It is now possible to achieve prolonged remission of malignant lymphoma and certain cancers with high-dose chemotherapy followed by autograft with haematopoietic stem cells. We tested such a protocol, evaluating haematologic recovery, in order to determine the total cost of hospitalization.

Agents with broad cytotoxic activity, steep linear log dose-response relationships, relative non-cross-resistance, and nonoverlapping nonhematologic toxicities can be combined to create new high-dose combination regimens. We have previously reported phase I dose-escalation studies of ifosfamide, carboplatin, and the combination of the two. Etoposide has reported synergism with these alkylators and produces mucositis as its dose-limiting toxicity. The current study was designed to define the maximum tolerated doses of high-dose combination ifosfamide/carboplatin/etoposide (ICE), with stem cell support for amelioration of hematologic toxicity. Forty-eight adults with advanced malignancy received ICE chemotherapy by 96-hour continuous infusion. Initially, etoposide was added to fixed-dose ifosfamide and carboplatin, then the maximum tolerated dose of etoposide was fixed while doses of the alkylators were individually escalated. Autologous marrow, with or without peripheral blood progenitor cells, was reinfused 3 days after completing chemotherapy. The maximum tolerated doses of ifosfamide, carboplatin, and etoposide were identified as 16 g/m2, 1.8 g/m2, and 1.2 g/m2, respectively. Mortality was 4%. Patients who had prior cisplatin exposure were at increased risk for renal toxicity. If serum creatinine levels (monitored twice daily) rose sharply during chemotherapy, ifosfamide and carboplatin were immediately stopped. Severe multiorgan toxicity developed in the few patients who experienced early renal toxicity. Early stopping enhanced the safety of this regimen. Interpatient differences in chemotherapy drug metabolism or reduced renal clearance may predispose individuals to severe toxicity by increasing overall drug exposure. It was concluded that the ICE regimen is well tolerated and warrants further exploration as treatment of patients with small cell lung cancer, ovarian and germ cell carcinomas, and lymphomas in phase II trials.

To determine the feasibility of escalating the hydrophilic topoisomerase I (topo I)-inhibitor topotecan (TPT) above myelosuppressive doses in adults with refractory or relapsed acute leukemias and to assess pharmacodynamic determinants of TPT action.

We treated 115 patients in a phase I/II dose-escalation study of ifosfamide/carboplatin/etoposide (ICE) followed by autologous stem cell rescue. Patients treated had a variety of diagnoses, including breast cancer (high-risk stage II disease with eight or more positive nodes, stage III disease, and responsive metastatic disease), non-Hodgkin's lymphoma, Hodgkin's disease, acute leukemia in first remission, and various solid tumors that were responsive to induction therapy. Patients received autologous bone marrow stem cells or peripheral blood stem cells primed by one of several methods. The maximum tolerated dose of ICE was determined to be ifosfamide 20,100 mg/m2, carboplatin 1,800 mg/m2, and etoposide 3,000 mg/m2 when administered as a 6-day regimen. The dose-limiting toxicities included acute renal failure, severe central nervous system toxicity, and "leaky capillary syndrome" with hypoalbuminemia, profound fluid overload, and pulmonary insufficiency. Analysis of hematologic recovery based on stem cell source and influence of hematopoietic growth factor administration was undertaken. Hematopoietic growth factor use significantly reduced neutrophil engraftment time for patients receiving bone marrow stem cells, with evidence of earlier recovery times for patients receiving granulocyte colony-stimulating factor compared with granulocyte-macrophage colony-stimulating factor. Neutrophil recovery times varied based on the source of stem cells used, with the earliest engraftment times seen for patients receiving peripheral blood stem cells primed with cyclophosphamide and granulocyte colony-stimulating factor. Platelet recovery times were not statistically different for any of the subsets. In conclusion, the maximum tolerated dose of ICE has been defined, and the source of stem cells and the use of hematopoietic growth factors influence hematopoietic recovery.

In order to evaluate the mobilization effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on peripheral blood stem cells (PBSCs), rhG-CSF was given to patients with urogenital malignancy before chemotherapy. Markers for the stem cells, such as colony forming unit-granulocyte/macrophage (CFU-GM) and burst forming unit-erythrocyte (BFU-E), were sequentially monitored in peripheral blood and leukapheresis samples. Five patients, including a 13-year-old boy, were given 5 micrograms/kg rhG-CSF subcutaneously: the pediatric case for four consecutive days and the adult cases for six consecutive days (53-72 years of age). None of the patients had received chemotherapy within the four weeks prior to the start of the rhG-CSF series. PBSC collections were performed on the fifth day in the pediatric case and on the fifth and seventh days in the adult cases. Progenitor cells were monitored by methyl-cellulose cell culture techniques. CFU-GM on day 5 of the rhG-CSF series in peripheral blood increased 14- to 53-fold compared with samples taken immediately before the series. CFU-GM in the leukapheresis products on day 5 was greatest (70 x 10(3)/kg) in the pediatric case and least (14 x 10(3)/kg) in the oldest patient's case. The totals of the CFU-GM collected by two phereses in the adult cases were 21-73 x 10(3)/kg and the totals of CD34 positive cells were 0.6 to 1.4 x 10(6)/kg. The data suggest rhG-CSF to induce sufficient PBSCs for bone marrow rescue into the peripheral blood without any preceding chemotherapy. The patient's age may, however, be a contributory factor in using this method.

To evaluate (1) the effect of granulocyte colony-stimulating factor (G-CSF) on peripheral-blood stem-cell (PBSC) mobilization; (2) the rate of hematopoietic recovery after G-CSF-mobilized PBSC transplantation; and (3) the outcome of high-dose myeloablative therapy and PBSC transplantation in patients with relapsed or refractory lymphoma.

A recent randomized multicentric French study has shown that intensification with stem cell rescue improves the response rate and progression-free survival in multiple myeloma. Transplantation with primed peripheral blood stem cells (PBSC) displays a faster hematological recovery, especially for platelets, as compared with a bone marrow stem cell graft. In multiple myeloma, the optimal mobilization method for PBSC is unknown. The present study compares mobilization with cyclophosphamide 4 g/m2 + G-CSF 5 micrograms/kg versus G-CSF 5 micrograms/kg alone versus G-CSF 10 micrograms/kg alone in two cases of multiple myeloma, using an intrapatient controlled evaluation of the amount of CD34-positive cells obtained during each leukapheresis. In both cases, the highest CD34-positive cells yield was obtained with G-CSF at 10 micrograms/kg. Despite the low number of cases, this method, devoid of life-threatening toxicity, might be of greatest interest in multiple myeloma.

Interleukin-1 alpha (IL-1 alpha) can act as both a hematopoietic growth factor and a stimulant of cellular and humoral immune responses. To promote acceleration of hematologic recovery and induce immune antitumor activity, we initiated a phase I/II dose escalation trial of 6-hour daily infusions of recombinant human IL-1 alpha after autologous transplantation. Forty patients with Hodgkin's disease (n = 9) and non-Hodgkin's lymphoma (n = 31) transplanted with unmobilized autologous peripheral blood stem cells or bone marrow stem cells received daily 6-hour infusions of IL-1 alpha (day 0 to day +13) at daily doses between 0.1 to 10 micrograms/m2/d; 7 patients received only 7 planned days of IL-1 alpha (day 0 through 6). Most patients received all 14 days of therapy, although 5 patients discontinued treatment early (after 1 to 6 doses) because of fever and severe chills. Toxicity included IL-1 alpha-related fever (occurring on a median of 9 of 14 treatment days), fatigue, and severe chills. Hypotension was dose-limiting and led to discontinuation of IL-1 alpha in both patients receiving 10 micrograms/m2/d. IL-1 alpha-treated patients receiving 3.0 micrograms/m2/d (the maximum tolerated dose) achieved neutrophil recovery (absolute neutrophil count greater than 500/microL) significantly earlier (median, 12 days; range, 11 to 27) than untreated control patients or those receiving IL-1 alpha at 0.1 to 1.0 micrograms/m2/d (median, 27; range, 9 to 63; P < .0001). In addition, the IL-1 alpha patients' bone marrows at day +14 were significantly enriched with committed myeloid progenitor cells. Strong trends to earlier freedom from red blood cell (P = .06) and platelet (P = .09) transfusions were also noted after IL-1 alpha treatment. This earlier hematopoietic engraftment after 3.0 micrograms/m2/d IL-1 alpha allowed earlier hospital discharge (median, 25 v 37 days for control or low-dose IL-1 alpha patients [P < .0001]) and a concomitant reduction (by $38,000) in median hospital charges (P = .01). The clinical toxicities of IL-1 alpha infusion are substantial, though not life-threatening. The accelerated hematopoiesis and immune response activation observed in this trial suggest the value of its further investigation in controlled trials and perhaps in combination with other hemopoietins after transplantation.

Our cumulative experience continues to validate the fetal nucleated erythrocyte as the target fetal cell type of choice, primarily because it reflects the cytogenetic status of the current pregnancy. Additional cell types, such as the granulocyte, await further study. Quantitative PCR is a sensitive and useful new method that can facilitate rapid comparisons between cell separation methods or different monoclonal antibodies. It can also be used on patient material to determine final purity of the enriched maternal samples. If the purity is too low, FISH studies will be complicated by the presence of thousands of maternal cells. Our planned studies include an analysis of why aneuploid pregnancies appear to have a higher number of fetal cells in the maternal circulation. We are also studying the timing of the fetomaternal transfer of cells with qPCR analysis of sorted maternal samples drawn weekly from well-dated women. We are continuously improving our methods (both in separations and antibodies) to reach a fetal cell purity of at least 20% for cytogenetic diagnosis by FISH studies. With the knowledge obtained thus far by us and by others, such a goal appears to be achievable within the near future.

The aim of this study was to examine the effect of G-CSF given after salvage chemotherapy on the mobilisation of peripheral blood progenitor cells (PBPC) in pretreated patients. Seven patients with relapsed or refractory non-Hodgkin's lymphoma (NHL) were treated with methotrexate, cyclophosphamide, cytarabine, etoposide and dexamethasone. G-CSF was given at a dose of 3.8-7.2 micrograms/kg (1-2 ampoules) daily by subcutaneous injection from the onset of neutropenia (< 1.0 x 10(9)/L). A median of 3 leukaphereses was performed when the white cell count was recovering. The median number of granulocyte-macrophage colony-forming cells (GM-CFC) collected was 99 x 10(4)/kg per leucapheresis (range 19-800) or 260 x 10(4)/kg in total per patient (110-1800). Six patients underwent myeloablative chemotherapy with PBPC rescue. No autologous bone marrow or growth factors post-PBPC infusion were administered. The median duration of severe neutropenia (< 0.5 x 10(9)/L) was 8.5 days (range 5-10) and to recovery of neutrophils post-PBPC infusion was 11.5 days (10-15). Severe thrombocytopenia (< 20 x 10(9)/L) was present for 4 days (range 1-5) and the median number of days post-infusion to platelet-transfusion independence was 9 (6-12). In conclusion, G-CSF combined with chemotherapy mobilised large numbers of PBPC for subsequent autotransplantation in pretreated patients with NHL. A single leukapheresis procedure may be sufficient following this protocol.

To investigate the feasibility of peripheral blood CD34+ cell selection and to analyze CD34+ cell-mediated engraftment after high-dose chemotherapy, we performed a phase I/II trial in 21 patients with advanced malignancies. The rationale for the selection of CD34+ cells from peripheral blood progenitor cell (PBPC) collections is based on the observation that contaminating tumor cells can be depleted approximately 3 logs using this procedure. CD34+ cells from chemotherapy+granulocyte colony-stimulating factor-mobilized PBPCs were positively selected with an avidin-biotin immunoadsorption column (CEPRATE SC system). One leukapheresis product with a median number of 2.8 x 10(6) CD34+ cells/kg was labeled with a biotinylated anti-CD34 monoclonal antibody and subsequently processed over the column. The yield of selected CD34+ cells was 73% +/- 24.6%. The purity of the CD34+ cell fraction was 61.4% +/- 19.7%. CD34+ cells were shown to represent predominantly committed progenitors coexpressing CD33, CD38, and HLA-DR molecules (lin+). They gave rise to myeloid as well as erythroid and multilineage colonies in vitro. In addition, positively selected CD34+ cells also comprised early hematopoietic progenitor cells, as shown by the presence of CD34+/lin- cells. Transfusion of positively selected CD34+ cells (2.5 x 10(6) CD34+/kg; range, 0.45 to 5.1) after high-dose VP16 (1,500 mg/m2), ifosfamide (12 g/m2), carboplatin (750 mg/m2), and epirubicin (150 mg/m2) (VIC-E) in 15 patients resulted in a rapid and stable engraftment of hematopoiesis without any adverse events. As compared with 13 historical control patients reconstituted with a comparable number of unseparated PBPCs, time to neutrophil and platelet recovery was identical in both groups (absolute neutrophil count > 500/microL, day + 12; platelet count > 50,000/microL, day + 15). These data indicate that autologous peripheral blood CD34+ cells and unseparated PBPCs mediate identical reconstitution of hematopoiesis after high-dose VIC-E chemotherapy. Because positive selection of CD34+ cells from mobilized blood results in a median 403-fold depletion of T cells, allogeneic CD34+ cells from mobilized blood should be investigated as an alternative to bone marrow cells for allotransplantation.

Only a minority of patients with chronic myeloid leukaemia (CML) benefit from allogeneic bone marrow transplantation (BMT), a potentially curative therapy, or from treatment with interferon alpha, which prolongs survival in cytogenetic responders. In Genoa a programme has been initiated in which CML patients are autografted with Ph-negative peripheral stem cells. To assess the pattern of marrow reconstitution, we studied the clonality of haemopoiesis in five females who engrafted and were Philadelphia chromosome negative. This was performed by evaluating the methylation patterns of the X-linked hypervariable DXS255 locus with the probe M27 beta. All four analysable women showed polyclonal methylation patterns in both granulocytes and T lymphocytes, suggesting that marrow reconstitution occurred from normal residual stem cells.

This study assessed the feasibility and effect of blood progenitors as the only source of haemopoietic support for myeloablative therapy for patients with primary resistant multiple myeloma and markedly infiltrated bone marrow. 17 patients with advanced, primary resistant myeloma received a priming regimen of cyclophosphamide (3 g/m2) and etoposide (900 mg/m2) with GM-CSF. During haematological recovery, at least 2 x 10(6) CD34+ mononuclear cells/kg were collected from each patient with 4-12 leukaphereses. High-dose chemotherapy was then given which consisted of thiotepa (750 mg/m2), busulfan (10 mg/kg) and cyclophosphamide (120 mg/kg) followed by reinfusion of the blood progenitors. Haemopoietic reconstitution was rapid with recovery of granulocytes to > 1.0 x 10(9)/l after a median of 10 d and of platelets to 50 x 10(9)/l after a median of 29 d. The myeloma responded in 10/17 patients for a projected median duration of at least 12 months. Survival was prolonged significantly in comparison with the outcome of control patients who did not receive intensive treatment. Blood progenitors, assessed from the number of CD34+ cells, produced early haemopoietic recovery after myeloablative therapy that induced sustained control of advanced and resistant multiple myeloma.

To assess the "in vivo" effect of 13-cis-retinoic acid and low dose Ara-C in MDS as well as to establish "in vitro" advantage of retinoid dose-related growth pattern on bone marrow cultures as defined by culture timing and CFU-GM proliferative response.

We investigated the question whether lymphokine-activated killer precursor (pre-LAK) cells are induced by OK-432 in vivo, in hepatocellular carcinoma (HCC). Ten patients with HCC were randomly divided into two groups. In group A (n = 5), OK-432 was pre-operatively administered via the hepatic artery. The group B patients (n = 5) served as controls. Tumor-infiltrating lymphocytes (TILs) were collected from the resected tumors. The cytotoxicity of TILs against natural killer (NK)-sensitive K562 cells and NK-insensitive Raji cells was examined by phenotypic analysis with flow cytometry. Freshly isolated TILs, whether treated with OK-432 or not, showed low cytotoxicity against both tumor cells. However, OK-432 pretreatment increased the T-lymphocyte population of TILs, particularly with interleukin-2 (IL-2) receptor positive cells. When TILs were co-cultured with recombinant interleukin-2 (rIL-2), the cytotoxicity was significantly activated in the OK-432 treated group, while untreated TILs showed no activation (P < 0.05). We postulate that pre-LAK cells are induced by OK-432 in TILs, mainly from the T-lymphocyte population. The possibility that LAK cells can be endogenously induced in HCC if OK-432 and rIL-2 are concomitantly administered needs to be considered for immunotherapy to HCC.

This study attempted to determine the use of a single cycle of high dose cyclophosphamide (60 mg/kg/day x 2) with (N = 16) and without granulocyte macrophage colony stimulating factor (GM-CSF) (N = 12) followed by intensive treatment and autologous stem cell transplantation (ASCT) in patients with relapsed disease.

Folylpolyglutamate synthetase (FPGS) is responsible for the metabolism of natural folates and a broad range of folate antagonists to polyglutamate derivatives. Recent studies indicated increased accumulation of methotrexate (MTX) polyglutamates (MTX-PG) in blast cells as a predictor of favorable treatment outcome in childhood acute lymphoblastic leukemia (ALL). We determined the expression of FPGS activity in blasts from children with ALL at diagnosis and after treatment with MTX as a single agent, before conventional remission induction therapy. The levels of enzyme activity in ALL blasts at diagnosis (median of 689 pmol/h/mg protein) were significantly higher (P = .003) than those found in acute nonlymphoblastic leukemia (ANLL) blasts (median of 181 pmol/h/mg protein). Comparable lineage differences in normal lymphoid versus nonlymphoid cells suggest a lineage-specific control of FPGS expression, FPGS activity increased in ALL blasts after in vivo exposure to MTX. The median increase in FPGS activity was significantly higher (P = .003) in B-lineage ALL (188%) than in T-lineage ALL (37%). Likewise, the percentage of intracellular long chain MTX-PG (Glu3-6) was significantly higher (P = .02) in B-lineage ALL (92%) than in T-lineage ALL (65%), consistent with higher FPGS activity in B-lineage blasts. This finding could explain, at least in part, the superior outcome in children with B-lineage ALL treated with antimetabolite therapy.

Eighteen patients with chronic myelogenous leukemia (CML) in chronic (9 patients) or advanced phases (9 patients) underwent autologous bone marrow transplantation (BMT) with a preparative regimen using high doses of cyclophosphamide, etoposide and total body irradiation (CY-VP16-TBI): cyclophosphamide 60 mg/kg daily on days 1 and 2; VP16 250 mg/m2 twice daily on days 1-3 and TBI 1020 cGy in six fractionated doses on days 5-7. Autologous marrow cells were reinfused on day 8. Three of the 8 patients in late chronic phase Philadelphia (Ph) chromosome-positive CML (37%) achieved a cytogenetic response, with Ph suppression to 25%, 29% and 44% Ph-positive metaphases, respectively, and lasting for 11, 1 and 3 months, respectively. The median duration of chronic phase following BMT was 26+ months (range 2-33+ months). One patient with Ph-negative, BCR-rearranged, chronic phase CML had a decrease of the BCR-rearranged band to 10% of pretreatment levels, which persisted for 6 months. None of the 9 patients with advanced CML phases (5 in second chronic, 1 in blastic, 3 in accelerated) achieved meaningful cytogenetic responses. Their median survival was 7 months from the time of BMT. Toxicities were mostly related to myelosuppression, particularly thrombocytopenia. Febrile episodes developed in 16 patients (89%). Treatment-related deaths occurred in 2 patients (11%). In summary, autologous BMT using a TBI-containing regimen had acceptable toxicity. Future investigations will evaluate the additional benefit of purged autologous stem cell transplantation in patients with chronic phase CML.