An IkappaBalpha inhibitor causes leukemia cell death through a p38 MAP kinase-dependent, NF-kappaB-independent mechanism.

Treatment of U937 cells with an IkappaBalpha phosphorylation inhibitor, Bay 11-7085, induced a rapid phosphorylation of p38 mitogen-activated protein (MAP) kinase, significant apoptosis, extensive necrosis, and a weak phosphorylation of MAP kinase kinase. Bay 11-7085 had no effect on the basal levels of phosphorylated IkappaBalpha but completely inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of IkappaBalpha. Although Bay 11-7085 prevented phorbol 12-myristate 13-acetate-induced NF-kappaB nuclear translocation, SN50, a specific inhibitor of nuclear translocation and function of NF-kappaB, did not induce any significant nuclear/DNA fragmentation, caspase 3 activation, or cell death. The p38 MAP kinase-specific inhibitor, SB203580, completely inhibited the phosphorylation of p38 MAP kinase and significantly decreased Bay 11-7085-induced apoptosis. In contrast, the MAP kinase kinase-specific inhibitor PD98059 had no effect on Bay 11-7085-induced apoptosis. Caspase-specific inhibitor, z-Val-Ala-Asp-fluoromethyl ketone prevented Bay 11-7085-induced activation of caspase 3 but was not able to block Bay 11-7085-induced phosphorylation of p38 MAP kinase. These data suggest that Bay 11-7085 induces apoptosis through a p38 MAP kinase-dependent, NF-kappaB-independent mechanism.

Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation.

PURPOSE: The present study evaluates the clinical significance of detection of cytokeratin 19 (K19) in the bone marrow of patients with breast cancer undergoing high-dose chemotherapy (HDCT) and autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: We studied retrospectively cryopreserved bone marrow aspirates from 83 patients with high-risk stage II, III, and IV breast cancer obtained before bone marrow harvest but after induction chemotherapy. All samples were histologically negative for metastases. Polymerase chain reaction (PCR) for K19 was performed according to methods described previously and results were correlated with the probability of relapse following HDCT and ABMT. RESULTS: The incidence of occult metastases as defined by PCR for K19 message was 52% for 19 stage II, 57% for 14 stage III, and 82% for 50 stage IV patients (two-tailed P = .0075, chi 2 test). The probability of relapse at 3 years after ABMT was 32% and 94% for K19-positive stage II/III and stage IV patients, respectively, versus 10% and 14% for K19-negative stage II/III and stage IV patients, respectively. The difference was significant for stage IV patients (two-tailed P = .0002). CONCLUSION: It has been shown that PCR is a highly sensitive method to detect K19 message in the bone marrow. The incidence of K19 positivity in bone marrow increases significantly with advancing stage. In patients with breast cancer, especially metastatic breast cancer, undergoing HDCT and ABMT, the presence of K19 is associated with a poor prognosis.

Maximum-tolerated doses of ifosfamide, carboplatin, and etoposide given over 6 days followed by autologous stem-cell rescue: toxicity profile.

PURPOSE: A phase I dose-escalation study of ifosfamide, carboplatin, and etoposide (ICE) with autologous stem-cell rescue (ASCR) was conducted to determine the maximum-tolerated dose (MTD) of ICE given over 6 days. PATIENTS AND METHODS: One hundred fifty-four patients with a variety of poor-prognosis malignancies received escalating doses of ifosfamide 6,000 to 24,000 mg/m2, carboplatin 1,200 to 2,100 mg/m2, and etoposide 1,800 to 3,000 mg/m2 divided over 6 days. Mesna was administered in a dose equal to ifosfamide. ASCR was performed 48 hours after the completion of ICE. The source of stem cells was bone marrow (BM) in patients without BM micrometastases and peripheral-blood stem cells (PBSC) in patients with BM micrometastases. Patients were evaluated for hematologic and nonhematologic toxicities, as well as response to therapy. RESULTS: The MTD of the ICE regimen is 20,100 mg/m2 of ifosfamide, 1,800 mg/m2 of carboplatin, and 3,000 mg/m2 of etoposide. The dose-limiting toxicities of ICE were CNS toxicity and acute renal failure. Additionally, reversible elevations of serum creatinine levels were noted in 29% of patients treated at the upper dose levels. Forty-six patients were treated at the MTD. Severe, reversible mucositis and enteritis were the major nonhematologic toxicities seen at the MTD (78% and 33%, respectively). The overall mortality rate was 8% for all dose levels (4% at the MTD). At the MTD, the median times to an absolute neutrophil count > or = 0.5 x 10(9)/L, to a platelet count > or = 20 x 10(9)/L, and to discharge were 18, 22, and 24 days, respectively. The overall response rate was 40% for 77 patients with assessable disease at the time of treatment. CONCLUSION: ICE is well tolerated, with acceptable hematopoietic side effects and predictable organ toxicity.

The migration of hematopoietic cells: an in vitro study system.

An in vitro migration assay system for the study of hematopoietic cell migration was established. Using a large well modification of earlier described migration chambers, it was found that, in the presence of whole murine serum from either normal or aplastic mice, a heterogeneous population of cells was stimulated to migrate through limited size pores (8 mu) in a thin (5 mu) polycarbonate filter. In dilution studies, serum obtained from animals that had been rendered aplastic by total body irradiation provided a stronger migration stimulus than serum from normal animals. This observation is consistent with observations of hematopoietic cell migration in vivo. Primitive spleen colony-forming cells and in vitro granulocyte/macrophage colony-forming cells were present in the migrating population at a comparable fraction to that found in untreated bone marrow. These studies demonstrate the feasibility of studying hematopoietic stem cell migration under controlled experimental conditions.

Low antigen density leukemia cells: selection and comparative resistance to antibody-mediated marrow purging.

Low tumor-associated antigen (TAA) expressing tumor cells present an obstacle to effective antibody directed purging of tumor cells from bone marrow. In this study, a comparison was made of the efficiency with which low TAA expressing leukemia cells could be depleted using two monoclonal antibody (MoAb) directed purging techniques: 1) complement (C)-mediated cytolysis, and 2) physical separation using magnetic microspheres. Low TAA sublines were selected from a cultured human leukemia cell line by growing out cells remaining after treatment with anti-TAA and C, or after immunomagnetic (IM) purging. IM-selected sublines showed lower TAA expression than did C-selected sublines, and sublines resulting from multiple selections expressed less TAA than those that had only been through one selection. These sublines were then examined for sensitivity to C or IM purging. The highly selected, lowest TAA expressing sublines were markedly resistant to both IM and C. Less selected sublines were resistant to C, but not to IM. In both techniques, addition of MoAbs against a second TAA restored the efficiency of purging to that observed with the parental line. When low TAA subline cells were seeded into simulated bone marrow and subjected to purging, C-mediated lysis removed less than 40% of leukemia cells, whereas IM purging removed 85% of the cells. These results indicate that there are low antigen density cells that are resistant to C-mediated purging, but which retain sensitivity to IM removal.

The CD34+ cell fraction in bone marrow and blood is not universally predictive of CFU-GM.

We have measured the presence of granulocyte-macrophage colony forming cells (CFU-GM) and CD34+ cells in blood and bone marrow. We have compared these hematopoietic cell assays using regression analysis. We have found that under the limited and specific case of blood cells from an individual recovering from myelo-suppressive chemotherapy, the fraction of cells that is CD34 positive is predictive of the number of granulocyte-macrophage colonies (CFU-GM) which will grow. This result is in agreement with published data. We have found, however, that in bone marrow aspirates, or in the blood of individuals recovering from cyclophosphamide chemotherapy and receiving either granulocyte-macrophage colony stimulating factor (G-CSF) or folinic acid (FA) therapy, there is poor correlation between CD34+ cell fraction and CFU-GM. Accordingly, the use of CD34+ fraction cannot be relied upon to substitute for the CFU-GM assay in assessing the hematopoietic cell content of blood or bone marrow samples.

Peripheral blood and bone marrow hematopoietic stem cells: are they the same?

Hematopoietic stem cells collected from the peripheral blood (PBSCs) have been successfully used for transplantation. Originally, these cells, collected from patients with chronic myelogenous leukemia, were used to restore chronic phase of the disease. More recently, PBSCs have been used principally for autologous transplantation for nonhematologic malignancies, although there are a few recorded cases of allogeneic transplantation with PBSCs. The process of obtaining peripheral blood stem cells is tedious in comparison to the process of harvesting bone marrow for transplantation, requiring multiple lengthy apheresis procedures to collect sufficient cells for transplant. Moreover, the risks associated with PBSC collection (anesthesia risk for catheter placement, risk of introducing infection during collection) are just as great as those associated with marrow harvesting. However, for the patient whose bone marrow is unharvestable due to hypocellularity or fibrosis, PBSC transplantation may reopen the option of treatment by high-dose cytotoxic therapy and autologous hematopoietic stem cell rescue. Peripheral blood stem cell transplantation may also afford the transplant option to the patient whose marrow has been infiltrated with disease. While it has not been demonstrated, it is hypothetically likely that the probability of tumor cell contamination of circulating blood is quite low, due to the lack of adherence necessary for colonization. Recent reports have indicated that autologous transplantation with PBSCs may result in more rapid engraftment relative to autologous transplantation with bone marrow. This has only been reported in programs wherein PBSCs are collected under mobilizing conditions. If it is the case that PBSCs produce more rapid engraftment, however, then the additional effort and cost associated with PBSC collection might be outweighed by the reduced morbidity, mortality, and cost associated with early return of granulocytes in the transplant patient. The biology of the early engraftment phenomenon should be studied, as it is possible that it is related to the mobilizing conditions under which the cells are collected and not due to an intrinsic quality of PBSCs.

Non-specific cell binding characteristics of para-magnetic polystyrene microspheres used for antibody-mediated cell selection.

The binding of cells to paramagnetic polystyrene microbeads in the absence of coupling antibodies was measured. Cells from normal bone marrow, from an acute lymphoblastic leukemia (ALL) cell line or from a neuroblastoma cell line, were labeled with the fluorochrome Hoechst 33342 and incubated with microbeads by rotation at 4 degrees C. Following this incubation, the microbeads with all attached cells were collected using an externally applied magnetic field and visualized by microscopic examination under ultraviolet illumination. The incubation variables included the protein content of the medium, and the period of rotation. Normal bone marrow was found to adhere sparingly to the microbeads; less than 1.0% of the total nucleated cell population was recovered with the beads, whereas greater than 5% of the ALL cells and greater than 30% of the neuroblastoma cells were found to bind non-specifically to the microspheres. Neither changes in the protein concentration of the medium or in the incubation period significantly altered the non-specific binding of the cell types examined. It is thus apparent that the use of these microspheres for positive selection of cells, such as the collection of hematopoietic stem cells for transplantation, would be compromised by a sizeable non-specific interaction. Modification of the surface of the microspheres to substantially reduce this interaction will be necessary before efforts at positive selection using the magnetic microspheres can be fruitful.

Two novel high-dose treatment regimens for metastatic breast cancer--ifosfamide, carboplatin, plus etoposide and mitoxantrone plus thiotepa: outcomes and toxicities.

This report describes the results of two phase I/II dose escalation trials for the treatment of metastatic breast cancer. Successive groups of patients with metastatic breast cancer responsive to induction therapy following standard doses of chemotherapy were treated with escalating doses of ifosfamide (6,000 to 24,000 mg/m2), carboplatin (1,200 to 2,100 mg/m2), and etoposide (1,800 to 3,000 mg/m2) followed by autologous stem cell rescue. The maximum tolerated doses of these drugs were defined as ifosfamide 20,100 mg/m2, carboplatin 1,800 mg/m2, and etoposide 3,000 mg/m2. Major nonhematologic toxicity consisted of mucositis and enteritis, and the dose-limiting toxicities were central nervous system toxicity and acute renal failure. The overall treatment-related mortality rate was 4%. The event-free survival rate at 500 days for these patients was 31%. Patients with metastatic breast cancer refractory to all standard dose therapy were treated with escalating doses of mitoxantrone (45 to 105 mg/m2) and thiotepa (900 to 1,350 mg/m2) followed by autologous stem cell rescue. The maximum tolerated doses of these drugs were defined as mitoxantrone 90 mg/m2 and thiotepa 1,200 mg/m2 with mucositis and enteritis as the major nonhematologic toxicities and delayed myelosuppression as the dose-limiting toxicity. Twelve percent of the patients remain event free at 500 days and the treatment-related mortality rate for this group of heavily pretreated patients was 17%. These data suggest that patients with metastatic breast cancer may benefit from high-dose therapy and that treatment-related toxicity is tolerable.

High-dose ifosfamide/carboplatin/etoposide: maximum tolerable doses, toxicities, and hematopoietic recovery after autologous stem cell reinfusion.

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.

Relative efficiency of leukemic cell depletion using anti-murine-IgG1(Fc) or anti-murine-IgG coated immunomagnetic microbeads.

Microbeads for immunomagnetic bone marrow purging, to which sheep anti-mouse-IgG1(Fc) antibodies have been linked, and beads linked with antibodies against whole murine immunoglobulin were compared. Competitive binding studies, in which Fc fragments and Fab fragments were titrated onto the microbeads, followed by incubation with 125I-labeled whole mouse Ig, revealed that the beads linked with anti-mouse-IgG1(Fc) specifically bound the Fc region of the murine immunoglobulin molecules. The total amount of antibody of either IgG1 or IgG2 isotype that would adhere to microbeads linked with either type of antibody, as revealed by secondary binding with 125I rabbit antimouse Ig, was identical, suggesting that similar numbers of antibody binding sites were available. In cell depletion studies, it was found that if IgG1 isotype monoclonal antibodies were employed as binding intermediaries between the target cells and the microbeads, the efficiency of target cell depletion was superior with the anti-mouse-IgG1(Fc)-coated beads, even if the amount of MoAb coating the target cells was suboptimal. However, if the intermediary antibodies were of the IgG2 isotype, the efficiency of target cell depletion with these beads was inferior. These findings indicate that the efficiency of immunomagnetic bone marrow purging is dependent upon matching of the targeting MoAb and the secondary antibodies that link to the surface to the microbeads.

Phase I-II study of high-dose busulfan and cyclophosphamide followed by autologous peripheral blood stem cell transplantation for hematological malignancies: toxicities and hematopoietic recovery.

In a phase I-II study, we evaluated toxicities, tolerability, pace of engraftment, and tumor responses to high-dose bulsulfan and cyclophosphamide followed by autologous peripheral blood stem cell transplantation in patients with hematological malignancies. We treated 51 patients with various hematological malignancies involving the bone marrow with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) followed by reinfusion of autologous peripheral blood stem cells. Stem cells were previously collected during hematopoietic recovery after cyclophosphamide (100 mg/kg) and etoposide (600 mg/m2) followed by G-CSF (5 micrograms/kg/day). Neutrophil recovery (>0.5 x 10(9)/I) was rapid in the majority of patients (median 10 days after transplant, range 7-91 days), resulting in a low number of days with severe neutropenia (median 7 days, range 5-85 days) and with fever (median 5 days, range 1-13 days). Platelet recovery, however, was delayed in 60% of patients. There was one acute transplant-related death (2%). Four patients died of late, presumed infections, pulmonary complications (interstitial pneumonia). Tumor responses were documented in a significant proportion of these patients with high-risk hematological malignancies. We conclude that peripheral blood stem cell transplantation results in rapid recovery of neutrophils but variable recovery of platelets after high-dose busulfan and cyclophosphamide, when stem cells are harvested following priming with cyclophosphamide/etoposide and G-CSF. The regimen is well-tolerated with limited non-hematological toxicities and transplant-related mortality. While significant tumor responses were documented in this trial, the ultimate efficacy of the regimen needs to be further defined.

High-dose chemotherapy and autologous peripheral blood stem cell transplantation in patients with multiple myeloma and renal insufficiency.

Six patients with multiple myeloma and chronic renal insufficiency (serum creatinine >3.0 mg/dl), including four on dialysis, received high-dose busulfan and cyclophosphamide (BUCY) followed by autologous peripheral stem cell transplantation. Peripheral blood stem cells were collected after priming with cyclophosphamide, etoposide and G-CSF. Patterns of engraftment and toxicities were not apparently different from those seen in myeloma patients with normal renal function. There was one toxicity-related death, resulting from a massive spontaneous subdural hematoma. One patient died of disease progression 6 months after transplant, while the remaining four patients are alive and free of myeloma progression 6 to 39 months after high-dose therapy. Two of these patients have remained in complete remission for 28 and 39 months. Our experience suggests that high-dose therapy with BUCY and autologous peripheral blood stem cell rescue is feasible in patients with multiple myeloma and renal failure.

Intensive dose ifosfamide, carboplatin, and etoposide followed by autologous stem cell rescue: results of a phase I/II study in breast cancer patients.

We have recently treated 66 women with breast cancer with escalating doses of ifosfamide, carboplatin, and etoposide (ICE) followed by autologous stem cell rescue (ASCR). Patients received ifosfamide (6000-24,000 mg m-2), carboplatin (1200-2100 mg m-2), and etoposide (1800-3000 mg m-2) divided over 6 days with ASCR 48 h after completion of chemotherapy. Our patient population consisted of seven patients with stage II disease with eight or more positive nodes being treated in the adjuvant setting, 16 patients with a history of stage III or inflammatory breast cancer, and 43 patients with stage IV disease. Six patients were not evaluable for response due to early death from infection (three patients) and incomplete restaging (three patients). The overall response rate in patients with measurable metastatic disease was 50%. Of those patients with stage II disease, 85% remain alive and progression-free with a median follow-up of greater than one year. The two most frequent toxicities encountered were reversible elevations of liver function tests and mucositis/enteritis. The dose-limiting toxicities were central nervous system toxicity and nephrotoxicity.

Apheresis and transplant of hematopoietic progenitor cells (HPC) from allogeneic donors of age above 60 years.

Over the immediate past 4 years, our program has collected hematopoietic progenitor cells by apheresis from 48 individuals aged 61 and over (range 61-71 years of age). We have retrospectively analyzed the collection and transplant results associated with employing these donors, and have compared them with 175 donors aged 60 or less who were collected during the same time period. We have found no significant difference in venous access (P = 0.208), rate of post-transplant engraftment of neutrophils (P = 0.117) and platelets (P = 0.692), or in rate and grade of acute GVHD (P = 0.806). However, we have found that these older donors have a significantly lower mobilization of CD34 + cells as reflected in lower absolute counts of circulating CD34 + cells pre-apheresis (P = 0.016). This, in turn, results in lower CD34 + cell yields in apheresis products (P < 0.001), trending towards requiring more apheresis procedures (22.9 vs 13.7%, P = 0.095) to collect sufficient CD34 + cells for transplantation. We conclude that it is practical when necessary to employ donors aged 60 and above, as well as safe for both donor and intended recipient. However, concern over reduced CD34 + cell mobilization may be sufficient grounds to seek younger donors when possible.

Use of the Terumo SteriCell for the processing of bone marrow and peripheral blood stem cells.

The SteriCell cell processing instrument is a good choice for a stem cell processing laboratory that is of sufficient size that they cannot share an apheresis machine with the blood bank. It is a laboratory instrument, with no facility for patient connection. Because of its minimal size and weight, it is easily stored in a cramped laboratory. Its automated programs are appropriate for processing of bone marrow and peripheral blood stem cells, and it is quite easy to learn how to use (in our laboratory, most individuals have been completely facile with the SteriCell after fewer than six processings). Based on reported results from other instruments, the SteriCell provides cell yields that are comparable to competing instruments. Service (provided by Haemonetics) has been satisfactory, and support from Terumo has been excellent. We can recommend this instrument to any other laboratory.

Rate of non-adherent cell loss from long-term cultures of murine bone marrow: effect of medium conditioned by the adherent cell population.

The rate of random spontaneous cell loss from the non-adherent cell population of long-term cultures of murine bone marrow was determined. The measured rate of non-adherent cell loss is affected by previous conditioning of the medium in which the non-adherent cells are suspended. Specifically, the measured rate of non-adherent cell loss is significantly slowed when the non-adherent cells are suspended in medium conditioned in long-term haematopoietic cultures instead of fresh medium. It appears that a subset of the adherent cell population is the source of the factor or factors within the medium which result in this slower measured rate of non-adherent cell loss. This effect may be due to the stimulation to division of haematopoietic progenitor cells, offsetting the lysis of other non-adherent cells.