S. J. Noga

Purging of Autologous Peripheral-Blood Stem Cells Using CD34 Selection Does Not Improve Overall or Progression-Free Survival After High-Dose Chemotherapy for Multiple Myeloma: Results of a Multicenter Randomized Controlled Trial

PURPOSE Although high-dose chemotherapy supported by autologous peripheral-blood progenitor-cell (PBPC) transplantation improves response rates and survival for patients with multiple myeloma, all patients eventually develop progressive disease after transplantation. It has been hypothesized that depletion of malignant plasma cells from autografts may improve outcome by reducing infused cells contributing to relapse. PATIENTS AND METHODS A randomized phase III study using the CEPRATE SC System (Cellpro, Bothell, WA) to enrich CD34(+) autograft cells and passively purge malignant plasma cells was completed in 190 myeloma patients randomized to receive an autograft of CD34-selected or unselected PBPCs. RESULTS After CD34 selection, tumor burden was reduced by 1.6 to 6.0 logs (median, 3.1), with 54% of CD34-enriched products having no detectable tumor. Median time to count recovery, number of transfusions, transplantation-related mortality, and days in hospital were equivalent between the two transplantation arms. With a median follow-up of 37 months, 33 patients (36%) in the selected and 34 patients (35%) in the unselected arm had died (P =.784). Median overall survival in the selected arm was reached at 50 months and is not reached at this time in the unselected arm (P =.78). Median disease-free survival was 100 versus 104 weeks (P =.82), with 67% of patients in the selected arm and 66% of patients in the unselected arm relapsing. CONCLUSION This phase III trial demonstrates that although CD34 selection significantly reduces myeloma cell contamination in PBPC collections, no improvement in disease-free or overall survival was achieved.

Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing's sarcoma

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, 200 000 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.

CD34 selection using three immunoselection devices: comparison of T-cell depleted allografts

BACKGROUND 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. METHODS 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. RESULTS Yields of CD34(+) cells were comparable for the three devices. However, there were significant differences in purity (CEPRATE < Isolex 300i < CliniMACS) and time from start of fractionation to infusion (CEPRATE < CliniMACS < Isolex 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. DISCUSSION 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.

Collection of peripheral blood progenitor cells (PBPC) based on a rising WBC and platelet count significantly increases the number of CD34 + cells

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 × 106 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 × 106 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 <1% and 81.5% if ⩾1% (P < 0.001). collection of stem cells based on rising wbc and platelet counts significantly increased the number of cd34+ cells in leukaphereses and CD34-selected products in comparison with collection on a fixed day.

CD34+ stem cell augmentation of elutriated allogeneic bone marrow grafts : results of a phase II clinical trial of engraftment and graft-versus-host disease prophylaxis in high-risk hematologic malignancies

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 × 107 mononuclear cells/kg, 3.3 × 106 CD34+ cells/kg, 1.5 × 105 CFU-GM/kg and 5.5 × 105 CD3+ T cells/kg. Median time to engraftment of granulocytes (>500/μl) was 16 days and of platelets (>50 000/μl) 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 ⩾80 days, the incidence of clinically significant acute GVHD (>stage 1) and extensive, chronic GVHD was 5% and 11%, respectively. Peri-transplant (⩽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.

Phase I study of escalating doses of low-dose-rate, locoregional irradiation preceding Cytoxan-TBI for patients with chemotherapy-resistant non-Hodgkin’s or Hodgkin’s lymphoma

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 Hodgkins or non-Hodgkins 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.

Using point-of-care CD34 enumeration to optimize PBSC collection conditions

BACKGROUND 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. METHODS 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. RESULTS 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 value < or = 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 had < or = 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. DISCUSSION 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.

High incidence of graft failure in children receiving CD34+ augmented elutriated allografts for nonmalignant diseases.

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<0.001), but was not associated with CMV seropositivity, donor gender, or cell counts of the allograft. We conclude that although time to engraftment is similar to that seen with unmanipulated grafts, graft failure remains a significant problem in patients with hereditary, nonmalignant diseases. Future efforts will seek to preserve the benefits of elutriation with CD34+ selection by increasing immune ablation of the preparative regimen and/or increasing posttransplant immune suppression.

Chemotherapy does not nullify the ability of donor lymphocyte infusions to mediate graft-versus-host reactions.

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.

Human herpesvirus 8 (KSHV) contamination of peripheral blood and autograft products from multiple myeloma patients

Human herpesvirus 8 (HHV-8), also known as Kaposis 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.