Challenges in the use of allogeneic hematopoietic SCT for ectodermal dysplasia with immune deficiency.

Genetic mutations of proteins regulating nuclear factor of kappa-light polypeptide gene enhancer in B lymphocyte (NF-kappaB) activation result in heritable diseases of development and immunity. Hypomorphic, X-linked mutations in the IKBKG gene (NF-kappaB essential modulator (NEMO) protein), and hypermorphic, autosomal dominant mutations in the IKBA gene (inhibitor of NF-kappaB (IkappaB)-alpha protein), are associated with a phenotype of immune deficiency and often ectodermal dysplasia (ED-ID). ED-ID predisposes patients to recurrent and life-threatening infections and is typically fatal within the first few years of life. Allogeneic hematopoietic SCT (HSCT) may correct the immune deficiency associated with NEMO or IkappaBalpha mutations, but there is very little published data. We gathered clinical data on three ED-ID patients that had undergone HSCT. Conditioning regimens were variable, as were the stem cell sources. All three patients experienced engraftment difficulties as well as post transplant complications. These cases suggest that patients with immune deficiencies caused by NEMO or IkappaBalpha mutations may have intrinsic barriers to successful engraftment, which require further investigation.

Allogeneic bone marrow transplantation for children with acute leukemia: cytoreduction with fractionated total body irradiation, high-dose etoposide and cyclophosphamide.

Marrow-ablative chemo-radiotherapy followed by hematopoietic stem cell rescue from an allogeneic source improves outcomes for children with high-risk acute leukemia. The first effective pre-transplant preparative regimens consisted of high-dose cyclophosphamide (CY) and total body irradiation (TBI). Subsequent attempts have been made to improve leukemia-free survival, by adding other chemotherapy agents to these agents. In previous clinical studies of total body irradiation, etoposide, cyclophosphamide (TBI-VP-16-Cy) in adult allogeneic bone marrow transplantation, there has been a high incidence of severe regimen-related toxicity. In this study, we investigated the safety and efficacy of this combination in 41 children who received TBI (12-14 Gy), VP-16 (30 mg/kg), and CY (60 mg/kg x 2) and then either matched sibling or alternative donor transplants for acute leukemia. There was only one case of fatal regimen-related toxicity. The estimated 3-year event-free survival for patients with early or intermediate stage disease was 68% (53-88%). The estimated event-free survival of patients with advanced disease was 17% (5-59%). TBI-VP16-CY is safe in pediatric transplantation, and it has good efficacy for transplant recipients with less advanced disease. Bone Marrow Transplantation (2000) 25, 489-494.

Treatment of chronic myelogenous leukemia with autologous bone marrow transplantation followed by roquinimex.

Unmanipulated autologous bone marrow transplant (ABMT) offers patients with chronic myelogenous leukemia (CML) a long-term survival of 10%, at best. Immunotherapy has a role in the myeloid leukemias, and there is increasing evidence that of all hematopoietic neoplasms, CML may be the most susceptible to immune regulation. Roquinimex is known to enhance T cell, NK cell and macrophage activity. A phase II study was initiated in March 1992 to evaluate the role of roquinimex in Ph chromosome-positive CML post ABMT. Patients were conditioned with busulfan/ cyclophosphamide followed by reinfusion of unmanipulated Ph-positive bone marrow stem cells (>1 x 108 NBC/kg). When engraftment of neutrophils (ANC) reached 100/microl, patients received oral roquinimex twice weekly, escalating to a maximal dose of 0.2 mg/kg in 2 weeks. Seventeen patients have entered the study; 11 in first chronic phase (CP1); two in second chronic phase (CP2) and four in accelerated phase (AP). All required significant myelosuppressive therapy prior to ABMT to maintain stable blood counts and most had also received prior interferon therapy. All patients survived the transplant. Subsequent toxicity consisted mainly of musculoskeletal aches and peripheral edema. Additionally, specific skin changes were observed including graft-versus-host-like disease and eccrine sweat gland necrosis. Eight out of 17 patients are alive 28-60 months post ABMT. Of the nine patients who died, two were in CP2 and three in AP. All patients in CP1 went into a complete hematological remission post ABMT and seven of the 11 patients had at least a major cytogenetic response (greater than 65% Ph-negative metaphases) at 1 year or beyond and four of the 11 patients had a complete cytogenetic response at 2 years or beyond. Cytogenetic response post transplant often developed over time and did not simply represent post ABMT engraftment with Ph-negative cells. The clinical and cytogenetic data in these patients are encouraging and suggest that roquinimex may have significant activity when given post ABMT to patients with Ph-positive CML.

Autotransplantation for relapsed or refractory Hodgkin's disease: long-term follow-up and analysis of prognostic factors.

Seventy consecutive patients with refractory or relapsed Hodgkin's disease who received high-dose chemotherapy followed by autologous stem cell rescue were analyzed to identify clinically relevant predictors of long-term event-free survival. High-dose therapy consisted primarily of carmustine (BCNU), etoposide, cytarabine and cyclophosphamide (BEAC). The 5-year Kaplan-Meier event-free survival (EFS) for the entire cohort was 32% (95% confidence interval; 18-45%) with a median follow-up of 3.6 years (range 7 months-7.6 years). The most significant predictor of improved survival was the presence of minimal disease (defined as all areas < or =2 cm) at the time of transplant: the 5 years EFS was 46 vs 10% for patients with bulky disease (P = 0.0002). Other independent predictors identified by step-wise regression analysis included the presence of non-refractory disease and the administration of post-transplant involved-field radiotherapy (XRT). Treatment-related mortality occurred in 13 of 70 patients: nine patients (13%) died within the first 100 days, mainly from cardiopulmonary toxicity. However, only one of 24 patients (4%) transplanted during the last 4.5 years died from early treatment-related complications. While high-dose therapy followed by autotransplantation led to long-term EFS of 50% for patients with favorable prognostic factors, a substantial proportion of patients relapsed, indicating that new therapeutic strategies are needed.

Autotransplantation for relapsed or refractory non-Hodgkin's lymphoma (NHL): long-term follow-up and analysis of prognostic factors.

One hundred and thirty-six patients autografted for relapsed or refractory non-Hodgkin's lymphoma (NHL) were evaluated to assess long-term event-free survival and to identify important prognostic factors. High-dose therapy consisted primarily of carmustine (BCNU), etoposide, cytarabine, and cyclophosphamide (BEAC) followed by unpurged autologous stem cell rescue. The 5-year Kaplan-Meier event-free survival (EFS) for the entire cohort was 34% (95% confidence interval: 24-44%) with a median follow-up of approximately 3 years (range 0-7.5 years). For patients entering with minimal disease (defined as all areas < or = 2 cm), the 5-year EFS was 40 vs 26% for those entering with bulky disease (P = 0.0004). In the multivariate analysis, minimal disease on entry and administration of involved-field XRT post-transplant were significantly associated with improved EFS; the latter association was observed mainly in the cohort of patients with bulky disease. The overall 100-day treatment-related mortality rate was 4.4% (3% for the last 71 patients). New strategies are needed to reduce the high rate of relapse (50-60%) following auto-transplantation for relapsed or refractory NHL.

Patients > or = age 40 years undergoing autologous or allogeneic BMT have regimen-related mortality rates and event-free survivals comparable to patients < age 40 years.

To evaluate the safety and efficacy of marrow transplantation for older adults, the regimen-related mortality and event-free survival for patients > or = 40 years were compared with those for patients < 40 years. Of 148 consecutive patients receiving autotransplants for lymphoma or Hodgkin's disease, 70 were < 40 years and 78 were > or = 40 years at the time of transplant, including 40 who were > or = 50 years and 12 who were > or = 60 years. Eleven patients (16%) in the younger age group died from transplant-related complications compared with 4 (5%) in the older age group. The 4-year actuarial event-free survivals (EFS) for the younger and older age groups were 43% and 48%, respectively. After adjustment for covariates with prognostic significance, older age was marginally associated with improved event-free survival (P = 0.08). Of 92 consecutive patients undergoing allogeneic BMT during the same period, 62 patients were < 40 years and 30 patients were > or = 40 years, including 8 patients > or = 50 years, and 1 patient > 60 years. Non-relapse mortality (including deaths from GVHD) occurred in 28 of the younger patients (45%) and 9 of the older patients (30%). The 3-year actuarial EFS for the younger patients was 26% vs. 56% for the patients > or = 40 years (P = 0.057). However, this difference was mainly due to the higher proportion of patients with CML and early-stage leukemia in the older age group.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement depletion in vitro limits monoclonal antibody 6-19-dependent complement-mediated killing of tumor cells in bone marrow.

Selective killing of nonhematopoietic tumor cells in bone marrow harvested for autologous bone marrow transplantation was studied using cultured neuroblastoma cells, monoclonal antibody 6-19, and baby rabbit serum as a source of complement. Monoclonal antibody 6-19, a murine IgG2a antibody, was selected for binding to a cell surface antigen on cultured human neuroblastoma cells that is not expressed by hematopoietic cells. The antigen is an 80-kd sialoglycoprotein present on a wide variety of nonhematopoietic tumors, and it is expressed by normal fibroblast and endothelial cells. The effect of the presence or absence of bone marrow mononuclear cells on target cell survival was evaluated using Hoechst 33342-stained neuroblastoma cells, trypan blue exclusion, and fluorescence microscopy. More than 4 logs of neuroblastoma cells were destroyed in the presence of a more than tenfold excess of bone marrow cells following two incubations with monoclonal antibody 6-19 and complement for 30 min at 37 degrees C. Monoclonal antibody concentrations greater than 5-10 micrograms/ml did not increase cell lysis. The destruction of tumor cells was limited by depletion of complement activity. Tumor cell killing increased with complement concentration and incubation duration but there was no additional cell lysis with incubations greater than 30 min. The percentage of target cells killed was significantly decreased when the target cells were treated in the presence of increasing concentrations of bone marrow mononuclear cells. This decrease in target cell destruction is a result of additional depletion of complement activity by bone marrow mononuclear cells. These results suggest that optimal purging of tumor cells by antibody and complement will be achieved following multiple brief incubations with fresh antibody and complement.

Vascular anatomy and organ-specific tumor growth as critical factors in the development of metastases and their distribution among organs.

We have examined with 19 tumor cell lines the discrete roles that vascular anatomy and tumor-cell-organ-affinity play in the development of metastases and their distribution among organs. Spontaneous metastases of B16-G3.26 melanoma cells from a primary tumor growing in the foot pad of mice, or experimental metastases 21 days after intravenous tumor-cell injection resulted in tumor colonies only in the lungs. In contrast, when the lung microvasculature was bypassed, and the same cells given by systemic intra-arterial (s.i.a.) injection, large tumor colonies developed selectively in the ovaries, adrenal glands and bones, but rarely in the lungs. When animals injected i.v. were allowed to live with lung metastases for a long period of time, small tumor colonies began to develop in extra-pulmonary organs with a distribution identical to that seen after s.i.a. injection. Seven murine tumor cell lines (previously characterized by their ability to colonize primarily the lungs after i.v. injection) and 7 of the 8 studied human tumor cell lines colonized different specific extra-pulmonary organs after s.i.a. injection, frequently producing metastatic syndromes commonly described in patients with cancer, but rarely seen in animal models of metastasis. These results suggest that metastatic cells, even those capable of colonizing specific organs, do not freely circulate in the blood stream and lodge in specific tissues. In contrast, the cells must establish a vascular route of access to the target organ, e.g., through the systemic circulation from metastatic tumors in the lungs. Two cell lines considered to be tumorigenic but non-metastatic failed to colonize the lungs or extra-pulmonary organs after i.v. injection, but readily colonized specific organs after s.i.a. injection. Thus, tumor cells considered to be non-metastatic may be indeed metastatic if they are provided with vascular access to an organ more congenial to their growth requirements.

Cytokine effects and role of adhesive proteins and Fc receptors in human macrophage-mediated antibody dependent cellular cytotoxicity.

Mononuclear phagocytes participate in host immunological defense against tumors. We have investigated the role of selected recombinant cytokines on human macrophage-mediated tumor cytotoxicity in vitro utilizing a human colon cancer cell line target, SW1116, and murine monoclonal antibody 17-1A. Blood monocytes were kept in continuous culture to allow differentiation into macrophages. Maximum antibody dependent cellular cytotoxicity (ADCC) as measured in a 3H-thymidine release assay occurred after culturing the monocytes for 5-7 days. Human recombinant macrophage colony stimulating factor (CSF) (1,000 U/ml) did not increase ADCC above control levels whereas recombinant human granulocyte-macrophage colony stimulating factor, interleukin 4, and interleukin 3 were all capable of increasing ADCC. Antibodies to the CD11/CD18 integrin receptors did not significantly inhibit ADCC. When the ADCC incubation occurred in the presence of antibodies to the human Fc receptors, ADCC was inhibited significantly only by anti-FcRIII (3G8). A role for tumor necrosis factor alpha or other soluble mediators of cytotoxicity was not demonstrable in this system. These studies suggest avenues for manipulation and augmentation of macrophage-mediated antitumor ADCC.

An 80,000-kd glycoprotein cell surface antigen found only on nonhematopoietic cells in human bone marrow.

The murine IgG2a monoclonal antibody (MoAb) 6-19 binds to the surface of human nonhematopoietic cells but not to hematopoietic cells. As previously described, it can be used with complement to selectively kill nonhematopoietic cells prior to culture of human bone marrow. It is now shown that the 6-19 MoAb recognizes a specific antigenic determinant of apparent molecular mass 80 kd, detected by western blotting on nonhematopoietic tumor cell lines, endothelial cells, and bone marrow stromal cells. Mouse L cells were transfected with human DNA, and the 6-19 monoclonal antibody selected cells that expressed an antigen with similar characteristics. The antigenic determinant is absent in hematopoietic tumor cell lines, peripheral blood cells, and bone marrow hematopoietic cells. Investigation of the biochemical nature of the antigen, using various enzymes, lectin-binding studies, and western blotting suggests that the 6-19 epitope is at least partially carbohydrate and that the protein has N-linked sugars but not O-linked. The affinity (Ka approximately 10(9) M-1) of MoAb 6-19 binding is similar in tumor cells and normal fibroblasts and endothelial cells. The identification of a specific antigenic determinant of 80 kd may help to discriminate between hematopoietic and nonhematopoietic cells in human bone marrow.

Pathogenesis of vertebral metastasis and epidural spinal cord compression.

The authors have studied the sequential events in the process of vertebral metastasis that result in spinal cord compression. Different tumor cell lines were injected into the systemic arterial circulation of syngeneic or nude mice, and they were killed at timed intervals after injection or when they became paraplegic. The following observations were made. The tumor cells lodged and grew in the hematopoietic bone marrow of the vertebrae. Cancer cells in the vertebral marrow cavity invaded into the spinal canal through the foramina of the vertebral veins rather than destroying the cortical bone. Tumor cell lines that grew in an infiltrative fashion migrated toward a posterior location in the spinal canal, and compressed the spinal cord from a posterior direction. Tumor cell lines that grew as compact tumors formed a tumor mass at the same location from which the cells emerged from the vertebra, and compressed the cord predominantly from an anterior direction. Radiographic evidence of vertebral metastasis was a late event, and commonly associated with significant compression of the cord and extraosseous tumor. These experimental findings may help to establish better diagnostic and treatment strategies for patients with metastatic disease of the spine.

Characterization of human marrow stromal cells: role in progenitor cell binding and granulopoiesis.

Adherent cell layers and their associated extracellular matrices form when human marrow is incubated in cultures containing hydrocortisone and horse serum. These stromal layers contain cells positive for alkaline phosphatase; secrete collagens types I and III and fibronectin, bind the anti-actin monoclonal antibodies (MoAbs) HHF and CGA-7; stain with oil red O, and express the acetylated LDL receptor. Highly purified CD34 (My10)-positive progenitor cells attach to these stromal layers, and a 16-fold enrichment of CFU-GM in both stromal attachment and semisolid agar assays was observed. Granulopoiesis persisted up to 40 days (mean duration 25 days) after passaged stroma were recharged with stromal cell-depleted target cells in a two-stage liquid marrow culture system. Although equal to marrow fibroblasts in their ability to bind CD34+ myeloid progenitors, stromal layers were better at supporting granulopoiesis. This system provides an in vitro model to characterize the components of stroma and stroma-cytomatrix that enhance marrow progenitor cell localization and maintenance.

Lysis of human fibroblast colony-forming cells and endothelial cells by monoclonal antibody (6-19) and complement.

The murine IgG2a monoclonal antibody 6-19 binds to a wide variety of nonhematopoietic cells including human marrow-derived stromal cells but does not bind to marrow or peripheral blood cells. We studied the effects of this antibody and rabbit complement on marrow cells. Fibroblast colonies were eliminated from light density marrow cells by a single incubation with monoclonal antibody 6-19 and complement. The growth and composition of granulocytic and erythroid colonies were unaffected. Specific complement mediated cytotoxicity of the antibody was confirmed on passaged human fibroblasts derived from marrow (more than 99.6% of fibroblasts are killed by a single treatment). Similar results were obtained with human umbilical cord endothelial cells. In addition, such treatment abolished the initiation of Dexter culture stroma. Incubation of bone marrow cell suspensions with this antibody and complement will allow the study of stroma-free marrow cells in long-term liquid cultures.

Variables affecting the killing of cultured human neuroblastoma cells with monoclonal antibody and complement.

To determine incubation conditions that result in optimal in vitro killing of human tumor cells with monoclonal antibody and complement, variables affecting the killing of cultured human neuroblastoma cells with monoclonal antibody 6-19 and baby rabbit complement were studied. Neuroblastoma target cells were stained with the fluorescent dye Hoechst 33342, which enables rapid, sensitive detection of surviving cells in conjunction with trypan blue exclusion. Maximal cell lysis was obtained at an antibody concentration of 5-10 micrograms/ml. Greater than 5 logs of cell kill were obtained with appropriate treatment conditions. No antigenic modulation was detected. Complement activity was found to be the factor which limited the extent of cell kill. Specific cell lysis increased with increasing concentration of complement. As the reaction with antibody coated cells proceeded, complement activity was depleted. This resulted in the greatest cell kill occurring during the first 10-20 min of treatment. Generation of factor(s) inhibitory to complement activity was also found. However, the effect of inhibition on limiting the extent of cell kill was much less significant than the effect of complement depletion. When compared to a single incubation of the same total duration, the reduction in complement activity with duration of incubation resulted in greater killing by multiple brief incubations with fresh complement. The depletion of complement was found to increase at a greater cell concentration. This resulted in greater proportional survival as neuroblastoma cell concentration increased. Depletion of complement activity by aggregated monoclonal antibody was found to decrease cytotoxicity. This evaluation may provide a framework for optimization of target cell destruction using complement and other monoclonal antibodies and target cells.

A monoclonal anti-neuroblastoma antibody that discriminates between human nonhematopoietic and hematopoietic cell types.

A murine IgG2a monoclonal antibody, termed 6-19, was characterized in terms of its ability to bind to human cell lines and tissues. The hybridoma was selected for antibody binding to multiple human neuroblastoma cultured cell lines but not to peripheral blood mononuclear cells. 6-19 binds to the cell surface of all cultured human nonhematopoietic tumor cell lines tested, to cultured human fibroblasts and endothelial cells, and to nonhematopoietic tumors of many types. It does not bind detectably to any hematopoietic cells, leukemia cells, or lymphomas. In the presence of complement, 6-19 is very cytotoxic to cultured human neuroblastoma cells but not to bone marrow granulocyte-macrophage colony-forming cells. The 6-19 monoclonal antibody may prove useful in the identification or destruction of tumor and stromal cells in bone marrow.

A sensitive assay of cytotoxicity applicable to mixed cell populations.

The fluorescent vital dye, Hoechst 33342, was used to stain cultured cells prior to assay of antibody dependent complement mediated cytotoxicity. The fluorescence of nonviable dye stained cells is quenched by cellular uptake of trypan blue, but trypan blue excluding cells remain intensely fluorescent. Detection by fluorescence microscopy of one viable prestained cell per 10(5) unstained cells was accurate and reliable. The technique was found to have sensitivity equal to a clonogenic assay for measuring cytotoxicity. The dye stained cell assay may be used to measure depletion of a selected cell type, when those cells are stained prior to mixing with another cell population. This technique may prove useful to study model systems for depletion of tumor cells or T-cells from bone marrow.

Anti-neuroblastoma monoclonal antibodies which do not bind to bone marrow cells.

Three previously unreported mouse monoclonal antibodies are described. All three bind to cultured human neuroblastoma cells from six of six cell lines, but do not detectably bind to human bone marrow cells. The two IgG2a monoclonal antibodies bind to greater than 98% of the cultured cells from each cell line, bind to six of six human neuroblastoma tumors by immunoperoxidase histochemistry, and are cytotoxic with complement to cultured human neuroblastoma cells but not to human bone marrow granulocyte-macrophage colony-forming cells.