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Journal of Clinical Oncology, Vol 20, Issue 9 (May), 2002: 2344-2352
© 2002 American Society for Clinical Oncology

Autologous Transplantation for Aggressive Non-Hodgkin’s Lymphoma: Results of a Randomized Trial Evaluating Graft Source and Minimal Residual Disease

By Julie M. Vose, Graham Sharp, Wing C. Chan, Craig Nichols, Kevin Loh, David Inwards, Robert Rifkin, Philip J. Bierman, James C. Lynch, Dennis D. Weisenburger, Anne Kessinger, James O. Armitage

From the Departments of Internal Medicine, Anatomy and Cell Biology, Pathology and Microbiology, and Preventive and Societal Medicine, University of Nebraska Medical Center, Omaha, NE; Indiana University Medical Center, Indianapolis, IN; Queens Medical Center, Honolulu, HI; Mayo Clinic, Rochester, MN; and Rocky Mountain Cancer Center, Denver, CO.

Address reprint requests to Julie M. Vose, MD, Department of Internal Medicine, Section of Hematology/Oncology, University of Nebraska Medical Center, 600 S 42nd St, Omaha, NE 69198; email: jmvose{at}unmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine whether the source of autologous hematopoietic stem cells altered the clinical outcomes of patients undergoing high-dose chemotherapy and hematopoietic stem-cell transplantation (HSCT) for aggressive non-Hodgkin’s lymphoma (NHL).

PATIENTS AND METHODS: Of 105 high-risk, persistent, or relapsed NHL patients slated for an autologous HSCT entered onto this trial, 93 eligible patients were randomized to receive cytokine-naive autologous bone marrow transplantation (ABMT) (n = 46) or mobilized peripheral-blood stem-cell transplantation (PBSCT) (n = 47). All patients received carmustine, etoposide, cytarabine, and cyclophosphamide as the conditioning regimen. PBSCT patients also received identical mobilization with granulocyte colony-stimulating factor (G-CSF) 10 µg/kg/d, and both groups received G-CSF 5 µg/kg/d after the infusion of the stem-cell product until neutrophil engraftment.

RESULTS: PBSCT patients had significantly faster engraftment of all cell lineages: median time to absolute neutrophil count >= 500/µL, 10 days versus 13 days on the ABMT arm; median time to platelet count greater than 20,000/µL untransfused, 11 days versus 15 days on the ABMT arm; and median time to RBC transfusion independence, 8 days versus 16 days on the ABMT arm. The complete response rate was 72% for PBSCT and 54% for ABMT. The death rate before posttransplant day 100 was 2% on the ABMT arm and 6% on PBSCT arm. Event-free survival was 37% for PBSCT and 37% for ABMT. However, overall survival for PBSCT was 61% compared with 43% for ABMT.

CONCLUSION: Patients with aggressive NHL receiving HSCT randomized to PBSCT demonstrated improved neutrophil engraftment and platelet and RBC transfusion independence. The complete response rate and EFS were not statistically different by randomization arm. Patients whose harvests were positive for minimal residual disease by molecular analysis had poorer EFS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIGH-DOSE CHEMOTHERAPY and hematopoietic stem-cell transplantation (HSCT) from either autologous bone marrow or peripheral blood has now become the standard of care for patients with relapsed chemotherapy-sensitive aggressive non-Hodgkin’s lymphoma (NHL).1-3 In addition, some studies have evaluated this procedure in high-risk patients in first complete remission.4-6 Initially, peripheral-blood stem-cell transplantation (PBSCT) was used only for patients who were not eligible for bone marrow harvest because of contraindication to general anesthesia, extensive marrow fibrosis, or tumor involvement.7 As this procedure was used further, and mobilization of progenitor cells into the circulation by either chemotherapy,8 hematopoietic growth factors,9 or a combination of the two10 was refined, improved quality of the apheresis collections was possible. The information from these early PBSCT trials demonstrated a faster recovery of cell counts, less transplant-related morbidity, a shorter hospital stay, and a reduction of costs as compared with prior studies using autologous bone marrow transplantation (ABMT).11-13 Although a prospective randomized trial of PBSCT compared with ABMT has been performed, the major end point of that trial was engraftment.14 In a previous retrospective analysis, we observed that patients with otherwise comparable prognostic factors had an improved clinical outcome when undergoing PBSCT compared with ABMT.15 Therefore, we designed a prospective randomized trial to evaluate engraftment, tumor contamination, and disease outcome comparing ABMT with PBSCT in patients receiving high-dose chemotherapy for aggressive NHL. This study was performed at five transplant centers in the United States between 1993 to 1997 and now has close to a 5-year median follow-up.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Patients eligible for this trial were diagnosed with diffuse mixed-cell NHL, diffuse large-cell NHL, or immunoblastic NHL according to the Working Formulation classification system.16 They received high-dose chemotherapy and HSCT in first partial remission (PR) or complete response (CR) as intermediate/high- or high-risk patients according to the International Prognostic Index17 for either persistent disease after induction failure or relapsed NHL. High-risk patients were those who underwent transplantation in first PR or CR who had two or three of the risk factors from the age-adjusted International Prognostic Index identified at the time of initial diagnosis. Good-risk patients were those who underwent transplantation without a large mass more than 10 cm and only one of three characteristics: chemotherapy-resistant disease, an elevated lactate dehydrogenase (LDH) level above the upper limits of normal at the time of transplant, or having received three or more prior chemotherapy regimens. Poor-risk patients were those who underwent transplantation either with a large mass more than 10 cm and/or had more than one of the three characteristics mentioned above. This prognostic model was derived from a prior analysis of aggressive NHL transplants at the University of Nebraska Medical Center (UNMC).15

Study Design
Between November 1993 and June 1997, patients were entered onto this protocol at one of five transplant centers. Participating centers included the University of Nebraska Medical Center (J.M.V., P.J.B., A.K., and J.O.A.), Indiana University Medical Center (C.N.), Mayo Clinic (D.I.), Rocky Mountain Cancer Center (R.R.), or Queens Medical Center, University of Hawaii (K.L.). All patients signed written informed consent approved by the respective institutional review boards at the medical centers before randomization. The patients were entered onto the protocol at the time of initiation of the transplantation process and randomized to either unpurged autologous peripheral-blood stem cells or to unpurged autologous bone marrow as the hematopoietic stem-cell product. The patients were stratified by transplant center and prognosis according to our previously published prognostic system for transplantation.15 Central pathology review was performed at the University of Nebraska Medical Center (D.D.W. and W.C.C.).

Stem-Cell Collection
Patients randomized to ABMT underwent harvest under general anesthesia to obtain >= 2.0 x 108/kg mononuclear cells with no cytokine being administered before the collection. Patients randomized to PBSCT received 4 days of granulocyte colony-stimulating factor (G-CSF) at 10 µg/kg subcutaneously before the initiation of the apheresis procedure. The patients on the PBSCT arm had collections performed using a 12- to 20-L apheresis procedure to obtain a minimum of 1.5 x 106/kg CD34+ cells. If patients were unable to have adequate cells collected in their randomized cohort, they were considered harvest failures. Fresh samples from either the bone marrow or peripheral-blood harvest were shipped overnight to UNMC for analysis of minimal residual disease detection in the product according to the tests described below.

Chemotherapy and Transplantation
All patients who were able to go on to transplantation received the BEAC transplantation protocol consisting of carmustine 300 mg/m2 on day -7, etoposide 100 mg/m2 bid on days -6 to -3, cytarabine 100 mg/m2 bid on days -6 to -3, and cyclophosphamide 35 mg/kg/d on days -6 to -3. The patients received their autologous unpurged HSCT on day 0 using routine techniques. Patients were cared for in the participating transplant centers using their standard practices for antimicrobials, antivirals, and other clinical care. Transfusions were specified in the protocol as follows: irradiated RBC transfusions were to be given when the patient’s hemoglobin fell to less than 8.0 g/dL or as clinically indicated with blood loss, and irradiated platelets were to be transfused for a platelet count <= 20,000/µL or as clinically indicated for bleeding. Starting on day 0, all patients received G-CSF at 5 µg/kg subcutaneously until their absolute neutrophil count (ANC) was >= 500/µL for 3 consecutive days.

Engraftment
Days to reach an ANC >= 500/µL was defined as the first of 3 consecutive days when the ANC was >= 500/µL in the posttransplant period. Days to platelet transfusion independence was defined as the first day of 7 days with an unsupported platelet count of >= 20,000/µL in the posttransplant period. Days to RBC transfusion independence was defined as the first day of a 1-month period after transplantation without RBC transfusions.

Outcome Definitions
Patients were evaluated before transplantation with physical examination; computed tomography of the chest, abdomen, and pelvis; bone marrow biopsy; and other clinical tests as indicated for assessment of the disease. The response to transplant was assessed at day 100 after transplant by reexamination by physical examination, computed tomography, bone marrow biopsy, or other tests that were previously abnormal. The patients were defined as a CR if their abnormalities on tests or physical examination that could be attributed to the NHL had resolved. Patients were defined as a CR(u) if they had some minimal residual abnormalities on scans that were thought not to represent residual lymphoma. A PR was defined as more than 50% reduction in the bidimensional measurements of any known site of lymphoma. Patients who had less than a PR were defined as nonresponders.18 Early deaths were defined as deaths before day 100 after transplantation regardless of the cause.

Event-free survival (EFS) was defined as the time from transplantation until relapse or death from any cause. Overall survival was defined as the time from transplantation until death from any cause. These survivals are reported as 4-year estimates. The stratified log-rank test was used to compare the outcome of the randomization arms, with prognosis as the stratification factor. Cox proportional hazards regression was used for multivariate analysis of the risk factors for events or deaths, with the regression models stratified on prognosis.

Minimal Residual Disease Tests
Culture studies. Samples of peripheral blood and bone marrow were received before harvest, followed by either a sample from the bone marrow harvest or apheresis harvest, depending on treatment randomization, and processed as described previously.19 Briefly, samples were diluted with an equal volume of Hanks balanced salt solution, then layered over a cell separation medium (Accu-Prep Lymphocyte, Accurate Chemical and Scientific Corporation, Westbury, NY), and centrifuged at 400 x g for 20 minutes. The cells collected from the interface were washed once by centrifugation (400 x g for 7 minutes) and then resuspended in Tris-buffered ammonium chloride (pH 7.2) for 5 minutes to lyse mature red cells. An equal volume of complete medium (RPMI 1640 medium supplemented with 20% FCS, 1% penicillin/streptomycin, and 1% L-glutamine) was added to each tube and the suspensions were washed again. Cultures were established in 25-cm2 T-flasks using a modification of a technique described by Philip et al20 using 1% phytohemagglutinin-stimulated (Murex Diagnostics, Inc, Norcross, GA) human spleen cell–conditioned medium. Flasks containing 2.0 x 107 cells in 10 mL of medium were cultured at 37°C in 5% carbon dioxide in air. At the weekly change of half the medium, the supernatant cellularity of each culture was determined using a hemacytometer. Each flask was photographed weekly using a Nikon (Nikon Corporation, Tokyo, Japan) inverted phase-contrast microscope. A cytospin of the nonadherent cells was made for morphologic analysis and the harvested cells were then centrifuged at 400 x g for 5 minutes, the supernatant was removed, and the pellet was frozen for future DNA analysis. These parameters were used to determine whether there was any evidence of lymphoma in the infused harvest. The results were reported to the statistical core for analysis.

Molecular studies. DNA extraction of tissue sections from paraffin-embedded material was performed as described by Wright and Manos,21 and DNA extraction from fresh cell preparations was performed by the cell lysate method described by Gribben et al.22 A seminested polymerase chain reaction (PCR) procedure was used to amplify immunoglobulin H (IgH) complementary determining region III (CDRIII) using consensus primers as described by Wan et al.23 The PCR reactions were performed in an OmniGene Thermocycler (Hybaid Ltd, London, United Kingdom) with the following cycling parameters: annealing for 40 seconds at 55°C, extension for 40 seconds at 72°C, and denaturation for 40 seconds at 95°C. Both the initial and the nested rounds of amplification were performed for 30 cycles. Detection of bcl-2 translocation involving the major breakpoint region was performed using primers and reagents described by Gribben et al.24 The cycling conditions were similar to those used for amplifying IgH CDRIII. Sequencing of the tumor-specific IgH CDRIII was performed directly on the PCR product without prior cloning. If there was substantial contamination by IgH CDRIII products from normal B cells present in the tumor, pure clonal products could be obtained by either denaturing gradient gel electrophoresis of the PCR product or micromanipulation of the tumor sections before the PCR as described previously.25 The clonal IgH CDRIII product was recovered after gel purification and sequenced using the Sanger’s chain termination method with a cycle sequencing kit containing fluorescent dideoxynucleotide terminators according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). The sequencing gel was read using the Applied Biosystems 373A Sequenator. The V, D, and J regions in the CDRIII product were determined with the help of the GCG program and the sequence database (SAW) kindly supplied by Dr Schroeder, University of Alabama. The tumor-specific oligonucleotide primers and probes were then designed with the help of Olig 4.1 Software (National Biosciences, Plymouth, MN). In general, only one tumor-specific oligonucleotide primer could be obtained because of the short length of the CDRIII. In the detection of minimal residual disease, DNA was amplified using first-round consensus primers to the CDRIII region as described above. Two microliters of a 1:400 dilution of the PCR product from the first round of amplification was reamplified using either the consensus V- or J-nested primer coupled with the tumor-specific primer for that particular case. After the nested or seminested amplification, the PCR product is electrophoresed in agarose gel and Southern transferred to a nylon membrane. The membrane was then hybridized with the phosphorus-32–labeled tumor-specific probe at a temperature 10°C below the temperature maximum (Tm) calculated according to the formula Tm = 4(G + C) + 2(A + T) and washed at the appropriate stringency before autoradiography.26 The results were reported to the statistical core for analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients
One hundred five patients were randomized onto this protocol between November 1993 and June 1997. By central pathology review, three cases were determined to have ineligible histologies (one small lymphocytic, one mantle-cell lymphoma, and one small noncleaved-cell lymphoma). These cases were excluded from these analyses. One additional patient who was registered had no clinical information sent for data management. Eight eligible patients did not begin the transplant process because of progressive disease (seven patients) or patient preference (one patient). These patients have been excluded from the subsequent analyses, thereby leaving 93 eligible patients who began the transplantation process.

Of the 93 eligible patients, 46 were randomized to ABMT and 47 to autologous PBSCT. One patient died during the high-dose therapy and four patients were harvest failures. Three patients were unable to have adequate peripheral-blood stem cells harvested and underwent additional bone marrow harvesting. In addition, one patient was unable to have adequate cells harvested from the bone marrow and had supplemental peripheral stem cells harvested. Following the intent-to-treat principle, these patients were analyzed with their assigned randomized group.

Patient Characteristics
The median age of patients in the study was 47 years, with the population split evenly between male and female patients. The majority of patients were in the good-prognosis group. Few patients in either arm had a mass more than 10 cm at the time of transplantation. The majority of patients had chemotherapy-sensitive disease at the time of transplantation. The only characteristic that demonstrated an imbalance by randomization was the patients randomized to peripheral blood as their stem-cell source who were significantly more likely to have had three or more prior chemotherapy regimens. Further patient and disease characteristics are listed in Table 1.


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  		Table 1.  Patient Characteristics
 
Product Characteristics
Product characteristics are listed in Table 2. Patients randomized to PBSCT received significantly more mononuclear and nucleated cells (both P < .0001), but there was not a statistically significant difference in the number of CD34+ cells (P = .59). There was a trend toward the granulocyte-macrophage colony-forming unit being higher in the PBSCT group (P = .06).


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  		Table 2.  Product Characteristics by Randomization
 
Engraftment
Patients randomized to PBSCT had significantly faster engraftment in all cell lineages. The median time to achieve an ANC >= 500/µL was 10 days on the PBSCT arm and 13 days on the ABMT arm (P < .001) (Fig 1). The median time to a platelet count more than 20,000/µL with transfusion independence was 11 days on the PBSCT arm and 15 days on the ABMT arm (P = .023) (Fig 2). The median time to RBC transfusion independence was 8 days on the PBSCT arm and 16 days on the ABMT arm (P < .001).



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  		Fig 1. Time to achieve ANC >= 500/µL.

 


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  		Fig 2. Time to achieve platelet independence.

 
Outcome
The day-100 responses demonstrated a complete response rate, CR or CR(u), of 72% in patients randomized to PBSCT and 54% in patients randomized to ABMT (P = .09). The early death rate before day 100 was one of 46 (2%) on the ABMT arm and three of 47 (6%) on the PSCT arm (P = NS). The median follow-up of surviving patients who underwent transplantation is 55 months (range, 31 to 85 months). Progression of lymphoma has been documented in 26 (57%) patients on the ABMT arm and 27 (57%) patients on the PBSCT arm. Seven patients on the ABMT arm (hemorrhage, one; seizure, one; multisystem organ failure, two; car accident, one; congestive heart failure, one; and diffuse alveolar hemorrhage, one) and three patients on the PBSCT arm (pulmonary edema, one; congestive heart failure, one; and cardiac arrhythmia, one) died of causes other than lymphoma. Fourteen patients (three on the ABMT arm and 11 on the PBSCT arm) remain alive after progression of their lymphoma. Those alive after relapse tended to be patients with long first remissions and chemosensitive disease status before and after transplantation.

The relationship of randomization assignment to overall survival and EFS is shown in Figs 3 and 4. Patients randomized to PBSCT have longer overall survival compared with those patients randomized to ABMT: 4-year estimated overall survival for PBSCT, 61% (95% confidence interval [CI], 47% to 75%); for ABMT, 43% (95% CI, 29% to 58%) (P = .037) (Fig 3). However, the 4-year estimated EFS was not statistically different with PBSCT equal to 37% (95% CI, 23% to 51%) and ABMT equal to 37% (95% CI, 23% to 51%) (P = .39) (Fig 4). The relative risk of death for patients randomized to ABMT compared with patients randomized to PBSCT was 1.86 (95% CI, 1.03 to 3.36).



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  		Fig 3. Overall survival by randomization.

 


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  		Fig 4. EFS by randomization.

 
Minimal Residual Disease Testing
Molecular analysis. All specimens had molecular analysis attempted if their original lymphoma was informative by one of the techniques. Seven of the patients did not have adequate lymph node specimens to test. All remaining specimens from the patient’s original lymph node biopsy were tested for bcl-2, CDRIII, or T-cell receptor (TCR) as appropriate. Only 19 of the 46 patients randomized to the ABMT arm had specimens positive by PCR analysis. Of the 19 samples tested, nine (48%) were negative for bcl-2, CDRIII, or TCR as appropriate for their tumor. Five (26%) were positive for CDRIII, five (26%) were positive for TCR, and none were positive for bcl-2.

Twenty-two of the 47 peripheral-blood progenitor samples could be tested. Of the 22 tested, seven (32%) were negative for bcl-2, CDRIII, or TCR. Eight (36%) were positive for bcl-2, six were positive for CDRIII (27%), and one was positive for TCR (5%). Patients who were positive by one of these molecular techniques had a 28% (95% CI, 10% to 46%) 4-year EFS compared with a 56% EFS (95% CI, 32% to 81%) for those patients who tested negative (P = .039) (Fig 5). The majority of this difference was seen in the patients randomized to the ABMT arm, where the patients with molecularly negative harvests had a 4-year EFS of 68% compared with 30% for those patients with a molecularly positive harvest (P = .019). However, on the PBSCT arm, the 4-year EFS for patients with molecularly positive grafts was 28% compared with 42% for those patients with molecularly negative grafts (P = .45).



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  		Fig 5. EFS by molecular detection in graft product.

 
In vitro culture assay. All 46 of the bone marrow samples and 46 of 47 peripheral blood progenitor samples were assayed. Of the bone marrow culture assays, only two (4%) were scored as positive. In the peripheral-blood progenitor culture tumor assays, four (9%) were scored as positive. Although the numbers are small, this assay was positive in the same percentage of patients at UNMC as well as at the other participating centers. Patients whose in vitro culture assay was positive had a 4-year EFS of 33% (95% CI, 0% to 71%) compared with a 38% (95% CI, 27% to 48%) 4-year EFS in the patients whose culture assay was negative (P = NS) (Fig 6).



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  		Fig 6. EFS by in vitro culture growth in graft product.

 
Multivariate Analysis
The relationships of patient, disease, product source, biologic characteristics, and transplant center were analyzed for their effect on EFS. The Cox proportional hazards regression model, stratified on prognostic model, was used for multivariate analysis of the risk of an event. All characteristics with P < .20 according to bivariate analysis were considered as potential risk factors. In the multivariate analysis, an elevated LDH level was the only statistically significant independent predictor of the risk of an event (estimated relative risk, 3.2; 95% CI, 1.4 to 7.2; P = .006). After adjustment for elevated LDH level and using the stratified Cox proportional hazards model with all 93 patients, randomization to ABMT was still not significantly associated with the risk of an event (P = .34). The same analysis was used for relationship to overall survival. There were no statistically significant independent predictors of the risk of death in this analysis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
High-dose chemotherapy and autologous stem-cell transplantation has now become the standard of care for patients with relapsed chemotherapy-sensitive aggressive NHL.1 In addition, some studies have demonstrated an improved outcome for patients with high-risk aggressive NHL who receive autologous transplantation in first remission.4 Initially in these studies, autologous bone marrow was used as the hematopoietic stem-cell source. However, autologous hematopoietic progenitors from the peripheral blood were subsequently used when the patient’s marrow was hypocellular, fibrotic, or involved with tumor.7 Subsequently, as methods improved for the collection of peripheral-blood stem cells using hematopoietic cytokines, chemotherapy mobilization, or both, their use increased.8-10

It became apparent when comparing the engraftment times of patients undergoing PBSCT to historical control patients that neutrophil and platelet engraftment seemed to be faster than in those patients who had undergone ABMT.11-13 In addition, a matched-pair analysis from the European Bone Marrow Transplant registry was performed that analyzed patients undergoing ABMT or PBSCT for Hodgkin’s disease and NHL.27 In that study, there was no difference found in overall survival or progression-free survival in the NHL patients by graft source. However, as in the historical control studies, the engraftment was significantly better in the PBSCT group.

Subsequently, a randomized study in lymphoma patients by Schmitz et al14 observed median recovery times for neutrophil engraftment of 11 days versus 16 days in the PBSCT group compared with the ABMT group (P < .001). In addition, the time to platelet independence was 14 days in the PBSCT group compared with 23 days in the ABMT group (P < .001). A study by Damiani et al28 evaluated lymphoma patients who were randomized to receive either autologous G-CSF–primed bone marrow versus G-CSF–mobilized peripheral-blood stem cells for transplantation. In that study, 55 patients with NHL or Hodgkin’s disease were randomized to undergo one of these collection techniques. After high-dose ablative therapy, median time to neutrophil recovery was 12 days for G-CSF–stimulated ABMT and 11 days for G-CSF–mobilized PBSCT (P = .219). In addition, the median time to platelet recovery was 13 days for ABMT and 11 days for PBSCT (P = .242). The median number of transfusions was also the same in the two groups. The European Bone Marrow Transplant study demonstrated that either option of stimulating the hematopoietic stem cells produced similarly enhanced engraftment, compared with historical control patients receiving unprimed autologous bone marrow grafts.

In a previous retrospective analysis of the outcome in patients with aggressive NHL and otherwise similar prognostic factors undergoing ABMT versus PBSCT, patients undergoing PBSCT demonstrated an improved outcome.15 The reasons for this observation were not clear, and therefore this prospective randomized trial was designed to address the issues of engraftment and also evaluate the influence of minimal residual disease on clinical outcomes.

In addition to the engraftment of hematopoietic cells, it is always a concern with autologous transplantation that occult lymphoma cells could be present, which may influence the outcome for the patient. Consequently, the detection of minimal residual disease in hematopoietic stem-cell products is thought to be an important prognostic indicator and a criterion for patient selection for autologous transplant protocols.29

Various techniques are used to evaluate the presence of residual lymphoma cells in the graft products. Gribben et al22,24 have evaluated the nested PCR technique for the detection of bcl-2 in the transplant product. In the studies from the Dana-Farber Cancer Center, the outcome of patients who had detectable bcl-2 positivity in their graft after an antibody purging procedure had a significantly worse outcome than patients who received a negative graft.22

In addition to the molecular techniques, an in vitro tumor culture technique has been used extensively at UNMC on graft products for evaluation of residual tumor cells. In a previous study of this technique, 59 consecutive patients with aggressive NHL who were candidates for autologous transplantation had this technique applied to their harvested graft. The culture analysis demonstrated that 22 of these patients had occult lymphoma in their marrow. Results from the prior UNMC study demonstrated that those patients with occult lymphoma present in their harvest continued to relapse for up to 3 years after the transplantation, whereas no relapses were observed beyond 8 months in 13 patients receiving marrow that did not contain detectable lymphoma cells using the culture technique.30 A subsequent publication from UNMC demonstrated that 37% of cytokine-mobilized stem-cell grafts in patients with NHL were positive by the culture technique compared with 19% of nonmobilized peripheral-blood stem-cell grafts (P = .16). The presence of lymphoma cells did not have a significant impact on the clinical outcome in that analysis.31

In the prospective randomized study outlined in this article, patients with NHL who were otherwise transplant candidates were randomized to either autologous unpurged bone marrow or autologous unpurged peripheral-blood stem-cell transplantation. This study was designed to look not only at the engraftment of the patients selected by randomization, but also at the presence of occult tumor by molecular and culture techniques, and the outcome of the patients on the basis of this information. As was noted in the prior studies, patients undergoing the mobilized PBSCT demonstrated much improved engraftment in all cell lineages as compared with the patients randomized to unmobilized ABMT.

Although our previous retrospective analysis demonstrated that otherwise comparable patients undergoing PBSCT had an improved long-term EFS, this prospective randomized trial showed no difference in the EFS of patients by randomized graft source. Interestingly, the overall survival was improved on the PBSCT arm compared with the ABMT arm at the same time point. Evaluation of these patients who have relapsed after transplantation but remain alive demonstrates that these patients had long first duration of remissions before transplantation and chemotherapy-sensitive disease both before and after transplantation.

The outcome results derived from the molecular and culture results of the grafts demonstrate an improved outcome for patients with molecularly negative grafts as compared with positive ones. However, the result of the in vitro culture of the graft did not predict outcomes, although patients with culture-positive harvests tended to relapse sooner than patients with culture-negative harvests. It should be pointed out that only a subset of the grafts could be tested by molecular techniques because of patients not having a marker in their original tumor, or the lack of an adequate specimen. Also, the percentage of patients with culture-positive grafts in this study was substantially less than in our previous studies. This may reflect the fact that the majority of patients in this study were chemotherapy-sensitive good-prognosis patients, as compared with the poorer prognosis patients that were commonly underwent transplantation in the past.

As the benefits of mobilized PBSCT have been demonstrated over the past decade, the use of autologous bone marrow as a graft source has diminished substantially. This study confirms the improved engraftment of mobilized PBSCT; however, no additional long-term benefit of improved disease-free survival could be demonstrated. A small percentage of both PBSCT and ABMT products had minimal residual disease on the basis of molecular or culture techniques. Efforts should be directed at minimizing the lymphoma burden before peripheral-blood stem-cell collection to optimize the outcome with transplantation. On the basis of these results, mobilized PBSCT should remain the standard of care for patients undergoing autologous transplantation for lymphoma.


   ACKNOWLEDGMENTS
 
Supported by National Cancer Institute grant nos. CA61453 and CA61488.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Philip T, Guglielmi C, Hagenbeek A, et al: Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma. N Engl J Med 333: 1540-1545, 1995[Abstract/Free Full Text]

2. Mills W, Chopra R, McMillan A, et al: BEAM chemotherapy and autologous bone marrow transplantation for patients with relapsed or refractory non-Hodgkin’s lymphoma. J Clin Oncol 13: 588-595, 1995[Abstract/Free Full Text]

3. Stiff PJ, Dahlberg S, Forman SJ, et al: Autologous bone marrow transplantation for patients with relapsed or refractory diffuse aggressive non-Hodgkin’s lymphoma: Value of augmented preparative regimens—A Southwest Oncology Group trial. J Clin Oncol 16: 48-55, 1998[Abstract/Free Full Text]

4. Haioun C, Lepage E, Gisselbrecht C, et al: Survival benefit of high-dose therapy in poor-risk aggressive non-Hodgkin’s lymphoma: Final analysis of the prospective LNH87-2 protocol—A groupe d’Etude des lymphomes de l’Adulte study. J Clin Oncol 18: 3025-3030, 2000[Abstract/Free Full Text]

5. Nademanee A, Sniecinski I, Schmidt GM, et al: High-dose therapy followed by autologous peripheral-blood stem-cell transplantation for patients with Hodgkin’s disease and non-Hodgkin’s lymphoma using unprimed and granulocyte colony-stimulating factor–mobilized peripheral-blood stem cells. J Clin Oncol 12: 2176-2186, 1994[Abstract/Free Full Text]

6. Schenkein DP, Roitman D, Miller KB, et al: A phase II multicenter trial of high-dose sequential chemotherapy and peripheral blood stem cell transplantation as initial therapy for patients with high-risk non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 3: 210-216, 1997[Medline]

7. Kessinger A, Armitage JO, Smith DM, et al: High-dose therapy and autologous peripheral blood stem cell transplantation for patients with lymphoma. Blood 74: 1260-1265, 1989[Abstract/Free Full Text]

8. To LB, Shepperd KM, Haylock DN, et al: Single high doses of cyclophosphamide enable the collection of high numbers of hemopoietic stem cells from the peripheral blood. Exp Hematol 18: 442-447, 1990[Medline]

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Submitted September 28, 2001; accepted January 22, 2002.


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