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AUTOLOGOUS TRANSPLANTATION
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AUTOLOGOUS TRANSPLANTATION Purging and the Impact of Minimal Residual Disease Robert Vescio, MD, and James Berenson, MD
The use of myeloablative doses of chemotherapy and radiotherapy has led to improved survival and curative potential for many patients with hematologic malignancies. Bone marrow obtained from syngeneic or allogeneic donors and infused after intensive conditioning therapy will eventually regenerate the hematopoietic system and rescue the patient from bone marrow aplasia. Although this procedure has dramatically changed the outcome for conditions such as chronic myelogenous leukemia, the lack of donors and treatment-related toxicity limit the use of such transplantations. In contrast, autologous bone marrow and peripheral blood stem cells (PBSCs) are readily available, and the regeneration of host hematopoiesis avoids the toxicity that develops from graft-versus-host disease (GvHD). This reduction in toxicity is significant: the rate of treatment-related mortality remains at 20% to 30% for allogeneic transplantation but has fallen to 2% or less for patients undergoing autologous transplantation. In addition, autologous transplantation can be performed on older patients, an advantage in the treatment of diseases such as multiple myeloma that are more prevalent in this age group.34 The use of an autograft for hematopoietic support has two possible
From the Department of Medicine, University of California Los Angeles School of Medicine, and Veterans Affairs Medical Center-West Los Angeles, Los Angeles, California
HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 13 NUMBER 5 * OCTOBER 1999
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disadvantages, however. First, any beneficial graft-versus-tumor effect is eliminated. The importance of this immunologic destruction of tumor cells varies in different malignancies but may be critical for certain myeloproliferative or malignant conditions. As an example, for patients with cell-mediated lympholysis or chronic myelocytic leukemia (CML), the relapse rate following syngeneic transplantation is 50%, compared with 20% following HLA-matched related allogeneic tran~plantation.~ Second, bone marrow and peripheral blood stem cell autografts are frequently contaminated with malignant cells.3,21-23, 33 Although residual malignant cells within an autograft may contribute to disease recurrence, the overall effect of these contaminating cells is difficult to quantify. One potential method of quantifying this effect is to compare results between syngeneic and autologous transplantation. The efficacy of syngeneic transplantation is determined solely by the conditioning regimen, although some mild graft-versus-host and thus graft-versus-tumor effects may occur. Syngeneic transplantation can be thought of as equivalent to an optimally purged autologous transplantation, because the hematopoietic support is clearly tumor-free? Therefore, any apparent increase in relapse rates following autologous transplantation can be attributed to the reinfusion of malignant cells. Unfortunately, the data on relapse rates following syngeneic transplantation are scanty because of the small numbers of patients who have received syngeneic transplantation. Furthermore, disease severity at the time of transplantation may not be equivalent and therefore may not allow adequate comparisons between these two groups of patients. Nevertheless, the relapse rate following syngeneic transplantation delineates the potential efficacy of a tumor-purged autologous transplant procedure and, therefore, can serve as a guideline for estimating potential benefit. CAN TUMOR CELLS WITHIN THE AUTOGRAFT CONTRIBUTE TO DISEASE RELAPSE? Gene-Marking Studies
Molecular and immunohistochemical techniques have documented the presence of contaminating tumor cells in the autografts of most patients with hematologic malignancies and also within bone marrow It seems and PBSCs of patients with many types of solid tumors.6,26,31,x,44 likely that the reinfusion of large numbers of proliferative malignant cells contributes to disease recurrence, particularly in conditions in which a circulatory component of the disease is important. This phenomenon was conclusively demonstrated by Brenner et a1 in a study in which autografts from patients with acute myelogenous leukemia (AML) were
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marked with the neomycin resistance gene.8At the time of relapse, some of the leukemic cells contained the marker gene denoting their origin within the reinfused autograft specimen. A similar study was performed using autografts derived from patients with neurobla~toma.~~ The bone marrow autografts of eight patients were transduced with a neomycin resistance-containing retroviral vector. Subsequently, the three patients who developed progressive disease had evidence of genetically marked cells within the recurrent tumor. Results of Phase II and Retrospective Studies
A retrospective analysis of patients receiving an autologous bone marrow transplantation for AML was performed using patients registered in the European Cooperative Group for Blood and Marrow Transplantation (EBMT).I7Patients receiving transplants in first complete remission had relapse rates of 47% with unmanipulated autografts versus 35% with chemotherapeuticallypurged autografts ( P = 0.04). The results were even more pronounced when only those patients given autografts within 6 months of complete response were considered. For those patients, the relapse rate for unmanipulated autografts was 60%; for purged autografts it was 16% ( P < 0.0001). In an Italian study of 125 AML patients, 87 received autografts of unpurged bone marrow, 21 received bone marrow purged with standard-dose mafosfamide, and 17 received bone marrow purged with the dose of mafosfamide adjusted according to the sensitivity of the leukemic clone.3O The relapse rates were 54% in patients receiving the unpurged marrow, 43% in patients receiving autografts purged with standard-dose mafosfamide, and 32% in patients receiving autografts purged with adjusted-dose mafosfamide. The lower relapse rate for those patients receiving an autograft purged by dose-adjusted mafosfamide was statistically significant when compared with the outcome for patients receiving unpurged grafts ( P = 0.008). At Johns Hopkins Oncology Center, 45 patients with AML were reinfused with autografts treated with 4-hydroperoxycyclophosphamide (4-HC) following high-dose chemotherapy? The efficacy of the purging procedure was estimated using colony-forming unit-granulocyte macrophage (CFU-GM) reduction as a biomarker for cell killing. Those patients who received autografts with CFU-GM survival of less than 1% following 4-HC treatment had a lower relapse rate ( P = 0.01) and longer disease-free survival ( P = 0.006). Because the efficiency of the purging process was determined more by extrinsic factors unrelated to disease burden (e.g., red blood cell content),14this result suggests that purging of autografts for patients with AML may be beneficial.
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Autograft purging also appears to benefit patients with non-Hodgkin’s lymphoma. The most compelling data implying a beneficial effect from autograft manipulation were reported by Gribben and colleagues from Boston.19Patients with non-Hodgkin‘s lymphoma underwent autologous transplantation using autografts depleted of B cells by a combination of anti-B-cell antibodies and complement. Many of these tumor cells contained a bcZ-2 rearrangement, and this tumor marker was used to quantify tumor burden before and after purging. In the most recent update of these results, the relapse rate was 72% for those patients whose autografts contained detectable bcl-2 rearrangements versus 26% for those patients with tumor-free autografts (Fig. l).l*Survival rates were also significantly improved for patients who received autografts without detectable tumor. Although this finding suggests that effective autograft purging may significantly improve disease-free and overall survival for patients with non-Hodgkin’s lymphoma, some have questioned the conclusiveness of these results. The ineffectiveness of the antibody/complement purging process may reflect an adverse characteristic of non-Hodgkin’s lymphoma, which again could be a marker for poor prognosis. Therefore, the patients with antibody/complementsensitive tumors may also be the patients with chemosensitive disease and, thus, have a better response to the high-dose chemotherapy. This possibility seems unlikely, however, because pretransplant tumor bur-
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Years after ABMT Figure 1. Long-term follow-up of 114 patients in whom polymerase chain reaction (PCR)detectable lymphoma cells were detected in the harvested autologous bone marrow. Those patients whose bone marrow became PCR negative (PCR neg) after immunologic purging have improved outcome compared with those patients in whom residual lymphoma cells were still detectable after purging (PCR pos). (Dafa from Gribben JG: Contemporaty techniques for the detection of minimal residual disease. In The Role of Tumor Purging in Autologous Transplantation. Deerfield, IL, Discovery International, 1997)
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den, histology, or remission status were not prognostic factors for purging efficiency or for patient outcome. Similar studies in patients with non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL) and multiple myeloma have demonstrated that autograft tumor negativity is a good prognostic factor; again, however, these results do not conclusively prove that autograft tumor purging itself is important.16,~ 533, In a case-matched study of patients with non-Hodgkin's lymphoma enrolled in the EBMT registry, 270 patients receiving a purged bone marrow autograft were compared with 224 patients receiving an unpurged pr0duct.4~Overall survival was 54% for the patients receiving a purged autograft versus 48% for their case-matched controls (P = 0.18). Although there appeared to be a survival benefit from the purging of autografts derived from patients with low-grade disease, progressionfree survival was similar between groups. Results From the First Randomized Trial of Autograft Purging
Recently, a multi-institutional study was completed in which 193 patients with multiple myeloma were randomly assigned to receive an unmanipulated autograft or one purged of tumor cells by CD34 enrichment using the Ceprate SC device (CellPro, Inc., Bothell, WA). These patients were selected for study for two reasons. First, autologous transplantation has been proven beneficial for patients with multiple myeloma. Patients receiving high-dose therapy and autologous bone marrow transplantation have improved disease-free and overall survival rates compared with patients receiving standard chemotherapy4 Second, the malignant cells can be quantified and, thus, measured in the autografts before and after manipulation with the purging device. The rearranged immunoglobulin (Ig) gene in the myeloma cell can serve as a unique marker for the clonal population of cells in multiple myeloma patients.5,*sf 38 Unlike the Ig gene in patients with non-Hodgkin's lymphoma, the myeloma Ig gene does not undergo further somatic mutation, and therefore, no intraclonal Ig gene diversity exists. All of the multiple myeloma cells within a patient contain the same, unique, Ig gene sequence even years later when the disease recurs. The polymerase chain reaction (PCR) can therefore be used to measure the number of tumor cells within various cell populations, such as the autograft product. In this particular study, patients were eligible for enrollment if they had evidence of advanced multiple myeloma (Durie-Salmon stage I1 or stage 111disease or an elevated P2-microglobulin level) and had received no more than 6 months of chemotherapy. Cyclophosphamide, 2.5 g/m2,
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prednisone, 2 mg/kg/d x 4,and granulocyte colony-stimulating factor (G-CSF), 10 Fg/kg/d beginning on day 2, were given to mobilize sufficient stem cells for collection. Once stem cell collection began, the patients were randomly assigned to receive standard autologous PBSC collection or CD34 enrichment using the Ceprate SC device. All patients subsequently received high-dose chemotherapy with cyclophosphamide (120 mg/kg) and busulfan (14 mg/kg) followed by stem cell reinfusion. No posttransplant adjuvant therapy was allowed. Unfortunately, patient outcome remains immature, and no overall or disease-free survival benefits were demonstrable between arms at the most recent analysis with a median follow-up period of slightly more than 1 year (Fig. 2). Nevertheless, the study did meet its primary objectives: it demonstrated that the CD34 selection procedure is safe and results in a greater than 2-log reduction in autograft tumor burden.40 Only the initial 134 patients were eligible for tumor burden assessment; the final 59 patients were included to increase the power to detect 1.o
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Days Posttransplant Figure 2. Kaplan-Meierprobability of progression-freesurvival based on data as of January 1998 in the randomized phase 111 trial comparing CEPRATE SC enriched versus unselected peripheral blood progenitor cell transplantation in patients with multiple myeloma. Solid line = CD34 selected, dashed line = unselected. (From Vescio R, Schiller G,Stewart AK, et al: Multicenter phase 111 trial to evaluate CD34 + selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood 93:1858-1868, 1999; with permission.)
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differences in disease-free and survival outcome between groups. Using bone marrow collected immediately before mobilization chemotherapy, the myeloma Ig gene sequence was determined in 47 patients. Autograft tumor burden could then be determined in 44 of these patients using myeloma-specific Ig gene primers and quantitative PCR. The median level of tumor cells detected in the initial leukapheresis of patients who later received CD34-enriched autografts was 2.6 X lo6. In patients who received standard autologous PBSC autografts, it was 2.3 x lo6. Following CD34 enrichment, autograft tumor burden fell a median 3.1 logs, with most of these purged autografts containing no detectable tumor cells. Despite the greater than 1000-fold reduction in tumor burden, median time to neutrophil recovery (absolute neutrophil count [ANC] 2 500/p,L) was 12 days in each arm. Platelet recovery times were delayed by a median of 2 days for those receiving a purged product, although all patients achieved platelet transfusion independence by day 56. This delayed platelet recovery was attributed to a loss of approximately 40% of CD34 cells caused by the enrichment procedure. When patients receiving the threshold number of CD34 cells/kg deemed safe in the phase I1 study (2 x 106/kg) were compared on each arm, platelet recovery times were equivalent. Thus, autograft purging can be performed in a safe and effective manner. With these results, the Food and Drug Administration (FDA) approved use of the Ceprate SC device for autograft purging in treating multiple myeloma and other tumors that do not express the CD34 antigen. This broad approval may seem premature without demonstration of outcome efficacy, but the reasons behind this decision will be explained subsequently. ESTIMATING THE BENEFIT OF AUTOGRAFT PURGING Proof of Survival Benefit May Not Be Possible
Randomized trials comparing unmanipulated versus purged autografts for hematopoietic support are the optimal method for to determining the effectiveness and risk of the purging process. Even relatively large studies, however, may not be adequately powered to detect differences in survival that could be considered clinically relevant. A randomized phase I11 study would require enrollment of more than 1000 patients to have an 80% power (likelihood) to detect a 30% improvement in median survival time to a significance level of 0.05 (Table 1). This degree of improvement would certainly be clinically meaningful. Yet, a study of this magnitude could be performed only at significant cost and only for patients with the most common malignancies. Smaller gains in survival time could still be considered important
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Table 1. NUMBER OF PATIENTS REQUIRED PER GROUP FOR 80% POWER USING A SIGNIFICANCE LEVEL OF 0.05 (TWO-SIDED TEST)* _____~ ~~
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Anticipated improvement in Median Survival Time (%)
Number of Patients Required per Group
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3888 1098 548
344 244
*Assumption of median survival time of 18 months in the group with poorer prognosis and 48 months follow-up
and perhaps more likely to be achievable by autograft purging. Such results would be impossible to prove, however, given the large patient numbers required. The previously described CellPro studyMenrolling 193 patients, has only a 50% chance of documenting a statistically significant survival advantage, even if the median survival times are, in reality, improved by 12 months in the purged arm. In an example provided by Appelbaum? the relapse rate following syngeneic transplantation for AML is 50% compared with 58% for similar patients undergoing unpurged autologous transplantation? This small difference suggests that a small increase in relapse rates may be caused by AML tumor reinfusion. Although trials designed to assess changes in the proportion of patients cured by a procedure require fewer enrolled patients than outlined in Table 1, a randomized study enrolling 800 patients would be required to prove statistical significance, assuming that the purging process could render the autografts completely tumorfree in every case, which is clearly an optimistic assumption. Even greater patient numbers would therefore be required to prove autograft purging efficacy for a device with a more realistic 3- to 4-log efficiency of tumor cell removal.* Also, because the need for and efficiency of tumor cell purging will differ among different tumor types, one could argue that similar large studies for each malignant subtype would be necessary to prove efficacy for each unique condition. For these reasons, proof of benefit is unlikely to be demonstrated and, in fact, is unnecessary in certain circumstances that may be applicable to autograft purging. Before any procedure or treatment becomes acceptable, a demonstration of improved benefit versus risk should be made. This equation is the basis of clinical decision making and should be used to evaluate this autograft purging procedure. Because the benefit may be difficult if not impossible to prove, the FDA apparently accepted proof of the safety of the procedure coupled with demonstrable tumor cell autograft reduction as being sufficient for approval of the Ceprate SC device.
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Thus, the theoretical benefit of reducing autograft tumor load sufficed because there was only a negligible detriment to patient safety (in this case, a 2-day prolongation in platelet recovery time). Similarly, small proven benefits, such as demonstrated prolonged disease-free survival following autograft purging, should be sufficient for procedure acceptance provided that the added toxicity of purging is negligible. As the toxicity of the purging process increases, however, the need to demonstrate a benefit becomes more important. Some of the chemical and complement-mediated purging procedures impede hematologic engraftment for 1 to 2 weeks,', 35 a delay that is likely to increase both procedure-related mortality and hospital charges. These more toxic procedures, which may be more efficient at removing tumor cells, will have to demonstrate an improvement in either disease-free or overall survival more clearly before becoming widely used. Tumor Cell Sensitivity Determines the Need for Autograft Purging
Even without proven studies, the diseases for which autograft tumor purging will be most effective can be postulated. The ratio of autograft tumor cell burden to the systemic tumor cell burden following high-dose chemotherapy is likely to determine the benefit of autograft purging (Fig. 3). If a large number of viable tumor cells remain after
Figure 3. The impact of autograft tumor Cell infusion following high-dose chemotherapy that achieves a 2-log (A) or 5-log (13)tumor-cell kill. Autograft purging will be helpful only if autograft tumor burden is substantially greater than the viable tumor cells that remain following high-dose therapy (B).
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high-dose chemotherapy, the impact of reinfusing the small numbers of cancer cells within the autograft will be minimal. Consequently, for chemorefractory diseases, autograft purging will be of little value. In contrast, for diseases such as leukemia or high-grade lymphomas, which can be very chemosensitive, it becomes more important to reduce the number of tumor cells reinfused in the patient, because the reinfused cells may be a significant proportion of the overall tumor bulk after high-dose therapy. In the optimal situation, the high-dose chemotherapy completely eradicates residual tumor cells; then the reinfusion of viable tumor cells within the autograft could be a dramatic and potentially the sole cause of the eventual disease relapse. The benefit of autograft purging therefore depends on tumor sensitivity which at present can only be estimated in a general manner. Although most investigators would agree that acute leukemias and highgrade lymphomas are more chemosensitive than the majority of solid tumors, the ability to quantify this difference remains elusive, particularly for an individual patient. Because the patient most likely to benefit from a tumor-free autograft is one who has a chemosensitive tumor with minimal tumor bulk at the time of transplantation, a mechanism for assessing the effect of the high-dose chemotherapy on these patients is needed. New methods of monitoring minimal residual disease should improve this ability. As an example, the authors have developed a quantitative PCR assay that is capable of identifying myeloma cells with a sensitivity of 1:720,000. This allowed the authors to evaluate myeloma tumor burden within the peripheral blood of patients before and after the high-dose chemotherapy in the phase I11 trial described previously.4o Myeloma cell numbers within the peripheral blood were calculated using the Poisson distribution-based PCR assay on samples obtained before mobilization chemotherapy with cyclophosphamide, prednisone, and G-CSF and on the first day of leukapheresis collection, 10 to 14 days later. Preliminary results suggest that those patients with an increased number of circulating tumor cells following the mobilization therapy with cyclophosphamide have a greater chance of early relapse after tran~plantation.~~ Because the patients receive transplantation after cyclophosphamide and busulfan conditioning, circulating tumor response to cyclophosphamide mobilization chemotherapy may serve as an in vivo drug sensitivity test. It is quite possible that autograft tumor purging will only show benefit for those myeloma patients with more chemosensitive disease as evidenced by a complete remission before transplantation or by response to mobilization chemotherapy. Further follow-up of the patients in the phase I11 trial may resolve this issue. Of course, general statements about disease sensitivity should be continually reevaluated. As the effectiveness of high-dose chemotherapy improves and more chemorefractory diseases become treatable, autograft purging will take on a more important role.
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Necessity for Purging Increases With Multiple Rounds of Intensive Therapy
Many recent trials have used multiple rounds of intensive therapy6 As the number of cycles of high-dose therapy increases, the reinfusion of malignant cells within collected autografts becomes more relevant, as does the importance of autograft purging. Autografts are typically obtained before the first of multiple high-dose regimens and are divided for administration after each cycle of therapy. If a substantial tumor burden exists within this initial autograft, the reinfusion of these cells may become the most significant determinant of relapse. An example can demonstrate this point (Fig. 4). If a patient with an initial tumor load of 1 x lo8 cells received an autograft that contained 1 X lo5 malignant cells and then received three rounds of high-dose therapy, each accomplishing a 3-log reduction in tumor burden, that patient would be left with 3 X lo4 tumor cells following the final high-dose procedure. In contrast, if the initial autograft were purged free of tumor, then the patient would be tumor-free after the final round of treatment. Although this model is simplistic, it illustrates the concept that the benefit of autograft purging increases with the number of high-dose chemotherapy cycles delivered, because in each scenario a similar tumor burden (1 to 2 x lo5 cells) would have existed after the initial high-dose therapy cycle.
Figure 4. Example of patient tumor burden following successive rounds of high-dose chemotherapy, each achieving a 3-log tumor cell kill. Initial patient tumor burden = 108 cells. Unmanipulated autograft tumor burden = lo5 cells. Purged autograft = tumor-free. HD = viable tumor burden immediately following the first, second, and third cycle of chemotherapy. Post 1 , 2, and 3 = the tumor burden following the infusion of the autograft after each of these cycles. Square = Unpurged, circle = purged.
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AUTOGRAFT PURGING RISKS Delayed Engraftment
The potential benefits of autograft purging may sometimes be offset by adverse effects. Engraftment delay is the most obvious risk related to autograft purging. Typically, median times to neutrophil counts higher than 500/p,L and platelet counts higher than 2 x 104/p,Lare used to estimate engraftment quality, but these median times may not be the best indicator of overall treatment toxicity. Even if median neutrophil engraftment time is increased from 12 to 14 days by autograft purging, this 2-day prolongation may not be clinically relevant, because it is unlikely to correlate with significant changes in procedural risk or expense. In contrast, the few patients with truly delayed or incomplete engraftment may be markedly affected by poor autograft quality. These patients may need prolonged transfusion support and may not be able to tolerate future chemotherapy when indicated at the time of disease recurrence. The added expense and risks for these patients will probably overshadow any adversity incurred by a 1- or 2-day shift in median engraftment times. Consequently, there is a need for a standardized measurement that better encompasses these outlying patients as an indicator of the risks of hematopoietic engraftment. As an example, a comparison of the percentage of patients still requiring hematological support at days 15, 30, and 60 may better indicate clinically relevant differences in delayed engraftment between treatment arms. Autograft Purging and Impaired Immunity
Other less apparent adverse outcomes could result from autograft purging. Most of the purging methods significantly reduce the numbers of B and T cells in the autograft. This reduction could theoretically impair cell-mediated immunity and lead to an increased incidence of infectious complications after transplantation. A recently completed study using highly purified CD34'Thy1 'Lin - autografts as hematopoietic support substantiated this None of the eight evaluable patients receiving the purged autograft had recovered an absolute CD4 count higher than lOO/p,L after 100 days, and several patients died of posttransplant infection. In the CellPro Phase III study, CD4 cell recovery was also delayed for those patients receiving a CEPRATE SC selected autograft. Median CD4 cell counts were 211/kL in the selected autograft versus 298/p,L in the unselected autograft." Nevertheless, this statistically significant difference did not correlate with a higher incidence of infections in the posttransplantation period (56% for each arm, days 0
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to 100 post transplant). It is likely that a threshold number of lymphoid or lymphoid-precursor cells are required to maintain adequate immunological function after transplantation. This same process could also impair tumor cell immunity. This potential risk will be difficult to quantify and will probably have a variable effect depending on the tumor type. Nevertheless, it is possible that autograft purging could negatively affect disease-free survival by disrupting cell-mediated tumor control. Finally, incubation of autografts with chemotherapeutic agents such as 4-HC may lead to increased rates of treatment-related leukemia or myelodysplastic syndromes caused by the exposure of stem cells to these toxic This potential risk will be difficult to define without long-term follow-up of large randomized trials.
ASSESSMENT OF MINIMAL RESIDUAL DISEASE Measuring High-Dose Therapy Tumor Cell Kill
The majority of patients with acute leukemia and non-Hodgkin's leukemia receive autologous transplantation when they are in clinical remission. The inability to quantify the amount of residual disease before and after the transplantation procedure makes it difficult to determine the effectiveness of the therapy for an individual patient. Generalized comparisons of phase I1 study results are often the basis for high-dose therapy protocol design and administration, given the paucity of phase I11 studies. Obviously, differences in patient characteristics and supportive care measures make such comparisons problematic. The ability to measure the logarithmic cell kill of tumor cell burden by the high-dose regimen would be a significant advance and could allow more rapid and accurate comparisons. Furthermore, in certain instances maintenance therapy is given following the transplantation procedure in an effort to reduce further the residual disease burden (e.g., a-interferon in patients with multiple myel~ma.)'~ The inability to measure viable tumor burden in clinical remission disease states makes clinical decision making difficult, particularly for patients experiencing moderate side effects from this adjuvant therapy. Recent techniques have been developed that permit the detection of small numbers of residual tumor cells in patients in clinical remission. Techniques using PCR or in situ hybridization can detect levels as low as 1 malignant cell within a background of 1 X lo6 normal cells. Although most published studies assessing residual disease after transplantation have used semiquantitative assays, new quantitative PCR protocols have been developed. These methods are tools that investiga-
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tors can use to calculate the reduction of tumor burden in blood and bone marrow following hgh-dose chemotherapy. Correlation with Outcome After Autologous Transplantation
The persistence of minimal residual disease in acute leukemia patients following treatment has been predictive of disease relapse.", 20, 24, 37 In a prospective analysis of children with ALL, the relapse rate was 8% in patients without detectable residual leukemia after initial induction therapy versus 40% for the remaining patients ( P < 0.001). Similar adverse outcomes were noted for patients with residual disease following consolidation and intensification therapy. These results imply that minimal residual disease can be used to identify patients who appear to be in clinical remission but are at risk for disease relapse. These patients may benefit from the early use of alternative treatment such as transplantation.'O In a study performed at the Dana-Farber Cancer Institute, 10 of 31 assessable patients had residual chronic lymphoblastic leukemia (CLL) based upon Ig gene PCR amplification following allogeneic or autoloIn 5 of these 10 patients, PCR-detectable disease gous tran~plantation.~~ resolved within 6 months, and these 5 patients remain in clinical remission. The remaining 5 patients with persistent PCR-detectable disease have all subsequently relapsed. Of the 21 patients without detectable disease following transplantation, only 1 patient has relapsed. These numbers were too small to demonstrate statistical significance, but clearly a strong relationship between persistently detectable disease and disease outcome exists. Similar results are apparent for non-Hodgkin's lymphoma patients following dose-intensive treatment. The majority of patients with detectable bcI-2 or Ig gene rearrangements immediately following transplantation are destined to relapse.12,45 It is of interest that the elimination of detectable tumor cells may take months, because many non-Hodgkin's lymphoma and ALL patients with PCR-detectable disease approximately 1 month after transplantation subsequently remained in clinical and molecular remission. Similar results were noted in the authors' study of multiple myeloma patients after transplantation. Circulating and bone marrow myeloma cell tumor burden continues to fall for 3 to 6 months following autologous transp l a n t a t i ~ n Unlike . ~ ~ patients with non-Hodgkin's lymphoma and ALL, the majority of multiple myeloma patients with detectable tumor before transplantation continue to have disease after transplantation that is measurable with sensitive PCR techniques. Because autologous transplantation is not curative in multiple myeloma, these findings are not
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surprising. Nevertheless, increases in circulating tumor burden from pretransplant levels predict disease progression?* Even blood samples collected at the time of patient discharge may reflect transplant efficacy. Of 33 evaluable patients, 4 patients had increased circulating myeloma cell burden on the day of engraftment, compared with levels before mobilization chemotherapy. All four patients developed progressive disease, three within only 6 months. In contrast, the relapse rate at 24 months was 48% for those patients whose circulating multiple myeloma cell concentration had decreased from pretransplant levels.
SUMMARY
Is a Theoretical Benefit Without Risk Enough to Justify Purging? Without a randomized trial any benefit of purging on patient survival will remain theoretical. Although molecular techniques may document that the autograft tumor burden is substantial when compared with the patient tumor load following conditioning therapy, a theoretical benefit may not be sufficient to persuade third-party payers to expend additional money for the purging procedure. For insurers, the equation to determine product usefulness does not consider risk as the only negative component, but a combination of risk and cost. When the amount that can be spent is limited, a demonstration that these additional costs will garner a true patient benefit becomes more important. Because it is difficult to assign a dollar amount for a theoretical benefit, third-party payors may be unwilling to accept the potential but unproven benefit from a purging procedure. Nevertheless, if the time to engraftment remains unchanged, the added costs of the purging procedure should be relatively minimal and, in the authors’ view, should not be a major impediment to its widespread use. Educated patients and physicians will probably demand that these procedures be performed in cases where autograft purging adds little if any risk and provides at least a significant potential for benefit.
References 1. Anderson KC, Barut BA, Ritz J, et al: Monoclonal antibody-purged autologous bone marrow transplantation therapy for multiple myeloma. Blood 77712-720, 1991 2. Appelbaum F R The Gulati/Romero/Ciavarella article reviewed. Oncology (Hunting) 8324-30, 1994 3. Atta J, Martin H, Bruecher J, et a t Residual leukemia and immunomagnetic bead
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purging in patients with bcr-abl-positive acute lymphoblastic leukemia. Bone Marrow Transplant 18:541-548, 1996 4. Attal M, Harousseau JL, Stoppa AM, et al: A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma patients. N Engl J Med 335:91, 1996 5. Bakkus MH, Heirman C, Van Riet I, et al: Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 80:232&2335, 1992 6. Barlogie B, Jagannath S, Vesole DH, et al: Superiority of tandem autologous transplantation over standard therapy for previously untreated multiple myeloma. Blood 89~789-793,1997 7. Bird JM, Luger S, Mangan P, et al: 4-Hydroperoxycyclophosphamide purged autologous bone marrow transplantation in non-Hodgkin’s lymphoma patients at high risk of bone marrow involvement. Bone Marrow Transplant 18:309-313, 1996 8. Brenner MK, Rill DR, Moen RC, et al: Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Lancet 341235436, 1993 9. Butturini A, Gale Rp: Clinical models of autotransplants in chronic myelogenous leukemia. Leuk Lymphoma ll(Supp1 1):255-257, 1993 10. CavC H, van der Werff ten Bosch J, Suciu S, et al: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. N Engl J Med 339:591598, 1998 11. Ciudad J, San Miguel JF, Lopez-Berges MC, et al: Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol 163774-3781, 1998 12. Corradini P, Astolfi M, Cherasco C, et al: Molecular monitoring of minimal residual disease in follicular and mantle cell non-Hodgkin’s lymphomas treated with high-dose chemotherapy and peripheral blood progenitor cell autografts. Blood 89:724-731, 1997 13. Cunningham D, Powles R, Malpas J, et al: A randomized trial of maintenance interferon following high-dose chemotherapy in multiple myeloma: Long-term follow-up results. Br J Haematol 102495-502, 1998 14. Douay L Negative selection and protection of normal progenitor cells for autografting. Bone Marrow Transplant 22423-430,1998 15. Gertz MA, Witzig TE, Pineda AA, et al: Monoclonal plasma cells in the blood stem cell harvest from patients with multiple myeloma are associated with shortened relapse-free survival after transplantation. Bone Marrow Transplant 19:337-342, 1997 16. Gorin NC, Aegerter P, Auvert B, et al: Autologous bone marrow transplantation for acute myelocytic leukemia in first remission: A European survey of the role of marrow purging. Blood 75:1606-1614, 1990 17. Gorin-NC, Labopin M, Meloni G, et al: Autologous bone marrow transplantation for acute myeloblastic leukemia in Europe: Further evidence of the role of marrow purging by mafosfamide. Leukemia 5:896, 1991 18. Gribben JG: Contemporary techniques for the detection of minimal residual disease. In Gribben J (ed):The Role of Tumor Purging in Autologous Transplantation. Deerfield, IL, Discovery International, 1997 19. Gribben JG, Freedman AS, Neuberg D, et al: Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N Engl J Med 325:1525-1533,1991 20. Gruhn B, Hongeng S, Yi H, et al: Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome. Leukemia 12675-681, 1998 21. Gupta D, Bybee A, Cooke F, et al: CD34 + -selected peripheral blood progenitor cell transplantation in patients with multiple myeloma: Tumor contamination and outcome. Br J Haematol 104166-177, 1999 22. Leonard BM, Hetu F, Busque L, et al: Lymphoma cell burden in progenitor cell grafts measured by competitive polymerase chain reaction: Less than one log difference between bone marrow and peripheral blood sources. Blood 91:331-339, 1998 23. McCann JC, Kanteti R, Shilepsky B, et al: High degree of occult tumor contamination in bone marrow and peripheral blood stem cells of patients undergoing autologous
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25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.
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transplantation for non-Hodgkin's lymphoma. Biol Blood Marrow Transplant 2:3743, 1996 Meloni G, Diverio D, Vignetti M, et a1 Autologous bone marrow transplantation for acute promyelocytic leukemia in second remission: Prognostic relevance of pretransplant minimal residual disease assessment by reverse-transcription polymerase chain reaction of the PML/RAFh fusion gene. Blood 90:1321-1325, 1997 Mizuta S, Kohno A, Tanimoto M, et a1 Quantitative molecular analysis of the efficacy of ex vivo purging predicts clinical outcome after autologous BMT in patients with Blineage acute lymphoblastic leukemia. Blood 88(Suppl 1):73a, 1996 Moss TJ, Cairo M, Santana VM, et al: Clonogenicity of circulating neuroblastoma cells: Implications regarding peripheral blood stem cell transplantation. Blood 83:3085 3089, 1994 Provan D, Bartlett-Pandite L, Zwicky C, et al: Eradication of polymerase chain reactiondetectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation. Blood 88:222€&2235,1996 Ralph QM, Brisco MJ, Joshua DE, et a1 Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new B-cell clone: Evidence from the Ig heavy chain gene. Blood 82:202-206, 1993 Rill DR, Santana VM, Roberts WM, et a1 Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 84:380-383,1994 Rizzoli V, Mangoli L, Carlo-Stella C: Autologous bone marrow transplantation in acute myelogenous leukemia. Leukemia 61101-1106, 1992 Ross AM, Cooper BW, Lazarus HM, et a1 Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques. Blood 822605-2610, 1993 Rowley SD, Jones RJ, Piantadosi S, et al: Efficacy of ex vivo purging for autologous bone marrow transplantation in the treatment of acute nonlymphoblastic leukemia. Blood 74~501-506, 1989 Sharp JG, Kessinger A, Mann S, et al: Outcome of high-dose therapy and autologous transplantation in non-Hodgkin's lymphoma based on the presence of tumor in the marrow or infused hematopoietic harvest. J Clin Oncol 14214-219, 1996 Siege1 DS, Jagannath S, Desikan KR, et al: Feasibility of high dose chemotherapy with peripheral blood stem cell support for multiple myeloma patients over the age of 70. Blood 88(Suppl 1):130a, 1996 Treleaven J, Powles R, Singhal S, et al: Autografting with CD52 monoclonal antibodypurged marrow for acute lymphoblastic leukemia. Blood 88(Suppl 1):225a, 1996 Tricot G, Gazitt Y, Leemhuis T, et a1 Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma. Blood 91:4489-4495, 1998 van Dongen JJ, Seriu T, Panzer-Grumayer ER, et a1 Prognostic value of minimal residual disease in acute lymphoblastic leukemia in childhood. Lancet 352:1731-1738, 1998 Vescio RA, Cao J, Hong CH, et al: Myeloma Ig heavy chain V region sequences reveal prior antigeneic selection and marked somatic mutation but no intraclonal diversity. J IITUINUIO~ 155:2487-2497, 1995 Vescio RA, Han EJ, Schiller GJ, et a1 Quantitative comparison of multiple myeloma tumor contamination in bone marrow harvest and leukapharesis autografts. Bone Marrow Transplant 18:10>110, 1996 Vescio R, Schiller G, Stewart AK, et a1 Multicenter phase I11 trial to evaluate CD34+ selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood 93:1858-1868, 1999 Vescio RA, Schiller GJ, Stewart AK, et a1 Quantitative assessment of myeloma peripheral blood and bone marrow tumor burden in patients undergoing autologous transplantation. Blood 88(Suppl 1):641a, 1996 Vescio RA, Stewart AK, Ballester 0,et a1 Circulating multiple myeloma cell burden predicts disease relapse following autologous transplantation. Blood 92(Suppl 1):97a, 1998
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43. Williams CD, Goldstone AH, Pearce RM, et al: Purging of bone marrow in autologous bone marrow transplantation for non-Hodgkin’s lymphoma: A case-matched comparison with unpurged cases by the European Blood and Marrow Transplant Lymphoma Registry. J Clinical Oncol 142454-2464, 1996 44. Witzig TE, Gertz MA, Pineda AA et al: Detection of monoclonal plasma cells in the peripheral blood stem cell harvests of patients with multiple myeloma. Br J Haematol 89640-612, 1995 45. Zwicky CS, Maddocks AB, Andersen N, et a1 Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in non-Hodgkin’s lymphoma is associated with decreased relapse after autologous bone marrow transplantation. Blood 8833314-3322, 1996 Address reprint requests to
Robert Vescio, MD DVA West Los Angeles Bldg 500 Room 4237 (111H) 11301 Wilshire Boulevard Los Angeles, CA 90073
0889-8588/99 $8.00
+ .OO
AUTOLOGOUS TRANSPLANTATION Purging and the Impact of Minimal Residual Disease Robert Vescio, MD, and James Berenson, MD
The use of myeloablative doses of chemotherapy and radiotherapy has led to improved survival and curative potential for many patients with hematologic malignancies. Bone marrow obtained from syngeneic or allogeneic donors and infused after intensive conditioning therapy will eventually regenerate the hematopoietic system and rescue the patient from bone marrow aplasia. Although this procedure has dramatically changed the outcome for conditions such as chronic myelogenous leukemia, the lack of donors and treatment-related toxicity limit the use of such transplantations. In contrast, autologous bone marrow and peripheral blood stem cells (PBSCs) are readily available, and the regeneration of host hematopoiesis avoids the toxicity that develops from graft-versus-host disease (GvHD). This reduction in toxicity is significant: the rate of treatment-related mortality remains at 20% to 30% for allogeneic transplantation but has fallen to 2% or less for patients undergoing autologous transplantation. In addition, autologous transplantation can be performed on older patients, an advantage in the treatment of diseases such as multiple myeloma that are more prevalent in this age group.34 The use of an autograft for hematopoietic support has two possible
From the Department of Medicine, University of California Los Angeles School of Medicine, and Veterans Affairs Medical Center-West Los Angeles, Los Angeles, California
HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 13 NUMBER 5 * OCTOBER 1999
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disadvantages, however. First, any beneficial graft-versus-tumor effect is eliminated. The importance of this immunologic destruction of tumor cells varies in different malignancies but may be critical for certain myeloproliferative or malignant conditions. As an example, for patients with cell-mediated lympholysis or chronic myelocytic leukemia (CML), the relapse rate following syngeneic transplantation is 50%, compared with 20% following HLA-matched related allogeneic tran~plantation.~ Second, bone marrow and peripheral blood stem cell autografts are frequently contaminated with malignant cells.3,21-23, 33 Although residual malignant cells within an autograft may contribute to disease recurrence, the overall effect of these contaminating cells is difficult to quantify. One potential method of quantifying this effect is to compare results between syngeneic and autologous transplantation. The efficacy of syngeneic transplantation is determined solely by the conditioning regimen, although some mild graft-versus-host and thus graft-versus-tumor effects may occur. Syngeneic transplantation can be thought of as equivalent to an optimally purged autologous transplantation, because the hematopoietic support is clearly tumor-free? Therefore, any apparent increase in relapse rates following autologous transplantation can be attributed to the reinfusion of malignant cells. Unfortunately, the data on relapse rates following syngeneic transplantation are scanty because of the small numbers of patients who have received syngeneic transplantation. Furthermore, disease severity at the time of transplantation may not be equivalent and therefore may not allow adequate comparisons between these two groups of patients. Nevertheless, the relapse rate following syngeneic transplantation delineates the potential efficacy of a tumor-purged autologous transplant procedure and, therefore, can serve as a guideline for estimating potential benefit. CAN TUMOR CELLS WITHIN THE AUTOGRAFT CONTRIBUTE TO DISEASE RELAPSE? Gene-Marking Studies
Molecular and immunohistochemical techniques have documented the presence of contaminating tumor cells in the autografts of most patients with hematologic malignancies and also within bone marrow It seems and PBSCs of patients with many types of solid tumors.6,26,31,x,44 likely that the reinfusion of large numbers of proliferative malignant cells contributes to disease recurrence, particularly in conditions in which a circulatory component of the disease is important. This phenomenon was conclusively demonstrated by Brenner et a1 in a study in which autografts from patients with acute myelogenous leukemia (AML) were
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marked with the neomycin resistance gene.8At the time of relapse, some of the leukemic cells contained the marker gene denoting their origin within the reinfused autograft specimen. A similar study was performed using autografts derived from patients with neurobla~toma.~~ The bone marrow autografts of eight patients were transduced with a neomycin resistance-containing retroviral vector. Subsequently, the three patients who developed progressive disease had evidence of genetically marked cells within the recurrent tumor. Results of Phase II and Retrospective Studies
A retrospective analysis of patients receiving an autologous bone marrow transplantation for AML was performed using patients registered in the European Cooperative Group for Blood and Marrow Transplantation (EBMT).I7Patients receiving transplants in first complete remission had relapse rates of 47% with unmanipulated autografts versus 35% with chemotherapeuticallypurged autografts ( P = 0.04). The results were even more pronounced when only those patients given autografts within 6 months of complete response were considered. For those patients, the relapse rate for unmanipulated autografts was 60%; for purged autografts it was 16% ( P < 0.0001). In an Italian study of 125 AML patients, 87 received autografts of unpurged bone marrow, 21 received bone marrow purged with standard-dose mafosfamide, and 17 received bone marrow purged with the dose of mafosfamide adjusted according to the sensitivity of the leukemic clone.3O The relapse rates were 54% in patients receiving the unpurged marrow, 43% in patients receiving autografts purged with standard-dose mafosfamide, and 32% in patients receiving autografts purged with adjusted-dose mafosfamide. The lower relapse rate for those patients receiving an autograft purged by dose-adjusted mafosfamide was statistically significant when compared with the outcome for patients receiving unpurged grafts ( P = 0.008). At Johns Hopkins Oncology Center, 45 patients with AML were reinfused with autografts treated with 4-hydroperoxycyclophosphamide (4-HC) following high-dose chemotherapy? The efficacy of the purging procedure was estimated using colony-forming unit-granulocyte macrophage (CFU-GM) reduction as a biomarker for cell killing. Those patients who received autografts with CFU-GM survival of less than 1% following 4-HC treatment had a lower relapse rate ( P = 0.01) and longer disease-free survival ( P = 0.006). Because the efficiency of the purging process was determined more by extrinsic factors unrelated to disease burden (e.g., red blood cell content),14this result suggests that purging of autografts for patients with AML may be beneficial.
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Autograft purging also appears to benefit patients with non-Hodgkin’s lymphoma. The most compelling data implying a beneficial effect from autograft manipulation were reported by Gribben and colleagues from Boston.19Patients with non-Hodgkin‘s lymphoma underwent autologous transplantation using autografts depleted of B cells by a combination of anti-B-cell antibodies and complement. Many of these tumor cells contained a bcZ-2 rearrangement, and this tumor marker was used to quantify tumor burden before and after purging. In the most recent update of these results, the relapse rate was 72% for those patients whose autografts contained detectable bcl-2 rearrangements versus 26% for those patients with tumor-free autografts (Fig. l).l*Survival rates were also significantly improved for patients who received autografts without detectable tumor. Although this finding suggests that effective autograft purging may significantly improve disease-free and overall survival for patients with non-Hodgkin’s lymphoma, some have questioned the conclusiveness of these results. The ineffectiveness of the antibody/complement purging process may reflect an adverse characteristic of non-Hodgkin’s lymphoma, which again could be a marker for poor prognosis. Therefore, the patients with antibody/complementsensitive tumors may also be the patients with chemosensitive disease and, thus, have a better response to the high-dose chemotherapy. This possibility seems unlikely, however, because pretransplant tumor bur-
100
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80 60 40
20 I
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Years after ABMT Figure 1. Long-term follow-up of 114 patients in whom polymerase chain reaction (PCR)detectable lymphoma cells were detected in the harvested autologous bone marrow. Those patients whose bone marrow became PCR negative (PCR neg) after immunologic purging have improved outcome compared with those patients in whom residual lymphoma cells were still detectable after purging (PCR pos). (Dafa from Gribben JG: Contemporaty techniques for the detection of minimal residual disease. In The Role of Tumor Purging in Autologous Transplantation. Deerfield, IL, Discovery International, 1997)
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den, histology, or remission status were not prognostic factors for purging efficiency or for patient outcome. Similar studies in patients with non-Hodgkin's lymphoma, acute lymphoblastic leukemia (ALL) and multiple myeloma have demonstrated that autograft tumor negativity is a good prognostic factor; again, however, these results do not conclusively prove that autograft tumor purging itself is important.16,~ 533, In a case-matched study of patients with non-Hodgkin's lymphoma enrolled in the EBMT registry, 270 patients receiving a purged bone marrow autograft were compared with 224 patients receiving an unpurged pr0duct.4~Overall survival was 54% for the patients receiving a purged autograft versus 48% for their case-matched controls (P = 0.18). Although there appeared to be a survival benefit from the purging of autografts derived from patients with low-grade disease, progressionfree survival was similar between groups. Results From the First Randomized Trial of Autograft Purging
Recently, a multi-institutional study was completed in which 193 patients with multiple myeloma were randomly assigned to receive an unmanipulated autograft or one purged of tumor cells by CD34 enrichment using the Ceprate SC device (CellPro, Inc., Bothell, WA). These patients were selected for study for two reasons. First, autologous transplantation has been proven beneficial for patients with multiple myeloma. Patients receiving high-dose therapy and autologous bone marrow transplantation have improved disease-free and overall survival rates compared with patients receiving standard chemotherapy4 Second, the malignant cells can be quantified and, thus, measured in the autografts before and after manipulation with the purging device. The rearranged immunoglobulin (Ig) gene in the myeloma cell can serve as a unique marker for the clonal population of cells in multiple myeloma patients.5,*sf 38 Unlike the Ig gene in patients with non-Hodgkin's lymphoma, the myeloma Ig gene does not undergo further somatic mutation, and therefore, no intraclonal Ig gene diversity exists. All of the multiple myeloma cells within a patient contain the same, unique, Ig gene sequence even years later when the disease recurs. The polymerase chain reaction (PCR) can therefore be used to measure the number of tumor cells within various cell populations, such as the autograft product. In this particular study, patients were eligible for enrollment if they had evidence of advanced multiple myeloma (Durie-Salmon stage I1 or stage 111disease or an elevated P2-microglobulin level) and had received no more than 6 months of chemotherapy. Cyclophosphamide, 2.5 g/m2,
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prednisone, 2 mg/kg/d x 4,and granulocyte colony-stimulating factor (G-CSF), 10 Fg/kg/d beginning on day 2, were given to mobilize sufficient stem cells for collection. Once stem cell collection began, the patients were randomly assigned to receive standard autologous PBSC collection or CD34 enrichment using the Ceprate SC device. All patients subsequently received high-dose chemotherapy with cyclophosphamide (120 mg/kg) and busulfan (14 mg/kg) followed by stem cell reinfusion. No posttransplant adjuvant therapy was allowed. Unfortunately, patient outcome remains immature, and no overall or disease-free survival benefits were demonstrable between arms at the most recent analysis with a median follow-up period of slightly more than 1 year (Fig. 2). Nevertheless, the study did meet its primary objectives: it demonstrated that the CD34 selection procedure is safe and results in a greater than 2-log reduction in autograft tumor burden.40 Only the initial 134 patients were eligible for tumor burden assessment; the final 59 patients were included to increase the power to detect 1.o
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Days Posttransplant Figure 2. Kaplan-Meierprobability of progression-freesurvival based on data as of January 1998 in the randomized phase 111 trial comparing CEPRATE SC enriched versus unselected peripheral blood progenitor cell transplantation in patients with multiple myeloma. Solid line = CD34 selected, dashed line = unselected. (From Vescio R, Schiller G,Stewart AK, et al: Multicenter phase 111 trial to evaluate CD34 + selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood 93:1858-1868, 1999; with permission.)
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differences in disease-free and survival outcome between groups. Using bone marrow collected immediately before mobilization chemotherapy, the myeloma Ig gene sequence was determined in 47 patients. Autograft tumor burden could then be determined in 44 of these patients using myeloma-specific Ig gene primers and quantitative PCR. The median level of tumor cells detected in the initial leukapheresis of patients who later received CD34-enriched autografts was 2.6 X lo6. In patients who received standard autologous PBSC autografts, it was 2.3 x lo6. Following CD34 enrichment, autograft tumor burden fell a median 3.1 logs, with most of these purged autografts containing no detectable tumor cells. Despite the greater than 1000-fold reduction in tumor burden, median time to neutrophil recovery (absolute neutrophil count [ANC] 2 500/p,L) was 12 days in each arm. Platelet recovery times were delayed by a median of 2 days for those receiving a purged product, although all patients achieved platelet transfusion independence by day 56. This delayed platelet recovery was attributed to a loss of approximately 40% of CD34 cells caused by the enrichment procedure. When patients receiving the threshold number of CD34 cells/kg deemed safe in the phase I1 study (2 x 106/kg) were compared on each arm, platelet recovery times were equivalent. Thus, autograft purging can be performed in a safe and effective manner. With these results, the Food and Drug Administration (FDA) approved use of the Ceprate SC device for autograft purging in treating multiple myeloma and other tumors that do not express the CD34 antigen. This broad approval may seem premature without demonstration of outcome efficacy, but the reasons behind this decision will be explained subsequently. ESTIMATING THE BENEFIT OF AUTOGRAFT PURGING Proof of Survival Benefit May Not Be Possible
Randomized trials comparing unmanipulated versus purged autografts for hematopoietic support are the optimal method for to determining the effectiveness and risk of the purging process. Even relatively large studies, however, may not be adequately powered to detect differences in survival that could be considered clinically relevant. A randomized phase I11 study would require enrollment of more than 1000 patients to have an 80% power (likelihood) to detect a 30% improvement in median survival time to a significance level of 0.05 (Table 1). This degree of improvement would certainly be clinically meaningful. Yet, a study of this magnitude could be performed only at significant cost and only for patients with the most common malignancies. Smaller gains in survival time could still be considered important
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Table 1. NUMBER OF PATIENTS REQUIRED PER GROUP FOR 80% POWER USING A SIGNIFICANCE LEVEL OF 0.05 (TWO-SIDED TEST)* _____~ ~~
~~
Anticipated improvement in Median Survival Time (%)
Number of Patients Required per Group
10 20 30 40 50
3888 1098 548
344 244
*Assumption of median survival time of 18 months in the group with poorer prognosis and 48 months follow-up
and perhaps more likely to be achievable by autograft purging. Such results would be impossible to prove, however, given the large patient numbers required. The previously described CellPro studyMenrolling 193 patients, has only a 50% chance of documenting a statistically significant survival advantage, even if the median survival times are, in reality, improved by 12 months in the purged arm. In an example provided by Appelbaum? the relapse rate following syngeneic transplantation for AML is 50% compared with 58% for similar patients undergoing unpurged autologous transplantation? This small difference suggests that a small increase in relapse rates may be caused by AML tumor reinfusion. Although trials designed to assess changes in the proportion of patients cured by a procedure require fewer enrolled patients than outlined in Table 1, a randomized study enrolling 800 patients would be required to prove statistical significance, assuming that the purging process could render the autografts completely tumorfree in every case, which is clearly an optimistic assumption. Even greater patient numbers would therefore be required to prove autograft purging efficacy for a device with a more realistic 3- to 4-log efficiency of tumor cell removal.* Also, because the need for and efficiency of tumor cell purging will differ among different tumor types, one could argue that similar large studies for each malignant subtype would be necessary to prove efficacy for each unique condition. For these reasons, proof of benefit is unlikely to be demonstrated and, in fact, is unnecessary in certain circumstances that may be applicable to autograft purging. Before any procedure or treatment becomes acceptable, a demonstration of improved benefit versus risk should be made. This equation is the basis of clinical decision making and should be used to evaluate this autograft purging procedure. Because the benefit may be difficult if not impossible to prove, the FDA apparently accepted proof of the safety of the procedure coupled with demonstrable tumor cell autograft reduction as being sufficient for approval of the Ceprate SC device.
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Thus, the theoretical benefit of reducing autograft tumor load sufficed because there was only a negligible detriment to patient safety (in this case, a 2-day prolongation in platelet recovery time). Similarly, small proven benefits, such as demonstrated prolonged disease-free survival following autograft purging, should be sufficient for procedure acceptance provided that the added toxicity of purging is negligible. As the toxicity of the purging process increases, however, the need to demonstrate a benefit becomes more important. Some of the chemical and complement-mediated purging procedures impede hematologic engraftment for 1 to 2 weeks,', 35 a delay that is likely to increase both procedure-related mortality and hospital charges. These more toxic procedures, which may be more efficient at removing tumor cells, will have to demonstrate an improvement in either disease-free or overall survival more clearly before becoming widely used. Tumor Cell Sensitivity Determines the Need for Autograft Purging
Even without proven studies, the diseases for which autograft tumor purging will be most effective can be postulated. The ratio of autograft tumor cell burden to the systemic tumor cell burden following high-dose chemotherapy is likely to determine the benefit of autograft purging (Fig. 3). If a large number of viable tumor cells remain after
Figure 3. The impact of autograft tumor Cell infusion following high-dose chemotherapy that achieves a 2-log (A) or 5-log (13)tumor-cell kill. Autograft purging will be helpful only if autograft tumor burden is substantially greater than the viable tumor cells that remain following high-dose therapy (B).
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high-dose chemotherapy, the impact of reinfusing the small numbers of cancer cells within the autograft will be minimal. Consequently, for chemorefractory diseases, autograft purging will be of little value. In contrast, for diseases such as leukemia or high-grade lymphomas, which can be very chemosensitive, it becomes more important to reduce the number of tumor cells reinfused in the patient, because the reinfused cells may be a significant proportion of the overall tumor bulk after high-dose therapy. In the optimal situation, the high-dose chemotherapy completely eradicates residual tumor cells; then the reinfusion of viable tumor cells within the autograft could be a dramatic and potentially the sole cause of the eventual disease relapse. The benefit of autograft purging therefore depends on tumor sensitivity which at present can only be estimated in a general manner. Although most investigators would agree that acute leukemias and highgrade lymphomas are more chemosensitive than the majority of solid tumors, the ability to quantify this difference remains elusive, particularly for an individual patient. Because the patient most likely to benefit from a tumor-free autograft is one who has a chemosensitive tumor with minimal tumor bulk at the time of transplantation, a mechanism for assessing the effect of the high-dose chemotherapy on these patients is needed. New methods of monitoring minimal residual disease should improve this ability. As an example, the authors have developed a quantitative PCR assay that is capable of identifying myeloma cells with a sensitivity of 1:720,000. This allowed the authors to evaluate myeloma tumor burden within the peripheral blood of patients before and after the high-dose chemotherapy in the phase I11 trial described previously.4o Myeloma cell numbers within the peripheral blood were calculated using the Poisson distribution-based PCR assay on samples obtained before mobilization chemotherapy with cyclophosphamide, prednisone, and G-CSF and on the first day of leukapheresis collection, 10 to 14 days later. Preliminary results suggest that those patients with an increased number of circulating tumor cells following the mobilization therapy with cyclophosphamide have a greater chance of early relapse after tran~plantation.~~ Because the patients receive transplantation after cyclophosphamide and busulfan conditioning, circulating tumor response to cyclophosphamide mobilization chemotherapy may serve as an in vivo drug sensitivity test. It is quite possible that autograft tumor purging will only show benefit for those myeloma patients with more chemosensitive disease as evidenced by a complete remission before transplantation or by response to mobilization chemotherapy. Further follow-up of the patients in the phase I11 trial may resolve this issue. Of course, general statements about disease sensitivity should be continually reevaluated. As the effectiveness of high-dose chemotherapy improves and more chemorefractory diseases become treatable, autograft purging will take on a more important role.
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Necessity for Purging Increases With Multiple Rounds of Intensive Therapy
Many recent trials have used multiple rounds of intensive therapy6 As the number of cycles of high-dose therapy increases, the reinfusion of malignant cells within collected autografts becomes more relevant, as does the importance of autograft purging. Autografts are typically obtained before the first of multiple high-dose regimens and are divided for administration after each cycle of therapy. If a substantial tumor burden exists within this initial autograft, the reinfusion of these cells may become the most significant determinant of relapse. An example can demonstrate this point (Fig. 4). If a patient with an initial tumor load of 1 x lo8 cells received an autograft that contained 1 X lo5 malignant cells and then received three rounds of high-dose therapy, each accomplishing a 3-log reduction in tumor burden, that patient would be left with 3 X lo4 tumor cells following the final high-dose procedure. In contrast, if the initial autograft were purged free of tumor, then the patient would be tumor-free after the final round of treatment. Although this model is simplistic, it illustrates the concept that the benefit of autograft purging increases with the number of high-dose chemotherapy cycles delivered, because in each scenario a similar tumor burden (1 to 2 x lo5 cells) would have existed after the initial high-dose therapy cycle.
Figure 4. Example of patient tumor burden following successive rounds of high-dose chemotherapy, each achieving a 3-log tumor cell kill. Initial patient tumor burden = 108 cells. Unmanipulated autograft tumor burden = lo5 cells. Purged autograft = tumor-free. HD = viable tumor burden immediately following the first, second, and third cycle of chemotherapy. Post 1 , 2, and 3 = the tumor burden following the infusion of the autograft after each of these cycles. Square = Unpurged, circle = purged.
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AUTOGRAFT PURGING RISKS Delayed Engraftment
The potential benefits of autograft purging may sometimes be offset by adverse effects. Engraftment delay is the most obvious risk related to autograft purging. Typically, median times to neutrophil counts higher than 500/p,L and platelet counts higher than 2 x 104/p,Lare used to estimate engraftment quality, but these median times may not be the best indicator of overall treatment toxicity. Even if median neutrophil engraftment time is increased from 12 to 14 days by autograft purging, this 2-day prolongation may not be clinically relevant, because it is unlikely to correlate with significant changes in procedural risk or expense. In contrast, the few patients with truly delayed or incomplete engraftment may be markedly affected by poor autograft quality. These patients may need prolonged transfusion support and may not be able to tolerate future chemotherapy when indicated at the time of disease recurrence. The added expense and risks for these patients will probably overshadow any adversity incurred by a 1- or 2-day shift in median engraftment times. Consequently, there is a need for a standardized measurement that better encompasses these outlying patients as an indicator of the risks of hematopoietic engraftment. As an example, a comparison of the percentage of patients still requiring hematological support at days 15, 30, and 60 may better indicate clinically relevant differences in delayed engraftment between treatment arms. Autograft Purging and Impaired Immunity
Other less apparent adverse outcomes could result from autograft purging. Most of the purging methods significantly reduce the numbers of B and T cells in the autograft. This reduction could theoretically impair cell-mediated immunity and lead to an increased incidence of infectious complications after transplantation. A recently completed study using highly purified CD34'Thy1 'Lin - autografts as hematopoietic support substantiated this None of the eight evaluable patients receiving the purged autograft had recovered an absolute CD4 count higher than lOO/p,L after 100 days, and several patients died of posttransplant infection. In the CellPro Phase III study, CD4 cell recovery was also delayed for those patients receiving a CEPRATE SC selected autograft. Median CD4 cell counts were 211/kL in the selected autograft versus 298/p,L in the unselected autograft." Nevertheless, this statistically significant difference did not correlate with a higher incidence of infections in the posttransplantation period (56% for each arm, days 0
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to 100 post transplant). It is likely that a threshold number of lymphoid or lymphoid-precursor cells are required to maintain adequate immunological function after transplantation. This same process could also impair tumor cell immunity. This potential risk will be difficult to quantify and will probably have a variable effect depending on the tumor type. Nevertheless, it is possible that autograft purging could negatively affect disease-free survival by disrupting cell-mediated tumor control. Finally, incubation of autografts with chemotherapeutic agents such as 4-HC may lead to increased rates of treatment-related leukemia or myelodysplastic syndromes caused by the exposure of stem cells to these toxic This potential risk will be difficult to define without long-term follow-up of large randomized trials.
ASSESSMENT OF MINIMAL RESIDUAL DISEASE Measuring High-Dose Therapy Tumor Cell Kill
The majority of patients with acute leukemia and non-Hodgkin's leukemia receive autologous transplantation when they are in clinical remission. The inability to quantify the amount of residual disease before and after the transplantation procedure makes it difficult to determine the effectiveness of the therapy for an individual patient. Generalized comparisons of phase I1 study results are often the basis for high-dose therapy protocol design and administration, given the paucity of phase I11 studies. Obviously, differences in patient characteristics and supportive care measures make such comparisons problematic. The ability to measure the logarithmic cell kill of tumor cell burden by the high-dose regimen would be a significant advance and could allow more rapid and accurate comparisons. Furthermore, in certain instances maintenance therapy is given following the transplantation procedure in an effort to reduce further the residual disease burden (e.g., a-interferon in patients with multiple myel~ma.)'~ The inability to measure viable tumor burden in clinical remission disease states makes clinical decision making difficult, particularly for patients experiencing moderate side effects from this adjuvant therapy. Recent techniques have been developed that permit the detection of small numbers of residual tumor cells in patients in clinical remission. Techniques using PCR or in situ hybridization can detect levels as low as 1 malignant cell within a background of 1 X lo6 normal cells. Although most published studies assessing residual disease after transplantation have used semiquantitative assays, new quantitative PCR protocols have been developed. These methods are tools that investiga-
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tors can use to calculate the reduction of tumor burden in blood and bone marrow following hgh-dose chemotherapy. Correlation with Outcome After Autologous Transplantation
The persistence of minimal residual disease in acute leukemia patients following treatment has been predictive of disease relapse.", 20, 24, 37 In a prospective analysis of children with ALL, the relapse rate was 8% in patients without detectable residual leukemia after initial induction therapy versus 40% for the remaining patients ( P < 0.001). Similar adverse outcomes were noted for patients with residual disease following consolidation and intensification therapy. These results imply that minimal residual disease can be used to identify patients who appear to be in clinical remission but are at risk for disease relapse. These patients may benefit from the early use of alternative treatment such as transplantation.'O In a study performed at the Dana-Farber Cancer Institute, 10 of 31 assessable patients had residual chronic lymphoblastic leukemia (CLL) based upon Ig gene PCR amplification following allogeneic or autoloIn 5 of these 10 patients, PCR-detectable disease gous tran~plantation.~~ resolved within 6 months, and these 5 patients remain in clinical remission. The remaining 5 patients with persistent PCR-detectable disease have all subsequently relapsed. Of the 21 patients without detectable disease following transplantation, only 1 patient has relapsed. These numbers were too small to demonstrate statistical significance, but clearly a strong relationship between persistently detectable disease and disease outcome exists. Similar results are apparent for non-Hodgkin's lymphoma patients following dose-intensive treatment. The majority of patients with detectable bcI-2 or Ig gene rearrangements immediately following transplantation are destined to relapse.12,45 It is of interest that the elimination of detectable tumor cells may take months, because many non-Hodgkin's lymphoma and ALL patients with PCR-detectable disease approximately 1 month after transplantation subsequently remained in clinical and molecular remission. Similar results were noted in the authors' study of multiple myeloma patients after transplantation. Circulating and bone marrow myeloma cell tumor burden continues to fall for 3 to 6 months following autologous transp l a n t a t i ~ n Unlike . ~ ~ patients with non-Hodgkin's lymphoma and ALL, the majority of multiple myeloma patients with detectable tumor before transplantation continue to have disease after transplantation that is measurable with sensitive PCR techniques. Because autologous transplantation is not curative in multiple myeloma, these findings are not
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surprising. Nevertheless, increases in circulating tumor burden from pretransplant levels predict disease progression?* Even blood samples collected at the time of patient discharge may reflect transplant efficacy. Of 33 evaluable patients, 4 patients had increased circulating myeloma cell burden on the day of engraftment, compared with levels before mobilization chemotherapy. All four patients developed progressive disease, three within only 6 months. In contrast, the relapse rate at 24 months was 48% for those patients whose circulating multiple myeloma cell concentration had decreased from pretransplant levels.
SUMMARY
Is a Theoretical Benefit Without Risk Enough to Justify Purging? Without a randomized trial any benefit of purging on patient survival will remain theoretical. Although molecular techniques may document that the autograft tumor burden is substantial when compared with the patient tumor load following conditioning therapy, a theoretical benefit may not be sufficient to persuade third-party payers to expend additional money for the purging procedure. For insurers, the equation to determine product usefulness does not consider risk as the only negative component, but a combination of risk and cost. When the amount that can be spent is limited, a demonstration that these additional costs will garner a true patient benefit becomes more important. Because it is difficult to assign a dollar amount for a theoretical benefit, third-party payors may be unwilling to accept the potential but unproven benefit from a purging procedure. Nevertheless, if the time to engraftment remains unchanged, the added costs of the purging procedure should be relatively minimal and, in the authors’ view, should not be a major impediment to its widespread use. Educated patients and physicians will probably demand that these procedures be performed in cases where autograft purging adds little if any risk and provides at least a significant potential for benefit.
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purging in patients with bcr-abl-positive acute lymphoblastic leukemia. Bone Marrow Transplant 18:541-548, 1996 4. Attal M, Harousseau JL, Stoppa AM, et al: A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma patients. N Engl J Med 335:91, 1996 5. Bakkus MH, Heirman C, Van Riet I, et al: Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 80:232&2335, 1992 6. Barlogie B, Jagannath S, Vesole DH, et al: Superiority of tandem autologous transplantation over standard therapy for previously untreated multiple myeloma. Blood 89~789-793,1997 7. Bird JM, Luger S, Mangan P, et al: 4-Hydroperoxycyclophosphamide purged autologous bone marrow transplantation in non-Hodgkin’s lymphoma patients at high risk of bone marrow involvement. Bone Marrow Transplant 18:309-313, 1996 8. Brenner MK, Rill DR, Moen RC, et al: Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Lancet 341235436, 1993 9. Butturini A, Gale Rp: Clinical models of autotransplants in chronic myelogenous leukemia. Leuk Lymphoma ll(Supp1 1):255-257, 1993 10. CavC H, van der Werff ten Bosch J, Suciu S, et al: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. N Engl J Med 339:591598, 1998 11. Ciudad J, San Miguel JF, Lopez-Berges MC, et al: Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol 163774-3781, 1998 12. Corradini P, Astolfi M, Cherasco C, et al: Molecular monitoring of minimal residual disease in follicular and mantle cell non-Hodgkin’s lymphomas treated with high-dose chemotherapy and peripheral blood progenitor cell autografts. Blood 89:724-731, 1997 13. Cunningham D, Powles R, Malpas J, et al: A randomized trial of maintenance interferon following high-dose chemotherapy in multiple myeloma: Long-term follow-up results. Br J Haematol 102495-502, 1998 14. Douay L Negative selection and protection of normal progenitor cells for autografting. Bone Marrow Transplant 22423-430,1998 15. Gertz MA, Witzig TE, Pineda AA, et al: Monoclonal plasma cells in the blood stem cell harvest from patients with multiple myeloma are associated with shortened relapse-free survival after transplantation. Bone Marrow Transplant 19:337-342, 1997 16. Gorin NC, Aegerter P, Auvert B, et al: Autologous bone marrow transplantation for acute myelocytic leukemia in first remission: A European survey of the role of marrow purging. Blood 75:1606-1614, 1990 17. Gorin-NC, Labopin M, Meloni G, et al: Autologous bone marrow transplantation for acute myeloblastic leukemia in Europe: Further evidence of the role of marrow purging by mafosfamide. Leukemia 5:896, 1991 18. Gribben JG: Contemporary techniques for the detection of minimal residual disease. In Gribben J (ed):The Role of Tumor Purging in Autologous Transplantation. Deerfield, IL, Discovery International, 1997 19. Gribben JG, Freedman AS, Neuberg D, et al: Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N Engl J Med 325:1525-1533,1991 20. Gruhn B, Hongeng S, Yi H, et al: Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome. Leukemia 12675-681, 1998 21. Gupta D, Bybee A, Cooke F, et al: CD34 + -selected peripheral blood progenitor cell transplantation in patients with multiple myeloma: Tumor contamination and outcome. Br J Haematol 104166-177, 1999 22. Leonard BM, Hetu F, Busque L, et al: Lymphoma cell burden in progenitor cell grafts measured by competitive polymerase chain reaction: Less than one log difference between bone marrow and peripheral blood sources. Blood 91:331-339, 1998 23. McCann JC, Kanteti R, Shilepsky B, et al: High degree of occult tumor contamination in bone marrow and peripheral blood stem cells of patients undergoing autologous
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transplantation for non-Hodgkin's lymphoma. Biol Blood Marrow Transplant 2:3743, 1996 Meloni G, Diverio D, Vignetti M, et a1 Autologous bone marrow transplantation for acute promyelocytic leukemia in second remission: Prognostic relevance of pretransplant minimal residual disease assessment by reverse-transcription polymerase chain reaction of the PML/RAFh fusion gene. Blood 90:1321-1325, 1997 Mizuta S, Kohno A, Tanimoto M, et a1 Quantitative molecular analysis of the efficacy of ex vivo purging predicts clinical outcome after autologous BMT in patients with Blineage acute lymphoblastic leukemia. Blood 88(Suppl 1):73a, 1996 Moss TJ, Cairo M, Santana VM, et al: Clonogenicity of circulating neuroblastoma cells: Implications regarding peripheral blood stem cell transplantation. Blood 83:3085 3089, 1994 Provan D, Bartlett-Pandite L, Zwicky C, et al: Eradication of polymerase chain reactiondetectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation. Blood 88:222€&2235,1996 Ralph QM, Brisco MJ, Joshua DE, et a1 Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new B-cell clone: Evidence from the Ig heavy chain gene. Blood 82:202-206, 1993 Rill DR, Santana VM, Roberts WM, et a1 Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 84:380-383,1994 Rizzoli V, Mangoli L, Carlo-Stella C: Autologous bone marrow transplantation in acute myelogenous leukemia. Leukemia 61101-1106, 1992 Ross AM, Cooper BW, Lazarus HM, et a1 Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques. Blood 822605-2610, 1993 Rowley SD, Jones RJ, Piantadosi S, et al: Efficacy of ex vivo purging for autologous bone marrow transplantation in the treatment of acute nonlymphoblastic leukemia. Blood 74~501-506, 1989 Sharp JG, Kessinger A, Mann S, et al: Outcome of high-dose therapy and autologous transplantation in non-Hodgkin's lymphoma based on the presence of tumor in the marrow or infused hematopoietic harvest. J Clin Oncol 14214-219, 1996 Siege1 DS, Jagannath S, Desikan KR, et al: Feasibility of high dose chemotherapy with peripheral blood stem cell support for multiple myeloma patients over the age of 70. Blood 88(Suppl 1):130a, 1996 Treleaven J, Powles R, Singhal S, et al: Autografting with CD52 monoclonal antibodypurged marrow for acute lymphoblastic leukemia. Blood 88(Suppl 1):225a, 1996 Tricot G, Gazitt Y, Leemhuis T, et a1 Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma. Blood 91:4489-4495, 1998 van Dongen JJ, Seriu T, Panzer-Grumayer ER, et a1 Prognostic value of minimal residual disease in acute lymphoblastic leukemia in childhood. Lancet 352:1731-1738, 1998 Vescio RA, Cao J, Hong CH, et al: Myeloma Ig heavy chain V region sequences reveal prior antigeneic selection and marked somatic mutation but no intraclonal diversity. J IITUINUIO~ 155:2487-2497, 1995 Vescio RA, Han EJ, Schiller GJ, et a1 Quantitative comparison of multiple myeloma tumor contamination in bone marrow harvest and leukapharesis autografts. Bone Marrow Transplant 18:10>110, 1996 Vescio R, Schiller G, Stewart AK, et a1 Multicenter phase I11 trial to evaluate CD34+ selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood 93:1858-1868, 1999 Vescio RA, Schiller GJ, Stewart AK, et a1 Quantitative assessment of myeloma peripheral blood and bone marrow tumor burden in patients undergoing autologous transplantation. Blood 88(Suppl 1):641a, 1996 Vescio RA, Stewart AK, Ballester 0,et a1 Circulating multiple myeloma cell burden predicts disease relapse following autologous transplantation. Blood 92(Suppl 1):97a, 1998
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43. Williams CD, Goldstone AH, Pearce RM, et al: Purging of bone marrow in autologous bone marrow transplantation for non-Hodgkin’s lymphoma: A case-matched comparison with unpurged cases by the European Blood and Marrow Transplant Lymphoma Registry. J Clinical Oncol 142454-2464, 1996 44. Witzig TE, Gertz MA, Pineda AA et al: Detection of monoclonal plasma cells in the peripheral blood stem cell harvests of patients with multiple myeloma. Br J Haematol 89640-612, 1995 45. Zwicky CS, Maddocks AB, Andersen N, et a1 Eradication of polymerase chain reaction detectable immunoglobulin gene rearrangement in non-Hodgkin’s lymphoma is associated with decreased relapse after autologous bone marrow transplantation. Blood 8833314-3322, 1996 Address reprint requests to
Robert Vescio, MD DVA West Los Angeles Bldg 500 Room 4237 (111H) 11301 Wilshire Boulevard Los Angeles, CA 90073