow samples from normal donors.

To determine the effect of G-CSF administration on both the total number of CD34+ cells and the primitive CD34+ subsets in bone marrow (BM), we have analyzed BM samples serially obtained from 10 normal donors in steady-state and during G-CSF treatment. Filgrastim was administered subcutaneously at a dosage of 10 microg/kg/day (n = 7) or 10 microg/kg/12 h (n = 3) for 4 consecutive days. Peripheral blood sampling and BM aspirates were performed on day 1 (just before G-CSF administration), day 3 (after 2 days of G-CSF), and day 5 (after 4 days of G-CSF). During G-CSF administration, a significant increase in the total number of BM nucleated cells was observed. The percentage (range) of CD34+ cells decreased in BM from a median of 0.88 (0.47-1.44) on day 1 to 0.57 (0.32-1.87), and to 0.42 (0.16-0.87) on days 3 and 5, respectively. We observed a slight increase in the total number of BM CD34+ cells on day 3 (0.66 x 10(9)/l (0.13-0.77)), and a decrease on day 5 (0.23 x 10(9)/l (0.06-1.23)) as compared with steady-state (0.40 x 10(9)/l (0.06-1.68)). The proportion of primitive BM hematopoietic progenitor cells (CD34+CD38-, CD34+HLA-DR-, CD34+CD117-) decreased during G-CSF administration. In parallel, a significant increase in the total number of CD34+ cells in peripheral blood was observed, achieving the maximum value on day 5. These results suggest that in normal subjects the administration of G-CSF for 5 days may reduce the number of progenitor cells in BM, particularly the most primitive ones.

Progenitor cell yields are frequently poor in patients with histologically indolent lymphomas especially when mobilized within 6 months of previous chemotherapy.

High-dose therapy with peripheral blood stem cell (PBSC) support is a frequently used treatment option in younger patients with poor prognosis histologically indolent (low-grade) non-Hodgkin's lymphoma (NHL), usually at the time of second or subsequent response to conventional-dose therapy. We have undertaken PBSC collection in 57 patients with histologically indolent NHL mobilized with either cyclophosphamide 1.5 g/m2 or the ESHAP regimen, followed by daily G-CSF. Progenitor cell yields were determined by quantification of CD34+ cells and GM-CFC. Twelve patients (21%) failed to achieve the minimum progenitor cell requirements of 1 x 10(6)/kg CD34+ cells or 1 x 10(5)/kg GM-CFC in their pooled harvests and 40 patients (70%) failed to achieve the optimal harvest thresholds of 3.5 x 10(6)/kg CD34+ cells or 3.5 x 10(5)/kg GM-CFC. This high failure rate is significantly higher than that in patients with histologically aggressive NHL or Hodgkin's disease. A multivariate analysis was performed to identify factors contributing to the low stem cell yields in this group. This identified the time interval from the last chemotherapy to the priming chemotherapy as the most important predictive factor. With respect to CD34 and GM-CFC numbers, on the single harvest on the day the white cell count first exceeded 5 x 10(9)/l the P values were 0.0078 and 0.0065, respectively, and for the progenitor cell values on the pooled harvests the P values were 0.004 for CD34+ cells and 0.015 for GM-CFC. Progenitor cell yields may therefore be improved in patients with low grade lymphoma by harvesting at diagnosis if no marrow disease is present, or by delaying mobilization for 6 months post-chemotherapy in patients in first or subsequent remission.

Influence of rhG-CSF scheduling on megakaryocytopoietic recovery following 5-fluorouracil-induced hematotoxicity in splenectomized B6D2F1 mice.

Recombinant human granulocyte colony-stimulating factor, rhG-CSF, is widely applied to ameliorate neutropenia following chemotherapy. However, rhG-CSF can exert negative effects on megakaryocytopoiesis that might cause a delay of megakaryocyte recovery. Therefore, the present study was designed to test different rhG-CSF administration protocols with regard to their megakaryocytic inhibitory potential in a 5-fluorouracil (5-FU)-induced experimental model system. Splenectomized B6D2F1 mice received a single injection of 5-FU (150 mg/kg) on day 0 followed by 50 micrograms/kg/day rhG-CSF given daily for either zero, four, or eight days. Five days after 5-FU, bone marrow and blood hematopoiesis were reduced significantly when compared with controls, independent of whether or not animals received rhG-CSF. However, nine days after 5-FU, granulopoietic recovery from 5-FU-induced toxicity was faster for rhG-CSF-treated versus untreated mice as demonstrated by higher values for colony forming unit-granulocyte macrophage (CFU-GM) and granulocytes (CFU-GM: 7.2 +/- 0.4 versus 5 +/- 0.6 x 10(4)/femur, granulocytes: 4.3 +/- 2 versus 1.4 +/- 0.4 x 10(5)/ml, respectively). Furthermore, significant mobilization of CFU-megakaryocyte (CFU-Meg) and CFU-GM into the peripheral blood was induced by the eight-day administration of rhG-CSF following 5-FU (day 9: 911 +/- 102 CFU-Meg/ml, 2330 +/- 152 CFU-GM/ml). However, megakaryocytic cells in these same mice were considerably lower when compared with those of animals receiving no rhG-CSF (CFU-Meg: 2.7 +/- 0.2 x 10(3) versus 4.2 +/- 0.2 x 10(3)/femur; small acetylcholinesterase positive (SAChE+) cells: 4.9 +/- 0.3 x 10(3) versus 7.3 +/- 0.9 x 10(3)/femur; megakaryocytes: 2.5 +/- 0.2 x 10(3) versus 4.1 +/- 0.7 x 10(3)/femur; platelets: 2.67 +/- 0.5 x 10(9) versus 3.1 +/- 0.5 x 10(9)/ml, respectively). On the other hand, the shortening of the rhG-CSF treatment from eight to four days caused a rapid granulopoietic recovery comparable to animals receiving eight days of G-CSF with no significant delay in megakaryocytic recovery when compared with mice treated with 5-FU alone; however, with four days of rhG-CSF, the mobilization of CFU into the peripheral blood was significantly less effective. Taken together, the results showed that a shortening of rhG-CSF treatment after chemotherapy is capable of ameliorating neutropenia without negatively affecting megakaryocytopoietic recovery. If, however, maximum recruitment of CFU into the peripheral blood circulation by rhG-CSF for subsequent harvest and transplantation is needed, any shortening of rhG-CSF administration is not advisable.

Effects of G-CSF administration and peripheral blood progenitor cell collection in 20 healthy donors.

The effects of both daily G-CSF administration and subsequent peripheral blood progenitor cell collection (PBPCC) by apheresis on 20 healthy adult donors were studied. All received daily G-CSF (filgrastim) 10 micrograms/kg for 5-7 days by subcutaneous injection. G-CSF administration was well tolerated, except for moderate bone pain and headache. Peak values of CD34+ cells were observed on days 5 (n = 12) or 6 (n = 8). In all donors a significant increase in CD3+, CD4+, CD8+, CD19+, and NK cells was observed on day 5 in relation to the baseline values. CD4/CD8 lymphocyte ratio was unmodified by G-CSF. None of the donors required a central venous line for PBPCC. Immediately after PBPCC, a platelet count below 100 x 10(9)/1 was observed in nine of 18 cases, although in all donors platelet counts were over 100 x 10(9)/1 7 days later. A lymphocytopenia on day 7 following PBPCC was observed, although there was a tendency to achieve baseline values 30-90 days after the procedure. Mean numbers ( +/- SD) of collected cells x 10(6)/kg after a median of two (1-4) apheresis sessions and a median of 20 1 (10-40) processed were: CD34+ 5.5 ( +/- 2.3), CD3+ 326 ( +/- 105), CD4+ 207 ( +/- 64), CD8+ 164 ( +/- 60), CD19+ 88 ( +/- 32), and NK cells 32 ( +/- 14). We conclude that G-CSF administration to healthy donors is a well-tolerated procedure which is associated with (a) obtaining a high number of hematopoietic progenitor cells, and (b) a significant increase in T, B, and NK cells in donors' blood. In addition, PBPCC by apheresis results in a moderate, rapidly reversible, and clinically irrelevant thrombocytopenia and a moderate lymphocytopenia, which tends to resolve within 3 months following the procedure.

248 Growth factors in combination: Phase I study of dose intensified carboplatin (CB), cyclophosphamide (CT) and etoposide (VP) in patients (PTS) with advanced, refractory cancer

This phase I study was designed in order to verify the toxicity of CB. CTX, VP given at high doses, in combination with GCSF alone or GCS and GMCSF given sequentially. The two lymphokines, with different properties, might have their ideal integration in the sequential administration. A group of 10 heavily pretread patients (PTS) with stage IV disease (2 breast cancer, 2 small cell and 1 non small cell lung cancer, 1 sarcoma, 1 colorectal cancer, 1 ependymoblastorna, 1 signet-ring cell bladder cancer, 1 neuroendocrine prostate cancer) were enrolled into the study between August and September 1994. Median (M) PS (ECOG) was 1, M age 41 years (22–65). Previous treatments: 7 PTS had surgery, 4 PTS had radiotherapy, all PTS had received previously a median of 4. 5 chemotherapy courses (4–12). CB-CTX-VP combination was administered over 3 days. Each patient, received two courses of the same CT, followed, randomly, either by 14 days of GCSF (arm A) or by 7 days of GCSF and 7 days of GMCSF (arm B) with cross over in the second CT course. Both growth factors were given sq at the dose of 5μg/kg/day. CB dose was calculated according to Calvert et al. (JCO 1989,7:1748) and expressed with the area under the concentration versus time curve (AUC). All patients received an outpatient treatment. The maximum tolerated doses of CTX and Vp, found in a previous work (Ann Oncol 1994, 5:90,), were, respectively, 1500 and 400mg/m2, while CB doses ranged from 5 to 8 AUC. Twenty chemotherapy courses over 20 are evaluable. Absolute neutrophil count (ANC)<1000μL for 54 days in arm A versus 68 days in arm B (<i>P</i>=0.02); platelets (PLT) count<25.000μL: 57 days arm A versus 30 days in arm B (<i>P</i>=0.03); days of hospitalisation 35 in arm Aversus 16 in arm B (<i>P</i>=0.75); platelets transfusion: 107 Vs 58 (<i>P</i>=0.02); PRBCS 15 vs 5 (<i>P</i>=0.25). No treatment related death occurred. At the present time, eight patients had responses and are alive. These data indicate that dose intensified CT may be delivered safely; GCSF alone shortens days of neutropenia, the combination of the 2 cytokines shortens the time of thrombocytopenia and decreases the number of platelets transfusions.