Effects of three cytokine regimens on hematologic recovery and progenitor cell mobilization after high-dose cyclophosphamide, etoposide, and cisplatin

The aim of this study was to compare both the effects on hematologic recovery and circulating progenitor cell mobilization and the toxicity of three cytokine regimens administered after high-dose non-myeloablative chemotherapy with cyclophosphamide 5 g/m(2), etoposide 1.5 g/m(2) and cisplatin 150 mg/m(2). Thirty-five consecutive patients were non-random sequentially allocated to one of three treatment groups: (1) granulocyte colony-stimulating factor alone (n = 15); (2) granulocyte-macrophage colony-stimulating factor alone (n = 10), and (3) sequential interleukin-3 and granulocyte-macrophage colony-stimulating factor (n = 10). Neutrophil recovery in group 1 was significantly hastened as compared to the two other groups (median 2 days, p < 0.005), while no significant differences were observed between groups 2 and 3. CD34+ cells peaked about 2 days earlier in group 1 compared to the other groups (p = 0.0001), whereas the median peak value of CD34+ cells was similar in the three groups. In all patients, the toxicity related to cytokine administration was low and easily manageable with nonsteroidal anti-inflammatory drugs.     

Multiple cycles of high-dose doxorubicin and cyclophosphamide with G-CSF mobilized peripheral blood progenitor cell support in patients with metastatic breast cancer

Background: In a previous study we applied doxorubicin and cyclophosphamide in a dose-intensive regimen with GM-CSF to patients with metastatic breast cancer (MBC). That treatment failed to prolong the remission duration compared to conventional-dose chemotherapy. In the present study we escalated the dosages of the same agents to: 1) determine the maximum tolerated dosages (MTD) when given for three cycles with G-CSF mobilised peripheral blood progenitor cell (PBPC) reinfusion and 2) evaluate the antitumour effect of this regimen.Patients and methods: For mobilisation of PBPC, G-CSF 15 µg/kg/day was given subcutaneously (s.c.), and in subsequent cohorts leucapheresis was started on days 3, 4 or 6. The intention was to treat MBC patients with three cycles of doxorubicin and cyclophosphamide at a starting dose of doxorubicin 90 mg/m² and cyclophosphamide 1000 mg/m². Dosages were then escalated in subsequent cohorts of at least three patients. In case of dose-limiting mucositis, only the dose of cyclophosphamide was escalated in the next cohort.Results: Twenty-one patients entered this protocol, of which 18 patients received high-dose chemotherapy. The mobilisation of PBPC using G-CSF only was sufficient for three cycles of high-dose chemotherapy in 10 of 21 (47%) patients. Mucositis precluded dose escalation of doxorubicin beyond 110 mg/m². The MTD in this combination was 110 mg/m² for doxorubicin, and 4 g/m² for cyclophosphamide, with haemorrhagic cystitis being the dose-limiting toxicity. The overall response rate was 78% (95% confidence interval (95% CI): 57%–97%), with 22% (95% CI: 3%–41%) complete responses.Conclusion: The MTD of this three cycle high-dose regimen was doxorubicin 110 mg/m² and cyclophosphamide 4 g/m² with mucositis and cystitis being dose-limiting toxicities. Although the primary aim was not the evaluation of antitumour effect, this high-dose regimen does not appear to provide an improvement of treatment results in comparison with our previous study with the same drugs at moderately high-dosages without stem cell support.     

Salvage treatment with epirubicin and/or paclitaxel in metastatic breast cancer patients relapsed after high-dose chemotherapy with peripheral blood progenitor cells

AIMS AND BACKGROUND: To evaluate feasibility and efficacy of paclitaxel as a single agent or in combination with epirubicin in breast cancer taxane-naive patients who have failed previous high-dose chemotherapy. METHODS: Since February 1995, we have treated 32 patients in first relapse or progression after high-dose chemotherapy. Nineteen patients had metastatic breast cancer, 12 more than 3 involved axillary lymph nodes, and 1 inflammatory breast cancer at inclusion to the program. The median time to relapse after high-dose chemotherapy was 12 months (range, 2-43). At relapse, 12 patients were treated with epirubicin (90 mg/m2) plus paclitaxel (175 mg/m2) administered on day 1 every 21 days. In 20 patients who had previously received more than 350 mg/m2 of a cumulative dose of epirubicin and in one patient pretreated with chemotherapy containing mitoxantrone, we employed paclitaxel (175 mg/m2) alone. A median number of five courses was administered (range, 2-10). RESULTS: The overall response rate after 3 courses (29 of 32 patients were assessable) was 55% and after 6 courses (21 of 32 patients were assessable) was 57%. The median time to progression was 7 months (95% CI, 5.7-9.2), and median survival was 27.5 months (95% CI, 17.8-37.0). Toxicity was recorded for 180 cycles (epirubicin + paclitaxel for 62 cycles and paclitaxel alone for 118 cycles). The main toxicity in both regimens was hematologic. We observed WHO grade 3-4 neutropenia (in 8 patients, 25%), for which G-CSF (5 microg/kg/day s.c.) was employed. WHO grade 3-4 thrombocytopenia occurred in 2 patients (6%) and WHO grade 3 anemia in 1 patient (3%). CONCLUSIONS: Our study showed that paclitaxel (alone or in combination with epirubicin) is feasible as salvage treatment in heavily pretreated patients.     

Proto-oncogene expression during early myeloid differentiation of normal peripheral blood progenitor cells

Peripheral blood mononuclear cells cultured in liquid medium with fetal calf serum undergo differentiation to myeloblasts (7 days) and then mature granulocytes (14-21 days). This culture system was used to study proto-oncogene expression during pre-myeloblast myeloid differentiation. c-myc mRNA was present in the peripheral blood mononuclear cells placed into culture, fell during the first 2-4 days of culture and then rose between days 2-4 and day 7 of culture, prior to and coincident with the appearance of myeloblasts. Histone H3 mRNA was absent or present at very low levels at initiation of cultures, and then rose throughout the first 7 days of culture. c-fms mRNA was absent at initiation of cultures, and appeared on days 2-5 of culture, prior to the appearance of myeloblasts. c-fos mRNA was not detected during differentiation of peripheral blood mononuclear cells to myeloblasts. Elucidation of patterns of proto-oncogene expression during normal myeloid differentiation is a prerequisite for interpretation of proto-oncogene expression in myeloid leukemia cells.     

Bone marrow reconstitution after high-dose chemotherapy and autologous peripheral blood progenitor cell transplantation: Effect of graft size

BACKGROUND: Peripheral blood progenitor cell transplantation is rapidlyreplacing autologous bone marrow transplantation as hematologicalsupport after high-dose chemotherapy for lymphoma or solid tumors.Controversy exists concerning the number of progenitor cellsrequired for rapid and sustained bone marrow recovery, and asto which of the widely available methods for estimating thisnumber should be employed. METHODS: Forty consecutive patients with solid tumors or lymphomas receivedhigh-dose chemotherapy followed by autologous peripheral stemcell reinfusion. All stem cell harvests had been performed aftermobilization with standard-dose chemotherapy followed by 300µg G-CSF daily. Hematopoietic reconstitution was studiedin relation to pertinent patient characteristics, to the sizeof the graft (in terms of the total number of mononuclear cells(MNC), the number of granulocyte/macrophage colony-forming units(CFU-GM) and the number of CD34⁺ cells, and to the use of G-CSFafter stem cell reinfusion. RESULTS: Both the numbers of CFU-GM and CD34⁺ cells reinfused, but notthose of the MNC, correlated with granu-locyte and plateletrecovery. Patients who received at least 5 x 10⁶ CD34⁺ cells/kgbody weight achieved platelet transfusion independence on day12 after reinfusion (range: day 7–37), significantly earlierthan patients who had received less (p     

Harvesting of autologous blood stem cells after a mobilising regimen with low-dose cyclophosphamide

Although high-dose cyclophosphamide (HD-CTX) is commonly used as a mobilising regimen for autologous peripheral blood stem cell (PBSC) collection, significant morbidity and insufficient harvesting may complicate the procedure. Alternative regimens and lower doses of cyclophosphamide (CTX) have been investigated as possible ways of overcoming these difficulties. Low-dose CTX (1.5 g/m2) was administered to 102 lymphoma patients as an autologous PBSC mobilising regimen. The collection of 6 x 10(6) CD34+ cells/kg was chosen as the target of the apheresis sessions, whereas 3 x 10(6)/kg were considered the minimum necessary to perform autologous stem cell transplantation (ASCT) safely. The apheretic sessions were started a median of eight days after CTX administration; a median of two aphereses was required. More than 6 x 10(6) CD34+ cells/kg were collected from 78 patients, between 3 and 6 x 10(6)/kg from 19, and fewer than 3 x 10(6)/kg from 5, two of whom underwent bone marrow harvesting and one a successful second PBSC harvesting session using the same mobilising regimen. Eighty-two patients underwent autografting, six of whom received a second transplant after relapse (five using autologous PBSCs coming from the first apheretic course). Low-dose CTX proved to be a safe and effective regimen for autologous PBSC mobilization and also compared favourably with alternative regimens in terms of the rate of harvesting insufficiency. This does not imply that low-dose CTX is the best mobilising regimen for all patients, and the identification of prognostic factors predicting mobilising potential may help in choosing the best individualised regimen.     

Transplantation of autologous peripheral blood progenitor cells procured after high-dose cytarabine-based consolidation chemotherapy for adults with secondary acute myelogenous leukemia in first remission.

Patients with acute myelogenous leukemia secondary to an antecedent hematologic disturbance or cytotoxic chemotherapy are considered to have a very low likelihood of leukemia-free survival regardless of the form of post-remission therapy. The purpose of this study is to evaluate, on the basis of intention to treat, the feasibility and efficacy of high-dose cytarabine/anthracycline consolidation chemotherapy followed by autologous transplantation of chemotherapy/rHuG-CSF-mobilized peripheral blood progenitor cells for seventeen adult patients (median age 63, range 27 to 68) with secondary acute myelogenous leukemia in first remission. Ten eligible patients underwent autologous transplantation of peripheral blood progenitor cells procured following high-dose cytarabine/mitoxantrone consolidation chemotherapy used as a method of in vivo purging. A median of 5 collections (range 2 to 13) were required to procure a median of 9.27 x 10(8) total mononuclear cells/kg (range 2.35 to 21.44 x 10(8) per kg). The median number of CD34-positive progenitor cells was 1.18 x 10(6) kg (range 0.34 to 30.9 x 10(6) kg). After preparative conditioning with 11.25 Gy total body radiation and cyclophosphamide (120 mg/kg) and autologous transplantation, the median time to neutrophil and platelet recovery were 18 days (range 12 to 29 days) and 25 days (range 8 to 158+ days), respectively. After a median follow-up for surviving patients of 33.4 months (range 7.5 to 54 months), 9 of 17 patients (53%) remain alive with 7 in continued first remission. The median remission duration is 13 months (3 to 53 months) and actuarial leukemia-free survival at 3 years is 51+/-25%. Toxicity of autologous peripheral blood progenitor cell transplant included serious liver and pulmonary toxicity in 2 and 1 patient, respectively. Our results demonstrate that a postremission program of high-dose cytarabine-based consolidation chemotherapy followed by autologous transplantation of chemotherapy-mobilized peripheral blood progenitor cells is feasible for patients with secondary acute myelogenous leukemia producing prolonged leukemia-free survival with minimal toxicity.     

Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells

Fibroblast growth factor (FGF) belongs to a family of heparin-binding polypeptides and shows multiple functions including cell proliferation, differentiation, survival and motility. The expression of FGF receptors is widely distributed on different hematopoietic progenitor cells and stromal cells, and FGFs play an important role in hematopoietic stem cell homeostasis. FGFs have been shown to sustain the proliferation of hematopoietic progenitor cells, maintaining their primitive phenotype. Basic FGF (bFGF, FGF-2) stimulates the formation of an adherent stromal cell layer in human long-term bone marrow cultures, and promotes hematopoietic cell development. FGF-2 has also been shown to synergize with other hematopoietic growth factors to enhance in vitro colony formation by several classes of hematopoietic progenitor cells. Results of ex vivo expansion and clinical trials to date suggest that hematopoietic cells cultured under stroma-free cytokine combination conditions may be insufficient to restore hematopoiesis after a myeloablative conditioning regimen, although some recent trials demonstrated an improvement in engraftment and a reduction of the period of pancytopenia, especially neutrophils and platelets, after transplantation. A recent study by our group demonstrated that FGF-2 is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion. These observations could be useful in reducing the long period of severe thrombocytopenia that occurs frequently after umbilical/placental cord blood transplantation. The development of more effective amplifying systems for hematopoietic stem/progenitor cells can be expected since FGFs have multiple functions in hematopoiesis.     

Intramedullary spinal cord recurrence after high-dose chemotherapy and autologous peripheral blood progenitor cell transplantation for limited-disease small cell lung cancer

Intramedullary spinal cord metastasis is very rare in small-cell lung cancer (SCLC), and develops in only 2% of neurological disorders associated with SCLC according to previous reports. We describe here a patient with SCLC who developed intramedullary spinal cord recurrence after high-dose chemotherapy (HDCT) followed by autologous blood progenitor cell transplantation (ABPCT). A 59-year-old Japanese male was referred to us for diagnosis and treatment of an abnormal shadow on a chest radiograph. Based on transbronchial biopsy and staging procedures, he was diagnosed with limited-disease (LD)-SCLC. He received concurrent chemoradiotherapy followed by late intensification with HDCT supported by ABPCT. He achieved complete response and was discharged after receiving prophylactic cranial irradiation (PCI). However 6 months later, he noticed rapidly progressive weakness of the left lower extremity and bowel/bladder dysfunction. Magnetic resonance imaging (MRI) of the spinal cord disclosed an intramedullary tumor exhibiting an enhancement effect with Gd-DTPA at the 11-12th level of the thoracic vertebra. Immediately, radiotherapy to the spinal cord metastasis was given at a dose of 30 Gy, and his neurological disorders were completely resolved. At this time of reporting, he is doing well without recurrence. This case indicates that intramedullary spinal cord is one of the recurrence sites implicated after HDCT and PCI in LD-SCLC.     

Expanding the role of blood progenitor cells

Five years ago the haemopoietic growth factors were introducedto clinical practice with the aim of reducing the depth andduration of chemotherapy induced neutropenia. Now, they havea wider remit, with important roles in supporting dose intensivetreatments and mobilising BPC. Similarly, BPC themselves haveuntil now been predominantly used in autologous transplantationfollowing myeloablative treatments. In the next five years wecan expect to see BPC from novel sources manipulated to featurein many new roles, including allogeneic transplantation, multicyclicdose-intensive chemotherapy and gene therapy blood progenitor cells, mobilisation, transplantation     

A study of ovarian cancer patients treated with dose-intensive chemotherapy supported with peripheral blood progenitor cells mobilised by filgrastim and cyclophosphamide.

We have shown that large numbers of haemopoietic progenitor cells are mobilised into the blood after filgrastim [granulocyte colony-stimulating factor (G-CSF)] alone and filgrastim following cyclophosphamide chemotherapy in previously untreated patients with ovarian cancer. These cells may be used to provide safe and effective haemopoietic rescue following dose-intensive chemotherapy. Using filgrastim alone (10 micrograms kg-1), the apheresis harvest contained a median CFU-GM count of 45 x 10(4) kg-1 and 2 x 10(6) kg-1 CD34+ cells. Treatment with filgrastim (5 micrograms kg-1) following cyclophosphamide (3 g m-2) resulted in a harvest containing 66 x 10(4) kg-1 CFU-GM and 2.4 x 10(6) kg-1 CD34+ cells. There was no statistically significant difference between these two mobilising regimens. We have also demonstrated that dose-intensive carboplatin and cyclophosphamide chemotherapy can be delivered safely to patients with ovarian cancer when supported by peripheral blood progenitor cells and filgrastim. Carboplatin (AUC 7.5) and cyclophosphamide (900 mg m-2) given at 3 weekly intervals with progenitor cell and growth factor support was well tolerated in terms of haematological and systemic side-effects. Double the dose intensity of chemotherapy was delivered compared with our standard dose regimen when the treatment was given at 3 weekly intervals. Median dose intensity could be further escalated to 2.33 compared with our standard regimen by decreasing the interval between treatment cycles to 2 weeks. However, at this dose intensity less than a third of patients received their planned treatment on time. All the delays were due to thrombocytopenia.     

Murine stromal cells producing hyper-interleukin-6 and Flt3 ligand support expansion of early human hematopoietic progenitor cells without need of exogenous growth factors.

Expansion of primitive hematopoietic progenitor cells (HPC) is a major challenge in stem cell biology. Stimulation by growth factors (GF) is essential for proliferation of HPC, while the role of stromal cell coculture for maintenance of progenitor/stem cell potential is unclear. We evaluated the potential of a murine stromal cell layer providing hematopoietic GF to support expansion of human CD34(+) cells. Murine MS-5 cells were transfected with the cDNA encoding huFlt3 ligand and the interleukin6/sinterleukin-6R fusion protein hyper-IL-6. Expansion of CFC and week6 CAFC was at least as efficient in transfected clones compared to control cocultures supported with exogenous GF. Cell numbers reached 17.5- to 62.3- (day 14) and 17.4- to 92.4-fold (day 21) of input cells. Expansion of CFU-GM/Mix was 4.0- to 12.8-fold (day 14) and 4.9- to 11.7-fold (day 21). Primitive week6 CAFC were expanded up to 6.5-fold (day 14) and 6.2-fold (day 21) without exogenous GF. When direct contact of HPC and stromal cells was inhibited, a loss of CFC and much more of CAFC potential was observed with unaffected overall cell proliferation. Here, we show the generation of GF producing murine stromal cells which efficiently support early hematopoiesis without exogenous GF. Direct stromal cell-HPC contact is advantageous for maintenance of differentiation potential.     

Different mechanisms for anti-tumor effects of low- and high-dose cyclophosphamide

It is known that, besides its direct cytotoxic effect as an alkylating chemotherapeutic agent, cyclophosphamide also has immuno-modulatory effects, such as depletion of CD4+CD25+ regulatory T cells. However, its optimal concentration has not yet been fully elucidated. Therefore, we first compared the effects of different doses of cyclophosphamide on T cell subsets including CD4+CD25+ T cells in mice. Cyclophosphamide (20 mg/kg) decreased the numbers of splenocytes, CD4+ and CD8+ T cells by approximately 50%, while a decline in CD4+CD25+ T cell number was more profound, leading to the remarkably lower ratios of CD4+CD25+ T cells to CD4+ T cells. In contrast, 200 mg/kg cyclophosphamide severely decreased the numbers of all the T cell subsets by > 90% although the decreased ratios of CD4+CD25+ T cells to CD4+ T cells were still observed. Next, low-dose cyclophosphamide significantly inhibited in vivo growth of murine hepatoma MH129 tumor in immuno-competent but not immuno-deficient mice. This anti-tumor effect was abolished by CD4+CD25+ T cell repletion. In contrast, high-dose cyclophosphamide exhibited similar anti-tumor effects in both mice. In addition, contrary to antibody-mediated CD4+CD25+ T cell depletion, administration of low-dose cyclophosphamide after tumor inoculation was more efficacious than the prior administration. Our data show that low-dose cyclophosphamide selectively depletes CD4+CD25+ T cells, leading to enhanced anti-tumor effects against pre-existing tumors, while the anti-tumor effect of high-dose cyclophosphamide is solely attributed to its direct cytotoxicity. These findings appear to be highly crucial in a clinical setting of combined chemotherapy and immunotherapy for cancer treatment.     

Impact of autologous bone marrow infusion on hematopoietic recovery after high-dose cyclophosphamide, etoposide, and cisplatin

Because of potential tumor contamination and inadequacy of current purging technique of bone marrow in patients with solid tumors, we investigated an alternative approach to high-dose therapy without autologous bone marrow (ABM) infusion. Three levels of nonmyeloablative doses of cyclophosphamide 4.5 to 5.25 g/m2, etoposide 750 to 1,200 mg/m2, and cisplatin 120 to 165 mg/m2 (CVP) were administered to patients with metastatic solid tumors. Patients were randomized to ABM (n = 46) or no-ABM (NABM) (n = 46) infusion after CVP to study the impact of ABM on hematopoietic recovery, morbidity, and mortality. All patients had ABM harvested, underwent conventional chemotherapy, and then received CVP. Seventy-three patients received two courses of similar doses. The following were the median days to absolute neutrophil count (ANC) of 0.1 x 10(9)/L: for the ABM arm, 19, 21, and 19 and for the NABM arm, 23, 20, and 21 at levels 1, 2, and 3, respectively, during course 1 (P = .01, .80, and .01, respectively). During course 2, ANCs to 0.1 x 10(9)/L and 0.5 x 10(9)/L were attained significantly faster at levels 1 and 3 in the ABM arm. ANC to 1.0 x 10(9)/L was comparable in both arms. Incidence of infection and duration of fever were similar in both arms. Although mortality and the incidence of delayed hematopoietic recovery were more frequent in the NABM arm, this was not statistically significant. Platelet recovery was consistently prolonged in course 2 in both arms, with demonstrable benefit of ABM in course 2 when dose levels were collectively considered. We conclude that (1) ABM enhanced recovery of ANC to 0.1 x 10(9)/L; (2) ABM did not decrease the incidence of infections and the duration of fever; and (3) CVP can be safely given without ABM to carefully selected patients.     

Peripheral 5-FU blood concentration after high-dose injection into the hepatic artery: potential preventive effect on extrahepatic metastatic foci]

To clarify the effect of high-dose 5-FU injection into the hepatic artery (1,000 mg/m2 weekly) on liver metastases of colorectal cancer, the peripheral venous 5-FU concentration was measured in two groups of patients, one which had undergone hepatectomy and the other which had not. The area under the concentration-time curve (AUC) was calculated and the preventive effect of 5-FU on extrahepatic lesions was examined. The peripheral venous 5-FU concentration and AUC were higher in patients who received the drug via the hepatic artery after hepatectomy, and 5-FU was effective for the prevention of extrahepatic lesions as well as against recurrence in the residual liver.     

Radiation-induced lung fibrosis after treatment of small cell carcinoma of the lung with very high-dose cyclophosphamide

Twenty-five previously untreated patients with small cell carcinoma of the lung were treated with cyclophosphamide 160 to 200 mg/kg (with autologous bone marrow support) followed by radiotherapy (4000 cGy) to the primary site and mediastinum. No other treatment was given until relapse occurred. Nineteen patients were assessable at least 4 months after radiotherapy; of these, 15 (79%) developed radiologic evidence of fibrosis, which was symptomatic in 14 (74%). The time of onset of fibrosis was related to the volume of lung irradiated. A retrospective analysis was made of 20 consecutive patients treated with multiple-drug chemotherapy and an identical radiotherapy regimen as part of a randomized trial. Radiologic and symptomatic fibrosis was one half as frequent (35%) as in the high-dose cyclophosphamide group. Very high-dose cyclophosphamide appears to sensitize the lung to radiotherapy and promotes the production of fibrosis.     

联合化疗加粒细胞集落刺激因素动员外周造血干细胞的作用

目的研究大剂量联合化疗(HDCT)加粒细胞集落刺激因子(G-CSF)对外周血造血干细胞的动员作用。方法8例非霍奇金淋巴瘤(NHL)多周期诱导化疗达完全缓解后,采用大剂量联合化疗,联合应用小剂量G-CSF进行外周造血干细胞的动员。结果动员后外周血WBC及MNC总数明显增加,与动员前比较,差异有显著性。冷冻前后,MNC计数、GFU-G集落总数无明显差异。预处理后,病人中性粒细胞、血小板恢复时间分别平均为(10.5±4)天及(11.5±6)天。结论大剂量联合化疗加小剂量G-CSF联合动员方案是安全有效的,值得推广。