- Email: [email protected]
Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer
Experimental Hematology 27 (1999) 615–623
Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer Carlos R. Bachiera, Erhan Gokmenb, Judy Tealeb, Sophie Lanzkrona, Craig Childsa, Wilbur Franklinc, Elizabeth Shpallc, Judith Douvilled, Stephanie Weberd, Thomas Mullerd, Douglas Armstrongd, and Charles F. LeMaistrea a South Texas Cancer Institute, San Antonio, TX; bUniversity of Texas Health Science Center, San Antonio, TX;cUniversity of Colorado, Denver, CO; dAastrom Biosciences, Inc., Ann Arbor, MI
(Received 23 June 1998; revised 19 November 1998; accepted 24 November 1998)
The use of hematopoietic growth factors, stromal monolayers, and frequent medium exchange allows the expansion of hematopoietic progenitors ex-vivo. We evaluated the use of exvivo expanded progenitor cells for hematopoietic reconstitution following high dose chemotherapy (HDC) in breast cancer patients. Patients with high-risk Stage II or metastatic breast carcinoma underwent bone marrow aspirations using general anesthesia. A total of 675–1125 3 106 mononuclear cells (MNC) were seeded for ex-vivo expansion for 12 days in controlled perfusion bioreactors (Aastrom Biosciences, Inc.). The bone marrow cultures, which included the stromal cells collected with the aspirate, were supplemented with erythropoietin, granulocyte-macrophage-colony stimulating factor (GMCSF)/IL-3 fusion protein (PIXY 321), and flt3 ligand. Stem cell transplant was performed with expanded cells after HDC. A median bone marrow volume of 52.9 mL (range 42–187 mL) was needed to inoculate the bioreactors. Median fold expansion of nucleated cells (NC) and colony forming unit granulocytemacrophage (CFU-GM) was 4.9 and 9.5, respectively. The median fold expansion of CD341lin2and long-term culture–initiating culture (LTC-IC) was 0.42 and 0.32, respectively. Five patients were transplanted with ex-vivo expanded NC. Median days to an absolute neutrophil count . 500/mL was 18 (range 15–22). Median days to a platelet count . 20,000/ml was 23 (range 19–39). All patients had sustained engraftment of both neutrophils and platelets. Immune reconstitution was similar to that seen after HDC and conventional stem cell transplantation. We conclude that ex-vivo expansion of progenitor cells from perfusion cultures of small volume bone marrow aspirates, allows hematopoietic reconstitution after HDC. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.
Offprint requests to: Carlos R. Bachier, M.D., South Texas Cancer Institute, 7700 Floyd Curl Drive, San Antonio, Texas 77229; E-mail: cbachier @txdirect.net
Keywords: Ex-vivo expansion—Growth factors—Stem cell transplant
Introduction Hematopoietic growth factors and peripheral blood stem cells (PBSC) have decreased the time to recovery of platelets and white blood cells in patients treated with high dose chemotherapy. Even with these advances there is still an obligatory time to recovery of neutrophils and platelets of about 9 and 14 days, respectively [1–6]. Furthermore, PBSC mobilization and collection can be associated with complications and side effects including catheter related infections, pneumothorax, fevers, bone pain and allergic reactions [7]. Finally, PBSC grafts can be contaminated with malignant cells [8–10] that may contribute to cancer recurrence posttransplant [11–13]. Developments in hematopoietic stem cell biology now allow the maintenance and growth of hematopoietic progenitor cells ex-vivo. Ex-vivo hematopoietic stem cell expansion could potentially be used to further decrease the time to engraftment, for tumor purging and to decrease the risks and side effects associated with bone marrow harvest or collection of PBSC. It could also be applied to graft engineering strategies including gene therapy and expansion of subsets of lympho-hematopoietic cells. Strategies aimed at expanding progenitor cells ex-vivo include incubation of mononuclear or CD341 selected cells in the presence of hematopoietic growth factors [14–21]. Some culture conditions also include the use of autologous plasma and stromal monolayers in the ex-vivo expansion mixture [22]. Differences in the efficacy and yield of expansion in these studies are likely due to variations in the exvivo expansion conditions and the progenitor cell assay
0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(98)0 0 0 8 5 - X
616
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
used to measure fold expansion. The optimal conditions for expansion of these progenitor cells has not been defined. Furthermore, most of the current ex-vivo expansion strategies are associated with differentiation of hematopoietic progenitors into mature cells and loss of early precursors as measured by CD34 subsets and long-term culture–initiating culture (LTC-IC) assays. Initial human studies showed effective purging of chronic myelogenous leukemia cells in long-term culture [23]. No growth factors were used and, therefore, there was loss of progenitor cells during culture. More recently, Brugger et al. [19] ex-vivo expanded CD341 peripheral blood cells in tissue culture flasks. The ex-vivo expanded cells were infused after nonmyeloablative intensive chemotherapy. Rapid engraftment was achieved in all four patients transplanted with expanded cells. Koller and colleagues [24,25] evaluated the role of different culture conditions on the ex-vivo expansion of hematopoietic progenitors. Ex-vivo expansion of LTC-IC cells was dependent on the presence of autologous stroma and frequent medium exchanges. Incubation of bone marrow progenitors with IL-1, IL-3, IL-6, GM-CSF, G-CSF, stem cell factor (SCF), erythropoietin (EPO), and interferon g in different combinations was evaluated for optimal expansion conditions. In stromal-free, CD34-enriched cell cultures, the combination of IL-3/GM-CSF/EPO/SCF was found to be as efficient on expanding LTC-IC progenitors as the addition of other growth factors to this combination. Furthermore, decreasing cell enrichment of CD341 lin2cells was associated with a three- to fivefold increase in NC, CFU-GM, and LTC-IC. Based on these results, the cell production system (CPS) was developed by Aastrom Biosciences for the ex-vivo expansion of bone marrow nucleated cells (NC). Preliminary clinical studies published by Champlin et al. [26] demonstrated the safety of transplanting bone marrow NC ex-vivo expanded with the CPS together with a standard bone marrow harvest. In the current trial, we used ex-vivo expanded bone marrow cells as the only source of stem cells for hematopoietic reconstitution following myeloablative chemotherapy for breast cancer.
Methods Patients Patients with Stage II breast cancer with metastasis to more than ten axillary lymph nodes and Stage IV breast cancer in sensitive relapse were enrolled in this trial after signing an Institutional Review Board approved informed consent. All patients in this study underwent bone marrow harvest, ex-vivo expansion, and transplant at a single center (South Texas Cancer Institute). Other entry criteria included a maximum of two prior chemotherapy regimens, $30% bone marrow cellularity (core bone marrow biopsy prior to enrollment), no bone marrow involvement by tumor determined by H & E stains of bone marrow biopsies, and adequate end organ func-
tion. Early in the trial, $35 3 106/mL bone marrow NC concentration at the time of harvest and $2500 3 106 NC post-expansion were required for transplant with ex-vivo expanded stem cells. Bone marrow aspiration Patients were taken to the operating room and placed under general anesthesia. Using 16-inch gauge Illinois needles and 10 mL syringes, initial 3–5 mL of bone marrow per aspirate were obtained from the superior posterior iliac crests and collected in 150mL bags containing 5000 units of heparin. A median of 85.7 mL was collected (range 45–187). The harvest was continued until a standard back-up bone marrow collection of at least 1 3 108 NC/ kg was obtained. Ex-vivo expansion Device description. The single-use, closed, disposable, sterile cell culture device that is part of the Aastrom CPS consists of two compartments separated by a gas-permeable, liquid-impermeable membrane. The lower cell culture chamber is continuously perfused by growth medium at 378C. The Aastrom CPS incubator contains a cold compartment for media storage during the 12-day cell culture (reagent perfusion begins on Day 3), and a warm (378C) compartment in which the cell cassette containing the cell culture device is placed for the duration of the culture. In the incubator, the cells grow on the tissue culture plastic surface of the cell culture bed in the cell culture device, developing a dense stromal layer. The upper cell culture chamber of the cell culture device is provided with a constant flow of oxygen, nitrogen and CO2, such that oxygenation of the cells is accomplished by diffusion across the membrane and through the culture medium. CO2 is removed by the same diffusion mechanism. The medium used to perfuse the cultured cells is stored in a closed vessel in an adjacent compartment of the same incubator at 48C. The vessel’s only external connection is by medical grade tubing. A computer system allows the operator to monitor the status of the cell production. Cell culture conditions. Bone marrow mononuclear cells were obtained after Ficoll Hypaque separation. A total of 675–1100 3 106 MNC were inoculated in three or four bioreactors. The hematopoietic cells were suspended in tissue culture medium composed of Iscove’s Modified Dulbecco’s Medium supplemented with 10% fetal bovine serum, 10% horse serum, hydrocortisone (5 mM), PIXY 321 (5 ng/mL), L-glutamine (4 mM), erythropoietin (0.1 U/ mL), Flt3-L (25 ng/mL), gentamicin sulfate (5 mg/mL), vancomycin (20 mg/mL). PIXY 321, Flt3-L, and erythropietin were all clinical grade human recombinant proteins. The cells are incubated in the Aastrom CPS for 12 days at 378C with the tissue culture medium continuously replaced with fresh medium. Sampling of the culture medium, via a septum in the medium waste line, is carried out 48 hours prior to harvest to test for bacterial and fungal contaminants. After 12 days of incubation in this closed system, the nonadherent cell fraction was removed by draining the growth medium from the cell culture device into the harvest bag. The chamber was then rinsed with 100 mL of 0.04% Trypsin-EDTA solution. This was followed by gentle, automated agitation of the cell culture device and collection of the rinse into the harvest bag. The adherent layer was detached from the cell culture bed surface by sterile addition of 100 mL of 0.04% Trypsin-EDTA solution. This was followed by agitation of the cell culture device and collection of the rinse into the harvest bag. The expanded cell product containing
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623 Table 1. Patient characteristics Patient Number
Age (years) Weight (kg) Stage Prior Chemotherapy Regimens
1
2
3
4
5
6
7
46 63 II 1
42 70 II 1
42 69 II 1
41 58 IV 1
44 82 IV 2
53 92 II 1
39 59 II 1
both adherent and non-adherent cells was washed using the COBE 2991 Cell Processor. The final cell product was suspended in approximately 250 mL of Normosol and 0.5% human serum albumin for immediate infusion. Tumor contamination assay Tumor contamination was analyzed according to the immunocytochemistry method described by Franklin et al. [27]. Mononuclear cells before and after ex-vivo expansion were washed once with medium-199 (Gibco Laboratories; Grand Island, NY) containing 10% heparin and resuspended with 25% FBS and DPBS (Hyclone Lab.; Logan, UT) at a concentration of 2.5 3 106 cells/mL. Cytospins were prepared using a Cyto-Tek centrifuge (Miles Scientific; Elkhart, IN). Two hundred microliters of cell suspension (5 3 105 cells) were added to the cytocentrifuge slide holder loaded with a silicone-coated glass slide and 1 mL filter paper. Cells were then centrifuged onto the slide at 500 rpm (160 3 g) for 5 minutes and air dried for a minimum of 30 minutes. Slides were fixed for 20 minutes using acetone/methanol/formalin (45%/45%/10%). Fixed slides were stained in 200 mL containers according to a modification of the alkaline phosphatase antialkaline phosphatase (APAAP) technique and were counterstained with hematoxylin. A total of ten stained slides were examined using a standard binocular light microscope with a low power (103) objective. This assay was able to detect three tumor cells in a background of 106 normal cells. Progenitor cell assays The efficacy of expansion was measured by analyzing NC numbers, CFU-GM and LTC-IC colonies, and flow cytometry for CD341/CD341 lin2subsets. For CFU-GM assays, 1.5 3 104 cells per mL of culture medium (Methylcellulose supplemented with PIXY 321, Erythropoietin and G-CSF) were plated into 35-mm nontissue cultured treated plates. Plates were then incubated at 378C in 5% CO2 and 95% O2
617
for 14 days. For LTC-IC assays, bone marrow stroma was prepared by incubating bone marrow mononuclear cells in tissue culture flasks containing Dexter medium. Stromal cells were grown in Dexter medium at 378C in 5% CO2 and 95% O2. Stromal cells were then trypsinized, washed, and exposed to 15–20 Gy of gamma irradiation. Irradiated stromal cells were then plated in 96-well plates (1 3 104 cells per well at a concentration of 1 3 105 cells per mL of Dexter medium). Patient samples before and after ex-vivo expansion were diluted in Dexter medium at four different cell concentrations and incubated on plates containing irradiated stroma in a humidified incubator at 338C in 5% CO2 and 95% O2. LTC-IC cultures were maintained by replacing 50% of the medium in each well with fresh Dexter medium once weekly. These cultures were maintained for 5 weeks in liquid culture before harvesting for progenitor cell assays. Lin2 cell subtypes were obtained using the following antibodies: CD3, CD11b, CD15, CD20. Due to the presence of CD341 expressing stromal cells, total CD341 NC was not analyzed in the exvivo expanded product. Clinical design Patients underwent bone marrow harvest and inoculation of the CPS 12 days prior (Day 212) to the infusion of ex-vivo expanded cells. On Day 26 they were admitted to the hospital and on Day 25 they began their high dose chemotherapy regimen consisting of: Cyclophosphamide 1.875 mg/m2/d 3 3; Cisplatinum 55 mg/m2/d 3 3; BCNU 600 mg/m2/d 3 1. On Day 0, or 12 days after bone marrow harvest and inoculation of the CPS bioreactors, patients received the infusion of ex-vivo expanded cells. On Day 11 they started G-CSF at 10 mcg/kg per day until white blood cell engraftment. Analysis of immune reconstitution Peripheral blood samples were obtained from four patients at 5, 6, 7, and 12 months post-transplant. The fifth patient died of relapsed disease at Day 159 post-transplant prior to immunological evaluation. Peripheral blood mononuclear cells obtained by Ficoll-Hypaque density gradient sedimentation were stained for flow cytometry and analyzed using FACS (Becton Dickinson; Mountain View, CA). The fluorochrome-labeled mouse monoclonal antiCD3, CD4, CD8, CD19, CD16, CD56, CD45RA, and CD45RO antibodies, all purchased from Becton Dickinson, were used. For background staining, irrelevant mouse antibodies of IgG1 and IgG2a were used. CD41 CD45RA1, CD41 CD45RO1, CD81 CD45RA1, and CD81 CD45RO1 subsets were defined as fractions of gated lymphocyte population co-staining for CD4 and CD45RA, CD4 and CD45RO, CD8 and CD45RA, and CD8 and CD45RO,
Table 2. Pre-expansion bone marrow cell product
Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Mean
Volume of BM* (mL)
NC 3 106/mL
CD341 3 106/kg (%)
CD341LIN2 3 105/kg (%)
LTC-IC 3 103/kg
187.0 87.6 85.7 113.0 45.6 52.2 61.5 90.4
19.3 44.5 77.0 23.7 47.4 23.0 40.3 39.4
0.53 (4.9) 0.14 (1.1) 0.38 (2.9) 0.81 (4.3) 0.61 (5.6) 0.23 (3.3) 0.81 (7.0) 0.50 (4.2)
2.6 (2.4) 1.4 (1.1) 2.8 (2.2) 3.0 (1.6) 2.0 (1.9) 0.8 (1.1) 5.7 (4.9) 2.9 (2.2)
1.31 0.33 0.43 0.48 0.47 ND 1.19 0.70
*Initial volume of the bone marrow (BM) aspirated. A mean of 60 mL needed to inoculate bioreactors; Pt. 5 patient.
618
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
Table 3. Tumor cell detection Pt. #4 *Tumor cell concentration (Pre-expansion) † Total number of tumor cells (Pre-expansion) Total number of tumor cells (Post-expansion)
1.8
Pt. #5
66.5
1620
59,850
0
0
*Number of tumor cells 3 106 hematopoietic cells. † Number of tumor cells in 900 3 106 cells inoculated into bioreactors.
respectively. The CD45RA marker has been demonstrated on “naive” T cells, whereas the CD45RO marker is found on activated or memory T cells [28,29]. Absolute numbers of lymphocyte subsets were calculated by multiplying the percentage of cells staining positive with the relevant antibodies by the absolute number of lymphocytes determined by a Coulter counter analysis (Miami, FL) and a manual differential count. Serum immunoglobulins were also quantified at 5 to 12 months post-transplant.
Results Patient characteristics Seven patients were enrolled including five patients with Stage II and two patients with Stage IV breast cancer (Table 1). Median age was 44 years (range 41–53 years). Six patients had one, and one patient had two prior chemotherapy regimens. Their median weight was 69 kg (range 58–92 kg). Cells were expanded for all seven patients enrolled, however, only five patients were transplanted with the expanded product. Pre-expansion bone marrow cell product A median volume of 85.7 mL (range 45–187) of bone marrow per patient was aspirated from the superior posterior iliac crests (Table 2). The median recovery yield after Ficoll centrifugation was 46.6% (range 23%–60.6%). From the initial bone marrow volume, a median of 52.9 mL per patient (range 42–187 mL) was needed to inoculate the bioreactors with 675–1125 3 106 MNC. The median NC concentration of bone marrow prior to Ficoll centrifugation was
40 3 106/mL (range 19–77 per mL). The median numbers of CD341 MNC, CD341lin2and LTC-IC in the cell inoculum was 0.53 3 106/kg (range 0.14–0.81 3 106/kg), 2.6 3 105/kg (range 0.8–5.7) and 0.47 3 103/kg (range 0.33– 1.31 3 103/kg), respectively. Tumor cell purging Of the seven patients, only patients with Stage IV breast cancer (patients 4 and 5) were found to have tumor cells detected (Table 3) by immunocytochemical assays in their bone marrow samples. The total number of tumor cells inoculated into the bioreactors prior to expansion were 1620 and 59850 in these two patients. No tumor cells were seen in any of the bone marrow samples (n 5 7) after ex-vivo expansion. Post-expansion bone marrow cell product Median NC post-expansion (Table 4) was 0.50 3 108/kg (range 0.26–0.63 3 108/kg). Median CFU-GM post-expansion was 1.9 3 105/kg (range 1.2–12.2 3 105/kg). Median fold expansion of NC and CFU-GM was 4.9 and 9.5, respectively. The median number of CD341lin2 post-expansion was 1.15 3 105/kg (range 0.28–2.07 3 105/kg). The median fold expansion of CD341lin2 was 0.42. The median number of LTC-IC post-expansion was 0.33 3 103/kg (range 0.04–0.68 3 103/kg). The median fold expansion of LTC-IC was 0.32 (range 0.25–2.05). Clinical outcome Five patients were transplanted with ex-vivo expanded NC (Table 5). Patients 1 and 3 underwent stem transplant with their “back up” bone marrow NC due to concerns early in the trial on the adequate number of NC needed for engraftment pre and post ex-vivo expansion. Median days to an absolute neutrophil count . 500/ml was 18 (range 15–22). Median days to a platelet count . 20,000/ml was 23 (range 19–39). Kinetics of white blood cell and platelet engraftment are shown in Fig. 1. Median number of febrile days (temperature $38.38C) were 3 (range 0–8 days). Median number of transfusion of PRBC’s and platelets was 2 (range 2–8) and 4 (range 3–8), respectively. Average length of stay was 14.4 days. All patients had sustained engraftment of both white blood cell and platelets with a median follow-up
Table 4. Post-expansion cell product
Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Mean
NC 3 108/kg (fold expansion)
CFU-GM 3 105/kg (fold expansion)
CD341LIN2 3 105/kg (fold expansion)
LTC-IC 3 103/kg (fold expansion)
0.36 (5.0) 0.63 (6.5) 0.26 (2.5) 0.58 (3.6) 0.50 (5.4) 0.28 (4.9) 0.54 (4.6) 0.45 (4.7)
12.2 (30.7) 5.0 (12.6) 1.9 (9.5) 1.6 (5.0) 1.2 (3.7) 1.5 (8.1) 5.1 (54.7) 4.1 (17.6)
1.15 (0.67) 1.26 (1.18) 1.43 (0.64) 0.58 (0.20) 0.65 (0.37) 0.28 (0.40) 2.37 (0.42) 1.10 (0.55)
0.32 (0.25) 0.68 (2.05) 0.12 (0.29) 0.14 (0.29) 0.33 (0.70) 0.04 0.40 (0.34) 0.29 (0.56)
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
of 183 days (range 156–338). Patients 4 and 5 relapsed at 211 and 149 days post-transplant, respectively. Patient 5 died of breast cancer 159 days post-transplant. Patient 4 is in partial remission after additional chemotherapy. Patients 1 and 3 underwent stem transplant with their “back up” bone marrow NC due to concerns early in the trial on the adequate number of NC needed for engraftment pre and post ex-vivo expansion. Patients 1 and 3 received 1.6 3 108 NC/kg and 2.5 3 108 NC/kg respectively on Day 0. They received the same high dose chemotherapy regimen and post-transplant supportive care as patients undergoing transplant with ex-vivo expanded bone marrow cells. Days to neutrophil and platelet engraftment were 12/40 (patient 1) and 11/23 (patient 3), respectively.
Immune reconstitution Patients 2, 4, 6, and 7 were evaluated for immune reconstitution at 12, 7, 6, and 5 months post-transplant, respectively. Patient 5, who died of relapsed disease at Day 159 posttransplant, and two patients who received nonexpanded bone marrow grafts were not studied. No opportunistic infectious complications were observed in any of the patients. As shown in Table 6, serum immunoglobulin levels were within adult normal range in all patients at 5 to 12 months post-transplant. None of the patients received intravenous immunoglobulin. Total lymphocyte counts as well as number of B lymphocytes and natural killer cells had returned to normal or near-normal levels in three patients at 5 to 12 months (Table 6). While the absolute numbers of CD81 T-cell subset were normal at the time of evaluation in all patients, CD41 T-cell counts remained low as long as 12 months post-transplant. The CD4/CD8 ratios were inverted in three patients similar to that observed after conventional hematopoietic stem cell transplantation [30–33]. CD45 isoform expression on the emerging T cells was also addressed. CD45RO co-expression was more common among both CD41 and CD81 T-cell subsets than CD45RA except in one patient who had similar numbers of CD81 cells coexpressing CD45RA and CD45RO. These results are also similar to that observed after conventional hematopoietic stem cell transplantation [32–33].
619
Discussion Preclinical studies by Koller et al. [24,25] showed that exvivo expansion of bone marrow cells using continuous perfusion of media and growth factors along with autologous stroma was associated with expansion of early and late hematopoietic cells including NC, CFU-GM, and LTC-IC. Based on these results, a Phase I trial was initiated to determine if expansion of small volumes of bone marrow could provide enough progenitor cells for lymphohematopoietic reconstitution following high dose chemotherapy in patients with breast cancer. Aspiration of small volumes of bone marrow for expansion was performed under general anesthesia to allow for the collection of additional hematopoietic cells as back-up for patients with failure to engraft. There were no engraftment failures and none of the patients required infusion of back-up cells after transplant with ex-vivo expanded cells. Patients 1 and 3 underwent transplant with an engrafting dose of unmanipulated bone marrow cells due to concerns early in the trial about the low quantity of NC pre or post ex-vivo expansion. Patient 1 had what was considered a low NC concentration in the pre-inoculation product. Patient 3 had a low number of NC post-expansion. As more preclinical and clinical information was obtained, the threshold for the use of expanded cells was modified and all other patients underwent transplant with expanded hematopoietic cells as the only source of stem cells. The use of growth factor priming prior to the aspiration of bone marrow cells may further decrease the volume of bone marrow required for inoculation of the bioreactors without significantly affecting the quality or quantity of the expansion product. The mean fold expansion of NC and CFU-GM was 4.9 and 9.5, respectively. Nevertheless we had an average of approximately 50% loss of CD 341lin2 and LTC-IC after expansion. This suggests a differentiation of these early subsets of progenitor cells in our expansion conditions. These results contrast Stiff et al. [34] using the same CPS conditions. In their experience, a 1.23-fold expansion in CD 341lin2 was obtained after ex-vivo expansion. Despite the loss of early progenitors, all patients in our trial showed adequate hematopoietic reconstitution up to a year post-transplant. Furthermore, the time to engraftment of neutrophils
Table 5. Hematopoietic reconstitution after ex-vivo expanded stem cell transplant Patient number 2 4 5 6 7 Mean
Days to ANC $ 500/mL 15 22 18 20 16 18.2
Days to PLT . 20,000/mL 19 39 26 23 20 25.4
Days of febrile neutropenia 0 6 3 0 4 2.6
# of PRBC’s/Platelet transfusions 2/3 10/8 2/8 2/4 4/3 4/5
Patients 1 and 3 underwent transplant with unmanipulated bone marrow mononuclear cells due to failure to achieve adequate MNC concentration (patient 1) and to suboptimal expansion (patient 3).
620
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
Figure 1. Hematopoietic recovery following high dose chemotherapy and ex-vivo expanded progenitor cell transplant. (A) Post-transplant white blood cell recovery; (B) Post-transplant neutrophil recovery; (C) Post-transplant platelet recovery.
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
621
Table 6. Analysis of post-transplant immune reconstitution Adult normal ranges Months post-transplant IgM (mg/dL) IgG (mg/dL) IgA (mg/dL) Total lymphocyte count/mm3 CD31 T cells/mm3 CD191 cells/mm3 CD161 CD561 cells/mm3 CD41 T cells/mm3 CD41 CD45RA1 T cells (%CD3) CD41 CD45RO1 T cells (% CD3) CD81 T cells/mm3 CD81 CD45RA1 T cells (% CD3) CD81 CD45RO1 T cells (% CD3) CD4/CD8 ratio
(40–248 mg/dL) (680–1445 mg/dL) (71–374 mg/dL) (1000–4800/mm3) (632–2464/mm3) (40–399/mm3) (60–654/mm3) (323–1546/mm3)
(170–1154/mm3)
(1.0–2.5)
and platelets was similar to the reported Loyola University experience [34]. Studies have shown that contamination of breast cancer cells in grafts used for hematopoietic reconstitution is associated with a higher incidence of relapse when compared to patients with no evidence of tumor cells in their bone marrow or peripheral blood grafts [11–13]. Preclinical studies using hematopoietic ex-vivo expansion have shown up to 2–4 log reduction in tumor cells in the bone marrow from patients with breast cancer [35]. This reduction in tumor cell number is achieved by: (a) a smaller volume of starting cells used for expansion compared to the volume required for a standard bone marrow or peripheral blood stem cell transplant; and (b) passive purging of tumor cells during the process of ex-vivo expansion. In our trial, evaluation of tumor cell contamination pre- and post-expansion demonstrated effective purging in two patients with tumor cell contamination prior to ex-vivo expansion. Due to a dilution effect, the starting number of tumor cells after expansion in patient 4 was below the lower limits of sensitivity for our tumor detection assay. Therefore, we can not exclude the presence of tumor cells after ex-vivo expansion in patient 4. These data suggest that the CPS ex-vivo expansion device may be an effective purging method in breast cancer. A larger number of patients will be required to confirm this advantage and to make statistically significant clinical correlates with overall and disease free survival. The median days to neutrophil and platelet engraftment were 18 and 23 with sustained engraftment now documented for more than 6 months in all patients. This represents an approximate 1 week delay in engraftment when compared to peripheral blood stem cell transplants, but is similar to the engraftment kinetics seen with bone marrow transplants. The incidence of infection, febrile days, average length of stay in the hospital, and requirement of PRBC and platelet transfusion was similar to patients undergoing peripheral blood stem cell transplant at our institution.
Patient 2
Patient 4
Patient 6
Patient 7
12 165 1496 229 960 787 48 115 125 5% 27% 557 26% 40% 0.22
7 93 1186 252 1275 561 ND ND 280 2% 48% 258 22% 24% 1.08
6 186 1441 205 900 702 36 162 261 8% 32% 214 18% 46% 0.58
5 123 910 177 510 290 81 132 71 4% 30% 450 18% 40% 0.33
It was important for several reasons to test in this study the capacity of the ex-vivo expanded grafts in supporting lymphopoiesis post-transplant. The available in-vitro assays that are used to asses the efficacy of in-vitro expansion in preclinical and clinical studies, namely the CFC and LTC-IC assays, are not informative of the grafts capacity in supporting lymphopoiesis. The cytokines used for expansion in this study (Flt3-L, IL-3/GM-CSF, EPO) and in most other studies either lack or exhibit limited proliferative potential for lymphoid lineage cells [36–38]. This is supported by the FACS analysis of the post-expansion product, which typically reveals ,1%–3 % mature B and T cells and lymphoid progenitors (data not shown). Lymphoid engraftment data have not been reported in other clinical studies where exvivo expanded grafts were used in combination with conventional grafts. Therefore, the true multilineage engraftment potential of ex-vivo expanded grafts has not yet been fully addressed. Clinically, no patient in this study experienced any opportunistic infection. It is interesting that serum immunoglobulin levels were normal as early as 6 months post-transplant, which compare favorably to those observed in some studies of conventional hematopoietic stem cell transplants [30]. Although the number of patients studied are too small to reach firm conclusions, numerical reconstitution of lymphocyte subsets appeared to be similar to that seen after conventional transplants. Faster return of CD81 cells to normal levels, inverted CD4/CD8 ratios within the first year post-transplant, delayed recovery of CD41 cells, and more frequent expression of CD45RO isoform than CD45RA on emergent lymphocytes are also well documented after conventional hematopoietic stem cell transplants [30–33]. The latter observation most likely reflects slower generation of CD45RA1 “naive” T-helper cells in adult patients; a process that appears to be thymus-dependent. It is generally accepted that a thymic-independent pathway exists in the generation of CD81 CD45RA1 T cells [39–41] consistent with
622
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
their faster recovery post-transplant compared with CD41 CD45RA1 T cells. Whether the regeneration of lymphocytes in these patients is solely driven from the stem cells and progenitors in the ex-vivo expanded grafts is unknown. Survival of host-stem cells and progenitors after high dose chemotherapy can not be excluded. It is also possible that a small number of mature lymphocytes are maintained during the ex-vivo expansion process. In this case, a relatively restricted receptor repertoire would be expected. Preliminary results of our ongoing studies indicate that B- and T-cell receptor repertoires are complex in diversity and that they exhibit characteristics similar to that seen after conventional transplants [42]. Overall, these results suggest that ex-vivo expanded cells have similar capacity in supporting lymphopoiesis post-transplant when compared to that of conventional grafts. Longer follow-up and larger studies will be needed to confirm these results and demonstrate the durability of the grafts. This trial demonstrates the feasibility of using ex-vivo expanded progenitor cells for hematopoietic reconstitution following myeloablative chemotherapy. With optimization in culture conditions, engraftment times may approach those of peripheral blood stem cell transplants. Potential applications of this system include expansion of lymphohematopoietic subsets for cell therapies during or after stem cell transplants, gene therapy, and growth of dendritic cells.
References 1. Kessinger A, Armitage JO, Landmark JD, Smith DM, Weisenburger D (1988) Autologous peripheral hematopoietic stem cell transplantation restores hematopoietic function following marrow ablative therapy. Blood 71:723 2. Juttner CA, To LB, Ho JQ, Bardy PG, Dyson PG (1988) Early lymphohematopoietic recovery after autografting using peripheral blood stem cells in acute nonlymphoblastic leukemia. Transplant Proc 20:40 3. Sheridan W, Begley CG, Juttner CA, Szer J, To LB (1992) Effect of peripheral blood progenitor cells mobilized by filgrastim (G-CSF) on platelet recovery after high dose chemotherapy. Lancet 1:640 4. Juttner CA, To LB, Roberts MM, Haylock D, Dyson PG (1992) Comparison of hematologic recovery, toxicity, and supportive care of autologous PBSC, autologous BM, and allogeneic BM transplants. Int J Cell Cloning 10:160 5. Ager S, Scott MA, Magendra P (1995) Peripheral blood stem cell transplantation after high dose chemotherapy in patients with malignant lymphoma: a retrospective comparison with autologous bone marrow transplantation. Bone Marrow Transplant 16:79 6. Schmitz N, Linch DC, Dreger P (1996) Randomized trial of filgrastimmobilized peripheral progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients. Lancet 347:353 7. Alexander HR (1993) Vascular access and other specialized techniques of drug delivery. In VT Devita, S Hellman, SA Rosenberg (eds) Cancer: Principles and Practice of Oncology. Philadelphia: JB Lippincott Co 8. Gribben J, Neuberg D, Freedman A, Gimmi C, Pesek K, Barber M, Saporito L, Woo S. Coral F, Spector N, Rabinowe S, Grossbard B, Ritz J, Nadler L (1993) Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood 81:3449
9. Brugger W, Bross KJ, Glatt M, Weber F, Mertelsmann R, Kanz L (1994) Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 83:636 10. Vredenburgh J, Silva O, de Sombre K, Tyer K, Abou-Ghalia A, Cook M, Layfielld L, Peters WP, Bast RC (1996) A comparison of immunohistochemistry, two-color immunofluorescence, and flow cytometry with cell sorting for the detection of micrometastasic breast cancer in the bone marrow. J Hematotherapy 5:57 11. Brockstein BE, Ross AA, Moss TJ, Kahn DG, Hollingsworth K, Williams SF (1996). Tumor cell contamination of bone marrow harvest products: clinical consequences in a cohort of advanced-stage breast cancer patients undergoing high-dose chemotherapy. J Hematother 6:617 12. Vredenburgh JJ, Silva O, Broadwater G, Berry D, DeSombre K, Tyer C, Petros WP, Peters WP, Bast RC (1997) The significance of tumor contamination in the bone marrow from high risk primary breast cancer patients treated with high dose chemotherapy and hematopoietic support. Biol Blood Marrow Transplant 3:91 13. Schulze R, Schulze M, Wischnik A, Ehnle S, Doukas K, Behr W, Ehret W, Schlimok G (1997) Tumor contamination of peripheral blood stem cell transplants and bone marrow in high risk breast cancer patients. Bone Marrow Transplant 12:1223 14. Henschler R, Brugger W, Luft T, Frey R, Mertelsmann R, Kanz L (1994) Maintenance of transplantation potential in ex-vivo expanded CD341-selected human peripheral blood progenitor cells. Blood 84:2898 15. Sandstrom C, Bender J, Papoutsakis E, Miller W (1995) Effects of CD 341 cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells. Blood 86:958 16. Smith S, Bender J, Maples P (1993) Expansion of neutrophil precursors and progenitors in suspension cultures of CD 341 cells enriched from human bone marrow. Exp Hematol 21:870 17. Lill M, Lynch M, Fraser J (1994) Production of functional myeloid cell from CD34-selected hematopoeitic progenitor cells using a clinically relevant ex vivo expansion system. Stem Cells 12:626 18. Sato M, Sawada K, Koizumi K (1993) In vitro expansion of human peripheral blood CD341 cells. Blood 82:3600 19. Brugger W, Heimfeld S, Berenson F, Merstelsmann R, Kanz L (1995) Reconstitution of hematopoiesis after high dose chemotherapy by autologous progenitor cells generated ex-vivo. N Engl J Med 333:283 20. Haylock D, To L, Dowse T, Juttner C, Simmons P (1992) Ex vivo expansion and maturation of peripheral blood CD 341 cells into the myeloid lineage. Blood 80:1405 21. Shapiro F, Yao T, Raptis G, Reich L, Norton L, Moore M (1994) Optimization of conditions for ex vivo expansion of CD341 cells from patients with Stage IV breast cancer. Blood 84:3567 22. Sandstrom C, Bender J, Miller W, Papoutsakis E (1996) Comparison of perfused and static peripheral blood cell cultures with and without stroma using a modified culture chamber for non-adherent cell retention. Biotech Bioeng 50:493 23. Udomsakdi C, Eaves C, Swolin B, Teid D, Barnett M, Eaves A (1992) Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level. Proc Natl Acad Sci USA 89:6192 24. Koller M, Emerson S, Palsson B (1993) Large-scale expansion of human stem cell and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures. Blood 82:378 25. Koller M, Palsson B, Manchel I, Palsson B (1995) Long-term culture initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects. Blood 86:1784 26. Champlin RE, Mehra R, Gajewski J (1995) Ex-vivo expanded progenitor cell transplantation in patients with breast cancer. Blood 86:295a 27. Franklin WA, Shpall EJ, Archer P, Johnston CS, Garza-Williams S, Hami L, Bitter MA, Bast RC, Jones RB (1996) Immunocytochemical detection of breast cancer in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support. Breast Cancer Res Treat 41:1
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623 28. Sanders ME, Makgoba MW, Shaw S (1998) Human naive and memory T cells. Immunol Today 9:195 29. Swain SL, Craft M, Dubey C, Haynes L, Rogers P, Zhang X, Bradley LM (1996) From naive to memory T cells. Immunol Rev 150:142 30. Atkinson K (1990) Reconstitution of the haemopoietic and immune systems after bone marrow transplantation. Bone Marrow Transplant 5:209 31. Store J, Ehitherspoon RP, Storb J (1995) T cell reconstitution after bone marrow transplantation into adult patients does not resemble T cell development in early life. Bone Marrow Transplant 16:413 32. Koehne G, Zeller W, Stockschlaeder M, Zander AR (1997) Phenotype lymphocyte subsets after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 19:149 33. Talmadge JE, Reed E, Ino K, Kessinger A, Kunszyski C, Heimann D, Varney M, Jackson J, Vose JM, Bierman PJ (1997) Rapid immunologic reconstitution following transplantation with mobilized peripheral blood stem cells as compared to bone marrow. Bone Marrow Transplant 19:161 34. Stiff P, Oldenberg D, His E, Chen B, Smith A, Bayer R, Malhotra D, Kerger C, Armstrong RD, Muller T (1997) Transplantation of ex vivo expanded cells grown in Aastrom stromal-based perfusion bioreactors from small marrow aliquots (40 ml) produces durable hematopoietic reconstitution after ablative chemotherapy. Blood 90:395a 35. Lundell BI, Vredenburgh JJ, Tyer C, DeSombre K, Smith AK (1997) Ex-vivo perfusion culture expansion tumor positive bone marrow from breast cancer patients results in passive purging during the culture period. Blood 90:216a
623
36. Namikawa R, Muench MO, de Vries JE, Roncarob MG (1996) The Flk2/Flt3 Ligand synergies with Interleukin-7 in promoting stromalcell-independent expansion and differentiation of human fetal pro-B cells in-vitro. Blood 87:1881 37. Pribyl JR, Dittel BN, LeBien TW (1995) In-vitro studies of human B lymphopoiesis. Ann NY Acad Sci 764:9 38. Ito M, Anan K, Misawa M, Kai S, Hara H (1996) In vitro differentiation of murine sca-11lin2 cells into myeloid, B cells and T cell lineages. Stem Cells 14:412 39. Roux E, Helg C, Dumont-Girard F, Chapuis B, Jeannet M, Roosnek E (1996) Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts. Blood 87:3984 40. Mackall C, Granger L, Sheard MA, Cepeda R, Gress RE (1993) T-cell regeneration after bone marrow transplantation: differential CD45 isoform expression on thymic-derived versus thymic-independent progeny. Blood 82:2585 41. Heitger A, Neu N, Kern H, Panzer-Grumayer ER, Greinidx H, Nachbaur D, Niederwieser, Fink FM (1997) Essential role of the thymus to reconstitute naïve (CD45RA1) T-helper cells after human allogeneic bone marrow transplantation. Blood 90:850 42. Gokmen E, Raaphorst FM, Bachier C, LeMaistre CF, Boldt DH, Teale JM (1997) B- and T-cells obtained from ex-vivo expanded bone marrow mononuclear cell transplant recipients exhibit Ig H and TCR Vb CDR3 repertoires similar to that seen after conventional hematopoietic stem cell transplantation. Blood 90:539a
Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer Carlos R. Bachiera, Erhan Gokmenb, Judy Tealeb, Sophie Lanzkrona, Craig Childsa, Wilbur Franklinc, Elizabeth Shpallc, Judith Douvilled, Stephanie Weberd, Thomas Mullerd, Douglas Armstrongd, and Charles F. LeMaistrea a South Texas Cancer Institute, San Antonio, TX; bUniversity of Texas Health Science Center, San Antonio, TX;cUniversity of Colorado, Denver, CO; dAastrom Biosciences, Inc., Ann Arbor, MI
(Received 23 June 1998; revised 19 November 1998; accepted 24 November 1998)
The use of hematopoietic growth factors, stromal monolayers, and frequent medium exchange allows the expansion of hematopoietic progenitors ex-vivo. We evaluated the use of exvivo expanded progenitor cells for hematopoietic reconstitution following high dose chemotherapy (HDC) in breast cancer patients. Patients with high-risk Stage II or metastatic breast carcinoma underwent bone marrow aspirations using general anesthesia. A total of 675–1125 3 106 mononuclear cells (MNC) were seeded for ex-vivo expansion for 12 days in controlled perfusion bioreactors (Aastrom Biosciences, Inc.). The bone marrow cultures, which included the stromal cells collected with the aspirate, were supplemented with erythropoietin, granulocyte-macrophage-colony stimulating factor (GMCSF)/IL-3 fusion protein (PIXY 321), and flt3 ligand. Stem cell transplant was performed with expanded cells after HDC. A median bone marrow volume of 52.9 mL (range 42–187 mL) was needed to inoculate the bioreactors. Median fold expansion of nucleated cells (NC) and colony forming unit granulocytemacrophage (CFU-GM) was 4.9 and 9.5, respectively. The median fold expansion of CD341lin2and long-term culture–initiating culture (LTC-IC) was 0.42 and 0.32, respectively. Five patients were transplanted with ex-vivo expanded NC. Median days to an absolute neutrophil count . 500/mL was 18 (range 15–22). Median days to a platelet count . 20,000/ml was 23 (range 19–39). All patients had sustained engraftment of both neutrophils and platelets. Immune reconstitution was similar to that seen after HDC and conventional stem cell transplantation. We conclude that ex-vivo expansion of progenitor cells from perfusion cultures of small volume bone marrow aspirates, allows hematopoietic reconstitution after HDC. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.
Offprint requests to: Carlos R. Bachier, M.D., South Texas Cancer Institute, 7700 Floyd Curl Drive, San Antonio, Texas 77229; E-mail: cbachier @txdirect.net
Keywords: Ex-vivo expansion—Growth factors—Stem cell transplant
Introduction Hematopoietic growth factors and peripheral blood stem cells (PBSC) have decreased the time to recovery of platelets and white blood cells in patients treated with high dose chemotherapy. Even with these advances there is still an obligatory time to recovery of neutrophils and platelets of about 9 and 14 days, respectively [1–6]. Furthermore, PBSC mobilization and collection can be associated with complications and side effects including catheter related infections, pneumothorax, fevers, bone pain and allergic reactions [7]. Finally, PBSC grafts can be contaminated with malignant cells [8–10] that may contribute to cancer recurrence posttransplant [11–13]. Developments in hematopoietic stem cell biology now allow the maintenance and growth of hematopoietic progenitor cells ex-vivo. Ex-vivo hematopoietic stem cell expansion could potentially be used to further decrease the time to engraftment, for tumor purging and to decrease the risks and side effects associated with bone marrow harvest or collection of PBSC. It could also be applied to graft engineering strategies including gene therapy and expansion of subsets of lympho-hematopoietic cells. Strategies aimed at expanding progenitor cells ex-vivo include incubation of mononuclear or CD341 selected cells in the presence of hematopoietic growth factors [14–21]. Some culture conditions also include the use of autologous plasma and stromal monolayers in the ex-vivo expansion mixture [22]. Differences in the efficacy and yield of expansion in these studies are likely due to variations in the exvivo expansion conditions and the progenitor cell assay
0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(98)0 0 0 8 5 - X
616
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
used to measure fold expansion. The optimal conditions for expansion of these progenitor cells has not been defined. Furthermore, most of the current ex-vivo expansion strategies are associated with differentiation of hematopoietic progenitors into mature cells and loss of early precursors as measured by CD34 subsets and long-term culture–initiating culture (LTC-IC) assays. Initial human studies showed effective purging of chronic myelogenous leukemia cells in long-term culture [23]. No growth factors were used and, therefore, there was loss of progenitor cells during culture. More recently, Brugger et al. [19] ex-vivo expanded CD341 peripheral blood cells in tissue culture flasks. The ex-vivo expanded cells were infused after nonmyeloablative intensive chemotherapy. Rapid engraftment was achieved in all four patients transplanted with expanded cells. Koller and colleagues [24,25] evaluated the role of different culture conditions on the ex-vivo expansion of hematopoietic progenitors. Ex-vivo expansion of LTC-IC cells was dependent on the presence of autologous stroma and frequent medium exchanges. Incubation of bone marrow progenitors with IL-1, IL-3, IL-6, GM-CSF, G-CSF, stem cell factor (SCF), erythropoietin (EPO), and interferon g in different combinations was evaluated for optimal expansion conditions. In stromal-free, CD34-enriched cell cultures, the combination of IL-3/GM-CSF/EPO/SCF was found to be as efficient on expanding LTC-IC progenitors as the addition of other growth factors to this combination. Furthermore, decreasing cell enrichment of CD341 lin2cells was associated with a three- to fivefold increase in NC, CFU-GM, and LTC-IC. Based on these results, the cell production system (CPS) was developed by Aastrom Biosciences for the ex-vivo expansion of bone marrow nucleated cells (NC). Preliminary clinical studies published by Champlin et al. [26] demonstrated the safety of transplanting bone marrow NC ex-vivo expanded with the CPS together with a standard bone marrow harvest. In the current trial, we used ex-vivo expanded bone marrow cells as the only source of stem cells for hematopoietic reconstitution following myeloablative chemotherapy for breast cancer.
Methods Patients Patients with Stage II breast cancer with metastasis to more than ten axillary lymph nodes and Stage IV breast cancer in sensitive relapse were enrolled in this trial after signing an Institutional Review Board approved informed consent. All patients in this study underwent bone marrow harvest, ex-vivo expansion, and transplant at a single center (South Texas Cancer Institute). Other entry criteria included a maximum of two prior chemotherapy regimens, $30% bone marrow cellularity (core bone marrow biopsy prior to enrollment), no bone marrow involvement by tumor determined by H & E stains of bone marrow biopsies, and adequate end organ func-
tion. Early in the trial, $35 3 106/mL bone marrow NC concentration at the time of harvest and $2500 3 106 NC post-expansion were required for transplant with ex-vivo expanded stem cells. Bone marrow aspiration Patients were taken to the operating room and placed under general anesthesia. Using 16-inch gauge Illinois needles and 10 mL syringes, initial 3–5 mL of bone marrow per aspirate were obtained from the superior posterior iliac crests and collected in 150mL bags containing 5000 units of heparin. A median of 85.7 mL was collected (range 45–187). The harvest was continued until a standard back-up bone marrow collection of at least 1 3 108 NC/ kg was obtained. Ex-vivo expansion Device description. The single-use, closed, disposable, sterile cell culture device that is part of the Aastrom CPS consists of two compartments separated by a gas-permeable, liquid-impermeable membrane. The lower cell culture chamber is continuously perfused by growth medium at 378C. The Aastrom CPS incubator contains a cold compartment for media storage during the 12-day cell culture (reagent perfusion begins on Day 3), and a warm (378C) compartment in which the cell cassette containing the cell culture device is placed for the duration of the culture. In the incubator, the cells grow on the tissue culture plastic surface of the cell culture bed in the cell culture device, developing a dense stromal layer. The upper cell culture chamber of the cell culture device is provided with a constant flow of oxygen, nitrogen and CO2, such that oxygenation of the cells is accomplished by diffusion across the membrane and through the culture medium. CO2 is removed by the same diffusion mechanism. The medium used to perfuse the cultured cells is stored in a closed vessel in an adjacent compartment of the same incubator at 48C. The vessel’s only external connection is by medical grade tubing. A computer system allows the operator to monitor the status of the cell production. Cell culture conditions. Bone marrow mononuclear cells were obtained after Ficoll Hypaque separation. A total of 675–1100 3 106 MNC were inoculated in three or four bioreactors. The hematopoietic cells were suspended in tissue culture medium composed of Iscove’s Modified Dulbecco’s Medium supplemented with 10% fetal bovine serum, 10% horse serum, hydrocortisone (5 mM), PIXY 321 (5 ng/mL), L-glutamine (4 mM), erythropoietin (0.1 U/ mL), Flt3-L (25 ng/mL), gentamicin sulfate (5 mg/mL), vancomycin (20 mg/mL). PIXY 321, Flt3-L, and erythropietin were all clinical grade human recombinant proteins. The cells are incubated in the Aastrom CPS for 12 days at 378C with the tissue culture medium continuously replaced with fresh medium. Sampling of the culture medium, via a septum in the medium waste line, is carried out 48 hours prior to harvest to test for bacterial and fungal contaminants. After 12 days of incubation in this closed system, the nonadherent cell fraction was removed by draining the growth medium from the cell culture device into the harvest bag. The chamber was then rinsed with 100 mL of 0.04% Trypsin-EDTA solution. This was followed by gentle, automated agitation of the cell culture device and collection of the rinse into the harvest bag. The adherent layer was detached from the cell culture bed surface by sterile addition of 100 mL of 0.04% Trypsin-EDTA solution. This was followed by agitation of the cell culture device and collection of the rinse into the harvest bag. The expanded cell product containing
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623 Table 1. Patient characteristics Patient Number
Age (years) Weight (kg) Stage Prior Chemotherapy Regimens
1
2
3
4
5
6
7
46 63 II 1
42 70 II 1
42 69 II 1
41 58 IV 1
44 82 IV 2
53 92 II 1
39 59 II 1
both adherent and non-adherent cells was washed using the COBE 2991 Cell Processor. The final cell product was suspended in approximately 250 mL of Normosol and 0.5% human serum albumin for immediate infusion. Tumor contamination assay Tumor contamination was analyzed according to the immunocytochemistry method described by Franklin et al. [27]. Mononuclear cells before and after ex-vivo expansion were washed once with medium-199 (Gibco Laboratories; Grand Island, NY) containing 10% heparin and resuspended with 25% FBS and DPBS (Hyclone Lab.; Logan, UT) at a concentration of 2.5 3 106 cells/mL. Cytospins were prepared using a Cyto-Tek centrifuge (Miles Scientific; Elkhart, IN). Two hundred microliters of cell suspension (5 3 105 cells) were added to the cytocentrifuge slide holder loaded with a silicone-coated glass slide and 1 mL filter paper. Cells were then centrifuged onto the slide at 500 rpm (160 3 g) for 5 minutes and air dried for a minimum of 30 minutes. Slides were fixed for 20 minutes using acetone/methanol/formalin (45%/45%/10%). Fixed slides were stained in 200 mL containers according to a modification of the alkaline phosphatase antialkaline phosphatase (APAAP) technique and were counterstained with hematoxylin. A total of ten stained slides were examined using a standard binocular light microscope with a low power (103) objective. This assay was able to detect three tumor cells in a background of 106 normal cells. Progenitor cell assays The efficacy of expansion was measured by analyzing NC numbers, CFU-GM and LTC-IC colonies, and flow cytometry for CD341/CD341 lin2subsets. For CFU-GM assays, 1.5 3 104 cells per mL of culture medium (Methylcellulose supplemented with PIXY 321, Erythropoietin and G-CSF) were plated into 35-mm nontissue cultured treated plates. Plates were then incubated at 378C in 5% CO2 and 95% O2
617
for 14 days. For LTC-IC assays, bone marrow stroma was prepared by incubating bone marrow mononuclear cells in tissue culture flasks containing Dexter medium. Stromal cells were grown in Dexter medium at 378C in 5% CO2 and 95% O2. Stromal cells were then trypsinized, washed, and exposed to 15–20 Gy of gamma irradiation. Irradiated stromal cells were then plated in 96-well plates (1 3 104 cells per well at a concentration of 1 3 105 cells per mL of Dexter medium). Patient samples before and after ex-vivo expansion were diluted in Dexter medium at four different cell concentrations and incubated on plates containing irradiated stroma in a humidified incubator at 338C in 5% CO2 and 95% O2. LTC-IC cultures were maintained by replacing 50% of the medium in each well with fresh Dexter medium once weekly. These cultures were maintained for 5 weeks in liquid culture before harvesting for progenitor cell assays. Lin2 cell subtypes were obtained using the following antibodies: CD3, CD11b, CD15, CD20. Due to the presence of CD341 expressing stromal cells, total CD341 NC was not analyzed in the exvivo expanded product. Clinical design Patients underwent bone marrow harvest and inoculation of the CPS 12 days prior (Day 212) to the infusion of ex-vivo expanded cells. On Day 26 they were admitted to the hospital and on Day 25 they began their high dose chemotherapy regimen consisting of: Cyclophosphamide 1.875 mg/m2/d 3 3; Cisplatinum 55 mg/m2/d 3 3; BCNU 600 mg/m2/d 3 1. On Day 0, or 12 days after bone marrow harvest and inoculation of the CPS bioreactors, patients received the infusion of ex-vivo expanded cells. On Day 11 they started G-CSF at 10 mcg/kg per day until white blood cell engraftment. Analysis of immune reconstitution Peripheral blood samples were obtained from four patients at 5, 6, 7, and 12 months post-transplant. The fifth patient died of relapsed disease at Day 159 post-transplant prior to immunological evaluation. Peripheral blood mononuclear cells obtained by Ficoll-Hypaque density gradient sedimentation were stained for flow cytometry and analyzed using FACS (Becton Dickinson; Mountain View, CA). The fluorochrome-labeled mouse monoclonal antiCD3, CD4, CD8, CD19, CD16, CD56, CD45RA, and CD45RO antibodies, all purchased from Becton Dickinson, were used. For background staining, irrelevant mouse antibodies of IgG1 and IgG2a were used. CD41 CD45RA1, CD41 CD45RO1, CD81 CD45RA1, and CD81 CD45RO1 subsets were defined as fractions of gated lymphocyte population co-staining for CD4 and CD45RA, CD4 and CD45RO, CD8 and CD45RA, and CD8 and CD45RO,
Table 2. Pre-expansion bone marrow cell product
Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Mean
Volume of BM* (mL)
NC 3 106/mL
CD341 3 106/kg (%)
CD341LIN2 3 105/kg (%)
LTC-IC 3 103/kg
187.0 87.6 85.7 113.0 45.6 52.2 61.5 90.4
19.3 44.5 77.0 23.7 47.4 23.0 40.3 39.4
0.53 (4.9) 0.14 (1.1) 0.38 (2.9) 0.81 (4.3) 0.61 (5.6) 0.23 (3.3) 0.81 (7.0) 0.50 (4.2)
2.6 (2.4) 1.4 (1.1) 2.8 (2.2) 3.0 (1.6) 2.0 (1.9) 0.8 (1.1) 5.7 (4.9) 2.9 (2.2)
1.31 0.33 0.43 0.48 0.47 ND 1.19 0.70
*Initial volume of the bone marrow (BM) aspirated. A mean of 60 mL needed to inoculate bioreactors; Pt. 5 patient.
618
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
Table 3. Tumor cell detection Pt. #4 *Tumor cell concentration (Pre-expansion) † Total number of tumor cells (Pre-expansion) Total number of tumor cells (Post-expansion)
1.8
Pt. #5
66.5
1620
59,850
0
0
*Number of tumor cells 3 106 hematopoietic cells. † Number of tumor cells in 900 3 106 cells inoculated into bioreactors.
respectively. The CD45RA marker has been demonstrated on “naive” T cells, whereas the CD45RO marker is found on activated or memory T cells [28,29]. Absolute numbers of lymphocyte subsets were calculated by multiplying the percentage of cells staining positive with the relevant antibodies by the absolute number of lymphocytes determined by a Coulter counter analysis (Miami, FL) and a manual differential count. Serum immunoglobulins were also quantified at 5 to 12 months post-transplant.
Results Patient characteristics Seven patients were enrolled including five patients with Stage II and two patients with Stage IV breast cancer (Table 1). Median age was 44 years (range 41–53 years). Six patients had one, and one patient had two prior chemotherapy regimens. Their median weight was 69 kg (range 58–92 kg). Cells were expanded for all seven patients enrolled, however, only five patients were transplanted with the expanded product. Pre-expansion bone marrow cell product A median volume of 85.7 mL (range 45–187) of bone marrow per patient was aspirated from the superior posterior iliac crests (Table 2). The median recovery yield after Ficoll centrifugation was 46.6% (range 23%–60.6%). From the initial bone marrow volume, a median of 52.9 mL per patient (range 42–187 mL) was needed to inoculate the bioreactors with 675–1125 3 106 MNC. The median NC concentration of bone marrow prior to Ficoll centrifugation was
40 3 106/mL (range 19–77 per mL). The median numbers of CD341 MNC, CD341lin2and LTC-IC in the cell inoculum was 0.53 3 106/kg (range 0.14–0.81 3 106/kg), 2.6 3 105/kg (range 0.8–5.7) and 0.47 3 103/kg (range 0.33– 1.31 3 103/kg), respectively. Tumor cell purging Of the seven patients, only patients with Stage IV breast cancer (patients 4 and 5) were found to have tumor cells detected (Table 3) by immunocytochemical assays in their bone marrow samples. The total number of tumor cells inoculated into the bioreactors prior to expansion were 1620 and 59850 in these two patients. No tumor cells were seen in any of the bone marrow samples (n 5 7) after ex-vivo expansion. Post-expansion bone marrow cell product Median NC post-expansion (Table 4) was 0.50 3 108/kg (range 0.26–0.63 3 108/kg). Median CFU-GM post-expansion was 1.9 3 105/kg (range 1.2–12.2 3 105/kg). Median fold expansion of NC and CFU-GM was 4.9 and 9.5, respectively. The median number of CD341lin2 post-expansion was 1.15 3 105/kg (range 0.28–2.07 3 105/kg). The median fold expansion of CD341lin2 was 0.42. The median number of LTC-IC post-expansion was 0.33 3 103/kg (range 0.04–0.68 3 103/kg). The median fold expansion of LTC-IC was 0.32 (range 0.25–2.05). Clinical outcome Five patients were transplanted with ex-vivo expanded NC (Table 5). Patients 1 and 3 underwent stem transplant with their “back up” bone marrow NC due to concerns early in the trial on the adequate number of NC needed for engraftment pre and post ex-vivo expansion. Median days to an absolute neutrophil count . 500/ml was 18 (range 15–22). Median days to a platelet count . 20,000/ml was 23 (range 19–39). Kinetics of white blood cell and platelet engraftment are shown in Fig. 1. Median number of febrile days (temperature $38.38C) were 3 (range 0–8 days). Median number of transfusion of PRBC’s and platelets was 2 (range 2–8) and 4 (range 3–8), respectively. Average length of stay was 14.4 days. All patients had sustained engraftment of both white blood cell and platelets with a median follow-up
Table 4. Post-expansion cell product
Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Mean
NC 3 108/kg (fold expansion)
CFU-GM 3 105/kg (fold expansion)
CD341LIN2 3 105/kg (fold expansion)
LTC-IC 3 103/kg (fold expansion)
0.36 (5.0) 0.63 (6.5) 0.26 (2.5) 0.58 (3.6) 0.50 (5.4) 0.28 (4.9) 0.54 (4.6) 0.45 (4.7)
12.2 (30.7) 5.0 (12.6) 1.9 (9.5) 1.6 (5.0) 1.2 (3.7) 1.5 (8.1) 5.1 (54.7) 4.1 (17.6)
1.15 (0.67) 1.26 (1.18) 1.43 (0.64) 0.58 (0.20) 0.65 (0.37) 0.28 (0.40) 2.37 (0.42) 1.10 (0.55)
0.32 (0.25) 0.68 (2.05) 0.12 (0.29) 0.14 (0.29) 0.33 (0.70) 0.04 0.40 (0.34) 0.29 (0.56)
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
of 183 days (range 156–338). Patients 4 and 5 relapsed at 211 and 149 days post-transplant, respectively. Patient 5 died of breast cancer 159 days post-transplant. Patient 4 is in partial remission after additional chemotherapy. Patients 1 and 3 underwent stem transplant with their “back up” bone marrow NC due to concerns early in the trial on the adequate number of NC needed for engraftment pre and post ex-vivo expansion. Patients 1 and 3 received 1.6 3 108 NC/kg and 2.5 3 108 NC/kg respectively on Day 0. They received the same high dose chemotherapy regimen and post-transplant supportive care as patients undergoing transplant with ex-vivo expanded bone marrow cells. Days to neutrophil and platelet engraftment were 12/40 (patient 1) and 11/23 (patient 3), respectively.
Immune reconstitution Patients 2, 4, 6, and 7 were evaluated for immune reconstitution at 12, 7, 6, and 5 months post-transplant, respectively. Patient 5, who died of relapsed disease at Day 159 posttransplant, and two patients who received nonexpanded bone marrow grafts were not studied. No opportunistic infectious complications were observed in any of the patients. As shown in Table 6, serum immunoglobulin levels were within adult normal range in all patients at 5 to 12 months post-transplant. None of the patients received intravenous immunoglobulin. Total lymphocyte counts as well as number of B lymphocytes and natural killer cells had returned to normal or near-normal levels in three patients at 5 to 12 months (Table 6). While the absolute numbers of CD81 T-cell subset were normal at the time of evaluation in all patients, CD41 T-cell counts remained low as long as 12 months post-transplant. The CD4/CD8 ratios were inverted in three patients similar to that observed after conventional hematopoietic stem cell transplantation [30–33]. CD45 isoform expression on the emerging T cells was also addressed. CD45RO co-expression was more common among both CD41 and CD81 T-cell subsets than CD45RA except in one patient who had similar numbers of CD81 cells coexpressing CD45RA and CD45RO. These results are also similar to that observed after conventional hematopoietic stem cell transplantation [32–33].
619
Discussion Preclinical studies by Koller et al. [24,25] showed that exvivo expansion of bone marrow cells using continuous perfusion of media and growth factors along with autologous stroma was associated with expansion of early and late hematopoietic cells including NC, CFU-GM, and LTC-IC. Based on these results, a Phase I trial was initiated to determine if expansion of small volumes of bone marrow could provide enough progenitor cells for lymphohematopoietic reconstitution following high dose chemotherapy in patients with breast cancer. Aspiration of small volumes of bone marrow for expansion was performed under general anesthesia to allow for the collection of additional hematopoietic cells as back-up for patients with failure to engraft. There were no engraftment failures and none of the patients required infusion of back-up cells after transplant with ex-vivo expanded cells. Patients 1 and 3 underwent transplant with an engrafting dose of unmanipulated bone marrow cells due to concerns early in the trial about the low quantity of NC pre or post ex-vivo expansion. Patient 1 had what was considered a low NC concentration in the pre-inoculation product. Patient 3 had a low number of NC post-expansion. As more preclinical and clinical information was obtained, the threshold for the use of expanded cells was modified and all other patients underwent transplant with expanded hematopoietic cells as the only source of stem cells. The use of growth factor priming prior to the aspiration of bone marrow cells may further decrease the volume of bone marrow required for inoculation of the bioreactors without significantly affecting the quality or quantity of the expansion product. The mean fold expansion of NC and CFU-GM was 4.9 and 9.5, respectively. Nevertheless we had an average of approximately 50% loss of CD 341lin2 and LTC-IC after expansion. This suggests a differentiation of these early subsets of progenitor cells in our expansion conditions. These results contrast Stiff et al. [34] using the same CPS conditions. In their experience, a 1.23-fold expansion in CD 341lin2 was obtained after ex-vivo expansion. Despite the loss of early progenitors, all patients in our trial showed adequate hematopoietic reconstitution up to a year post-transplant. Furthermore, the time to engraftment of neutrophils
Table 5. Hematopoietic reconstitution after ex-vivo expanded stem cell transplant Patient number 2 4 5 6 7 Mean
Days to ANC $ 500/mL 15 22 18 20 16 18.2
Days to PLT . 20,000/mL 19 39 26 23 20 25.4
Days of febrile neutropenia 0 6 3 0 4 2.6
# of PRBC’s/Platelet transfusions 2/3 10/8 2/8 2/4 4/3 4/5
Patients 1 and 3 underwent transplant with unmanipulated bone marrow mononuclear cells due to failure to achieve adequate MNC concentration (patient 1) and to suboptimal expansion (patient 3).
620
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
Figure 1. Hematopoietic recovery following high dose chemotherapy and ex-vivo expanded progenitor cell transplant. (A) Post-transplant white blood cell recovery; (B) Post-transplant neutrophil recovery; (C) Post-transplant platelet recovery.
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
621
Table 6. Analysis of post-transplant immune reconstitution Adult normal ranges Months post-transplant IgM (mg/dL) IgG (mg/dL) IgA (mg/dL) Total lymphocyte count/mm3 CD31 T cells/mm3 CD191 cells/mm3 CD161 CD561 cells/mm3 CD41 T cells/mm3 CD41 CD45RA1 T cells (%CD3) CD41 CD45RO1 T cells (% CD3) CD81 T cells/mm3 CD81 CD45RA1 T cells (% CD3) CD81 CD45RO1 T cells (% CD3) CD4/CD8 ratio
(40–248 mg/dL) (680–1445 mg/dL) (71–374 mg/dL) (1000–4800/mm3) (632–2464/mm3) (40–399/mm3) (60–654/mm3) (323–1546/mm3)
(170–1154/mm3)
(1.0–2.5)
and platelets was similar to the reported Loyola University experience [34]. Studies have shown that contamination of breast cancer cells in grafts used for hematopoietic reconstitution is associated with a higher incidence of relapse when compared to patients with no evidence of tumor cells in their bone marrow or peripheral blood grafts [11–13]. Preclinical studies using hematopoietic ex-vivo expansion have shown up to 2–4 log reduction in tumor cells in the bone marrow from patients with breast cancer [35]. This reduction in tumor cell number is achieved by: (a) a smaller volume of starting cells used for expansion compared to the volume required for a standard bone marrow or peripheral blood stem cell transplant; and (b) passive purging of tumor cells during the process of ex-vivo expansion. In our trial, evaluation of tumor cell contamination pre- and post-expansion demonstrated effective purging in two patients with tumor cell contamination prior to ex-vivo expansion. Due to a dilution effect, the starting number of tumor cells after expansion in patient 4 was below the lower limits of sensitivity for our tumor detection assay. Therefore, we can not exclude the presence of tumor cells after ex-vivo expansion in patient 4. These data suggest that the CPS ex-vivo expansion device may be an effective purging method in breast cancer. A larger number of patients will be required to confirm this advantage and to make statistically significant clinical correlates with overall and disease free survival. The median days to neutrophil and platelet engraftment were 18 and 23 with sustained engraftment now documented for more than 6 months in all patients. This represents an approximate 1 week delay in engraftment when compared to peripheral blood stem cell transplants, but is similar to the engraftment kinetics seen with bone marrow transplants. The incidence of infection, febrile days, average length of stay in the hospital, and requirement of PRBC and platelet transfusion was similar to patients undergoing peripheral blood stem cell transplant at our institution.
Patient 2
Patient 4
Patient 6
Patient 7
12 165 1496 229 960 787 48 115 125 5% 27% 557 26% 40% 0.22
7 93 1186 252 1275 561 ND ND 280 2% 48% 258 22% 24% 1.08
6 186 1441 205 900 702 36 162 261 8% 32% 214 18% 46% 0.58
5 123 910 177 510 290 81 132 71 4% 30% 450 18% 40% 0.33
It was important for several reasons to test in this study the capacity of the ex-vivo expanded grafts in supporting lymphopoiesis post-transplant. The available in-vitro assays that are used to asses the efficacy of in-vitro expansion in preclinical and clinical studies, namely the CFC and LTC-IC assays, are not informative of the grafts capacity in supporting lymphopoiesis. The cytokines used for expansion in this study (Flt3-L, IL-3/GM-CSF, EPO) and in most other studies either lack or exhibit limited proliferative potential for lymphoid lineage cells [36–38]. This is supported by the FACS analysis of the post-expansion product, which typically reveals ,1%–3 % mature B and T cells and lymphoid progenitors (data not shown). Lymphoid engraftment data have not been reported in other clinical studies where exvivo expanded grafts were used in combination with conventional grafts. Therefore, the true multilineage engraftment potential of ex-vivo expanded grafts has not yet been fully addressed. Clinically, no patient in this study experienced any opportunistic infection. It is interesting that serum immunoglobulin levels were normal as early as 6 months post-transplant, which compare favorably to those observed in some studies of conventional hematopoietic stem cell transplants [30]. Although the number of patients studied are too small to reach firm conclusions, numerical reconstitution of lymphocyte subsets appeared to be similar to that seen after conventional transplants. Faster return of CD81 cells to normal levels, inverted CD4/CD8 ratios within the first year post-transplant, delayed recovery of CD41 cells, and more frequent expression of CD45RO isoform than CD45RA on emergent lymphocytes are also well documented after conventional hematopoietic stem cell transplants [30–33]. The latter observation most likely reflects slower generation of CD45RA1 “naive” T-helper cells in adult patients; a process that appears to be thymus-dependent. It is generally accepted that a thymic-independent pathway exists in the generation of CD81 CD45RA1 T cells [39–41] consistent with
622
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623
their faster recovery post-transplant compared with CD41 CD45RA1 T cells. Whether the regeneration of lymphocytes in these patients is solely driven from the stem cells and progenitors in the ex-vivo expanded grafts is unknown. Survival of host-stem cells and progenitors after high dose chemotherapy can not be excluded. It is also possible that a small number of mature lymphocytes are maintained during the ex-vivo expansion process. In this case, a relatively restricted receptor repertoire would be expected. Preliminary results of our ongoing studies indicate that B- and T-cell receptor repertoires are complex in diversity and that they exhibit characteristics similar to that seen after conventional transplants [42]. Overall, these results suggest that ex-vivo expanded cells have similar capacity in supporting lymphopoiesis post-transplant when compared to that of conventional grafts. Longer follow-up and larger studies will be needed to confirm these results and demonstrate the durability of the grafts. This trial demonstrates the feasibility of using ex-vivo expanded progenitor cells for hematopoietic reconstitution following myeloablative chemotherapy. With optimization in culture conditions, engraftment times may approach those of peripheral blood stem cell transplants. Potential applications of this system include expansion of lymphohematopoietic subsets for cell therapies during or after stem cell transplants, gene therapy, and growth of dendritic cells.
References 1. Kessinger A, Armitage JO, Landmark JD, Smith DM, Weisenburger D (1988) Autologous peripheral hematopoietic stem cell transplantation restores hematopoietic function following marrow ablative therapy. Blood 71:723 2. Juttner CA, To LB, Ho JQ, Bardy PG, Dyson PG (1988) Early lymphohematopoietic recovery after autografting using peripheral blood stem cells in acute nonlymphoblastic leukemia. Transplant Proc 20:40 3. Sheridan W, Begley CG, Juttner CA, Szer J, To LB (1992) Effect of peripheral blood progenitor cells mobilized by filgrastim (G-CSF) on platelet recovery after high dose chemotherapy. Lancet 1:640 4. Juttner CA, To LB, Roberts MM, Haylock D, Dyson PG (1992) Comparison of hematologic recovery, toxicity, and supportive care of autologous PBSC, autologous BM, and allogeneic BM transplants. Int J Cell Cloning 10:160 5. Ager S, Scott MA, Magendra P (1995) Peripheral blood stem cell transplantation after high dose chemotherapy in patients with malignant lymphoma: a retrospective comparison with autologous bone marrow transplantation. Bone Marrow Transplant 16:79 6. Schmitz N, Linch DC, Dreger P (1996) Randomized trial of filgrastimmobilized peripheral progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients. Lancet 347:353 7. Alexander HR (1993) Vascular access and other specialized techniques of drug delivery. In VT Devita, S Hellman, SA Rosenberg (eds) Cancer: Principles and Practice of Oncology. Philadelphia: JB Lippincott Co 8. Gribben J, Neuberg D, Freedman A, Gimmi C, Pesek K, Barber M, Saporito L, Woo S. Coral F, Spector N, Rabinowe S, Grossbard B, Ritz J, Nadler L (1993) Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood 81:3449
9. Brugger W, Bross KJ, Glatt M, Weber F, Mertelsmann R, Kanz L (1994) Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 83:636 10. Vredenburgh J, Silva O, de Sombre K, Tyer K, Abou-Ghalia A, Cook M, Layfielld L, Peters WP, Bast RC (1996) A comparison of immunohistochemistry, two-color immunofluorescence, and flow cytometry with cell sorting for the detection of micrometastasic breast cancer in the bone marrow. J Hematotherapy 5:57 11. Brockstein BE, Ross AA, Moss TJ, Kahn DG, Hollingsworth K, Williams SF (1996). Tumor cell contamination of bone marrow harvest products: clinical consequences in a cohort of advanced-stage breast cancer patients undergoing high-dose chemotherapy. J Hematother 6:617 12. Vredenburgh JJ, Silva O, Broadwater G, Berry D, DeSombre K, Tyer C, Petros WP, Peters WP, Bast RC (1997) The significance of tumor contamination in the bone marrow from high risk primary breast cancer patients treated with high dose chemotherapy and hematopoietic support. Biol Blood Marrow Transplant 3:91 13. Schulze R, Schulze M, Wischnik A, Ehnle S, Doukas K, Behr W, Ehret W, Schlimok G (1997) Tumor contamination of peripheral blood stem cell transplants and bone marrow in high risk breast cancer patients. Bone Marrow Transplant 12:1223 14. Henschler R, Brugger W, Luft T, Frey R, Mertelsmann R, Kanz L (1994) Maintenance of transplantation potential in ex-vivo expanded CD341-selected human peripheral blood progenitor cells. Blood 84:2898 15. Sandstrom C, Bender J, Papoutsakis E, Miller W (1995) Effects of CD 341 cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells. Blood 86:958 16. Smith S, Bender J, Maples P (1993) Expansion of neutrophil precursors and progenitors in suspension cultures of CD 341 cells enriched from human bone marrow. Exp Hematol 21:870 17. Lill M, Lynch M, Fraser J (1994) Production of functional myeloid cell from CD34-selected hematopoeitic progenitor cells using a clinically relevant ex vivo expansion system. Stem Cells 12:626 18. Sato M, Sawada K, Koizumi K (1993) In vitro expansion of human peripheral blood CD341 cells. Blood 82:3600 19. Brugger W, Heimfeld S, Berenson F, Merstelsmann R, Kanz L (1995) Reconstitution of hematopoiesis after high dose chemotherapy by autologous progenitor cells generated ex-vivo. N Engl J Med 333:283 20. Haylock D, To L, Dowse T, Juttner C, Simmons P (1992) Ex vivo expansion and maturation of peripheral blood CD 341 cells into the myeloid lineage. Blood 80:1405 21. Shapiro F, Yao T, Raptis G, Reich L, Norton L, Moore M (1994) Optimization of conditions for ex vivo expansion of CD341 cells from patients with Stage IV breast cancer. Blood 84:3567 22. Sandstrom C, Bender J, Miller W, Papoutsakis E (1996) Comparison of perfused and static peripheral blood cell cultures with and without stroma using a modified culture chamber for non-adherent cell retention. Biotech Bioeng 50:493 23. Udomsakdi C, Eaves C, Swolin B, Teid D, Barnett M, Eaves A (1992) Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level. Proc Natl Acad Sci USA 89:6192 24. Koller M, Emerson S, Palsson B (1993) Large-scale expansion of human stem cell and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures. Blood 82:378 25. Koller M, Palsson B, Manchel I, Palsson B (1995) Long-term culture initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects. Blood 86:1784 26. Champlin RE, Mehra R, Gajewski J (1995) Ex-vivo expanded progenitor cell transplantation in patients with breast cancer. Blood 86:295a 27. Franklin WA, Shpall EJ, Archer P, Johnston CS, Garza-Williams S, Hami L, Bitter MA, Bast RC, Jones RB (1996) Immunocytochemical detection of breast cancer in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support. Breast Cancer Res Treat 41:1
C.R. Bachier et al./Experimental Hematology 27 (1999) 615–623 28. Sanders ME, Makgoba MW, Shaw S (1998) Human naive and memory T cells. Immunol Today 9:195 29. Swain SL, Craft M, Dubey C, Haynes L, Rogers P, Zhang X, Bradley LM (1996) From naive to memory T cells. Immunol Rev 150:142 30. Atkinson K (1990) Reconstitution of the haemopoietic and immune systems after bone marrow transplantation. Bone Marrow Transplant 5:209 31. Store J, Ehitherspoon RP, Storb J (1995) T cell reconstitution after bone marrow transplantation into adult patients does not resemble T cell development in early life. Bone Marrow Transplant 16:413 32. Koehne G, Zeller W, Stockschlaeder M, Zander AR (1997) Phenotype lymphocyte subsets after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 19:149 33. Talmadge JE, Reed E, Ino K, Kessinger A, Kunszyski C, Heimann D, Varney M, Jackson J, Vose JM, Bierman PJ (1997) Rapid immunologic reconstitution following transplantation with mobilized peripheral blood stem cells as compared to bone marrow. Bone Marrow Transplant 19:161 34. Stiff P, Oldenberg D, His E, Chen B, Smith A, Bayer R, Malhotra D, Kerger C, Armstrong RD, Muller T (1997) Transplantation of ex vivo expanded cells grown in Aastrom stromal-based perfusion bioreactors from small marrow aliquots (40 ml) produces durable hematopoietic reconstitution after ablative chemotherapy. Blood 90:395a 35. Lundell BI, Vredenburgh JJ, Tyer C, DeSombre K, Smith AK (1997) Ex-vivo perfusion culture expansion tumor positive bone marrow from breast cancer patients results in passive purging during the culture period. Blood 90:216a
623
36. Namikawa R, Muench MO, de Vries JE, Roncarob MG (1996) The Flk2/Flt3 Ligand synergies with Interleukin-7 in promoting stromalcell-independent expansion and differentiation of human fetal pro-B cells in-vitro. Blood 87:1881 37. Pribyl JR, Dittel BN, LeBien TW (1995) In-vitro studies of human B lymphopoiesis. Ann NY Acad Sci 764:9 38. Ito M, Anan K, Misawa M, Kai S, Hara H (1996) In vitro differentiation of murine sca-11lin2 cells into myeloid, B cells and T cell lineages. Stem Cells 14:412 39. Roux E, Helg C, Dumont-Girard F, Chapuis B, Jeannet M, Roosnek E (1996) Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts. Blood 87:3984 40. Mackall C, Granger L, Sheard MA, Cepeda R, Gress RE (1993) T-cell regeneration after bone marrow transplantation: differential CD45 isoform expression on thymic-derived versus thymic-independent progeny. Blood 82:2585 41. Heitger A, Neu N, Kern H, Panzer-Grumayer ER, Greinidx H, Nachbaur D, Niederwieser, Fink FM (1997) Essential role of the thymus to reconstitute naïve (CD45RA1) T-helper cells after human allogeneic bone marrow transplantation. Blood 90:850 42. Gokmen E, Raaphorst FM, Bachier C, LeMaistre CF, Boldt DH, Teale JM (1997) B- and T-cells obtained from ex-vivo expanded bone marrow mononuclear cell transplant recipients exhibit Ig H and TCR Vb CDR3 repertoires similar to that seen after conventional hematopoietic stem cell transplantation. Blood 90:539a