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Background We developed a test to discern small numbers of residual leukemic progenitor cells in the bone marrow of patients with acute lymphoblastic leukemia (ALL) in remission. Preliminary studies revealed that before undergoing bone marrow transplantation such patients differed in their burden of leukemic progenitor cells. These observations suggested that the burden of these cells might influence the risk of relapse after transplantation.
Methods The number of residual leukemic progenitor cells before bone marrow transplantation was determined for 83 patients with high-risk ALL. We combined multiparameter flow cytometry and cell sorting with assays for leukemic progenitor cells in a quantitative method for the detection of minimal residual disease.
Results The count of leukemic progenitor cells in bone marrow specimens from patients in remission varied markedly between patients, ranging from 0 to 12,546 cells per million mononuclear cells, or from 0 to 1.255 percent (median, 51 leukemic progenitor cells per million mononuclear cells, or 0.005 percent). Patients whose count of leukemic progenitor cells exceeded the median value had a higher likelihood of relapse than did patients with values below the median (relapse rate at one year, 100 percent vs. 41 percent; P<0.001). There was a statistically significant inverse relation between the leukemic progenitor-cell content of bone marrow before transplantation and the duration of remission after transplantation (P<0.001). The estimated risk of relapse for patients with
Conclusions The count of residual leukemic progenitor cells is a powerful predictor of relapse after autologous bone marrow transplantation, particularly among male patients. Its measurement may be useful for analyzing and improving the treatment of patients with high-risk ALL in remission.
51 leukemic progenitor cells per million mononuclear cells was more than 3.5 times the risk for patients with lower counts, after adjustment for the effects of other covariates (P = 0.005).
Methods
Study Patients and Transplantation Protocol
Eighty-three patients with high-risk ALL in complete remission were enrolled in a clinical study of autologous bone marrow transplantation in a single institution. The study protocol was approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota, and written informed consent was obtained from all patients or their legal guardians according to the guidelines of the Department of Health and Human Services. The characteristics of the patients are shown in Table 1. Bone marrow was obtained with the patients under general anesthesia by multiple aspirations from the iliac crests in sufficient amounts to yield a minimum of 4.5 x 108 nucleated cells per kilogram of body weight. Autografts from patients with B-lineage ALL were purged with either BA-1,2,3 monoclonal antibodies plus complement in combination with 4-hydroperoxycyclophosphamide (4-HC) (42 patients) or B43-pokeweed antiviral protein immunotoxin plus 4-HC (22 patients), according to previously published protocols6,7. Autografts from patients with T-lineage ALL were purged with anti-CD5 plus anti-CD7 ricin immunotoxins plus 4-HC (19 patients), according to previously published protocols5. Three consecutive conditioning regimens were used before bone marrow transplantation (Table 1), the details of which have been published elsewhere5,6,7,8.
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We used a quantitative assay system to detect minimal residual disease in patients with ALL. The system combines flow cytometry and cell sorting with assays of leukemic progenitor-cell colonies to measure the residual burden of leukemia in patients with ALL in remission. A detailed description of the assay system and its ability to detect residual clonogenic blasts in bone marrow samples from patients with high-risk ALL in remission has been published elsewhere5,6.
Statistical Analysis
The data were analyzed by standard statistical methods with the BMDP-90 software program (University of California Press, Berkeley). The probability of relapse was estimated by the Kaplan-Meier product-limit method9. Univariate analyses with the Mantel-Cox log-rank test10,11 were conducted to determine the relation between the probability of relapse after bone marrow transplantation and the characteristics of the patient. When appropriate, continuous covariates, such as the white-cell count at diagnosis and the number of leukemic progenitor cells per million mononuclear cells in bone marrow before transplantation, were analyzed, both with Cox regression models (with the risk factors treated as continuous variables) and with Kaplan-Meier analysis based on dichotomous groupings of covariates. Given the wide variation in white-cell counts at diagnosis and in leukemic progenitor cells, natural logs were applied to these covariates before their evaluation in the univariate Cox regression analyses. Multivariate analysis of relapse with known or suspected covariates was performed with the Cox proportional-hazards model.
Results
Residual Leukemic Progenitor Cells before Transplantation
The leukemic progenitor-cell content of the bone marrow specimens obtained during remission varied markedly between patients, ranging from 0 to 12,546 per million mononuclear cells (0 to 1.255 percent; median, 51 leukemic progenitor cells per million mononuclear cells, or 0.005 percent) (Table 1). Younger patients (P = 0.07) and those in their second or a subsequent remission (P = 0.02) tended to have more residual leukemic progenitor cells in their bone marrow (Table 2). Six of seven patients with very high burdens (exceeding 1000 leukemic progenitor cells per million mononuclear cells) were less than 18 years of age, and all seven were in their second or a subsequent remission. Among patients with two or more remissions, there was a trend toward a higher count of leukemic progenitor cells if the first remission had lasted less than 18 months (P = 0.11). By comparison, the patient's sex, previous extramedullary disease, white-cell count at diagnosis, immunophenotype, or percentage of lymphoblasts in bone marrow did not correlate with the number of leukemic progenitor cells (Table 2).
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Fifty-eight patients (70 percent) relapsed 27 days to 25 months after bone marrow transplantation. Among these patients, the median time to relapse was 106 days. As shown in Figure 1A, the Kaplan-Meier estimates of the probabilities of relapse were 72 percent (95 percent confidence interval, 61 to 83 percent) at one year and 83 percent (95 percent confidence interval, 73 to 93 percent) at two years after bone marrow transplantation. Fifteen patients (18 percent) died of complications with no evidence of leukemia 15 to 980 days (median, 72) after bone marrow transplantation. Ten patients remained alive without evidence of leukemia 11 to 54 months (median, 40) after transplantation.
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As shown in Table 3, female patients relapsed sooner and more frequently than male patients (P = 0.001). Patients in first remission at the time of transplantation were significantly less likely to have a relapse than those in a second or subsequent remission (P = 0.02) (Table 3). Among the 72 patients in a second or subsequent remission at the time of bone marrow transplantation, those whose first remissions had lasted less than 18 months were more likely to relapse than those whose first remissions had been longer (P = 0.02). Patients whose white-cell count at diagnosis was 50,000 per cubic millimeter were significantly more likely to relapse than those with a lower count (P = 0.01) (Table 3). Patients whose count of leukemic progenitor cells exceeded the median value of 51 per million mononuclear cells had a higher likelihood of relapse at one year than patients with lower values (100 percent vs. 41 percent, P<0.001) (Table 3). Regression analysis using the residual burden of leukemic progenitor cells as a continuous variable also showed a statistically significant inverse relation between the count of leukemic progenitor cells in bone marrow before transplantation and the duration of remission after transplantation (P<0.001). There were no statistically significant differences in regard to relapse between groups defined according to the other risk factors examined in this study.
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The patients were subsequently divided into three groups according to the number of leukemic progenitor cells in their bone marrow. As shown in Figure 2, those with 200 leukemic progenitor cells per million mononuclear cells (28 patients) had the highest probability of relapse, those with 10 to 199 cells per million mononuclear cells (29 patients) had a lower probability of relapse, and those with fewer than 10 cells per million mononuclear cells (26 patients) had the lowest probability of relapse. Seventeen patients had no leukemic progenitor cells in their bone marrow. Among these patients, the relapse rate at one year was only 30 percent.
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Cox proportional-hazards regression analyses were conducted for further evaluation of the role of the leukemic progenitor-cell count as a predictor of relapse after bone marrow transplantation. First, regression models were constructed to assess the interactions between the leukemic progenitor-cell burden and the other covariates. No significant interactions were found between the burden and either the number of remissions (one vs. two or more, P = 0.69) or the white-cell count at diagnosis (<50,000 vs. 50,000 per cubic millimeter, P = 0.76). The interaction between the burden of leukemic progenitor cells and the length of the first remission (<18 vs. 18 months) approached statistical significance (P = 0.07). However, the interaction between the burden of leukemic progenitor cells and the sex of the patient was statistically significant (P = 0.02). Female patients were likely to have a relapse regardless of their burden of leukemic progenitor cells, whereas male patients were more likely to remain in remission when they had a low burden (data not shown).
Two Cox regression models were evaluated (Table 4). In the first model, which includes patients in first remission, all covariates except the number of remissions were significantly related to the risk of relapse, and a residual burden of leukemic progenitor cells in bone marrow was found to be the overwhelming predictor of relapse after adjustment for the effects of other predictors. The estimated risk of relapse for patients with 51 leukemic progenitor cells per million mononuclear cells was more than 3.5 times the risk for patients with lower counts, even after adjustment for the effects of other covariates (P = 0.005).
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Discussion
We compared the count of residual leukemic progenitor cells in bone marrow samples obtained during remission before bone marrow transplantation from 83 high-risk patients with ALL, using a quantitative assay system to detect minimal residual disease5,6. A high count of leukemic progenitor cells in bone marrow obtained before transplantation was a strong predictor of relapse after transplantation. Multivariate analysis of potentially confounding covariates with the Cox proportional-hazards model established the count of residual leukemic progenitor cells as an important predictor of relapse after autologous bone marrow transplantation.
Early and frequent relapses during the first year after transplantation6,8 demonstrate that residual leukemic progenitor cells in high-risk patients with ALL in remission can avoid the lethal effects of intensive conditioning regimens used before transplantation. Possible explanations for the failure of conditioning regimens currently used to eradicate the residual burden of leukemia include intrinsic resistance to radiation, drug resistance associated with leukemic progenitor cells, or both; a high percentage of dormant blasts among leukemic progenitor cells; and rapid self-renewal and repopulation of leukemic progenitor cells in the bone marrow after transplantation. Until more effective conditioning regimens are developed, a high burden of leukemic progenitor cells during remission before transplantation is likely to result in a substantial proportion of cells unharmed by radiochemotherapy, leading to early and frequent relapses. The fact that there is an inverse relation between the burden of leukemic progenitor cells before bone marrow transplantation and the length of remission after transplantation suggests that the determination of minimal residual disease based on this relation could be very useful as a guide to treatment. For example, it might allow one to decide when the residual burden of leukemic progenitor cells in high-risk patients with ALL is sufficiently small that one might expect to be successful in the use of high-dose chemotherapy or radiochemotherapy as conditioning for patients awaiting transplantation.
An unexpected finding of the present analysis was the interaction between the burden of leukemic progenitor cells and the patient's sex. Female patients were at high risk for relapse even when they had minimal burdens, whereas male patients with minimal burdens were more likely to remain in remission. These results indicate that the residual leukemia burden of female patients may be particularly resistant to radiochemotherapy. Female sex was a significant predictor of relapse even after adjustment for other potentially confounding prognostic factors. The underlying cause of the influence of sex on the probability of relapse after transplantation remains unclear.
In the present study, 44 of 53 patients with autografts who were evaluated after purging by the assay for minimal residual disease had no residual leukemic progenitor cells. These patients had a very high relapse rate, indicating that the association between a high count of leukemic progenitor cells in the autografts obtained before purging, a measure of the in vivo burden of such cells in the patients, and the outcome after bone marrow transplantation was not the result of a reinfusion of autografts contaminated with higher numbers of residual leukemic progenitor cells.
In comparison with cytogenetic or molecular genetic analyses, the assay for minimal residual disease does not require the presence of clonal chromosomal abnormalities or probes specific to a particular clone; this feature may allow routine analysis of minimal residual disease in bone marrow samples obtained from patients with ALL in remission. A search for residual leukemic progenitor cells in such samples using this assay may yield more biologically relevant information about the quality of remission than qualitative analyses that do not discriminate between ALL blasts of differing clonogenic or proliferative potential, such as PCR amplification of clonotypic DNA sequences. Perhaps the combined use of an assay for minimal residual disease and a highly sensitive PCR method will ultimately yield the most reliable and biologically important information about the quality of remission in patients with B-lineage ALL.
Supported in part by grants (CA 21737, CA 42633, CA 42111) from the National Institutes of Health, by the Children's Cancer Research Fund, and by the Bone Marrow Transplant Research Fund. Dr. Uckun is a Scholar of the Leukemia Society of America. Dr. Kersey is the recipient of an Outstanding Investigator Grant (CA 41729) from the National Cancer Institute.
Source Information
From the Bone Marrow Transplantation Program (F.M.U., J.H.K., R.H., D.W., M.E.N., N.K.C.R.) and the Departments of Therapeutic Radiology-Radiation Oncology (F.M.U., M.E.N.), Pediatrics (F.M.U., J.H.K., M.E.N., N.K.C.R.), and Medicine (D.W.), University of Minnesota Health Sciences Center, Minneapolis.
Address reprint requests to Dr. Uckun at the University of Minnesota Hospital, Box 356 UMHC, 420 Delaware St. S.E., Minneapolis, MN 55455.
References
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