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Paucity of Leukemic Progenitor Cells in the Bone Marrow of Pediatric B-Lineage Acute Lymphoblastic Leukemia Patients with an Isolated Extramedullary First Relapse¹

ALL Biology Reference Laboratory, Children’s Cancer Group, Parker Hughes Cancer Center, Hughes Institute, St. Paul, Minnesota 55113 [F. M. U., M. B. S.]; Department of Hematology-Oncology, Children’s Hospital, Los Angeles, California 90027 [P. S. G.]; Department of Preventive Medicine, University of Southern California, Los Angeles, California 90033 [D. O. S.]; Children’s Cancer Group, Arcadia, California 91066 [D. O. S., M. G. S.]; Department of Pediatric Hematology/Oncology, Methodist Children’s Hospital of South Texas, San Antonio, Texas 78229 [K. H. L.]; and Princess Margaret Hospital, Perth, Western Australia, Australia 6001 [M. W.]

	ABSTRACT

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Isolated extramedullary relapse in childhood acute lymphoblastic leukemia (ALL) may be accompanied by occult bone marrow disease. We used a highly sensitive assay to quantify leukemic progenitor cells (LPCs) in the bone marrow of such patients. Multiparameter flow cytometry and blast colony assays were used to detect LPCs in the bone marrow of 31 pediatric B-lineage ALL patients with an isolated extramedullary first relapse. Sites of relapse were central nervous system (22 patients), testes (7 patients), and eye (2 patients). Bone marrow (BM) LPC counts ranged from 0/10⁶ mononuclear cells (MNCs) to 356/10⁶ MNCs (mean ± SE, 27.8 ± 13.1/10⁶ MNCs). LPCs were undetectable in 19 patients (61%). The BM LPC burden at the time of extramedullary relapse was similar, regardless of site (Wilcoxon P = 0.77) or time of relapse (Wilcoxon P = 0.80). Compared with higher risk, standard risk at initial diagnosis showed a trend for increased BM LPC burden (mean ± SE, 44.6 ± 17.1 versus 7.5 ± 3.3; Wilcoxon P = 0.22). After successful postrelapse induction chemotherapy, LPC counts in 21 evaluated patients ranged from 0/10⁶ to 175/10⁶ MNCs (mean ± SE, 15.9 ± 9.6/10⁶ MNCs). By comparison, LPC burden was higher after successful induction chemotherapy among children with an early BM relapse (range, 0 to 3262/10⁶ MNC; mean ± SE, 166 ± 107; Wilcoxon P = 0.11). Thus, not all patients with an extramedullary relapse have occult systemic failure with substantial involvement of the bone marrow, and after reinduction therapy, LPC counts were lower in these patients than in patients treated for an overt BM first relapse.

	INTRODUCTION

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Among children with ALL³ who were treated on contemporary intensive treatment programs of the Children’s Cancer Group, isolated extramedullary relapses in the CNS and testes occurred in 5% of all patients and 3% of boys, respectively (CCG).⁴ Similarly, rates of CNS relapse between 3% and 9% have been reported for recent trials (1981 through 1990) conducted by other groups (1, 2, 3, 4, 5, 6, 7) . The overall survival rate after an extramedullary relapse may depend on a number of factors. Survival after an isolated first testicular relapse appears to be better than survival after an isolated CNS relapse (8, 9, 10) , and survival after an isolated CNS relapse may depend both on prior systemic treatment and prior treatment administered to the CNS. For example, Gelber et al. (11) reported very poor outcomes for patients treated both initially and after relapse with cranial radiation, whereas other groups have reported survival rates approaching 50% or better for patients who received cranial radiation during salvage therapy, but not during initial therapy, or for patients who received radiation dose reductions after relapse based on their prior radiation exposure (9 , 12, 13, 14, 15) . Gaynon et al. (8) observed a 48% survival rate after CNS relapse among patients treated between 1983 and 1989 on the intensive, risk-adjusted CCG-100 series of studies. Ultimate failure in patients who experience an isolated CNS relapse is primarily due to a subsequent relapse, and these secondary relapses often occur in the BM (14 , 16 , 17) . This observation has motivated the hypothesis that at the time of isolated extramedullary first relapse, occult leukemic cells must have been present in the BM that appeared by standard morphological criteria to be free of disease (18) .

The detection of occult, or minimal residual, leukemia in remission BM specimens is a major focus in current leukemia research. Numerous methods, including the PCR (19, 20, 21) , multiparameter flow cytometry (22) , and fluorescent in situ hybridization (23) , are being tested for their abilities to predict relapse based on the quantification of MRD during complete remission (24) . Preliminary data from several small studies using PCR-based methods have suggested that residual leukemic cells were detectable in the BM of patients who experienced an isolated extramedullary relapse (25, 26, 27, 28, 29) .

We have developed a quantitative test that combines multiparameter flow cytometry, cell sorting, and blast colony assays (30 , 31) to discern small numbers of residual LPCs) in the BM of patients with ALL in remission (32) . Previously, we used this assay to compare the residual LPC burden in BM samples obtained from 83 high-risk ALL patients undergoing autologous BMT in remission (33) . In these patients, the LPC counts varied widely, ranging from 0 to 12546 LPCs/10⁶ MNCs or 0 to 1.3% (median, 51 LPCs/10⁶ MNCs or 0.005%). All patients whose LPC counts exceeded the median value of 51/10⁶ MNCs relapsed within 1 year, whereas only 41% of patients with lower LPC counts relapsed during the same time period (33) . The relative risk of relapse for patients with 51 LPCs/10⁶ MNCs was 3.7-fold higher than that of patients with lower LPC counts, after adjustment for the effects of other covariates (33) .

The inverse relationship we observed between the burden of LPCs before BMT and the length of remission after BMT suggested that the determination of LPC count in remission BMs could be useful as a guide to treatment allocation. Furthermore, this assay allows routine analysis of MRD burden in BM samples obtained from all patients with ALL in remission, because it does not require the presence of clonal chromosomal abnormalities or probes specific to a particular leukemic clone. These observations motivated the present study, in which we have used our assay system to determine whether LPCs were present in the BM of B-lineage ALL patients who experienced an isolated extramedullary first relapse after chemotherapy on frontline protocols.

	MATERIALS AND METHODS

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Patients.
This analysis involves 31 children with B-lineage ALL who experienced an isolated CNS, testicular, or ocular relapse as a first event and were enrolled between April 1997 and September 1998 on the CCG-1951 protocol. Patients with a combined CNS and ocular relapse also were eligible. For comparison, we also have included data from 100 children who experienced an early BM relapse and were treated on the CCG-1941 protocol between September 1995 and May 1998. Initial diagnosis of ALL was based on morphological, biochemical, and immunological features of the leukemic cells, including lymphoblast morphology on Wright-Giemsa-stained BM smears, negative staining for myeloperoxidase, and cell surface expression of two or more lymphoid differentiation antigens, as described previously (34 , 35) . In all cases, patients were classified as B-lineage ALL based on positivity for CD19 and/or CD24 in 30% of the leukemic cells and negativity for one or more of the T-cell-associated antigens CD2, CD3, CD5, or CD7. The CCG-1941 and 1951 protocols were approved by the National Cancer Institute and the Institutional Review Boards of the participating CCG-affiliated institutions. Informed consent was obtained from parents, patients, or both, as deemed appropriate, according to Department of Health and Human Services guidelines.

Treatment.
Prior to first isolated extramedullary relapse, patients were treated on either CCG risk-adjusted ALL protocols (n = 19) or on other frontline treatment programs (n = 12). Among patients treated on CCG studies, those with standard risk by National Cancer Institute criteria (2–9 years of age with WBC <50,000/µl; Ref. 36 ) were enrolled on CCG-1922 (37) or CCG-1952 and received systemic therapy based on previous low- and intermediate-risk ALL protocols (38 , 39) and intrathecal therapy without cranial radiation for presymptomatic treatment of the CNS. Higher risk patients (10 years of age and/or with WBC 50,000/µl; Ref. 36 ) with a rapid response to initial therapy received CCG intensive systemic therapy based on Nachman et al. (40) and intrathecal therapy alone; higher risk patients with a slow response to initial therapy received CCG-augmented intensive systemic therapy based on Nachman et al. (41) with both intrathecal drugs and cranial radiation therapy for CNS treatment. Infants (<12 months of age at initial diagnosis) received CCG/Pediatric Oncology Group intensive therapy without cranial radiation therapy (42) .

Treatment after relapse included a 5-week reinduction with etoposide, ifosfamide, dexamethasone, vincristine, L-asparaginase, i.v. methotrexate, and intrathecal triple therapy consisting of methotrexate, hydrocortisone, and 1-ß-D-arabinofuranosylcytosine. Additional details of therapy will be reported elsewhere. LPC determinations for this analysis were performed on specimens obtained at the time of relapse and at the end of postrelapse reinduction therapy.

Quantification of LPCs.
A detailed description of the assay system that combines multiparameter FACS and blast colony assays (30 , 31) and its validated ability to detect residual clonogenic blasts in BM samples from patients with ALL in remission has been published previously (32 , 33) . Briefly, CD19⁺/surface(s) IgM^- B-cell precursors were sorted using a FACS Vantage instrument (Becton Dickinson) at 1000–2000 cells/s from Ficoll-Hypaque-purified BM MNCs using two-color multiparameter flow cytometry with fluorescently labeled monoclonal antibodies directed against CD19 and sIgM (34) . The FACS Vantage instrument is equipped with an air-cooled argon laser (488 nm at 50mW). The anti-CD19 antibody was phycoerythrin-labeled B43 (33) and the anti-sIgM antibody was goat anti-human IgM µ chain (Biosource International, Camarillo, CA). The LPC fraction of these CD19⁺/sIgM^- precursors was quantified by counting the number of blast colonies that formed after 7 days of culture in conditions optimized for growth of B-lineage ALL cells (30 , 31 , 43) . Data are expressed as the number LPCs per 10⁶ MNCs isolated from the BM.

Statistical Methods.
Correlations of BM leukemic cell precursor burden with sites of relapse and other prognostic factors were performed using the method of Wilcoxon (44) . P < 0.05 was considered to be significant; P between 0.05 and 0.10 was considered to be of borderline significance.

	RESULTS

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Patient Characteristics and Timing and Sites of Relapse.
Clinical characteristics and relapse times for the 31 pediatric B-lineage ALL patients with an isolated extramedullary relapse are shown in Table 1 . Twenty-two patients relapsed in the CNS, 7 relapsed in the testes, and 2 relapsed in the eye. In 13 cases (CNS, 11; testicular, 1; ocular, 1), the relapse occurred early, i.e., <18 months (range, 4–17.6 months; median, 9 months) after first complete remission. In the remaining 18 patients, the relapse occurred late, i.e., 18 months after first complete remission (range, 18–112 months; median, 37 months). The majority of the patients were white (n = 24) and male (n = 18). By NCI risk classification criteria (36) , 17 patients had standard risk ALL (age, 1–9 years; white cell count, <50,000/µl), and 14 patients had high-risk ALL (age 10 years and/or white cell count 50,000/µl) at the time of their initial diagnosis.

LPC burden at relapse for the 22 patients with a CNS relapse ranged from 0 to 356/10⁶ MNCs (median, 0/10⁶ MNCs; mean ± SE, 26.5 ± 14.4/10⁶ MNCs); 8 of these patients had detectable LPCs, whereas 14 lacked detectable LPCs. LPC burden at relapse for the seven patients with a testicular relapse ranged from 0 to 164/10⁶ MNCs (median, 0/10⁶ MNCs; mean ± SE, 34.6 ± 11.2/10⁶ MNCs); three of these patients had detectable LPCs, and four lacked detectable LPCs. Of the two patients with an ocular relapse, one had an LPC burden at relapse of 37/10⁶ MNCs, and the other had undetectable LPCs. Comparison of these data showed no significant difference for LPC burden in the BM of patients who relapsed in either the CNS or testes or eye (Wilcoxon P = 0.77; Table 2 ).

LPC burden at relapse for the 17 patients classified as standard risk by NCI criteria (age, 1–9 years, and white cell count <50,000/µl) ranged from 0 to 356/10⁶ MNCs (median, 0/10⁶ MNCs; mean ± SE, 44.6 ± 17.1/10⁶ MNCs); 8 of these patients had detectable LPC, and 9 lacked detectable LPCs. LPC burden at relapse for the 14 patients classified as NCI high risk (age 10 years and white cell count 50,000/µl) was lower, ranging from 0 to 67/10⁶ MNCs (median, 0/10⁶ MNCs; mean ± SE, 7.5 ± 3.3/10⁶ MNCs); 3 of these patients had detectable LPCs, and 11 lacked detectable LPCs, but the comparison did not reach statistical significance (Wilcoxon P = 0.22; Table 2 ).

We also compared LPC data of the patients with an isolated extramedullary first relapse with LPC data obtained from 100 patients who experienced an early BM first relapse and were treated on the CCG-1941 protocol. At the time of relapse, LPC counts among these 100 patients ranged from 0/10⁶ MNCs to 47249/10⁶ MNCs (mean ± SE, 3356 ± 576/10⁶ MNCs; median, 1229/10⁶ MNCs), which was significantly higher than in the patients with an isolated extramedullary relapse (Wilcoxon P = 0.0001; Table 2 ). LPC data after postrelapse induction chemotherapy was available for 21 patients with an isolated extramedullary first relapse and 41 patients with an early BM relapse, all of whom achieved remission. Among the 21 patients with an isolated extramedullary relapse, LPC counts ranged from 0 to 175/10⁶ MNCs (mean ± SE, 15.9 ± 9.6/10⁶; median, 0/10⁶ MNCs); only 7 of the 21 patients had detectable LPCs. LPC counts were higher, ranging from 0/10⁶ MNCs to 3262/10⁶ MNCs (mean ± SE, 167 ± 107/10⁶ MNCs; median, 1/10⁶ MNCs) in the postinduction remission BMs of children with a BM relapse (Wilcoxon P = 0.11; Table 2 ).

	DISCUSSION

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Quantification of residual LPCs in remission BM samples from ALL patients as a measure of MRD is a powerful predictor of relapse after autologous BMT (33) . In the present study, we have quantified LPCs in the BMs of 31 children with B-lineage ALL who experienced an isolated extramedullary first relapse, either in the CNS, testes, or eye after intensive frontline therapy. LPCs were undetectable in the BMs of 19 of these patients (61%) and were detectable in the BMs of 12 of these patients (39%) at the time of isolated extramedullary first relapse. Among the patients with an extramedullary relapse, LPC counts ranged from 1 to 356/10⁶ MNCs, with a mean of 27.8 ± 13.1/10⁶ MNCs, which is lower than the LPC count predictive of relapse (51/10⁶ MNCs) in the BMT setting (33) . In contrast, LPC counts ranged from 1/10⁶ MNCs to 3262/10⁶ MNCs (mean ± SE, 167 ± 107/10⁶ MNCs) in the second remission BMs of 41 patients after a BM first relapse. These findings suggest that not all patients with an extramedullary relapse have occult systemic failure with substantial involvement of the BM.

The LPC burden at relapse was not correlated with site of relapse or time of relapse but may be higher for patients with standard risk versus higher risk ALL. This observation suggests that LPC burden may identify a subset of standard risk patients with a higher risk of secondary BM relapse. Previous studies have noted a significant correlation between time of first extramedullary or marrow relapse and subsequent outcome (8 , 9 , 14 , 17) . Thus, it will be of interest, when treatment outcome data are available, to determine whether LPC marrow burden is predictive of secondary relapse in the BM. Notably, LPC counts after postrelapse induction chemotherapy in children with an early BM relapse were higher than the postinduction LPC counts in children with an extramedullary relapse. These data indicate that the quality of remission may be better in patients with an isolated extramedullary relapse.

In earlier studies, survival after an extramedullary relapse was poor, often due to a subsequent relapse involving the marrow (10 , 16 , 17 , 45) , which led investigators to propose that occult disease must have been present in the BM at the time of isolated extramedullary first relapse (18) . In contrast, some more recent studies of extramedullary relapse, albeit with smaller numbers of patients, did not show high rates of secondary marrow relapses (12 , 13 , 15 , 46) . Direct comparison of the more recent and older studies is difficult, however, because the initial systemic treatment regimens differed in their overall intensities as well as in their methods for CNS prophylaxis. Most earlier studies used cranial radiation, or craniospinal radiation, for both frontline and retrieval therapy (10 , 11 , 16 , 17) . Later studies often used intensive intrathecal therapy initially, reserving cranial radiation for retrieval of patients with a CNS relapse, or reduced the dosage during salvage therapy for patients who had received radiation as part of the initial therapy (9 , 12 , 13) . In some cases, patients relapsed prior to receiving cranial radiation that was planned late in initial therapy but did receive radiation as salvage therapy. It should be noted, however, that the study by George et al. (45) found no difference in rates of secondary marrow relapse after an initial CNS relapse between patients who had or had not received initial cranial radiation therapy. Similarly, Winick et al. (14) reported a high rate of secondary marrow relapse among a large group of patients with first isolated CNS relapse, most of whom had not received prior radiation but did receive intrathecal therapy and radiation as part of their retrieval therapy. The true risk of hematological relapse subsequent to an isolated first extramedullary relapse among the current group of patients treated on CCG, intensive risk-adjusted protocols remains to be determined.

Using PCR-based assays, other investigators have reported higher frequencies of minimal disease in the BM of patients with an extramedullary relapse (25, 26, 27, 28) . In each of these studies, however, very small numbers of patients were studied. For example, Goulden et al. (28) reported PCR-detectable leukemia in the BMs of 12 of 13 patients with an extramedullary relapse. Similarly, O’Reilly et al. (25) reported PCR-detectable leukemia in the BMs of seven of seven patients studied who had experienced an isolated CNS relapse. In the present study, 19 of the 31 patients had undetectable LPCs; these data do not exclude the possibility, however, that highly sensitive methods such as nested PCR (21 , 47) or real time PCR (48, 49, 50) might detect residual disease in these patients. Comparison of the relative prognostic value of different methods for detection of MRD will require further follow-up of patients on this study. A similar investigation of occult BM disease in T-lineage ALL patients on this study is in progress and will be reported separately.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by CA-13539 from the National Cancer Institute, NIH. F. M. U. is a Stohlman Scholar of the Leukemia Society of America, New York, NY.

² To whom requests for reprints should be addressed, at Children’s Cancer Group, ALL Biology Reference Laboratory and Hughes Institute, 2665 Patton Road, Suite 300, St. Paul, MN 55113. Phone: (651) 697-9228; Fax: (651) 697-1042.

³ The abbreviations used are: ALL, acute lymphoblastic leukemia; CNS, central nervous system; CCG, Children’s Cancer Group; BM, bone marrow; BMT, BM transplantation; MRD, minimal residual disease; LPC, leukemic progenitor cell; FACS, fluorescence-activated cell sorting; MNC, mononuclear cell; NCI, National Cancer Institute.

⁴ H. Sather, personal communication.

Received 4/ 6/99; revised 6/14/99; accepted 6/14/99.

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