Ex-vivo expansion of hematopoietic progenitor cells: preliminary results in breast cancer

Ex-vivo expanded progenitor cells have been proposed as a source of cells to support high-dose chemotherapy and to decrease or eliminate the period of neutropenia following transplantation. To date, no clinical studies using ex vivo expanded cells, have demonstrated any decrease in the time to neutrophil or platelet recovery, although a number of clinical studies have been performed using a variety of growth factor cocktails and culture conditions. Over the past 6 years we have developed a static culture system that results in optimal expansion of myeloid progenitor cells. We have initiated a clinical study to evaluate this culture system in breast cancer patients receiving peripheral blood progenitor cells (PBPC) to support high-dose chemotherapy. CD34 selected cells were cultured for 10 days in 800 ml of defined media (Amgen Inc.) containing 100 ng/ml each of rhSCF, rhG-CSF and rhMGDF in 1L teflon bags (American Fluoroseal) at 20,000 to 50,000 cells per ml. After culture the cells were washed with 3 volumes of PBS to remove all media and growth factors and reinfused on day 0 of transplant followed by daily administration of rhG-CSF. On day +1 the patients received an unexpanded PBPC product to ensure the durability of the graft. Patients transplanted with expanded PBPC cells recovered neutrophil counts (ANC > 500/microl) as early as day 4 post transplant with a median of 6 days (range 4 to 14 days). In comparison, our historical control group of patients (N=175) had a median time to neutrophil engraftment of 9 days (range 7 to 24 days). A second cohort of patients were transplanted with expanded cells alone and a similar rapid engraftment was obtained. The first patients are now over 70 days post transplant with durable engraftment. No effect on platelet recovery has been observed in any patients to date. These data demonstrate that PBPC expanded under the conditions defined can significantly shorten the time to engraftment of neutrophils.     

Ex-vivo purging of hematopoietic progenitor cells

High-dose chemoradiation therapy with autologous hematopoietic progenitor cell support is effective treatment for patients with a variety of high-risk malignancies. In this setting, tumor cell contamination of the hematopoietic progenitor cell product is a frequently observed problem. The malignant cells in the autograft may be a source of relapse after transplant. Purging the hematopoietic cell grafts ex-vivo may eradicate malignant cells that may contaminate the autologous product. This chapter describes the major purging strategies that have been investigated clinically over the past decade. This investigation includes negative purging with pharmacologic agents and monoclonal antibodies, positive selection with immunoadsorption, immunomagnetic and flow cytometric sorting techniques, ex-vivo culture method, treatment with cytotoxic T cells, and virally mediated purging methods.     

Advanced breast cancer: high-dose chemotherapy and bone marrow autotransplants

The response of human breast cancer to drugs and radiation is dose-dependent, with higher doses producing increased response rates. However, dose escalation of several agents active against breast cancer is limited by bone marrow toxicity. This limitation can be overcome in some instances by transplantation of bone marrow cells. We evaluated 27 trials of bone marrow autotransplants in 172 patients who received single or multiple drug chemotherapy, radiation, or both. The overall response rate was 58%. Response rates were highest in trials involving multiple alkylating agents (76%) or previously untreated patients (81%). These data suggest that high-dose therapy and bone marrow autotransplants can produce remissions in patients with advanced breast cancer unresponsive to conventional therapy. A critical evaluation of this approach will require controlled trials in high-risk persons.     

Hematopoietic reconstitution after high-dose chemotherapy and autologous nonfrozen bone marrow rescue

Summary The kinetics of marrow engraftment were analyzed in 50 patient with acute leukemia (21), malignant lymphoma (15), and solid tumors (14) after high-dose multiagent chemotherapy followed by autologous bone marrow transplantation (ABMT) with nonfrozen bone marrow. Unseparated heparinized whole bone marrow was stored in 10% CPDA1 at 4°C for 72 h, then filtered and reinfused. The median number of nucleated cells reinfused was 1.6×10⁸/kg (range 0.5–3.8×10⁸/kg). All patients had a full hematopoietic reconstitution. Median time to achieve a neutrophil count > 500/l was 20 days (range 12–39) and median time to achieve an unsupported platelet count > 20.000/l was 20 days (range 10–55). The main factor associated with delayed engraftment was the number of prior chemotherapy cycles. We conclude that high-dose chemotherapy with nonfrozen ABMT is a safe procedure, without the requirement for costly cryopreservation facilities.     

Immune reconstitution following high-dose chemotherapy with stem cell rescue in patients with advanced breast cancer

The present study examines the nature of humoral and cellular immune reconstitution in 28 patients with advanced breast cancer following high-dose chemotherapy with stem cell rescue. Patients underwent testing of T, B, NK and dendritic cell function at serial time points until 1 year post transplant or until the time of disease progression. Abnormalities in T cell phenotype and function were observed following high-dose chemotherapy that persisted for at least 6-12 months. The vast majority of patients experienced an inversion of the CD4/CD8 ratio and demonstrated an anergic response to candida antigen. Mean T cell proliferation in response to PHA and to co-culture with allogeneic monocytes was significantly compromised. In contrast, mean IgG and IgA levels were normal 6 months post transplant and NK cell yields and function were transiently elevated following high-dose chemotherapy. Dendritic cells generated from peripheral blood progenitors displayed a characteristic phenotype and were potent inducers of allogeneic T cell proliferation in the post-transplant period. The study demonstrates that patients undergoing autologous transplantation for breast cancer experience a prolonged period of T cell dysfunction. In contrast, B, NK, and DC recover more rapidly. These findings carry significant implications for the design of post-transplant immunotherapy.     

Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer

The collection of small aliquots of bone marrow (BM), followed by ex vivo expansion for autologous transplantation may be less morbid, and more cost-effective, than typical BM or blood stem cell harvesting. Passive elimination of contaminating tumor cells during expansion could reduce reinoculation risks. Nineteen breast cancer patients underwent autotransplants exclusively using ex vivo expanded small aliquot BM cells (900-1200 x 10(6)). BM was expanded in media containing recombinant flt3 ligand, erythropoietin, and PIXY321, using stromal-based perfusion bioreactors for 12 days, and infused after high-dose chemotherapy. Correlations between cell dose and engraftment times were determined, and immunocytochemical tumor cell assays were performed before and after expansion. The median volume of BM expanded was 36.7 mL (range 15.8-87.0). Engraftment of neutrophils greater than 500/microL and platelets greater than 20,000/microL were 16 (13-24) and 24 (19-45) days, respectively; 1 patient had delayed platelet engraftment, even after infusion of back-up BM. Hematopoiesis is maintained at 24 months, despite posttransplant radiotherapy in 18 of the 19 patients. Transplanted CD34(+)/Lin(-) (lineage negative) cell dose correlated with neutrophil and platelet engraftment, with patients receiving greater than 2.0 x 10(5) CD34(+)/Lin(-) cells per kilogram, engrafting by day 28. Tumor cells were observed in 1 of the 19 patients before expansion, and in none of the 19 patients after expansion. It is feasible to perform autotransplants solely with BM cells grown ex vivo in perfusion bioreactors from a small aliquot. Engraftment times are similar to those of a typical 1000 to 1500 mL BM autotransplant. If verified, this procedure could reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which small stem cell doses are available, eg, cord blood transplants. (Blood. 2000;95:2169-2174)     

Peripheral progenitor cells (PBPC) in supportive care after high-dose chemotherapy in breast cancer

Hemopoietic growth factors (HGF) and leukapheresed peripheral progenitor cells (PBPC) are increasingly used for supportive care in high-dose chemotherapy (HDC) of solid tumors. Presently, therapeutic protocols with cyclic HDC plus PBPC support are successfuly used in breast cancer patients. Administration of PBPC significantly influences hemopoietic recovery in terms of shortening the pancytopenia period which reduces the risk of dangerous complications, especially the risk of infection. As a certain controversy exists about efficacy of this therapy, large randomized studies are conducted to find more accurate conclusions. In 1998 National Cancer Institute (NCI) gave top priority to four randomized studies of HDC with PBPC support. In recent years, rising yields of PBPC are obtained. The use of new combinations and dosages of hemopoietic growth factors leads to a significant increase of progenitor cells circulating in peripheral blood. Effective mobilization regimens combinations of chemotherapy and cytokines - enable to increase the numbers of circulating progenitors as much as 100-fold. Another aspect, how to minimize the risks is to reduce the transplant volume and so reduce the amount of cryoprotective agent DMSO (dimethyl sulfoxide) and hemolysed erythrocytes. This led to the idea to use only whole blood enriched for PBPC. At present it has been used also in our patients. The results show that enriched whole blood can be used as sufficient substitution for support in intensive cyclic chemotherapy in breast cancer patients.     

Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia

Cyclic neutropenia (CN) is a congenital hematopoietic disordercharacterized by remarkably regular oscillations of blood neutrophils from near normal to extremely low levels at 21-day intervals. Recurringepisodes of severe neutropenia lead to repetitive and sometimeslife-threatening infections. To investigate the cellular mechanism ofCN, the ultrastructure and the proliferative and survivalcharacteristics of bone marrow-derived CD34⁺ earlyprogenitors, CD33⁺/CD34 myeloid progenitors,and CD15⁺ neutrophil precursors from CN patients andhealthy volunteers were studied. The ultrastructural studies showedprofound apoptotic features in bone marrow progenitor cells in CN.Colony-forming assays demonstrated a 75% decrease in the number ofearly myeloid-committed colonies compared with controls. Long-termculture-initiating cell assays demonstrated a 6-fold increase inproduction of primitive progenitor cells in CN. To determine whetheraccelerated apoptosis might account for the underproduction of myeloidprogenitors, the hematopoietic subpopulations were labeled withfluorescein isothiocyanate-annexin V and analyzed by flow cytometry.Short-term culture of CN cells resulted in apoptosis of approximately65% of CD34⁺ cells, 80% ofCD33⁺/CD34 cells, and more than 70% ofCD15⁺ cells, as compared with 20%, 7%, and 15% apoptosisin respective control subpopulations. Evidence of accelerated apoptosisof bone marrow progenitor cells was observed in all 8 patientsparticipating in the study, regardless of the stage in the CN cycle inwhich bone marrow aspirations were obtained. Granulocytecolony-stimulating factor therapy of CN patients significantly improvedsurvival of bone marrow progenitor cells. These data indicate thatineffective production of neutrophils is due to accelerated apoptosisof bone marrow myeloid progenitor cells in CN.     

The role of high-dose chemotherapy with autologous bone marrow transplantation in the treatment of breast cancer

Autologous bone marrow reinfusion rapidly repopulates severely damaged bone marrow thus shortening the period of myelosuppression following high-dose chemotherapy programs. This strategy has been successfully employed in several hematologic malignancies such as acute leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and chronic myelogenous leukemia. More recently a number of clinical trials have investigated the role of high-dose chemotherapy with autologous bone marrow transplant in solid tumors. This strategy, when used in patients with advanced refractory metastatic breast cancer, results in a high objective response rate (30-70%) but most of these remissions are of short duration (3-4 months). When using high-dose single agents complete remissions are rare; with combination chemotherapy they are more frequent (20-50%). The utilization of high-dose chemotherapy with autologous marrow transplant as a consolidation after achieving a partial or complete remission with standard chemotherapy has shown more promising results with complete remissions approaching 70% in some series. The impact of any of these strategies on overall survival of patients with metastatic breast cancer remains to be demonstrated. The optimal patient selection criteria and strategies for additional development of this field are discussed.     

Costs of high-dose chemotherapy and peripheral blood progenitor cell autograft for breast cancer.

The aim of the study was to analyze the real cost of single or tandem high-dose chemotherapy (HDC) and peripheral blood progenitor cell autologous transplant (PBPCT) in patients with breast cancer. We analyzed the costs of 40 PBPCT performed in 20 patients. Tandem transplant was planned for each patient. Resources used and direct costs were identified for each patient. The study was carried out using the hospital perspective and monetary values were reported in 1999 Euro. The mean cost of whole procedure for single transplant was 20,816.63 Euro, while the mean cost of tandem transplant was 38,770.83 Euro. The cost distribution in the two groups was similar: the most expensive phase of procedure was the supportive phase post transplant (about 60% of total cost), with the categories of cost most represented being professional fees (about 28%) and pharmacy (about 35%). Awaiting more convincing trials of the clinical advantage of HDC in breast cancer, our analytical evaluation of transplant costs for different therapeutic options, single or tandem, permits identification of the most expensive categories in order to intervene for cost savings.     

CD4dull+ hematopoietic progenitor cells in murine bone marrow

CD4+ cells comprise approximately 3% to 6% of murine bone marrow (BM) cells. The majority are CD4dull+, but there are two distinct sub populations: CD4 brightly positive Gr-1- cells (CD4hiGr-1-) and CD4+ Gr- 1+ cells (CD4loGr-1lo). CD4hiGr-1- cells are considered to be mature T cells by cell surface antigen expression and morphology. CD4loGr-1lo cells, which comprise approximately 0.6% of the BM cells, express small amount of B220 and Thy1 antigens. Interestingly, colony-forming units (CFU)-spleen and CFU-C are not enriched in this population. However, when injected into lethally irradiated mice, CD4loGr-1lo cells were shown to differentiate into T-cell, B-cell, and myelo-monocyte lineages when assayed 26 weeks after transplantation. Furthermore, donor-derived CD4loGr-1lo cells were present in the recipients' BM at least 16 weeks after transplantation. These observations suggest that murine CD4loGr- 1lo cells in BM have self-renewal capability and retain the ability to differentiate into at least three lineages in long-term hematopoiesis.     

Crucial role of FLT3 ligand in immune reconstitution after bone marrow transplantation and high-dose chemotherapy

Almost 5 decades after the first clinical transplantations, delayed immune reconstitution remains a considerable hurdle in bone marrow transplantation, and the mechanisms regulating immune reconstitution after transplantation remain to be established. Whereas adult fms-like tyrosine kinase 3 ligand-deficient (FL(-/-)) mice have reduced numbers of early B- and T-cell progenitors, they sustain close to normal levels of mature B and T cells. Herein, we demonstrate that adult bone marrow cells fail to reconstitute B-cell progenitors and conventional B cells in lethally irradiated FL(-/-) recipients, which also display delayed kinetics of T-cell reconstitution. Similarly, FL is essential for B-cell regeneration after chemotherapy-induced myeloablation. In contrast, fetal progenitors reconstitute B lymphopoiesis in FL(-/-) mice, albeit at reduced levels. A critical role of FL in adult B lymphopoiesis is further substantiated by an age-progressive decline in peripheral conventional B cells in FL(-/-) mice, whereas fetally and early postnatally derived B1 and marginal zone B cells are sustained in a FL-independent manner. Thus, FL plays a crucial role in sustaining conventional B lymphopoiesis in adult mice and, as a consequence, our findings implicate a critical role of FL in promoting immune reconstitution after myeloablation and bone marrow transplantation.     

Secondary acute leukemia following mitoxantrone-based high-dose chemotherapy for primary breast cancer patients

The incidence of secondary myelodysplasia/acute myeloid leukemia (AML) was retrospectively assessed in an international joint study in 305 node-positive breast cancer patients, who received mitoxantrone-based high-dose chemotherapy (HDCT) followed by autologous stem cell support as adjuvant therapy. The median age of the patients was 57 years (range 22-67). In all, 268 patients received peripheral blood stem cells, and 47 patients received autologous bone marrow. After a median follow-up of 57 months (range 10-125), three cases of secondary AML (sAML) were observed, resulting in a cumulative incidence of 0.94%. One case of sAML developed 18 months after HDCT (FAB M3) The karyotype was translocation 15;17 and, after induction therapy, the patient underwent autologous stem cell transplantation, and is in complete remission (CR) of both breast cancer and AML. The second patient developed AML (FAB M4eo with inversion 16) 5 months after HDCT. This patient achieved CR after induction therapy, but died of infectious complication. A third patient developed AML (FAB M4) 6 months after HDCT. She achieved CR after induction therapy, but relapsed and expired 28 months after diagnosis of AML. sAML after mitoxantrone-based HDCT is a possible, but rare complication in breast cancer patients.     

Immune reconstitution following hematopoietic progenitor cell transplantation: challenges for the future

Successful hematopoietic progenitor cell transplantation requires rapid and complete transfer of the donor hematopoietic and immune systems to the host. Whereas the uncontrolled transfer of a nontolerant donor immune system results in GVHD in many cases, strategies which diminish GVHD also diminish immune reconstitution. Thus, the reliable, rapid and safe transfer of immunity from donor to host remains a major challenge for the field. Advances in the understanding of the biology of immune reconstitution have elucidated that thymic-dependent immune reconstitution can restore global immunity, but is especially vulnerable to toxicities associated with transplant. Alternatively, homeostatic peripheral expansion can be exploited for targeted immunity toward pathogens and tumors, but is difficult to manipulate without exacerbating GVHD risk. New translatable strategies are needed to safely augment one or both of these pathways in the setting of allogeneic hematopoietic progenitor cell transplantation.     

Coronavirus pneumonia following autologous bone marrow transplantation for breast cancer

Infectious bronchitis virus, otherwise known as coronavirus, can cause mild upper respiratory tract illnesses in children and adults. Rarely has coronavirus been linked, either by serology or nasal wash, to pneumonia. We report a case of a young woman who, following treatment for stage IIIA breast cancer using a high-dose chemotherapy regimen followed by autologous bone marrow and stem cell transplantation, developed respiratory failure and was found to have coronavirus pneumonia as diagnosed by electron microscopy from BAL fluid. We propose that coronavirus should be considered in the differential diagnosis of acute respiratory failure in cancer patients who have undergone high-dose chemotherapy and autologous hematopoietic support.     

Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures

Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.     

Streptococcal septicaemia following autologous bone marrow transplantation in children treated with high-dose chemotherapy

A total of 251 patients were given 326 courses of high-dose chemotherapy followed by autologous bone marrow transplantation between February 1979 and August 1988. Ninety-one cases of septicaemia developed in 84 patients, 33 of these cases (36%) were due to streptococci. The outcome was fatal for four patients (12.1%). No specific risk factors were identified which might account for this septicaemia and no clinical feature was significantly associated with these cases of bacterial infection. Pulmonary and neurologic septic complications, however, were of very poor prognosis since all patients with these complications died. The high rate of streptococcal septicaemia and the poor outcome for patients with prolonged and profound neutropenia led us to modify our choice of initial broad-spectrum antibiotic therapy.     

Economic analysis of conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer

We performed an economic analysis of data from 180 women in a clinical trial of conventional-dose chemotherapy vs high-dose chemotherapy plus stem-cell transplantation for metastatic breast cancer responding to first-line chemotherapy. Data on resource use, including hospitalizations, medical procedures, medications, and diagnostic tests, were abstracted from subjects' clinical trial records. Resources were valued using the Medicare Fee Schedule for inpatient costs at one academic medical center and average wholesale prices for medications. Monthly costs were calculated and stratified by treatment group and clinical phase. Mean follow-up was 690 days in the transplantation group and 758 days in the conventional-dose chemotherapy group. Subjects in the transplantation group were hospitalized for more days (28.6 vs 17.8, P=0.0041) and incurred higher costs (US dollars 84055 vs US dollars 28169) than subjects receiving conventional-dose chemotherapy, with a mean difference of US dollars 55886 (95% CI, US dollars 47298-US dollars 63666). Sensitivity analyses resulted in cost differences between the treatment groups from US dollars 36528 to US dollars 75531. High-dose chemotherapy plus stem-cell transplantation resulted in substantial additional morbidity and costs at no improvement in survival. Neither the survival results nor the economic findings support the use of this procedure outside of the clinical trial setting.     

Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells

Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disorder galactosialidosis (GS). PPCA(-/-) mice display a disease course similar to that of severe human GS, resulting in nephropathy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overexpressing the corrective enzyme in either erythroid cells or monocytes/macrophages has proven effective for the improvement of the phenotype, and encouraged the use of genetically modified BM cells for ex vivo gene therapy of GS. Here, we established stable donor hematopoiesis in PPCA(-/-) mice that received hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based, bicistronic retroviral vector overexpressing PPCA and the green fluorescent protein (GFP) marker. We observed complete correction of the disease phenotype in the systemic organs up to 10 months after transplantation. PPCA(+) BM-derived cells were detected in all tissues, with the highest expression in liver, spleen, BM, thymus, and lung. In addition, a lysosomal immunostaining was seen in nonhematopoietic cells, indicating efficient uptake of the corrective protein by these cells and cross-correction. Expression in the brain occurred throughout the parenchyma but was mainly localized on perivascular areas. However, PPCA expression in the central nervous system was apparently sufficient to delay the onset of Purkinje cell degeneration and to correct the ataxia. The long-term expression and internalization of the PPCA by cells of systemic organs and the clear improvement of the neurologic phenotype support the use of this approach for the treatment of GS in humans. (Blood. 2002;99:3169-3178)