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© 2003 American Society for Clinical Oncology Quantitation of GD2 Synthase mRNA by Real-Time Reverse Transcriptase Polymerase Chain Reaction: Clinical Utility in Evaluating Adjuvant Therapy in Neuroblastoma
From the Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY. Address reprint requests to Irene Y. Cheung, ScD, Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; email: cheungi{at}mskcc.org.
Purpose: Minimal residual disease (MRD) is one of the final hurdles to cancer cure. Because therapy (myeloablation, immunotherapy, or differentiation) for MRD is applied at the time of clinical remission, objective surrogate markers are needed to gauge treatment efficacy. Patients and Methods: Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) of GD2 synthase (ß1,4-N-acetylgalactosaminyltransferase, EC 2.4.1.92) mRNA, we evaluated MRD response to anti-GD2 monoclonal antibody 3F8 adjuvant therapy, namely, one cycle of radioimmunotherapy using iodine-131 (131I)-3F8 plus one cycle of unlabeled 3F8 in 45 stage 4 neuroblastoma patients (newly diagnosed or without prior relapse) on the N7 protocol at Memorial Sloan-Kettering Cancer Center. The prognostic effect of MRD in their bone marrows before and after this phase of adjuvant therapy on progression-free survival (PFS) and overall survival (OS) was also analyzed. Results: Before 3F8 treatment, 24 of 45 patients were in complete remission (CR), 12 were in very good partial remission (VGPR), and nine were in partial remission (PR), according to criteria from International Neuroblastoma Staging System plus 131I-3F8 scan; 71% had detectable tumor cells in marrow by real-time RT-PCR. Of the 32 positive patients, 20 became negative after therapy, with a 63% efficacy. When patients were stratified by CR/VGPR versus PR, GD2 synthase positivity was prognostic when detected before 3F8-targeted therapy (PFS, P = .045 and OS, P = .010). Persistent marker positivity was also predictive of PFS (P = .035) and OS (P = .027). Patients who succumbed to the disease had transcript levels four times higher than those who remain alive. Conclusion: GD2 synthase mRNA is a useful surrogate marker for evaluating adjuvant treatment efficacy in neuroblastoma with prognostic potential.
ONE OF the final hurdles to curing metastatic neuroblastoma is minimal residual disease (MRD), which escapes clinical detection leading to ultimate relapse and death. Finding an effective strategy to eradicate MRD has remained a formidable challenge.1 After induction, a common strategy employs myeloablative chemotherapy at the time of apparent clinical remission. This approach takes advantage of the linear dose-response relationship of individual chemotherapeutic drugs in specific tumors. Nevertheless, the value of such aggressive treatment, which is costly in both human and financial terms, remains debatable. Studies using myeloablative therapy with autologous stem-cell rescue have demonstrated a modest effect on outcome.2–5 Biologics (immunotherapy and differentiation therapy) are emerging rapidly as alternative methods of adjuvant therapy at the time of clinical remission,2,6–9 and are appealing because of their generally mild toxicity. However, the true worth of biologics in eradicating MRD is difficult to assess, because ascertaining antitumor efficacy of individual agents will necessitate lengthy patient follow-up to monitor clinical relapse or death. When disease is macroscopic, objective response can be easily measured. Unfortunately, when disease is below the threshold of clinical detection, efficacy measurement is often imprecise, lacking in objectivity, and difficult to verify. In fact, the inability to measure subclinical disease leaves no choice for cancer care professionals but to continue treatment (whether standard or experimental therapy) despite remission. This difficulty in quantifying MRD derives from the increasing demand of more stringent standards, where the unit of measure is an individual cell. Because of their low cell number, individual tumor cells escape detection by histological examination of bone marrow (BM) biopsies or aspirates.10,11 In contrast, tumor-specific or tumor-associated RNA or cDNA are signatures of viable tumor cells. This new level of detection is possible with reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR of specific gene transcripts serving as surrogate end points. In this report, we apply quantitative real-time RT-PCR of GD2 synthase mRNA to evaluate treatment efficacy at the time of MRD among stage 4 neuroblastoma (NB) patients undergoing N7 therapy at Memorial Sloan-Kettering Cancer Center (MSKCC).12 GD2 synthase (ß1,4-N-acetylgalactosaminyltransferase, EC 2.4.1.92) is the key enzyme required for GD2 synthesis.13 This transcript has been shown to have potential as a molecular marker of NB, in particular for the detection of rare NB cells.14–17 Recently, we validated a highly sensitive and specific quantitative RT-PCR assay that measures GD2 synthase mRNA. The transcript levels correlated well with the number of NB cells as measured by immunocytology.14 In the N7 protocol, anti-GD2 monoclonal antibody 3F8 was used to target radioimmunotherapy and immunotherapy to tumor cells when patients had achieved their minimal disease status. Iodine-131 (131I)-3F8 (hot antibody) directed a myeloablative dose of radiation to residual NB, whereas unlabeled 3F8 (cold antibody) delivered complement-mediated18,19 and antibody-dependent cell-mediated cytotoxicity.20–22 We measured disease response in the BM to one phase of the adjuvant therapy: one cycle of hot 3F8 plus one cycle of cold 3F8 among 45 patients; one patient did not receive cold 3F8 because of progressive disease (PD). Furthermore, we tested whether MRD had any prognostic effect on patient survival.
Patients Forty-five stage 4 NB patients on N7 protocol (36 newly diagnosed at MSKCC and nine prior-treated without relapse), all more than 1 year of age at initial presentation, were the subjects of this study. The N7 protocol12 used dose-intensive chemotherapy for induction, surgical resection, and 21 Gy hyperfractionated radiotherapy for local control; for consolidating remission, targeted radioimmunotherapy with 131I-3F8 and immunotherapy with unlabeled 3F8 were used. 131I-3F8 at 20 mCi/kg was myeloablative and required stem-cell support. Among patients with newly diagnosed NB entered onto N7, less than 10% developed PD during induction and were not eligible to receive 131I-3F8 or unlabeled 3F8 treatment. Written informed consent was obtained from the patients and/or their parents in accordance with guidelines of the institutional review board of MSKCC. Comprehensive extent-of-disease evaluation at MSKCC, in accordance with the International Neuroblastoma Staging System,23 included computed tomography and/or magnetic resonance imaging, technetium-99m bone scan, 131I- or 123I-metaiodobenylguanidine (MIBG) scan, urinary catecholamine metabolites measurements, and BM studies evaluated by histochemical examinations of bilateral biopsy specimens and of aspirates from bilateral anterior and bilateral posterior iliac crests. In addition, 131I-3F8 tumor imaging was carried out as previously described.24
Disease status was categorized according to the International Neuroblastoma Response Criteria:23 complete response (CR), no evidence of disease; very good partial response (VGPR), primary mass reduced by 90% to 99%, no evidence of distant disease except for skeletal residua, and catecholamines normal; partial response (PR), more than 50% decrease in measurable disease and
Immunocytology
Real-Time Quantitative RT-PCR
Statistical Analysis
Disease Status and Response Status of Patients Before Radioimmunotherapy Table 1  summarizes the disease status and response status of these patients when their BMs were sampled before radioimmunotherapy for real-time RT-PCR. The median time from diagnosis was 9.2 months. On the basis of six modalities of detection (computed tomography and/or magnetic resonance imaging, technetium-99m bone scan, 131I- or 123I-MIBG scan, urinary catecholamine metabolites measurements, BM biopsy and aspirate examination, and 131I-3F8 scan), 53% (24 of 45) of patients had no evidence of disease. Only five patients were positive in more than three detection modalities. In terms of response status, 80% (36 of 45) of the patients were in CR (n = 24) or VGPR (n = 12). Only nine patients were in PR before 131I-3F8 radioimmunotherapy.
Efficacy During This Phase of Adjuvant Therapy Even though these patients had good remission status as evidenced by the absence of disease detection in most of them and none had positive marrow by histological examination, 32 of 45 of their marrows before treatment were tested positive by real-time quantitative RT-PCR of GD2 synthase mRNA (Table 2 ). When compared with detection by immunocytology, nearly four times more samples were positive by RT-PCR. The difference between the two detection methods was much smaller for the posttreatment marrows. Twenty of the 32 RT-PCR–positive patients had no detectable GD2 synthase transcript in their marrow after this phase of 3F8-targeted antibody treatment (one hot plus one cold 3F8), which is consistent with a marrow response of 63% (20 of 32; Table 2).
Relationship Between Change in Transcript Level and Patient Survival Figure 1A illustrates the levels of GD2 synthase transcript when patients were stratified into three groups: those who had no detectable transcript (n = 13) versus those whose marrows became negative after treatment (n = 20) versus those whose marrows remained positive after this phase of immunotherapy. Patients who eventually died had transcript levels four times higher than those who remained alive (Fig 1B). In addition, among the 20 patients who eventually died, 45% (nine of 20) never had recurrence by histological examination in the BM. They died from PD or complications following relapse in the CNS (n = 4), CNS and lung (n = 1), primary site (n = 1), lymph node (n = 1), bone (n = 1), and treatment-related leukemia (n = 1).
Prognostic Importance of GD2 Synthase–Positive BMs Sampled Before Radioimmunotherapy on Patient Survival BMs that tested positive for GD2 synthase transcript before hot antibody treatment were associated with adverse PFS (P = .074) and OS (P = .021), respectively. When stratified by response status (CR/VGPR v PR) at the sampling time, patients with detectable GD2 synthase expression who were in partial remission at that time fared even worse, with P = .045 for PFS (Fig 2A and B ) and P = .010 for OS (Fig 3A and B). Similar analyses were performed for immunocytology. Positive immunocytology had no prognostic significance with respect to PFS and OS.
Prognostic Importance of GD2 Synthase–Positive BMs Sampled After Hot and One Cycle of Cold Antibody on Patient Survival The BM of these patients were tested after the completion of hot antibody and one cycle of cold antibody treatment. The median time from diagnosis was 16.7 months. Kaplan-Meier analyses indicated a trend toward a poorer outcome among patients with positive posttreatment marrow, although statistical significance was not reached (PFS, P = .065; OS, P = .064). Not surprisingly, patients whose BMs stayed positive for the GD2 synthase transcript had a significantly higher risk of disease progression (P = .035; Fig 4A ) and death (P = .027; Fig 4B).
Accurate MRD measurements can provide objective and rapid surrogate end points in evaluating the efficacy of adjuvant therapies. We hypothesize that MRD, being the likely final common pathway of inadequately treated or resistant cancer cells, will eventually be a dominant determinant of clinical outcome. After all, we expect that the adverse effects of tumor biology will continue to be neutralized by increasingly effective treatment strategies. If MRD can indeed independently predict patient outcome, an objective and measurable end point for adjuvant therapy may likely be realized. In this study, it was striking and somewhat surprising to find that marrows drawn before adjuvant treatment were positive in more than 70% of the patients by real-time RT-PCR, whereas only 20% of these patients had measurable disease by immunocytology, with both assays identifying GD2-positive cells. Patients who succumbed to NB had transcript levels four times higher than those who remain alive. On the completion of one cycle of unlabeled 3F8 immunotherapy, 63% of the positive marrows became negative by real-time RT-PCR. This surrogate end point would indeed suggest that the adjuvant therapy was efficacious. More importantly, the question of true efficacy must be addressed: Does GD2 synthase mRNA positivity correlate with clinical outcome, specifically to the gold standards of patient survival, both PFS and OS? In this phase of immunotherapy, GD2 synthase transcript was found to correlate with both PFS and OS with statistical significance. This was not so when immunocytology as a variable was analyzed. These Kaplan-Meier analyses generated several interesting observations. First, despite 71% marrow positivity before 3F8-targeted therapy, only 44% succumbed to their disease, strongly suggesting a potential efficacy of antibody-targeted adjuvant therapy on patient survival. Furthermore, 45% of the patients who eventually died did not have recurrence in the BM. Soft tissue relapse such as lung, CNS, and therapy-related acute myelogenous leukemia caused their death. Second, patients with persistently positive BMs detectable by RT-PCR before hot antibody treatment, after hot antibody treatment, and after one cycle of cold antibody treatment were more likely to progress and die from the disease. Fukuda et al26 reported that persistent tyrosine hydroxylase mRNA in the BM of stage 4 NB patients tested at more than 4 months after the start of chemotherapy was associated with poor prognosis. Thus, achieving and maintaining tumor-free marrow during this consolidation could be critical for improving patient outcome. Of note were patients who, despite a persistent positive marker in the marrow during this phase of adjuvant therapy, finally achieved molecular remission and remained disease-free. They may have become long-term survivors because of the anti-NB activity of treatment with subsequent additional cycles of 3F8. Third, despite their apparent clinical remission by International Neuroblastoma Staging System criteria plus 131I-3F8 scan, patients who were GD2 synthase–positive before 3F8-targeted immunotherapy had statistically poorer PFS and OS. This could be the result of either slow-responding or subclinical PD. Our finding is in agreement with a much larger series of stage 4 NB patients in the Children’s Cancer Group study, in which increasing tumor cell content in the BM as measured by immunocytology had an adverse effect on event-free survival.27 It was not surprising that GD2 synthase positivity was particularly adverse among patients who were in partial remission before adjuvant therapy. One could argue that improving the remission status before megatherapy consolidation might be necessary to improve patient outcome. However, whether marker positivity was because of slow-responding disease versus PD has important implications for future therapy design. Although slow responders may benefit from continued chemotherapy, PD demands, at the very least, change in chemotherapeutic strategy. Finally, posttreatment marrow RT-PCR positivity was associated with poorer PFS and OS, even though statistical significance was not reached. This could be because of the ability of subsequent cycles of antibody treatment to eradicate BM disease, thereby changing the long-term prognosis of GD2 synthase transcript–positive patients, or because the sensitivity of RT-PCR is still suboptimal and fails to identify the patients who are truly in complete remission. We expect that the application of additional molecular markers may further improve sensitivity, thus allowing us to predict outcome with greater certainty. We have previously shown that positive GD2 synthase mRNA in the BMs sampled at 24 months from diagnosis correlated strongly with PFS (P < .005) and OS (P < .001).14 Similar conclusions were drawn when another molecular marker, GAGE, was tested in the same cohort.28 These were clearly measurements of progression undetected by current modalities. Quantitation of GD2 synthase mRNA by real-time quantitative RT-PCR proved to have clinical relevance in evaluating adjuvant therapy in NB by detecting a subclinical level of tumor cells and, thus, was predictive of long-term patient outcome. This study, albeit small (n = 45), included only newly diagnosed or without prior relapse stage 4 patients treated in a single institution under the same protocol. Our survival analyses seemed to indicate the need to ensure that patients achieve both marrow remission by this technique (ie, the absence of molecular relapse) and clinical remission (CR/VGPR) before the onset of adjuvant therapy to achieve long-term cure. The question of MRD and clinical relevance may potentially be resolved on the completion of the Children’s Oncology Group phase III trials on the detection of MRD among high-risk NB patients performed at various times during therapy.29
Supported in part by grants from the National Institutes of Health (CA61017 and CA095742) and the Robert Steel Foundation, Hope Street Kids, Pediatric Cancer Foundation, and Katie’s Find A Cure Fund.
1. Cheung NKV, Kushner BH, LaQuaglia MP, et al: Treatment of advanced-stage neuroblastoma, in Raghavan D, Scher HI, Leibel SA, et al (eds): Principles and Practice of Genitourinary Oncology. Philadelphia, Lippincott-Raven, 1997, pp 1101–1111
2. Matthay KK, Villablanca JG, Seeger RC, et al: Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. N Engl J Med 341:1165–1173, 1999
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4. Pole JG, Casper J, Elfenbein G, et al: High-dose chemoradiotherapy supported by marrow infusions for advanced neuroblastoma: A Pediatric Oncology Group Study. J Clin Oncol 9:152–158, 1991 5. Ladenstein R, Philip T, Lasset C, et al: Multivariate analysis of risk factors in stage 4 neuroblastoma patients over the age of one year treated with megatherapy and stem-cell transplantation: A report from the European Bone Marrow Transplantation Solid Tumor Registry. J Clin Oncol 16:953–965, 1998[Abstract] 6. Cheung NKV, Kushner BH, Yeh SJ, et al: 3F8 monoclonal antibody treatment of patients with stage IV neuroblastoma: A phase II Study. Int J Oncol 12:1299–1306, 1998[Medline]
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8. Kushner BH, Kramer K, Cheung NKV: Phase II trial of the anti-G(D2) monoclonal antibody 3F8 and granulocyte-macrophage colony-stimulating factor for neuroblastoma. J Clin Oncol 19:4189–4194, 2001 9. Yu A, Uttenreuther-Fischer M, Huang C-S, et al: Phase I trial of a human-mouse chimeric anti-disialoganglioside monoclonal antibody ch14.18 in patients with refractory neuroblastoma and osteosarcoma. J Clin Oncol 16:2169–2180, 1998[Abstract] 10. Cheung NK, Heller G, Kushner BH, et al: Detection of metastatic neuroblastoma in bone marrow: When is routine marrow histology insensitive? J Clin Oncol 15:2807–2817, 1997[Abstract] 11. Cheung IY, Barber D, Cheung NKV: Detection of microscopic neuroblastoma in marrow by histology, immunocytology, and RT-PCR of multiple molecular markers. Clin Cancer Res 4:2801–2805, 1998[Abstract] 12. Cheung NK, Kushner BH, LaQuaglia M, et al: N7: A novel multi-modality therapy of high risk neuroblastoma (NB) in children diagnosed over 1 year of age. Med Pediatr Oncol 36:227–230, 2001[CrossRef][Medline]
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16. Hoon DSB, Kuo CT, Wen S, et al: Ganglioside GM2/GD2 synthetase mRNA is a marker for detection of infrequent neuroblastoma cells in bone marrow. Am J Pathol 159:493–500, 2001 17. Cheung IY, Lo Piccolo MS, Collins N, et al: Quantitation of GD2 synthase mRNA by real-time reverse transcription-PCR: Utility in bone marrow purging of neuroblastoma by anti-GD2 antibody 3F8. Cancer 94:3042–3048, 2002[CrossRef][Medline] 18. Cheung NKV, Walter EI, Smith-Mensah WH, et al: Decay-accelerating factor protects human tumor cells from complement-mediated cytotoxicity in vitro. J Clin Invest 81:1122–1128, 1988[Medline]
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24. Yeh SD, Larson SM, Burch L, et al: Radioimmunodetection of neuroblastoma with iodine-131-3F8: Correlation with biopsy, iodine-131-metaiodobenzylguanidine (MIBG) and standard diagnostic modalities. J Nucl Med 32:769–776, 1991 25. Cheung IY, Cheung NKV: Molecular detection of GAGE expression in peripheral blood and bone marrow: Utility as a tumor marker for neuroblastoma. Clin Cancer Res 3:821–826, 1997[Abstract] 26. Fukuda M, Miyajima Y, Miyashita Y, et al: Disease outcome may be predicted by molecular detection of minimal residual disease in bone marrow in advanced neuroblastoma: A pilot study. J Pediatr Hematol Oncol 23:10–13, 2001[CrossRef][Medline]
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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TOP
ABSTRACT
INTRODUCTION
1 positive BM site; mixed response, more than 50% decrease of any lesion with less than 50% decrease in any other; no response, less than 50% decrease but less than 25% increase in any existing lesion; and PD, new lesion or more than 25% increase in an existing lesion. 
