Autologous transplantation after high-dose chemo or radiotherapy is now frequently used for the treatment of patients with multiple myeloma (MM). The collection of peripheral blood stem cells (PBSC) has a theoretical advantage over autologous bone marrow collection as the malignant plasmacytic contamination is believed to be lower. However, the extent of B cell contamination in PBSC has not been extensively investigated. Using an immunoglobulin heavy chain gene 'fingerprinting' technique at diagnosis and during apheresis after one cycle of chemotherapy we detected a monoclonal population in 44% of PBSC samples (9 positives in 22 studied). There was no correlation between contamination and sex, age, Durie and Salmon classification, C-reactive protein and albumin. A significant correlation was observed with beta 2 microglobulin serum level (P = 0.02). Twenty one patients were grafted and up to the present, with a mean follow-up of 12 months, 6 patients have relapsed including 4 patients with contaminating B cells. Our results suggest that PBSC contamination, defines a 'poor risk' group of patients, with poor prognosis. However, we could not exclude reinitiation of the disease by plasmacyte stem cells after grafting.

We evaluated granulocyte colony-stimulating factor (G-CSF) as an adjunct to courses of conventional chemotherapy in 16 children with cancer. One course followed by G-CSF (20 episodes) was compared to identical courses without G-CSF (20 episodes) in the same patients. The mean duration of G-CSF therapy was 8.8 (5-13) days. The periods of neutropenia (4.8 days versus 16.5 days; p < 0.0001), days of hospitalization for febrile neutropenia (13 days versus 65 days; p = 0.02) and days on broad-spectrum antibiotics (13 days versus 95 days; p = 0.003) were significantly reduced. With the use of G-CSF the profound neutropenia could be prevented in 11 (55%) episodes. There were two episodes of fever and neutropenia in the G-CSF group as compared to 10 febrile neutropenias in the control group (p = 0.04). G-CSF was well tolerated and did not cause additional expenses when compared to the expenses needed for the treatment of febrile neutropenias. The cost benefit analyses showed that through using G-CSF a savings was realized in the amount of U.S. $20,650 for 20 cycles of chemotherapy, i.e., U.S. $1,033/chemotherapy cycle. We conclude that the use of G-CSF was efficacious and did not increase the total costs of therapy.

In 29 chemotherapy-naive patients with stage II-III breast cancer, peripheral blood stem cells (PBSCs) were mobilised following fluorouracil 500 mg m-2, epirubicin 90-120 mg m-2 and cyclophosphamide 500 mg m-2 (FEC) and granulocyte colony-stimulating factor (G-CSF; Filgrastim) 300 microgram s.c. daily. In all but one patient, mobilisation was successful, requiring three or fewer leucocytopheresis sessions in 26 patients; 28 patients subsequently underwent high-dose chemotherapy consisting of carboplatin 1600 mg m-2, thiotepa 480 mg m-2 and cyclophosphamide 6 g m-2 (CTC) followed by PBSC transplantation. Haemopoietic engraftment was rapid with a median time to neutrophils of 500 x 10(6) l(-1) of 9 days (range 8-10) in patients who received G-CSF after PBSC-transplantation; platelet transfusion independence was reached within a median of 10 days (range 7-16). Neutropenic fever occurred in 96% of patients. Gastrointestinal toxicity was substantial but reversible. Renal, neural or ototoxicity was not observed. Complications related to the central venous catheter were encountered in 64% of patients, with major vein thrombosis occurring in 18%. High-dose CTC-chemotherapy with PBSC-transplantation, harvested after mobilisation with FEC and G-CSF, is reasonably well tolerated without life-threatening toxicity and is a suitable high-dose strategy for the adjuvant treatment of breast cancer.

Thirty-three women with breast cancer have undergone high dose chemotherapy with autologous stem cell support at Huddinge Hospital. Twenty-eight patients had stage IV disease while five patients had disease stage II or III with involvement of > 10 axillary lymph nodes. Patients who received peripheral stem cells had a shorter duration of neutropenia than patients who received autologous bone marrow (p < 0.001). The transplant related mortality was 3 per cent. The calculated progression free survival was 39 per cent at 24 months after high dose therapy in women with stage IV chemosensitive breast cancer. Patients who got a complete remission after standard dose chemotherapy had a better survival rate than patients who got a partial remission. All women with refractory disease progressed within five months from therapy. Four out of five patients with disease stage II or III are progression free with the longest follow-up time of 46 months. High dose chemotherapy with stem cell support can be given with acceptable toxicity. The follow-up time is short but the results are promising, in particular for women who obtain a complete remission before the high dose therapy starts.

A preliminary study and related clinical trial were performed to evaluate the effects of granulosa-lutein cell co-culture on human embryo development and pregnancy rates for in-vitro fertilization (IVF). In the study, sibling two-pronuclear zygotes were randomly allocated to culture with (co-culture) or without (control) autologous granulosa-lutein cells. After 24 h, embryos were examined for blastomere number and degree of fragmentation. Co-culture had no effect on the average number of blastomeres per embryo at 24 h; however, fragmentation was significantly decreased in co-cultured embryos (0.7 +/- 0.1) compared with controls (1.3 +/- 0.2; P < 0.05). In the subsequent clinical trial, all two-pronuclear zygotes were co-cultured for 48 h prior to embryo transfer. The live birth rate per embryo transfer was 43.4% with an implantation rate per embryo of 17.6%. Of the untransferred embryos, 68% developed to the blastocyst stage and were cryopreserved. We conclude that the simple system of autologous granulosa-lutein cell co-culture improves embryo development, implantation and subsequent pregnancy rates for IVF.

High-dose etoposide was incorporated into a regimen of fractionated total-body irradiation (FTBI) and high-dose cyclophosphamide before autologous transplant with the goal to enhance the antitumor effect of the myeloablative regimen in poor-risk lymphoid malignancies.

With the aim of facilitating the ex vivo manipulation of peripheral blood hematopoietic progenitors (CPCs = circulating progenitor cells) collected by leukapheresis, we removed polymorphonuclear cells and monocytes that naturally adhere to nylon wool fibers. Leukapheresed cells harvested at the time of hematopoietic recovery after cancer therapy with high-dose cyclophosphamide plus hematopoietic growth factors were incubated with nylon wool fibers for 1 h at 37 degrees C. Evaluation of the cells non-adherent to the nylon wool in all experiments (n = 14) showed that the median recovery of nucleated cells and CPCs detected as CD34+ cells, CFU-GM and BFU-E was 16.4% (range 4.8%-34.0%), 60.0% (range 30.8-80.8%), 60.9% (range 33.4-74.5%) and 65.5% (range 30.8-69.2%), respectively. Therefore exposure to the nylon wool determined a selective removal of mature cells and a complementary enrichment of CPCs. The wide range of results depended on the significantly different cell compositions of the unmanipulated leukaphereses. The latter from patients receiving rhG-CSF (n = 10) comprised a median of 88.5% (range 77.8-93.8%) and 11.5% (range 6.2-22.2%) polymorphonuclear and mononuclear cells, respectively. In contrast, leukaphereses from patients receiving rhGM-CSF or PIXY321 (n = 4) comprised a median of 71.1% (range 55.4-85.0%) and 28.9% (range 15.0-44.6%) polymorphonuclear and mononuclear cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Although clinical trials are being used to evaluate economic outcomes of new agents, there are methodological problems. Decisions based on these analyses may lead to inefficient use of medical resources. Randomized clinical trials provide important information on the efficacy of new pharmaceutical agents for cancer patients. Policy makers are likely to require both economic and clinical data in order to approve pharmaceuticals for widespread use. Clinical trials provide an opportunity to evaluate economic outcomes for new agents. However, the interpretation of economic analyses of clinical trials raises issues related to perspective of the investigators, study design, collection of data on resource utilization, and generalizability of data to other settings. In this paper, we review these issues and illustrate problems associated with analyses of economic data from a recent phase III trial of hematopoietic growth factors. Clinical results were similar in both Paris and New York in this phase III trial. However, economic results differed markedly between the hospital in Paris and the hospital in New York. While significant savings in terms of fewer days in the hospital and fewer laboratory tests and radiographs for the granulocyte-macrophage colony-stimulating factor (GM-CSF) patients were noted at the New York hospital, resource savings were not identified at the hospital in France. Caution must be used when reimbursement policies are based on economic analyses of clinical trials. Policy decisions must be based on studies that are carefully conducted, analyzed, and interpreted from both a clinical and an economic perspective.

4-Hydroperoxycyclophosphamide (4-HC), a commonly used marrow-purging agent, is active against many tumors, but is also toxic to normal marrow progenitors. Amifostine (WR-2721) is a sulfhydryl compound with chemoprotectant activity. Preclinical studies using suspensions of bone marrow and breast cancer cells demonstrated that ex vivo treatment with amifostine followed by 4-HC resulted in protection of marrow progenitors, with no compromise in the antitumor effect of 4-HC. This fact stimulated the development of a clinical trial. Bone marrow was harvested from 15 poor-prognosis breast cancer patients and randomly assigned to ex vivo treatment with amifostine followed by 4-HC (amifostine + 4-HC), or treatment with 4-HC alone. High-dose chemotherapy was then administered followed by infusion of the purged autologous bone marrow support (ABMS). Leukocyte engraftment, defined as a white blood cell count > or = 1 x 10(9)/L, was achieved in an average of 26 days for patients whose marrow was purged with amifostine + 4-HC versus 36 days for patients whose marrow was purged with 4-HC alone (P = .032). The average number of platelet transfusions (12 v 29; P = .017) and days of antibiotic therapy (28 v 40; P = .012) were significantly less for patients whose marrow was exposed to amifostine + 4-HC, compared with 4-HC alone. Unpurged backup marrow fractions were infused into three patients whose marrow was purged with 4-HC alone, because of inadequate marrow recovery. None of the patients who received amifostine + 4-HC-purged marrow required a backup marrow fraction. Complete remissions were achieved in 83% of patients with measurable disease, with no difference between the two cohorts. Forty-three percent of patients remained alive and progression-free at a mean of 13 months posttransplant. There was no significant difference in the rate or pattern of relapse for patients whose marrow was purged with amifostine + 4-HC compared with those whose marrow was purged with 4-HC alone. Ex vivo treatment of marrow with amifostine significantly shortens the time to marrow recovery, thereby reducing the risk of myelosuppressive complications in breast cancer patients receiving high-dose chemotherapy and 4-HC-purged ABMS. Since supportive care requirements are also significantly decreased, amifostine may reduce the cost of such therapy.

Novel therapeutic agents in the form of recombinant human hematopoïetic cytokines have been developed. Two of them granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage factor (Gm-CSF) have become widely available commercially. A wholly new technique of supportive care became available to hematologists for patients from whom myelosuppressive chemotherapy represents the optimal treatment. These agents moved directly into large-scale prospective, randomized placebo-controlled studies. The data in support of glycosylated G-CSF as an adjunct to aggressive chemotherapy (ACVB) in lymphoma were provided by a randomized study on 162 patients. Reduction of duration of neutropenia < 500/microliter from 4 day to 1 day was observed during four cycles, as well as the reduction of the duration of infection and time in hospital. The same effects have been described with Gm-CSF and less intensive regimens. Hematopoietic growth factors are accepted as accelerating hematologic recovery after bone marrow grafting. In our randomized study with G-CSF, 315 patients were included. Neutrophil recovery > 1,000/microliter for 3 days was seen earlier in G-CSF treated patients (16 vs 27 d). Time to neutrophil > 500/microliter was reduced (14 vs 20 d). The difference was significant both in autograft and allograft patients. Patients had fewer days of infection and spent less time in hospital. In all randomized studies with G or Gm-CSF no difference in survival was observed at one year. If the only benefit of cytokines is to diminish toxicity, the next goals will be to determine the most appropriate situation in which these agents are most justified and to use these agents in novel approaches. Mobilization of peripheral blood stem cell with hematopoietic factors alone or in association with chemotherapy allows a further improvement in controlling toxicity after transplantation with a median time to neutrophil and platelets recovery of 12 days. Stem cells transplantation will become more easily integrated in dose-intensive treatment.

To evaluate the clinical value of growth factors (GFs) with peripheral-blood stem cells (PBSC) collected following mobilization with GFs, we randomized patients to receive or not to receive GFs following transplant.

We report the preliminary results of a study exploring the possibility of collecting circulating progenitor cells (PBSC) with a protocol based on the administration of single doses (4 g/m2) of cyclophosphamide and G-CSF (5 or 10-micrograms/kg) in 9 patients with non Hodgkin's lymphoma. The peak level of CD34+ cells occurred after a median of 10 days (range 8-11), generally coinciding with the median peak level of CFU-GM, with a mean 31.27 fold increase above basal levels. 3 (range 2-5) leukaphereses were required to harvest a median number of 25.1 x 10(4)/kg (8-105) CFU-GM and of 9.4 x 10(6)/kg (1.2-25) CD34+ cells. No difference was recorded between 5 and 10 micrograms/kg of G-CSF in terms of PBSC yield. In transplanted patients, a strong correlation was found between CD34+ cells infused/kg and platelet recovery (r = -0.8, p = 0.002). No toxicity was observed and apheretic procedures were regularly performed outpatiently. Our conclusion is that this protocol is particularly suitable for an outpatient treatment/collection program.

In vitro fertilization (IVF), blastomere biopsy of the 6-8 cell embryo, and single cell DNA diagnosis allows couples at risk of transmitting an X-linked or autosomal disease to start a pregnancy knowing their child will not be affected. We present a quick and reliable nested PCR strategy for sex determination at the single cell level by simultaneous amplification and subsequent restriction fragment analysis of the homologous but non-allelic ZFX and ZFY genes present on the X and Y chromosomes respectively. Amplified ZFX and ZFY sequences are of equal size and produce distinguishable HaeIII digestion products. In a randomized, blinded study of 194 individually isolated lymphoblasts, amniocytes, chorion villus cells, and blastomeres, 191 amplified successfully (98.4% sensitivity). None of the sample blanks showed any PCR product, all 90 of the karyotypically XY cells were correctly genotyped as ZFX/ZFY, all 83 of the 84 XX cells that amplified were correctly genotyped as ZFX only, and analyses of all same-embryo blastomeres were completely concordant (100% specificity). This strategy avoids a source of misdiagnosis observed in methods which detect only Y-specific sequences, where amplification failure in an XY cell results in an erroneous XX diagnosis. This rapid (6 hr) and simple method of analysis, when applied to preimplantation embryo diagnosis, allows the avoidance of offspring affected with an X-linked recessive disorder by transferring only female embryos for implantation and ensuing pregnancy.

We studied the utility of G-CSF for harvesting circulating haematopoietic stem cells in patients with leukaemia or lymphoma based on a comparative study in a single patient. Two successive cycles of leukapheresis following cytotoxic chemotherapy were performed in 22 patients as follows: the first cycle was performed with cytotoxic mobilization in all patients while the second cycle was randomized into two groups: cytotoxic (n = 10) and cytotoxic plus G-CSF (cytotoxic/G-CSF) (n = 12) mobilization. Repetitive cytotoxic mobilization did not alter the yields of mononuclear cells (MNC), myeloid (CFU-GM), and erythroid (BFU-E) progenitors. In contrast, cytotoxic/G-CSF mobilization produced significantly higher yields of MNC (2.6-fold), CFU-GM (5.5-fold), and BFU-E (3.9-fold) than did cytotoxic mobilization alone (P < 0.01). The ratio of CFU-GM to BFU-E was not affected by G-CSF. Furthermore, G-CSF led to an earlier peak of CFU-GM following chemotherapy. G-CSF is thus effective in expanding the pool of circulating haematopoietic progenitors.

Treatment of the central nervous system is crucial to the successful treatment of acute lymphoblastic leukemia in children. The intensity and timing of the therapy are based on the presence or predicted risk of central nervous system leukemia as assessed according to criteria that remain controversial.