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Summary: T-cell depletion of the marrow graft using counterflow centrifugal elutriation reduces the risk of graft-versus-host disease (GVHD). However, because of high rates of graft failure and relapse, elutriation alone has not improved survival. We have carried out a phase II clinical trial in 54 pediatric patients to determine if CD34+ selection to rescue pluripotent stem cells from the small lymphocyte fraction improves engraftment. The processed grafts contained a mean of 5.5 × 107 cells/kg IBW, 4.7 × 106 CD34+ cells/kg IBW, and 6.3 × 105 CD3+cells/kg IBW. Patients achieved an ANC >500 at a median of 16 days and platelet count >20 000 at a median of 28 days. The incidence of clinically significant GVHD was 19%. In total, 10 patients enrolled in this study experienced graft failure, with eight of the 14 patients transplanted for nonmalignant indications failing to engraft stably. Graft failure was statistically significantly associated with nonmalignant diagnosis (P more...

Summary. Between January 1987 and January 1997, 69 eligible patients with acute myeloid leukaemia (AML) in either second (CR2) or third (CR3) complete remission (CR2 = 60, CR3 = 9) underwent 4-hydroperoxycyclophosphamide-purged autologous bone marrow transplantation (BMT) at the Johns Hopkins Oncology Center. The patients' median age was 27 years (range 1–62) and all received busulphan and cyclophosphamide as their preparative regimen. The probability of event-free survival (EFS) at 5 years was 30%[95% Confidence Interval (CI): 19–42%] for CR2 patients and 22% (3–51%) for those in CR3, with a median follow up of 8 years in the surviving group. The median time to an absolute neutrophil count of 0·5 × 109/l was 45 d (range 20–185). Relapse was the major cause of failure with a relapse rate of 55% in CR2 and 44% in CR3, while the non-relapse, transplant-related mortality rate was 15% in CR2 and 33% in CR3. In univariate analysis, patient age, cytogenetics, white blood cell count at presentation, CR1 duration and the sensitivity of clonogeneic leukaemia (CFU-L) in the graft to 4HC were all prognostic for EFS. Using each of these significant variables in multivariate modelling, patient age and sensitivity of CFU-L to 4HC were determined to be predictors of EFS. 4HC-purged autologous BMT produced results similar to allogeneic BMT for AML patients beyond first remission. more...

A PBSC graft containing 4-5 x 10(6) CD34(+) cells/kg is considered optimal in terms of durable engraftment. Tracking CD34 kinetics via point-of-care testing during PBSC mobilization could determine which (and when) patients will yield an optimal product. We evaluated whether microvolume fluorimetry (MVF) would be useful in optimizing PBSC mobilization/harvest and if it will shorten our standard 6 h collection.Absolute CD34 values were obtained using the IMAGN 2000 and STELLer CD34 assay (50 microL sample volume). Peripheral blood (PB) CD34 values from 30 patients undergoing PBSC mobilization were used to generate a PB CD34-based algorithm that would predict collection day/duration of apheresis. The algorithm was then used prospectively to collect PBSC products on 50 hematologic malignancy (HM) patients.Using the algorithm, patients were assigned to either a 6 (11-20 CD34/microL), 4 (21-49 CD34/microL) or 2 (or = 50 CD34/microL) h collection. Patients with a CD34 valueor = 10/microL were re-tested. All patients (n = 43) predicted to mobilize reached the optimal CD34 (4-5 x 10(6)/kg) value with 1.0 apheresis procedure; seven patients hador = 10/microL (nonmobilizers). The majority (75%) had apheresis charges decreased by 33-66%; 47% only required a 2 h procedure and 28% required 4 h. All patients demonstrated rapid trilineage engraftment.Absolute PB CD34 measurement using MVF offers a rapid and reliable approach to obtaining optimal PBSC products with minimal technical expertise. Although not a replacement for conventional flow cytometry, it meets the requirements for a point-of-care procedure. more...

Many poor-risk neuroblastomas and tumours of the Ewing's sarcoma family (ET) recur despite autologous transplants. Recurrence may be due to tumor cells contained in the BM harvests or PBSC harvests. The objectives of this prospective study were to: (1) determine the incidence and degree of tumor cell contamination in paired BM and PBSC harvests; and (2) determine the efficacy of tumor cell purging by immunomagnetic CD34+ cell selection. 198 samples from 11 consecutive patients with neuroblastoma or Ewing's sarcoma were analyzed. We assayed tumor contamination by RT-PCR assay for PGP 9.5, plus immunohistochemistry for neuroblastoma-specific antigens (the latter in neuroblastoma only). None of these patients had tumor cells detected in their BM by clinical histology immediately before BM or PBSC harvests. However, 82% of PBSC and 89% of backup BM harvests were contaminated with tumor by RT-PCR and/or immunocytochemistry assays. Unselected PBSC and BM harvests contained similar quantities of tumor cells (median, approximately 200000 cells). Cyclophosphamide plus G-CSF mobilization did not affect the incidence or level of contamination in PBSC harvests, as compared to blood obtained before mobilization. Immunomagnetic CD34+ cell selection depleted tumor cells by a median of 3.0 logs for PBSC, and 2.6 logs for BM harvests. more...

To date, in utero bone marrow transplantation (BMT) has had limited success, largely because of poor donor engraftment. The poor engraftment is probably the result of performing the procedure late in gestation after significant fetal immunocompetence has developed and/or transplanting insufficient numbers of donor hematopoietic stem cells for competing successfully with ongoing fetal hematopoiesis. To overcome these problems, we performed in utero BMT on a fetus with globoid cell leukodystrophy during the first trimester of gestation using selected paternal bone marrow stem (CD34+) cells. CD34 selection allowed a substantially greater number of stem cells to be transplanted. Although the fetus died 7 weeks after the procedure (during the 20th week of gestation), full donor engraftment was established. Moreover, the cause of death appeared to be overwhelming donor engraftment and leukostasis with paternal myeloid cells infiltrating most tissues. The ability of in utero BMT to produce this degree of engraftment provides great promise for the use of this approach in the treatment of a variety of inherited disorders that can be diagnosed prenatally. more...

PURPOSE To evaluate tumor-cell contamination of peripheral-blood progenitor-cell (PBPC) collections obtained after priming with granulocyte colony-stimulating factor (G-CSF). PATIENTS AND METHODS Immunocytochemical (ICC) and tumor clonogenic (TCA) assays were used to analyze tumor-cell contamination of pretreatment peripheral-blood (PB) and bone marrow (BM) samples, and of PBPC collection samples obtained after priming with G-CSF 5 micrograms/kg/d for 5 or 7 days in 38 women with advanced breast cancer undergoing high-dose chemotherapy (HDC). Results were compared with 37 historical control patients who underwent PBPC mobilization with cyclophosphamide (4 g/m2) followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) 5 micrograms/kg/d for 14 days. RESULTS Before PBPC priming with G-CSF, only one of 37 (3%) PB and four of 36 (11%) BM samples had tumor cells detected by ICC. Tumor-cell contamination of PBPC collections obtained after 5 or 7 days of G-CSF priming was observed in only three of 38 patients (8%). All patients with tumor cells detected in the PBPC collection had stage IV disease. Cells with in vitro clonogenic potential were detected only in the pretreatment BM sample in one patient, and another two patients had ICC- and TCA-positive PBPC samples despite tumor-negative PB and BM before priming. These results are similar to those previously reported for PBPC primed with cyclophosphamide and GM-CSF. CONCLUSION In patients with advanced breast cancer responsive to cytotoxic chemotherapy, tumor-cell contamination is not increased in PBPC collected after 5 or 7 days priming with G-CSF and appears similar to that seen when PBPC are primed with cyclophosphamide followed by GM-CSF. more...

PURPOSE (1) To study the ability of mobilized peripheral-blood progenitor cells (PBPC) collected in a single large-volume leukapheresis performed on a predetermined date to accelerate engraftment after high-dose cyclophosphamide and thiotepa; (2) to establish the minimum dose of PBPC associated with early engraftment; and (3) to identify parameters predictive of collection of large numbers of PBPC. PATIENTS AND METHODS Twenty-three patients with breast cancer received cyclophosphamide (4 g/m2) and granulocyte-macrophage colony-stimulating factor ([GM-CSF] 5 micrograms/kg/d x 15 days) for PBPC mobilization. A single leukapheresis was performed 15 days after cyclophosphamide administration. Then, patients received high-dose cyclophosphamide and thiotepa followed by reinfusion of PBPC and 4-hydroperoxycyclophosphamide (4HC)-purged bone marrow. PBPC concentration was measured in serial peripheral-blood samples and in the leukapheresis product. Correlation analysis between PBPC dose and engraftment and between leukapheresis yield and patient characteristics was attempted. RESULTS A single leukapheresis processed a median 36 L (range, 24 to 46) blood and collected 5 x 10(6) CD34+ cells/kg (< 0.3 to 24) and 6.2 x 10(5) colony-forming units granulocyte-macrophage (CFU-GM)/kg (< 0.001 to 29). All sixteen patients (70%) reinfused with > or = 2.9 x 10(6) CD34+ cells/kg reached a level of greater than 1,000 leukocytes/microL by day 13 and greater than 50,000 platelets/microL by day 15. All of these patients had a percentage of peripheral-blood CD34+ cells > or = 0.5%, and all but one, a level of greater than 100,000 platelets/microL, on the day of leukapheresis. The bone marrow CD34+ cell percentage at study entry predicted the number of CD34+ cells collected after PBPC mobilization (R2 = .42, P = .002). All patients with > or = 2.5% bone marrow CD34+ cells experienced early engraftment. CONCLUSION Reinfusion of PBPC collected in a single leukapheresis accelerates engraftment in the majority of patients. Pretreatment bone marrow CD34+ cell content determines PBPC mobilization capacity and may help select hematopoietic rescue strategies. more...

The effect of priming on occult tumor cell involvement of peripheral blood (PB) and PB progenitor cell (PBPC) collections is poorly characterized. Using sensitive immunocytochemistry (ICC) and tumor clonogenic assays (TCA) specific for epithelial-derived tumor cells, hematopoietic specimens were analyzed for PBPC and occult tumor cell involvement in 28 patients with chemotherapy-sensitive stage IIIB or IV breast cancer. Before PBPC priming, tumor was detected by ICC in PB of 1 of 23 (4%) patients and in bone marrow (BM) harvests of 4 of 27 (15%) patients. Fifteen days after cyclophosphamide and granulocyte- macrophage colony-stimulating factor (GM-CSF) priming, 2 of 28 (7%) patients had ICC-positive PBPC collections. The median amplification of CD34+ PBPC during this time was over 19-fold (range, < 1 to 199). One patient had pretreatment tumor involvement of both PB and BM. One patient grew tumor colonies in TCA; the PB and BM were ICC- and TCA- positive, but the PBPC collection was ICC-positive and TCA-negative. After cytoreduction with conventional-dose chemotherapy, patients with advanced breast cancer and histologically negative BM biopsy specimens have rare tumor cell involvement of PB and BM. Despite effective PBPC priming with cyclophosphamide and GM-CSF, clonogenic breast cancer cells were not found in the PBPC collection performed on day 15. more...

In an attempt to reduce the incidence and severity of acute graft- versus-host disease (GVHD), we have decreased the number of bone marrow (BM) lymphocytes in the donor marrow graft before bone marrow transplantation (BMT) using counterflow centrifugal elutriation (CCE). In a phase I-II clinical trial, 23 patients received lymphocyte- depleted BM allografts from their HLA-identical, mixed lymphocyte culture (MLC)-nonreactive sibling donors. The patients entered in the study were deemed to be at high risk for treatment failure on the basis of age (greater than 30 years; median, 39 years) and the result of our skin explant assay predictive of acute GVHD. Patients predicted not to develop acute GVHD by this assay were excluded from this study. All patients received a standard lymphocyte dose of 0.5 x 10(6) morphologic lymphocytes per kilogram ideal body weight (IBW) in the marrow graft and were maintained on cyclosporine A (CsA) immunosuppression for 170 days after BMT. Prompt hematopoietic recovery occurred in 22 of 23 patients with a median time to an absolute neutrophil count (ANC) greater than or equal to 500/microL of 21 days. Donor cell engraftment was subsequently verified by cytogenetic and/or DNA analysis in all of 21 evaluable patients. No patient developed systemic acute GVHD. Only five (22%) developed cutaneous GVHD (clinical stage 1) that required steroid treatment, including one patient who failed to engraft. The median follow-up of the patients enrolled in this study is 14 months (range, 5 to 20 months). Actuarial survival 1 year after BMT is 83%. Thus, in two consecutive clinical trials using CCE to deplete donor BM of alloreactive lymphocytes (1.0 x 10(6) versus 0.5 x 10(6) lymphocytes/kg), we have demonstrated that the procedure does not interfere with BM engraftment and is effective in decreasing the incidence and severity of acute GVHD. Furthermore, comparison of these studies has revealed a differential dose response relationship between the number of graft lymphocytes, protection of engraftment, and induction of acute GVHD. Although there appears to be a modest relationship between lymphocyte dose and time to hematopoietic recovery, the 50% reduction in lymphocyte dose from that used in our previous trial resulted in a marked decrease in acute GVHD without compromising engraftment. more...

In an attempt to reduce the incidence and severity of acute graft- versus-host disease (GVHD), we have decreased the number of bone marrow (BM) lymphocytes in the donor marrow graft before bone marrow transplantation (BMT) using counterflow centrifugal elutriation (CCE). In a phase I-II clinical trial, 23 patients received lymphocyte- depleted BM allografts from their HLA-identical, mixed lymphocyte culture (MLC)-nonreactive sibling donors. The patients entered in the study were deemed to be at high risk for treatment failure on the basis of age (greater than 30 years; median, 39 years) and the result of our skin explant assay predictive of acute GVHD. Patients predicted not to develop acute GVHD by this assay were excluded from this study. All patients received a standard lymphocyte dose of 0.5 x 10(6) morphologic lymphocytes per kilogram ideal body weight (IBW) in the marrow graft and were maintained on cyclosporine A (CsA) immunosuppression for 170 days after BMT. Prompt hematopoietic recovery occurred in 22 of 23 patients with a median time to an absolute neutrophil count (ANC) greater than or equal to 500/microL of 21 days. Donor cell engraftment was subsequently verified by cytogenetic and/or DNA analysis in all of 21 evaluable patients. No patient developed systemic acute GVHD. Only five (22%) developed cutaneous GVHD (clinical stage 1) that required steroid treatment, including one patient who failed to engraft. The median follow-up of the patients enrolled in this study is 14 months (range, 5 to 20 months). Actuarial survival 1 year after BMT is 83%. Thus, in two consecutive clinical trials using CCE to deplete donor BM of alloreactive lymphocytes (1.0 x 10(6) versus 0.5 x 10(6) lymphocytes/kg), we have demonstrated that the procedure does not interfere with BM engraftment and is effective in decreasing the incidence and severity of acute GVHD. Furthermore, comparison of these studies has revealed a differential dose response relationship between the number of graft lymphocytes, protection of engraftment, and induction of acute GVHD. Although there appears to be a modest relationship between lymphocyte dose and time to hematopoietic recovery, the 50% reduction in lymphocyte dose from that used in our previous trial resulted in a marked decrease in acute GVHD without compromising engraftment. more...

Purpose In patients in whom bone marrow transplantation (BMT) fails, recurrence often occurs at sites known to have contained disease before initiating BMT. The purpose of this study was to find the maximal tolerable dose of locoregional irradiation (LRT) between 1000 and 2000 cGy that could be integrated with our Cytoxan-total body irradiation (TBI) BMT conditioning regimen in the treatment of lymphoma. Methods and materials Patients had Hodgkin’s or non-Hodgkin’s lymphoma in chemotherapy-refractory relapse. All patients received LRT to a maximum of three sets of fields encompassing either all current or all previously known sites of disease. Cytoxan-TBI consisted of cyclophosphamide 50 mg/kg daily for 4 days followed by TBI of 1200 cGy given in four fractions. Results Twenty-one patients were enrolled. Radiation Therapy Oncology Group Grade 3 in-field acute toxicity was observed in 1 patient at each dose level up to 1500 cGy and in 3 of 6 patients receiving 2000 cGy. Clinically evident late toxicities were limited to hypothyroidism and one second malignancy occurring outside the LRT fields. Conclusion Low-dose-rate, LRT with concurrent Cytoxan-TBI before BMT has acceptable rates of in-field toxicity for doses up to 1500 cGy in five fractions. This regimen safely permits the use of a total combined radiation dose of up to 2700 cGy during 2 weeks, with encouraging in-field response rates in treatment-refractory patients. more...

Within the last 5 years, the new field of somatic cell therapy (SCRx) has gone through exponential growth. This term was coined by the U.S.Food and Drug Administration (FDA) in guidance documents to encompass all materials of cellular or tissue origin used for therapeutic purposes [1]. This included autologous, allogeneic and xenogeneic materials. Initially, human bone marrow was one of the only products other than blood that was processed or manipulated [2]. New sources of stem cells capable of more rapid engraftment with fewer side effects soon followed. During this time period, various regulatory agencies began focusing on these new products, with the growth of both commercial and academic cell processing facilities. Agencies such as the FDA developed practice guidelines for processing these products. When the initial set of guidelines were proposed, it seemed logical that guidelines used for current good manufacturing practice (cGMP) be applied. With the advent of embryonic stem cell research and the literal explosion in new cellular tissues and components, the cGMP format became nearly unobtainable by most academic centers due to cost, physical requirements and regulatory/administrative issues. Now, most researchers hope to process under more generic, good tissue practice (GTP) standards, but even these are quite restrictive. more...

T-cell depletion of allografts markedly reduces the incidence of GvHD following BMT. The approach taken at our Center has utilized the physical separation method of counterflow centrifugal elutriation (CCE), augmented by recovery of stem cells from lymphocyte-rich fractions by immunoaffinity selection of CD34(+) stem cells. We wanted to compare the performance characteristics of three commercially available selection devices, as well as the clinical outcomes of patients who received allografts engineered by the different devices.BM allografts were prepared for patients undergoing BMT for hematologic malignancies. BM cells were separated into lymphocyte-rich and lymphocyte-depleted fractions using CCE, followed by recovery of CD34(+) cells from the lymphocyte-rich fraction using one of three immunoselection devices [CellPro CEPRATE, Nexell Isolex 300i (software version 2.5) and AmCell CliniMACS]. Allografts consisted of the lymphocyte-depleted fraction plus the CD34-selected fraction.Yields of CD34(+) cells were comparable for the three devices. However, there were significant differences in purity (CEPRATEIsolex 300iCliniMACS) and time from start of fractionation to infusion (CEPRATECliniMACSIsolex 300i). More technical problems were encountered with the Isolex 300i device. Allograft compositions were comparable. Transplant outcomes (engraftment and incidence of GvHD) also were comparable.Qualitatively and quantitatively, allografts prepared with the CEPRATE, Isolex 300i (v 2.5) and CliniMACS devices should be considered comparable for use in this setting and probably also for direct T-cell depletion of BM. more...

Although multiple myeloma is sensitive to both chemotherapy and RT, it remains incurable at present. However, treatment algorithms based on published data, as well as clinical experience, can be developed to optimize therapy. This includes not only therapy for the underlying disease but also supportive therapy to enhance quality of life. Because myeloma is incurable, these guidelines prominently identify the clinical settings appropriate for treatment of patients on clinical research protocols. more...

Cyclosporine (CsA) treatment following autologous and/or syngeneic bone marrow transplantation (BMT) results in the induction of an autoimmune syndrome similar to graft-vs-host disease (GVHD) after allogeneic BMT. This autoimmune process which generally occurs 14–28 days following discontinuation of CsA treatment is termed syngeneic GVHD. We evaluated if syngeneic GVHD could provide an immunotherapeutic advantage to eliminate residual tumor cells after autologous/syngeneic BMT in a rat myeloma model. The results indicated that syngeneic GVHD did provide an anti-tumor effect in this rat model and could be potentiated by administration of recombinant gamma interferon. Based on these results several clinical trials have been initiated. more...

A systematic approach to hematopoietic graft manipulation has minimized several of the variables inherent to allogeneic BMT. Through this approach, we have been able to significantly impact on morbidity and quality of life following allogeneic transplantation. Acute and chronic GVHD, blood product and antibiotic usage, in patient hospitalization, acuity, costs and survival (especially in patients older than 40) have been improved. The HLA barrier still presents a formidable obstacle to achieving a more widespread use of this therapy. The complications encountered in HLA matched/TCD grafts occur with even greater magnitude in the HLA-mismatched or unrelated donor setting. Several centers are now engaged in studies using TCD grafts that are augmented with high doses of CD34+ cells to ensure engraftment while reducing the incidence of GVHD (50-53). Mobilized allogeneic PBSC appear to be an excellent source of stem cells for BMT (5,6). The earlier reports showed decreased rates of GVHD, despite having T cell burdens 10 times higher than those found in unmanipulated bone marrow. However, several of these centers now report an unacceptably high incidence of chronic GVHD (along with its attendant morbidity) following allogeneic PBSC transplantation (54-55). Initial results of TCD in these PBSC grafts using CD34+ selection are disappointing in that recipients developed unexpectedly high incidences of both acute and chronic GVHD (56). No doubt, significant differences exist between marrow and PBSC ancillary cell populations. For example, two laboratories now report the presence of natural suppressor cells in these allogeneic PBSC products in both mice (57) and humans (58). Thus, the same, step-wise approach would be expected to improve graft performance when using PBSC, cord blood, fetal tissue, xenografts or genetically engineered products as a stem cell source. Indeed, there are new reports of improved clinical outcome (especially in the incidence of GVHD) in the PMRD setting using both CD34+ selected (59) and sequential CD34+/CD2+ selected (60) PBSC grafts. It is hoped that future graft engineering approaches will be as successful as previous studies and will extend this form of therapy to an even larger patient population. more...

Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34(+) stem cells contained RNA for ER beta and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER beta and AR transcripts. No ER alpha or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER beta protein was present in glycoprotein (GP) IIb(+) megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb(+) and GPIIb(-) cells derived from CD34(+) cells and in HEL cells. Western immunoblotting confirmed the presence of ER beta and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases. more...

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), has recently been identified within the bone marrow dendritic cells of multiple myeloma (MM) patients. This virus contains homologues to human cytokines such as IL-6 that could potentially stimulate myeloma cell growth and contribute to disease pathogenesis. Since mobilization chemotherapy may increase circulating dendritic cell numbers, we searched for HHV-8 in peripheral blood mononuclear cells (PBMCs) before and after mobilization chemotherapy given to MM patients. Furthermore, we determined if autograft purging using the CEPRATE SC device would reduce the percentage of HHV-8 infected stem cell products. Only two of the 39 PBMC samples collected prior to mobilization chemotherapy contained PCR detectable virus, yet nine of 37 PBMCs collected on the first day of leukapheresis had detectable HHV-8 (P = 0.016). HHV-8 was more frequently identified in autograft products before vs after Ceprate SC selection (40% vs 15%, P = 0.016). Although the role HHV-8 plays in myeloma pathogenesis remains unclear, these results imply that mobilization chemotherapy increases the numbers of circulating HHV-8-infected dendritic cells within the peripheral blood. In addition, CD34 selection of autograft products in MM patients may reduce the reintroduction of virally infected cells following high-dose chemotherapy. Bone Marrow Transplantation (2000) 25, 153-160. more...

A246 VMP Results in Fewer Bone Events and Greater ALP Increases Versus MP in the VISTA Study in Front-Line MM M Delforge,1 M Kropff,1 I Spicka,2 M Petrucci,3 PG Richardson,4 R Schlag,5 N Khuageva,6 MA Dimopoulos,7 O Shpilberg,8 O Samoilova,9 MV Mateos,10 K Liu,11 W Deraedt,12 H van de Velde,13 J San Miguel14 1University of Munster, Germany; 2University Hospital, Prague, Czech Republic; 3University La Sapienza, Rome, Italy; 4Dana-Farber Cancer Institute, Boston, MA; 5Praxisklinik Dr. Schlag, Wurzburg, Germany; 6SP Botkin Moscow City Clinical Hospital, Russian Federation; 7University of Athens, School of Medicine, Athens, Greece; 8Rabin Medical Center, Petah-Tiqva, Israel; 9Nizhnii Novgorod Region Clinical Hospital, Russian Federation; 10Hospital Universitario Salamanca, CIC, IBMCC (USAL-CSIC), Spain; 11Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan; 12Johnson & Johnson Pharmaceutical Research & Development, Beerse, Belgium; 13Johnson & Johnson Pharmaceutical Research & Development, Beerse, Belgium; 14Hospital Universitario Salamanca. CIC, IBMCC (USAL-CSIC), Salamanca, Spain more...

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function. more...

The kinetics of mobilization and optimal timing of peripheral blood progenitor cell (PBPC) collection were evaluated in 190 patients with multiple myeloma undergoing stem cell harvest after mobilization with cyclophosphamide, prednisone and G-CSF. There was a strong correlation between the WBC count and the number of CD34 + cells circulating in peripheral blood (r = 0.875). Initiating leukapheresis based on rising WBC and platelet counts rather than on a fixed day increased the mean number of CD34 + cells 115% (9.7 to 20.9 × 10 6 CD34 + cells/kg; P = 0.010) for the total of all leukaphereses and 59% for the total of all CD34-selected products (5.1 to 8.1 × 10 6 CD34 + cells/kg; P = 0.011). Although the yield and purity of the CD34-selected product were not significantly affected (P ≥ 0.071), the percentage of patients with concentrations of CD34 + cells in the initial leukapheresis of >1% increased from 47% to 70% (P = 0.004). The mean purity of the selected product was related to the starting percentage: 48.9% if more...

Hemorrhagic complications are frequently implicated clinically for the high morbidity and mortality of acute graft versus host disease (GVHD), however, only few reports characterize the incidence and timing of bleeding in relation to GVHD, and essentially no study has quantified the effect of bleeding on survival of allogeneic patients with GVHD. This study examines the association of bleeding with acute GVHD and the effect of both complications on survival.A total of 463 allogeneic patients transplanted at the Johns Hopkins Hospital, were included in the study. Bleeding evaluation was based on daily scores of intensity and blood transfusions. All bleeding sites were recorded. GVHD staging was defined by the extent of rash, serum bilirubin, diarrhea, and confirmatory histology.The incidence of GVHD was 27.4%, bleeding occurred in 40.2%. The incidence of bleeding was higher in patients with GVHD as compared with non-GVHD, and correlated with GVHD severity. The higher bleeding incidence in GVHD was due to gastrointestinal hemorrhage, hemorrhagic cystitis, and pulmonary hemorrhage. While the majority of bleeding (51/75) in non-GVHD patients initiated within 30 days after bone marrow transplantation (BMT), only 32.3% (21/65) of the bleeding in the GVHD group initiated within 30 days, and the risk for bleeding continued until day 100. Bleeding was a late event compared to GVHD, however, most bleeding episodes were associated with active GVHD. Both GVHD and bleeding were individually associated with reduced survival, with profound additive adverse effect: median survival in 221 nonbleeding non-GVHD was83.2 months, GVHD nonbleeding (39 patients) had median of 10.6 months, bleeding non-GVHD (99 patients) had median of 4.3 months, and median survival of the GVHD bleeding group (85 patients) was 3.2 months.Our results support an association of bleeding with acute GVHD, suggesting that GVHD is a risk factor for bleeding after BMT. The occurrence of bleeding clearly identified poor outcome subgroup within GVHD, suggesting further evaluation for clinical application of bleeding in the assessment of GVHD severity. more...

Although T cell depletion of allografts used in BMT has reduced GVHD, it has been associated with inferior engraftment and an increased risk of relapse. We have found that T cell depletion by counterflow centrifugal elutriation (CCE) also results in depletion of CD34+ stem cells. In order to determine if the discarded CD34+ cells would improve engraftment, we undertook a phase II trial of allogeneic BMT in which 110 patients (median age 43) with a variety of hematologic malignancies received CD34+ stem cell augmented, elutriated marrow grafts. The T cell-depleted grafts were tightly controlled and contained a mean of 4.3 x 10(7) mononuclear cells/kg, 3.3 x 10(6) CD34+ cells/kg, 1.5 x 10(5) CFU-GM/kg and 5.5 x 10(5) CD3+ T cells/kg. Median time to engraftment of granulocytes (>500/microl) was 16 days and of platelets (>50000/microl) was 25 days, comparable to that seen with unmanipulated marrow. No mixed hematopoietic chimerism was observed that was not associated with disease relapse. The four patients (3.6%) who failed to engraft were all at high risk because of prior donor transfusions or underlying marrow disorders. The incidence of GVHD was dependent on the duration of cyclosporin A (CsA) immunosuppression. In patients who received CsA for > or = 80 days, the incidence of clinically significant acute GVHD (>stage 1) and extensive, chronic GVHD was 5% and 11%, respectively. Peritransplant (< or = 100 day post-BMT) mortality for this group of patients was 15%. Event-free survival in selected subsets of patients compared favorably to previous studies in which patients received unmanipulated marrow allografts. more...

Two patients with multiple myeloma in relapse after allogeneic BMT received donor lymphocyte infusions (DLI) but later required chemotherapy for treatment of myeloma-related complications. In both patients, recovery from chemotherapy-induced aplasia was accompanied by manifestations of graft-versus-host reactions. The first patient developed grade II acute GVHD and a complete remission which has lasted >22 months. The second patient developed grade III acute GVHD but died with co-existing GVHD and extensive extramedullary myeloma. These results demonstrate that chemotherapy does not nullify the ability of donor lymphocytes to mediate graft-versus-host reactions. more...

T cell depletion (TCD) performed by elutriation has decreased the incidence of acute and chronic graft-versus-host disease (GvHD) following bone marrow transplantation (BMT). However, as with all forms of TCD, patients may experience graft failure (10%), delayed engraftment, and mixed chimerism. Because 66%-75% of the CD34+ cells coseparate with the small lymphocytes, which are removed by elutriation, we designed a phase I trial in HLA-identical siblings to determine if the readdition of these previously lost small CD34+ cells would improve elutriation's engraftment kinetics. CD34+ cells were isolated from the small cell fraction of 10 consecutive donor grafts and infused into the recipients along with the TCD graft. The positively selected product had a mean T cell content of 1.2 x 10(5)/kg and was 80% CD34+, doubling the CD34+ content of the graft. All patients engrafted promptly with a median time to 500 neutrophils/mm3, untransfused 50,000 platelets/mm3, and discharge from the hospital of 19 (range 10-25), 24 (14-52), and 24 (18-29) days, respectively. Acute GvHD occurred in 2 patients, and no patient had chronic GvHD. Augmenting stem cell dose may be an efficient and safe alternative for overcoming TCD-associated delayed engraftment and graft failure, rather than increasing immunosuppression. more...

PURPOSE The objective of this study was to characterize CD34+ cell grafts, obtained using a novel technique, from children undergoing autologous bone marrow transplantation (BMT) for cancer therapy. In particular, we wanted to determine if the CD34+ marrow cell grafts generated hematopoietic reconstitution, since a positive result would motivate further development and use of this methodology. PATIENTS AND METHODS This pilot feasibility clinical trial involved 13 patients < or = 25 years of age with advanced solid tumors, including seven children with neuroblastoma. Harvested bone marrow underwent immunomagnetic CD34+ selection. RESULTS In three of 13 enrolled patients, low purities of the CD34+ preparations disqualified the use of the CD34+ marrow grafts. Ten patients received myeloablative chemotherapy with etoposide, carboplatin, and cyclophosphamide, then were transplanted with CD34+ marrow grafts. In the 10 patients transplanted with CD34(+)-selected cells, the CD34+ cell purity (nucleated RBCs excluded) in the cell graft preparation was 91% total cell recovery from the starting light-density cells 2.2%, CD34+ cell recovery 38%, colony-forming unit-granulocyte-macrophage (CFU-GM) recovery 23%, and estimated tumor-cell depletion 2.6 logs (medians). The CD34+ marrow grafts administered to these patients contained a median of 2.3 x 10(6) nucleated cells, 1.4 x 10(6) CD34+ cells, and 1.3 x 10(4) CFU-GM per kilogram patient weight. Most patients experienced only the toxicities previously observed with this myeloblative chemotherapy regimen, although two unusual toxicities were observed. All 10 patients transplanted with CD34+ cell grafts engrafted. CONCLUSION The CD34+ purified grafts were enriched in stem/progenitor cells, with five of these 10 preparations containing > or = 94% CD34+ cells. Engraftment with CD34(+)-purified cell grafts as pure as 99% confirms that autologous CD34+ cells, alone, are sufficient to provide hematopoietic rescue for myeloablated patients. The best purification results were obtained on small marrow harvests from patients with neuroblastoma. The engraftment of highly purified CD34+ cells obtained by this technology and the antitumor effect of the transplant, by which two of 10 poor prognosis patients remain clinically free of tumor, have stimulated further clinical trials. more...

The murine erythropoietin-dependent erythroleukemia cell line, HCD-57, was employed to study the cell cycle-specific behavior of erythropoietin. Cell cycle duration for HCD-57 cells was approximately 12 hours and was uninfluenced by erythropoietin. Populations of HCD-57 cells synchronized in G1 by centrifugal elutriation were able to pass through one complete cell cycle in the absence of erythropoietin but, thereafter, arrested in G1 as identified by propidium iodide staining and flow cytometry. Analysis of cell cycle behavior using the metachromic dye acridine orange, however, revealed that HCD-57 cells pass through a G0 cell cycle phase and, like serum-deprived 3T3 cells, actually arrest in G0 when deprived of erythropoietin. Expression of the cell cycle regulatory protein p34cdc2 was invariant throughout the cell cycle in HCD-57 cells. p34cdc2 was constitutively phosphorylated in G0 cells, and this effect was not modified by erythropoietin. Erythropoietin receptor distribution was log normal in HCD-57 cells in each phase of the cell cycle. The affinity of these surface receptors for erythropoietin was essentially invariant throughout the cell cycle, but receptor expression was upregulated in G2M cells as compared with cells in G1 or S phase. Taken together, these data indicate that erythropoietin has an important role in the G0-G1 to S phase transition but, based on receptor expression, is involved in other phases of the cell cycle as well. more...

The effect of priming on occult tumor cell involvement of peripheral blood (PB) and PB progenitor cell (PBPC) collections is poorly characterized. Using sensitive immunocytochemistry (ICC) and tumor clonogenic assays (TCA) specific for epithelial-derived tumor cells, hematopoietic specimens were analyzed for PBPC and occult tumor cell involvement in 28 patients with chemotherapy-sensitive stage IIIB or IV breast cancer. Before PBPC priming, tumor was detected by ICC in PB of 1 of 23 (4%) patients and in bone marrow (BM) harvests of 4 of 27 (15%) patients. Fifteen days after cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF) priming, 2 of 28 (7%) patients had ICC-positive PBPC collections. The median amplification of CD34+ PBPC during this time was over 19-fold (range,1 to 199). One patient had pretreatment tumor involvement of both PB and BM. One patient grew tumor colonies in TCA; the PB and BM were ICC- and TCA-positive, but the PBPC collection was ICC-positive and TCA-negative. After cytoreduction with conventional-dose chemotherapy, patients with advanced breast cancer and histologically negative BM biopsy specimens have rare tumor cell involvement of PB and BM. Despite effective PBPC priming with cyclophosphamide and GM-CSF, clonogenic breast cancer cells were not found in the PBPC collection performed on day 15. more...

Tumor contamination of hematopoietic stem cell grafts may influence the outcome of breast cancer patients treated with high-dose chemotherapy. The goals of this study were: (a) to evaluate the prevalence of tumor contamination of bone marrow (BM) harvests in patients responding to systemic chemotherapy; (b) to evaluate reduction of BM tumor contamination by ex vivo purging with 4-hydroperoxycyclophosphamide (4HC); and (c) to compare the tumor contamination of peripheral blood progenitor cell collections and BM in advanced-stage breast cancer patients designated for peripheral blood progenitor cell infusion. We evaluated pre- and post-4HC purge BM specimens from 20 patients for tumor contamination using immunocytochemistry and for in vitro growth potential of tumor cells using a tumor cell clonogenic assay. Pre-4HC purge BM specimens from 15 of 20 (75%) patients were immunocytochemistry and tumor cell clonogenic assay negative. The remaining 5 BM specimens were immunocytochemistry positive, but only 3 of 5 specimens were tumor cell clonogenic assay positive. In vitro tumor colony growth was not observed in any post-4HC purge BM specimens. We also evaluated nine patients with bone or BM metastases from the start of induction chemotherapy. We found less tumor involvement of peripheral blood progenitor cell collections than of simultaneously obtained bone marrow aspirates. We conclude that bone marrow micrometastases occur with low frequency in women with chemotherapy-sensitive breast cancer and that ex vivo purging with 4HC may render tumor cells nonviable. more...

9010 Background: Platinum-induced peripheral neuropathy may cause pain, function loss, and can be a dose-limiting factor for cancer treatment. The neurotoxicity can be irreversible; therefore preve... more...

Stem cell contamination by tumor is common in many diseases for which autologous bone marrow transplantation is used. In in vitro models chemotherapeutic purging reduces contamination and may have an impact on clinical outcome. Purging, however, delays engraftment. Little is known about the ability of granulocyte colony-stimulating factor (G-CSF) to accelerate myelopoiesis after purged autologous bone marrow transplantation. We treated 22 women with metastatic breast cancer with high-dose cyclophosphamide and thiotepa and, following the infusion of 4-hydroperoxycyclophosphamide-purged marrow, administered G-CSF, 16 micrograms/kg daily, from day 0 to engraftment. Results were compared with a control population of 24 women with breast cancer who received identical chemotherapy and purged marrow but not growth factor. Neutrophil recovery was accelerated in the G-CSF-treated population. An absolute neutrophil count of 500 was reached in 19 days compared with 29 for the historic controls. The median number of days febrile was reduced (8 versus 5.5) as were the number of days of hospitalization from marrow infusion (33 versus 25). There was no difference in the number of days on antibiotics or time to last platelet transfusion. G-CSF was administered without any notable toxicity. G-CSF accelerates myelopoiesis following the infusion of 4-hydroperoxycyclophosphamide-purged autologous marrow and shortens hospitalization. more...

Despite its detrimental effects, graft-versus-host disease (GVHD) has antileukemic properties as evidenced by a lower relapse rate in patients who develop GVHD following allogenic bone marrow transplantation. Meaningful long-term survival may be achieved if this latter property can be retained at the same time that the deleterious immune sequelae of acute and chronic GVHD are diminished. This is the focus of several recent graft engineering protocols. Various bone marrow components (lymphocytes, hematopoietic stem cells, and committed progenitor cells) can now be isolated and then used to reformulate the marrow graft. Combined with host immunosuppression or cytokine augmentation, it now may be possible to modulate GVHD such that its beneficial properties are enhanced without incurring its life-threatening side effects. more...

Graft vs host disease (GVHD) remains a major complication following allogeneic bone marrow transplantation (BMT) [1]. The large number of peripheral blood T lymphocytes which contaminate the graft during bone marrow (BM) harvesting are thought to mediate GVHD by responding to foreign major and/or minor histocompatibility antigen differences of the recipient [2]. more...

Rhesus progenitor-enriched BM was exposed overnight to SIV and cultured in a limiting dilution assay where the potential for progenitor interaction with lymphocytes or macrophages was low. Virus was consistently isolated late in culture, detection being aided by coculture with CEM174 lymphoblasts. Although infected cells had reduced clonogenic activity, colonies were indistinguishable from those derived from uninfected BM with respect to proliferative potential, morphology, and longevity in culture. Primate immunodeficiency viruses, therefore, may infect immature BM populations, directly affecting hematopoietic activity. more...

It appears that part of the confusion surrounding the lineage of NS cells could be due, in part, to the presence of more than one cell population in normal BM. Whether other cell populations exist in other organ compartments, or can be induced, is presently unknown. This is of particular interest in allogeneic BMT where various lymphocyte depletion techniques have been employed to reduce the incidence of AGVHD. When CCE is used for depletion, the NS lymphocyte component is entirely removed. Since the incidence of AGVHD is significantly reduced with CCE lymphocyte-depleted rat and human BM, it appears that this subpopulation need not be present to abrogate AGVHD. Quite surprisingly, preliminary studies in rats indicates that this lymphocyte subpopulation may actually induce acute syngeneic GVHD (Fischer et al., 1989). That a cell(s) in the clonogenic compartment has the ability to suppress or down-regulate a variety of immune responses is not altogether surprising. This cell is better thought of as an auto-regulatory cell which has the ability to control the cellular interactions in its immediate micro-environment. Indeed, R/O NSCA can be augmented by GM-CSF, IL-3, and CsA (NoGa et al., 1988a). In vitro, this cell differentiates into the mono-myeloid series using a variety of stimulatory agents and can acquire tumoricidal activity. The ability to express NSCA is lost however, being present only during a brief window of early maturation. Only IL-3 can sustain NSCA in culture.(ABSTRACT TRUNCATED AT 250 WORDS) more...

We have used elutriation to deplete lymphocytes from the marrow allografts of 64 patients to date. The first phase I trial (1 x 10(6) lymphocytes/kg IBW) was designed to test the procedure for potential toxicities, most notably, graft failure. Study 2 (1 x 10(6) lymphocytes/kg, no CsA) was expected to reduce potential toxicity incurred from long term immunoprophylaxis while study 3 was aimed at reducing the incidence of GVHD by further reducing lymphocyte dose (5 x 10(5)/kg). Graft lymphocyte dose was based on morphologic determination and was subsequently confirmed by limiting dilution analysis and flow cytometry. Although grafts were standardized solely by lymphocyte dose, the product was more uniform than the original harvested BM with respect to other cell populations. Nearly all study I (n = 40) and study III (n = 20) patients engrafted with a median time to ANC greater than 500/ul of 19 days. Three of the 4 patients consecutively enrolled in study II failed to engraft, thus terminating the trial. While a moderate proportion of study I patients had AGVHD (44%) with attendant morbidity, only 20% of study III patients were found to have mild AGVHD (less than or equal to stage I). To date, this cohort has no organ or chronic GVHD and no GVHD-associated morbidity. Median follow-up times for patients in studies I and III are 27 and 11 months, respectively. Overall actuarial survival (n = 60) is 42% at 38 months (38% study I, 80% study III). Good prognosis study I patients experienced 45% actuarial survival versus 9% in the poor prognostic group. While lymphocyte depletion has been effective in reducing the incidence and severity of AGVHD, new strategies are needed to address the issue of disease relapse. As with other methods, lymphocyte depletion by elutriation caused an increased rate of leukemia relapse. The actuarial probability of remaining in remission for recipients of elutriated marrow containing 1 x 10(6) and 5 x 10(5) lymphocytes/kg, respectively, were 60% and 46% at 16 months. Elutriation provides a rapid, reproducible and flexible methodology for graft manipulation which has been effective in reducing the incidence and severity of AGVHD. However, if lymphocyte depletion is to fulfill its promise as a means of reducing the overall morbidity of allogeneic BMT, new strategies may be needed to address the issue of relapse. These may include changes in marrow ablative therapy and post-graft immunosuppression. Equally as important may be the ability to further manipulate accessory cells and lymphoid populations presently excluded from the graft. more...

17539 Background: Bortezomib (VELCADE; Vc) has substantial single agent activity in heavily pretreated patients. Response rates have been shown to improve when used in combination with other agents. Since some pts experience long durations of response with treatment-free periods, this open-label, single-arm study was designed to determine the utility of retreatment with Vc alone or in combination with dexamethasone, thalidomide or liposomal doxorubicin. Methods: Pts who previously responded to Vc alone or in combination are eligible for this study. Up to 78 pts will receive Vc alone or in combination at the finishing dose of their initial Vc treatment (0.7, 1.0, or 1.3 mg/m2) as a bolus, intravenous injection on days 1, 4, 8, and 11 of a 21-day treatment cycle. Responses are assessed by the investigators and also calculated from percentage reduction in serum or urine M-protein. Results: To date, 15 pts have been treated and 12 are evaluable for response (having received at least 2 cycles of therapy). 8/12 patients had dexamethasone added to their regimen. The population is representative of pts with relapsed and refractory MM: mean age, 68 years; 4 males, 8 females; median of 5 prior lines of therapy; and 67% IgG MM. Median treatment-free period between the end of first Vc treatment and the start of retreatment: 11.1 months (range 5–21 months). Investigator-assessed responses show that 50% of pts have achieved a PR or CR. Only 1 pt had progressive disease. One pt has developed new peripheral neuropathy, while pre-existing neuropathy has worsened in 4 pts. No Vc-related serious adverse events have been reported. Conclusions: Preliminary results show that retreatment with Vc alone or in combination has encouraging activity with manageable toxicity even in pts who are heavily pretreated. These data suggest that repeated use after a treatment break may achieve prolonged disease control. This is the first report of Vc retreatment for MM. [Table: see text] more...

We have previously found that the absolute peripheral blood (PB) CD34 measurement generated using IMAGN 2000 (50 μl sample size, 45 min turn-around) and the STELLer® CD34 assay correlated with the CD34+/kg cell dose. Further, an algorithm based on the A.M. PB CD34 value accurately predicted the time (2, 4, 6 hrs) interval required to obtain an optimal (5 × 106 CD34/kg) PBSC product. It is unknown whether PBSC kinetics differ among the HM and could affect the prediction of collection parameters. 40 patients [indolent lymphoma (IND) = 19, sensitive non-Hodgkin's lymphoma and Hodgkin's disease (SEN) = 11, multiple myeloma (MM) = 10] were enrolled on a critical pathway for PBSC mobilization which included priming with cyclophosphamide (CY, 2.5 g/m2) and G-CSF (10 mcg/kg). IND patients also received Rituxamab (375 mg/m2) 72 hrs prior to CY. The day/collection duration of apheresis were prospectively determined using the algorithm. Analysis of the entire 2, 4 or 6 hours cohort showed no difference in collection time and diagnosis, Med. day > 500 grans was 9, 11, 10; plat > 50 k: 11, 14, 18 days, for the 3 intervals, respectively. Engraftment was not influenced by diagnosis. We conclude that PB CD34 measurements can accurately dictate collection conditions irrespective of the specific HM while the inability to mobilize may be multifactorial. more...