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© 2000 American Society for Clinical Oncology In Vivo Cytoreduction Studies and Cell Sorting–Enhanced Tumor-Cell Detection in High-Risk Neuroblastoma Patients: Implications for Leukapheresis StrategiesFrom the Hematology-Oncology Service, Department of Pediatrics, University of Florence, Ospedale Pediatrico A. Meyer, Florence, and Oncology Service, Istituto G. Gaslini, Genoa, Italy. Address reprint requests to Lawrence B. Faulkner, MD, Sezione di Oncoematologia, Ospedale Meyer, Via L. Giordano 13, 50132 Firenze, Italy; email l.faulkner{at}ao-meyer.toscana.it
PURPOSE: To improve autologous leukapheresis strategies in high-risk neuroblastoma (NB) patients with extensive bone marrow involvement at diagnosis. PATIENTS AND METHODS: Anti-GD2 immunocytochemistry (sensitivity, 1 in 105 to 106 leukocytes) was used to evaluate blood and bone marrow disease at diagnosis and during the recovery phase of the first six chemotherapy cycles in 57 patients with stage 4 NB and bone marrow disease at diagnosis. A total of 42 leukapheresis samples from the same patients were evaluated with immunocytology, and in 24 of these patients, an anti-GD2 immunomagnetic enrichment step was used to enhance tumor-cell detection. RESULTS: Tumor cytoreduction was much faster in blood compared with bone marrow (3.2 logs after the first cycle and 2.1 logs after the first two cycles, respectively). Bone marrow disease was often detectable throughout induction, with a trend to plateau after the fourth cycle. By direct anti-GD2 immunocytology, a positive leukapheresis sample was obtained in 7% of patients after either the fifth or sixth cycle; when NB cell immunomagnetic enrichment was applied, 25% of patients had a positive leukapheresis sample (sensitivity, 1 in 107 to 108 leukocytes). CONCLUSION: Standard chemotherapy seems to deliver most of its in vivo purging effect within the first four cycles. In patients with overt marrow disease at diagnosis, postponing hematopoietic stem-cell collection beyond this point may not be justified. Tumor-cell clearance in blood seems to be quite rapid, and earlier collections via peripheral-blood leukapheresis might be feasible. Immunomagnetically enhanced NB cell detection can be highly sensitive and can indicate whether ex vivo purging should be considered.
MANY CURRENT protocols for chemosensitive high-risk malignancies incorporate one or more courses of high-dose therapy with autologous hematopoietic progenitor cell (HPC) support. In the future, the collection of large numbers of autologous leukocytes might be helpful not only for the purpose of hematopoietic reconstitution but also in the context of anticancer immunotherapies and/or gene therapies. However, the best timing for leukapheresis is still undefined and is probably the earliest that will allow the collection of tumor-free products. Early HPC collections have the advantage of providing better yields1-3 and allowing earlier dose intensification, which might be an important variable to improve tumor cytoreduction.4,5 In addition, because infused HPCs do contribute to long-term hematopoietic reconstitution,6 the potential for secondary myelodysplastic syndromes may be reduced by the collection of HPCs that have had limited exposure to chemotherapeutic agents.7,8 The use of relatively chemotherapy-naive leukaphereses might also be associated with an improved recovery rate of immunocompetent cells,9 which may hasten immunoreconstitution and optimize immunotherapy strategies.10 However, the inadvertent reinfusion of tumor cells with the graft is an increasingly recognized source of potential posttransplant relapse in neuroblastoma (NB).11,12 To minimize the likelihood of tumor-cell contamination, highly sensitive tumor-cell detection methods, as well as information on metastatic tumor-cell clearance kinetics during induction therapy, are needed. Gene-marking experiments have suggested that the minimum number of NB cells reinfused with the autologous hematopoietic graft that might be necessary to recolonize the host is in the 102 order of magnitude.11 Because the number of cells generally reinfused with the autologous rescue varies between 109 and 1010, for a clinically meaningful tumor-cell contamination analysis, detection sensitivity should extend into the range of 1 in 107 to 108 normal leukocytes. The currently available technologies of cell sorting and micrometastasis detection potentially allow this degree of sensitivity to be applied realistically to large sample numbers.13-15 As part of a collaboration between the Children’s Hospitals of Florence and Genoa, which was later extended to the Italian Cooperative Neuroblastoma Group, we studied blood and marrow samples from NB patients at different time points during induction therapy with high-sensitivity anti-GD2 immunocytology, as well as the application of immunomagnetically enhanced tumor-cell detection to leukapheretic products.
Patient Population After informed consent was given by the patients or their legal guardians, marrow and blood samples were obtained from a total of 57 patients with stage 4 (International Neuroblastoma Staging System) NB. Their median age was 3.8 years (range, 0.6 to 16.5 years). All patients were diagnosed as having NB according to standard criteria16 and had marrow invasion at diagnosis. Thus, all patients considered in this study had overt marrow disease at presentation. In 39 cases, this was documented both by immunocytology (  1% positivity; Table 1) and standard smear cytology; the remaining 18 patients had marrow immunocytology evaluations only after the initial diagnosis but had evident marrow invasion at diagnosis by standard marrow aspirate examination. Serial marrow and blood evaluations were performed as part of Italian Cooperative Neuroblastoma Study Protocol NB 97. The induction phase of the protocol consisted of two initial courses of ifosfamide 9 g/m2 and doxorubicin 75 mg/m2 administered in 3 days followed by two courses of carboplatin 1,000 mg/m2 and etoposide 300 mg/m2 administered in 2 days. Surgical resection was performed in patients who did not achieve a complete radiologic response. Another two courses of cyclophosphamide 3.6 g/m2 and etoposide 450 mg/m2 were administered over 3 days before consolidation with high-dose chemotherapy with autologous stem-cell rescue. Peripheral-blood stem cells were collected after the fourth, fifth, and/or sixth cycles. To enhance mobilization, filgrastim was used at 10 µg/kg/d in a single subcutaneous dose starting 5 days from the last chemotherapy administration day.
Immunocytology for Minimal Disease Detection For NB cell detection, anti-GD2 immunocytology was used (sensitivity, one NB cell in 105 to 106 leukocytes) in a total of 98 blood samples and 156 marrow samples obtained at diagnosis and during the first six chemotherapy cycles (Table 1). A total of 87 leukapheresis samples collected after the fifth and/or sixth chemotherapy cycles from 42 of the 57 patients were also evaluated. Mononuclear cells (MNCs) were obtained by Histopaque 1077 (Sigma, St Louis, MO) density separation at 600 x g for 15 minutes. Before density separation, peripheral-blood samples were diluted 1:1 and bone marrow samples were diluted 1:5 with calcium- and magnesium-free phosphate-buffered saline (PBS). The light-density MNC fraction was washed twice in calcium- and magnesium-free PBS. Leukapheretic products were processed without density separation. MNCs were resuspended at 1 x 106 cells/mL, and six cytospins containing up to 106 MNCs each on a single area 17 mm in diameter were prepared by centrifuging 0.5 to 1 mL of the MNC suspension at 400 x g for 10 minutes (Hettich Centrifuge, Tuttlingen, Germany) on regular glass slides. With this cytospin preparation and processing method, the actual number of cells recovered on the slides was at least 80% of the number layered initially. Cytospins were dried at room temperature overnight; three cytospins were processed immediately and three were frozen at -70°C. After fixation for 10 minutes in cold acetone, slides were rinsed and then incubated for 30 minutes at room temperature in a humid chamber with the primary anti-GD2 mouse monoclonal antibody (clone 3F8, provided by Nai-Kong V. Cheung, MD, PhD, Memorial Sloan-Kettering Cancer Center, New York, NY). The slides were then incubated for 30 minutes with a polyclonal biotinylated anti-mouse antibody, followed by a rinse, a wash in PBS for 10 minutes, drying for 5 minutes, and a third 10-minute incubation with a streptavidin–alkaline phosphatase complex (LSAB-2; Dako, Milan, Italy). After the wash, incubation with new fuchsin chromogen (Dako) for 8 minutes was used to stain bound alkaline phosphatase complexes. Incubation for 5 minutes with hematoxylin was used for counterstaining. For each evaluation, three cytospins were stained, one of which was used as a negative control in which PBS was substituted for the primary antibody. Thus, a large number of cells were screened in the negative controls—approximately half of those in the actual test—in order to minimize false-positive results related to nonspecific binding of the secondary antibody, avidin, biotin, or alkaline phosphatase. For each staining procedure, a positive control consisting of a cytospin prepared with an NB cell line (SK-N-FI; American Tissue Type Culture Collection, Rockville, MD) was used. A mean 1.4 x 106 MNCs/sample were screened under light microscopy, and positivity was assigned when a cell had a clearly recognizable nucleus and a completely stained cytoplasm. An interpretable test was defined as a test in which the positive control was brightly stained and the negative control had either no significant background staining such as to interfere with positive cell discrimination or no cells that would be scored as positive. The sensitivity and specificity of the method were verified in several scalar dilutions of NB cells (SK-N-FI) in normal donor MNCs down to one NB cell in 106 MNCs, and by comparison to data obtained from marrow smears and trephine biopsies performed concomitantly and evaluated blindly in different institutions.17 In fact, no bone marrow samples unequivocally positive by blinded centralized standard smear and trephine biopsy evaluation were negative by anti-GD2 immunocytology; thus we have no evidence for unequivocal false negativity of this method. No false positivity was found in the 20 peripheral-blood and bone marrow negative controls that were evaluated.
Immunomagnetic Extraction
Immunomagnetic enrichment was used for a total of 24 leukaphereses in 24 patients. At least 0.6 x 108 cells were processed (range, 0.6 to 5.01 x 108) (Table 3). This minimum cutoff number for cells was chosen in order to have a sensitivity of at least 1 in 107 normal leukocytes. In fact, when an immunomagnetic extraction efficiency of at least 50% is taken into account and when Poisson distribution statistics are allowed, at least six times the number of cells of the sensitivity target have to be evaluated.
Statistical Analysis Comparison between sample groups was performed using the Wilcoxon nonparametric test for pairs with two-tailed P values. The GraphPad Prism software package (GraphPad Software Inc, San Diego, CA) was used for statistical analysis, descriptive statistics, and plotting. The best-fitting curve in Fig 1 was obtained assuming one-phase exponential decay.
Blood and Marrow Tumor-Cell Clearance Kinetics The clearance of tumor cells was substantially faster in blood compared with bone marrow (Table 1 and Fig 1). Circulating NB cell frequency dropped by 3.2 logs after the first chemotherapy cycle, decreasing from a mean of 1,478 per 106 MNCs at diagnosis to the limit of detection of the assay after the first cycle. At diagnosis, 23 (79%) of 29 peripheral-blood samples tested were positive, whereas after the first cycle, one out of nine samples was positive, and after the second cycle, two (14%) out of 14 samples were positive, albeit at the limit of detection of the assay. Only one positive sample (5%) out of 20 was detected after the fourth cycle; this occurred in a patient with early progression who went off protocol. No circulating NB cells were detected in a total of 19 samples obtained after the fifth and sixth cycles. The bone marrow tumor-cell clearance rate was much slower, with a decrease of 2.1 logs at the evaluation performed after the second cycle and of another 1.4 logs at the evaluation performed after the fourth cycle, for an overall decrease of 3.5 logs from diagnosis to the recovery phase of the fourth cycle, with a tendency to plateau thereafter (Fig 1). The majority of bone marrow samples remained positive, albeit at different levels, throughout the first six chemotherapy cycles preceding hyperintensive consolidation with stem-cell rescue. By the Wilcoxon nonparametric test for pairs, relative to mononuclear leukocytes, NB cells were significantly less frequent in blood compared with bone marrow at diagnosis (P < .0001) and after the second cycle (P = .001). The bone marrow positivity decrement was significant between diagnosis and the second-cycle recovery phase (22 matched pairs, P < .0001) and between the second-cycle recovery phase and the fourth-cycle recovery phase (22 matched pairs, P = .0163). No further significant marrow clearance was detected that directly compared the recovery phases of the fourth and fifth cycles (10 matched pairs, P = .0781) and the fifth and sixth cycles (seven matched pairs, P = .8125). However, when GD2-positive cell frequencies of the fourth-cycle recovery phase were compared with those after the sixth cycle, a significant difference was found (24 matched pairs, P = .0093). This may be related to the relatively few evaluations available after cycle 5. The bone marrow clearance kinetics depicted in Fig 1 and summarized in Table 1 seem to suggest, however, that most of the further marrow tumor-cell kill possibly occurring after cycle 4 may be restricted to cycle 5.
Immunomagnetically Enhanced Tumor-Cell Detection To evaluate the possible interference of anti-GD2 immunomagnetic extraction with anti-GD2 staining, in four experiments, one of the cytospins of the postenrichment product was evaluated with directly fluoresceinated anti-NB84a.18 No significant difference with the anti-GD2 immunocytochemistry quantitative analysis was detected (paired samples t test, P = .4659). Testing the reliability of the anti-GD2 staining of the sorted product is particularly relevant, since GD2, to our knowledge, is the only antigen that meets the criteria of high and consistent expression in all NB cells. In fact, no GD2-negative tumor has been reported to date,17,19-25 with the possible exception of patients pretreated with anti-GD2 antibodies.26 In contrast, NB84a is expressed on SK-N-FI cells but may not be expressed on all NBs.27 When applying this highly sensitive methodology to clinical samples, an antigen that is consistently expressed on all NB cells is an absolute requirement. Of the many anti-GD2 antibodies available, 3F8, an immunoglobulin G3 monoclonal antibody, is probably the most extensively tested, both by immunocytology22 and immunoscintigraphy.19 Thus, 3F8 provides a unique opportunity for highly sensitive and specific tumor-cell detection. To evaluate the potential for false positivity of immunocytology applied to immunomagnetically sorted leukocytes, 21 negative controls consisting of 2 to 6 x 108 normal donor buffy coat MNCs were evaluated with the same procedure. In five instances (24%), nonspecifically stained material could be identified; this, however, could be discriminated from truly positive cells on morphologic grounds.
Leukapheresis Studies A total of 24 leukapheresis samples from 24 patients underwent immunomagnetic enrichment. At least 0.6 x 108 cells were processed (range, 0.6 to 5.01 x 108; Table 3). Six samples (25%) were found to contain positive cells in the sorted product, with tumor cell frequencies ranging from 0.8 to 111 per 108 leukocytes. When this number was translated into the transplantation cell dose order of magnitude, ie, 109 to 1010 leukocytes, and when an extraction efficiency in the 60% range was considered, even the lowest positivity might have been consistent with the potential reinfusion of approximately 10 to 100 NB cells.
In the rapidly growing field of cellular therapies, it is becoming increasingly important to develop guidelines for autologous leukapheresis timing and processing. Evaluating the minimum number of induction cycles after which it is potentially feasible to obtain virtually tumor-free leukocyte collections might have important implications. In fact, despite intensified treatments with autologous stem-cell support, more than 40% to 50% of pediatric patients with NB still relapse.28-32 One of the possible causes of treatment failure is the inadvertent reinfusion of tumor cells that contaminate autologous hematopoietic cell collections. The actual impact of these hypothetical "reinfusion relapses" is still a matter of debate. To address this matter, however, guidelines for leukapheresis timing and processing are needed, together with appropriately sensitive tumor-cell detection methodologies. To our knowledge, this is the first study to evaluate both immunocytology and high-sensitivity, immunomagnetically enhanced tumor-cell contamination studies of leukaphereses in the context of in vivo blood and marrow cytoreduction kinetics data. It has been shown that maximal NB tumor volume reduction may be achieved after the first three cycles.33 Our findings in terms of marrow tumor-cell clearance are consistent with a maximal cytoreductive effect during the first four to five cycles. In terms of peripheral-blood and marrow NB cell clearance, our data are consistent with data reported by Matthay et al34 showing that the great majority of peripheral-blood samples (201 of 215, ie, 93%) are negative at the 12th week of induction; however, in this study, peripheral blood was studied only at diagnosis and at the time of collection, ie, week 12 after initiation of therapy. We have previously demonstrated that in NB, blood is significantly less contaminated with tumor cells compared with marrow and that this difference is in the range of 2 logs,25 which suggests that a sufficient degree of stem-cell collection purity may be achieved earlier from peripheral blood compared with bone marrow. Saarinen et al35 studied 18 patients with serial anti-GD2 immunocytology marrow evaluations having a detection sensitivity of 1 in 104 MNCs. They found that only in five (28%) of them was negativity achieved after the second cycle. In our series, 14 (50%) of 28 patients had a tumor-cell frequency below 1 in 104 MNCs after the second cycle. In a larger evaluation reported by Matthay et al,34 in which immunocytology with a sensitivity of 1 in 105 MNCs was used, 80 (34%) out of 238 bone marrow samples were positive at 12 weeks. In our series, 19 (40%) out of 47 marrow samples had a positivity greater than 1 in 105 MNCs after the fourth cycle. We were not able to determine whether the discrepancy between having found no peripheral-blood positivities after the fifth cycle and yet having three out of 42 patients with positive leukaphereses performed at the same time point is due to the limited number of peripheral-blood samples tested after the fifth cycle (19 total) or rather to tumor-cell mobilization.36 To our knowledge, however, this phenomenon has not been reported in the context of NB. In our experience, anti-GD2 immunomagnetic tumor-cell enrichment has the potential to increase NB cell detection sensitivity to the 1 in 108 cell range. In other contexts, immunomagnetic enrichment techniques applied to tumor-cell detection have provided similar results.14,37 As mentioned, this level of detection might be in the range of what gene-marking experiments11 have suggested as being a possibly relevant sensitivity threshold. Considering an extraction efficiency in the 50% range, and allowing for Poisson distribution error statistics, at least six times the number of cells of the sensitivity target should be processed by this immunomagnetic enrichment method. This might actually be a substantial number of cells (up to one tenth of the whole hematopoietic graft); however, this drawback might be far outweighed by the advantages of avoiding extensive graft manipulations. In the context of low-level positivity, a second confirmatory detection method at the single-cell level might be useful. This could be based on the concomitant identification of a tumor-specific genetic lesion by in situ hybridization38 or by the detection of neuroblast-specific transcripts by reverse transcriptase polymerase chain reaction. The latter has been applied extensively in NB39-44; however, compared with immunocytology, this method is more labor-intensive, costly, and provides only approximate or no quantitative information. In addition, it might have potential specificity and sensitivity problems related to illegitimate transcription by leukocytes45,46 and/or variable mRNA expression by NB cells.47 Tumor-cell purging procedures might be justified in cases of proven or suspected positivity. This method might even be applied to thawed cells48 if the information regarding the suspicion of significant tumor-cell contamination is obtained after cryopreservation. The degree of purging needed, which might profoundly affect immune reconstitution, transplant-related morbidity,49,50 and costs, might be tailored to the degree of estimated tumor-cell contamination. In fact, purging procedures can vary widely in terms of extent of cell manipulation and final purification, eg, positive or negative purging or a combination of the two. Our findings would suggest that after maximal in vivo purging, in most instances a negative purging procedure that depletes at least 2 logs of tumor cells51-53 might suffice, thus avoiding the extensive immunocompetent cell depletion often associated with more aggressive purging procedures. If no positivity is documented at the level of 1 in 108 leukocytes, then it might be difficult to justify any purging procedure. Our patient group was selected for having overt bone marrow disease at diagnosis, and thus the same principles may not apply to the small proportion of patients who have high-risk features but lack evident marrow invasion, such as stage 3 MYCN-amplified or stage 4 bone marrow–negative patients. In conclusion, tumor-cell detection sensitivity can potentially be extended to 1 in 108 cells with immunomagnetic enrichment. This level of sensitivity may allow the administration of virtually tumor-free cell products. Combining this information with that regarding systemic NB tumor-cell clearance data might allow better leukapheresis timing and purging decision making. Given our data, we believe that there is probably little justification for postponing leukapheresis procedures in NB patients beyond the fourth or fifth induction cycle. In fact, a sufficient degree of in vivo circulating tumor-cell purging may be achievable sooner, and early leukapheresis might be justified provided that peripheral blood is negative by immunocytology and that highly sensitive, immunomagnetically enhanced tumor-cell detection is used to evaluate leukapheretic products and indicate whether ex vivo purging strategies should be considered.
Supported by the Associazione Italiana per la Lotta al Neuroblastoma, Genova, and the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy. We thank Nai-Kong V. Cheung, MD, PhD, at Memorial Sloan-Kettering Cancer Center, New York, NY, for providing the anti-GD2 antibody 3F8 for this study and for his helpful comments and suggestions on the manuscript.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
