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© 2003 American Society for Clinical Oncology Early Molecular Response of Marrow Disease to Biologic Therapy Is Highly Prognostic 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; e-mail: cheungi{at}mskcc.org.
Purpose: A promising treatment strategy for stage 4 neuroblastoma patients is the repeated application of anti-GD2 immunotherapy after activating myeloid effectors with granulocyte-macrophage colony-stimulating factor (GM-CSF). To use early marrow response as a prognostic marker is particularly relevant for patients not likely to benefit from this therapy. Patients and Methods: Eighty-six stage 4 neuroblastoma patients older than 1 year at diagnosis were classified in four clinical groups on protocol entry: complete remission or very good partial remission (n = 33), primary refractory (n = 33), secondary refractory (n = 10), and progressive disease (n = 10). Bone marrow samples collected before and following treatment were assayed for GD2 synthase mRNA by real-time reverse transcriptase polymerase chain reaction. Response and survival analyses were performed on posttreatment samples before the third cycle at 1.8 months from protocol entry. Results: GD2 synthase mRNA was evident in pretreatment marrow samples of the four clinical groups (42%, 52%, 60%, and 80% of samples, respectively), with median transcript level of 10.0, 16.6, 26.5, and 87.2, respectively. This marker became negative following antibody plus GM-CSF in 77% of complete remission or very good partial remission, 45% of primary refractory, 25% of secondary refractory, and 0% of progressive disease group. Progression-free survival was statistically different between responder and nonresponder groups (P < .0001). Among patients with minimal residual disease, molecular responders had a significantly lower risk of disease progression at a median follow-up of 29.8 months (P = .0001). Conclusion: GD2 synthase mRNA is a sensitive response marker of neuroblastoma in the bone marrow. It is particularly useful for minimal residual disease evaluation and may potentially be useful as an early predictor of resistance to antibody plus GM-CSF immunotherapy.
ALTHOUGH MULTIMODALITY treatment using dose-intensive chemotherapy, radiation, and surgery can potentially eradicate even the bulkiest and widespread neuroblastoma (NB), relapse in the majority of patients with stage 4 NB continues to be a therapeutic challenge. Most initial remissions are often incomplete because of occult minimal residual disease (MRD). Various novel treatment strategies are being developed to target MRD. Examples include the use of myeloablative therapy,1,2 differentiation therapy (13-cis-retinoic acid),2 as well as immunotherapy using antibody alone3 or in combination with cytokines such as interleukin-24 and granulocyte-macrophage colony-stimulating factor (GM-CSF).5–7 To measure the antitumor activity of these strategies, sensitive quantitative methods are needed to evaluate MRD. Measuring MRD response in the bone marrow (BM) should be of particular importance, since BM is typically involved at diagnosis and is often the first site of recurrence. An adjuvant treatment of interest is the application of anti-GD2 antibody therapy following the activation of myeloid effectors with GM-CSF. GM-CSF has been shown to potentiate the antitumor effects of anti-GD2 monoclonal antibody 3F8 both in vitro and in patients.7,8 This immunotherapy is administered every 1 to 2 months, and in some patients, for up to 2 years. To use early marrow response as a prognostic marker is particularly relevant for patients not likely to benefit from this therapy. We studied the molecular response of marrow NB using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) of GD2 synthase (ß1,4-N-acetylgalactosaminyltransferase, EC 2.4.1.92), the key enzyme required for the synthesis of ganglioside GD2,9 an antigen highly expressed in NB. GD2 has relatively little heterogeneity within tumors or among patients, and is rarely modulated or lost following 3F8 therapy.10 GD2 synthase mRNA has demonstrated potential as a molecular marker of NB in the BM.11–13 Its transcript levels correlate with the number of GD2-positive tumor cells in the BM,11 and has clinical utility in monitoring efficacy in marrow purging,14 as well as in radioimmunotherapy.15 In this article, we measured the transcript levels of patients with GD2-positive NB undergoing 3F8/GM-CSF therapy. While focusing on patients treated at the time of minimal disease (ie, with complete remission or very good partial remission [CR/VGPR]), we compared their marrow responses with those of patients with clinical evidence of disease (primary refractory, secondary refractory, or progressive disease [PD]) on protocol entry. Marrow NB response was assessed before the third cycle of 3F8 or GM-CSF treatment, at a median time of 1.8 months from the start of therapy. Here, we report the prognostic importance of early marrow nonresponse to this biologic therapy.
Patients Eighty-six stage 4 NB patients, all older than 1 year at diagnosis and enrolled onto protocol 94–18 at Memorial Sloan-Kettering Cancer Center (MSKCC), were the subjects of this analysis. They were staged according to the International Neuroblastoma Staging System,16 and grouped according to their disease status immediately before 3F8/GM-CSF treatment. Extent of disease work-up used computed tomography and/or magnetic resonance imaging, 99mTc- bone scan, 131I- or 123I-metaiodobenylguanidine scan, urinary catecholamine metabolites, as well as histologic examinations of BM aspirates and biopsy specimens. Thirty-three patients were in CR/VGPR; 33 patients had primary refractory disease, having failed to respond to induction chemotherapy; 10 patients had secondary refractory disease (ie, recurrent NB refractory to retrieval therapy); and 10 patients had PD. Written informed consent was obtained from the patients and/or their guardians in accordance to the guidelines of the institutional review board of MSKCC. Regimen for the administration of 3F8 and yeast-derived recombinant GM-CSF (Immunex Corp, Seattle, WA) was detailed previously.7 3F8/GM-CSF immunotherapy was administered at 1- to 2-month intervals, for up to 24 months from study entry. This treatment was continued until patients either developed PD or had a total of four cycles plus elevated human antimouse antibody titer.17 PD was defined in accordance with International Neuroblastoma Staging System response criteria.16
Histologic Examinations of Bone Marrow Samples
Real-Time Quantitative RT-PCR
Statistical Analysis
Relationship Between Clinical Groups and Patient Survival Patients (N = 86) were classified into four clinical groups according to their disease status before 3F8/GM-CSF treatment: CR/VGPR, primary refractory, secondary refractory, and PD. Kaplan-Meier survival analyses illustrated their probability of disease progression (Fig 1A  ) and death (Fig 1B).
Detection of Tumor Cells in Pretreatment BM by GD2 Synthase mRNA and Histologic Evaluation Molecular detection of tumor cells was evident in pretreatment BM of all four clinical groups (Table 1 ). The number of patients with positive GD2 synthase was 40% in the CR/VGPR group, 52% in the primary refractory group, 60% in the secondary refractory group, and 80% in the PD group. The median GD2 synthase transcript level ranged from 10.0 in the CR/VGPR group, to 87.2 in the PD group. This improvement in sensitivity of tumor cell detection was most notable among CR/VGPR patients, whose pretreatment BM samples were all negative by histologic evaluations. As shown in Table 2, 27 marrow samples were positive by RT-PCR but negative by histologic examination, whereas only four samples were positive by histologic analyses and negative by RT-PCR. Three samples were from the primary refractory group, and in one of these samplings, only a single cluster of NB was found after reviewing multiple marrow smears from all four sites. The fourth sample was from the secondary refractory group.
Early Response by GD2 Synthase mRNA and Histologic Evaluation Seventy-four of 86 patients were assessed for early response before treatment cycle 3 at 1.8 months from protocol entry; 22 were assessed after cycle 1; and 52 were assessed after cycle 2. Twelve CR/VGPR patients did not have posttreatment marrow samples before cycle 3 and were excluded from the response analysis. Molecular response was defined as follows: "responders" were patients whose pretreatment marrow samples were positive by RT-PCR, and whose posttreatment marrow samples were negative; "nonresponders" were patients whose posttreatment marrow samples were positive — a few nonresponders had marrow samples negative before therapy, while others’ remained positive before and after treatment. Patients whose pre- and posttreatment marrow samples were negative for GD2 synthase transcript were deemed nonassessable for response (Table 3 ). Among those with molecular evidence of NB, response was achieved in 77% of CR/VGPR group, 45% of primary refractory group, 25% of secondary refractory group, and 0% among patients with PD. In the CR/VGPR group, all but one patient had negative marrow histology before and after treatment, and was thus not assessable for histologic response. The molecular and histologic response rates in the other three clinical groups were comparable.
Prognostic Importance of Early Marrow Response on Patient Survival Outcome Seventy-four patients with marrow samples available for early response assessment were included in the Kaplan-Meier analysis (Fig 2 ). There was a highly significant advantage in PFS among patients who had molecular response, versus those who did not (P < .0001). In contrast, PFS curves of histologic responders and nonresponders were nearly superimposable (data not shown).
Prognostic Importance of Early Molecular Response of Marrow MRD on Progression-Free Survival Of particular interest were patients with marrow MRD (ie, their pretreatment BM samples were histologically negative). For early response analysis, these patients had their marrow samples evaluated before cycle 3. Their clinical disease statuses immediately before protocol entry were: CR/VGPR (n = 21), primary refractory (n = 23), secondary refractory (n = 5), and PD (n = 3). Molecular responders (median follow-up, 29.8 months) were significantly less likely to relapse than nonresponders (median follow-up, 7.7 months; P = .0001; Fig 3 ). Patients with negative transcript before and after 3F8/GM-CSF treatment had a median follow-up of 19.4 months.
Using GM-CSF to enhance anti-GD2 immunotherapy is a promising treatment strategy directed at microscopic stage 4 NB. This therapy has painful side effects because of cross-reactivity with peripheral pain fibers. The ability to use early marrow response as a prognostic marker can spare nonresponding patients from unnecessary discomfort, and use alternative treatment options in a more timely fashion. Furthermore, since the patients who are most likely to benefit from this therapy are those with CR/VGPR and without assessable disease, molecular detection of minimal residual NB in marrow can provide a surrogate measure of antitumor activity that is not possible with standard radiographic and histologic methods. MRD in the BM can be detected by immunologic and molecular methods. While conventional histologic examination has a detection sensitivity of 1/103; limit of detection for immunocytology is 1/105 to 1/106, whereas RT-PCR has sensitivity in the range of 1/105 to 1/107.20 Since examinations of BM biopsy and aspirates are a requisite part of extent-of-disease evaluation, histologic analysis has been the gold standard for assessing BM response in NB patients. However, to push sensitivity to new limits, techniques for detecting MRD with specificity for NB and no cross-reactivity with normal blood or marrow cells need to be developed and validated. Immunocytology can provide an excellent surrogate for response evaluation; however, freshly collected samples are required.18,21 In previous reports, we demonstrated that two molecular markers, GAGE19 and GD2 synthase,12 have superior sensitivity as well as specificity in the detection of MRD in NB. In addition, RT-PCR methods permit repeat testing of archived samples. Marker positivity was strongly correlated with poor clinical outcome.12,22 The development of a quantitative RT-PCR assay to measure GD2 synthase transcript has rendered this marker even more informative.11 Besides having a wide linear dynamic range, it has superior sensitivity and reproducibility, with the potential for high throughput capacity.
In this report, the percentage of patients whose marrow samples were positive for GD2 synthase was lowest among CR/VGPR patients (42%), and highest among those with PD (80%), representing the two ends of the disease spectrum. As one might expect, the median levels of GD2 synthase transcript also reflected this. However, since patients might have a relatively "clean" marrow despite refractory NB elsewhere in the body, the level of transcript in the BM was variable among patients. When compared with histologic detection on the aspirate samples, GD2 synthase RT-PCR was substantially more sensitive (Table 2 Most patients with favorable response at 1.8 months after study entry were from the CR/VGPR group. Although all patients were antibody-naive, the extent of disease and the proliferative tempo were different among the four clinical groups. These factors were expected to influence the response equation. We noted that patients with negative GD2 synthase before and following 3F8/GM-CSF treatment (though not assessable for response) had a more favorable clinical outcome than nonresponders. All of these patients had documented GD2 positivity in their tumor cells, as well as a history of marrow disease before their entry onto this protocol. Similar prognostic implication of persistent molecular negativity was found in our previous analysis of patients undergoing radioimmunotherapy, which was another novel treatment strategy.15 We reasoned that among these persistently negative patients, a small subset might have already been cured before they entered 3F8/GM-CSF therapy, while the large majority had residual disease, though beyond the current limit of detection. Additional sensitive tumor markers will probably be needed to measure disease in this latter subset, including the use of a well-established NB marker, tyrosine hydroxylase. Burchill et al23 have previously reported that the presence of this transcript in the peripheral blood of NB patients off treatment, who were clinically free of disease, was highly prognostic of their eventual outcome.
The utility of MRD measurement was best illustrated among patients in the CR/VGPR group, which was most likely to benefit from immunotherapy. Given their subclinical disease with pre- and posttreatment BM that was histologically negative, therapeutic efficacy was not assessable by conventional methods (Table 3 The importance of early response indicators echoes that found in childhood acute lymphoblastic leukemia, for which MRD can now be measured by PCR,24–26 and early response to induction chemotherapy is strongly predictive of long-term outcome.27–29 Ours is the first report demonstrating the prognostic importance of early molecular response in a metastatic pediatric solid tumor. It is also the first application of this technology to immunotherapy using antibody 3F8 in combination with cytokine GM-CSF. The quantitative nature of such measurements will allow more precise comparisons of antitumor activity among various modalities, agents, or schedules. Most importantly, the ability to identify a subset of patients who are unlikely to benefit from this adjuvant therapy and are at a great risk of relapse may provide the rationale for a more timely application of alternative treatment options.
The authors indicated no potential conflicts of interest.
We thank K. Kramer, MD; S. Modak, MD; E. Dantis, RN; C. Enero, RN; and L. Jones, RN for their clinical management. We also thank K. Yataghene, MD; K. Danis; and Y. Podzikowski for data management. For this clinical trial, GM-CSF was generously supplied by Immunex Corp, Seattle, WA.
Supported in part by grants from the National Institutes of Health (CA095742) and the Robert Steel Foundation, Hope Street Kids, the Pediatric Cancer Foundation, and the Katie-Find-a-Cure Fund.
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17. Cheung NK, Cheung IY, Canete A, et al: Antibody response to murine anti-GD2 monoclonal antibodies: Correlation with patient survival. Cancer Res 54:2228–2233, 1994 18. 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] 19. 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] 20. Ross AA: Minimal residual disease in solid tumor malignancies: A review. J Hematother 7:9–18, 1998[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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ABSTRACT
INTRODUCTION
5 was defined as negative. 
