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Long-Term Survivors of Advanced Neuroblastoma With MYCN Amplification: A Report of 19 Patients Surviving Disease-Free for More Than 66 Months

From The Study Group of Japan for Treatment of Advanced Neuroblastoma, Izumi, Chiba, Tsukuba, and Gunma, Japan.

Address reprint requests to Yoshiaki Tsuchida, MD, Gunma Children's Medical Center, 779 Shimohakoda, Hokkitsu, Seta-gun, Gunma 377-8577, Japan; email tuchida{at}gcmc.pref.gunma.jp

	ABSTRACT

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PURPOSE: According to initial reports, stage 4 neuroblastoma patients with amplification of the MYCN proto-oncogene developed progressive disease within 8 months. The prognosis for such patients, however, should now be reevaluated in light of recent results achieved with up-to-date combination chemotherapy.

PATIENTS AND METHODS: Patients with stage 3, 4, and 4S neuroblastoma and more than 10 copies of MYCN received induction chemotherapy, which from January 1985 to February 1991 consisted of regimen A₁ (cyclophosphamide 1,200 mg/m² on day 1, vincristine 1.5 mg/m² on day 1, pirarubicin 40 mg/m² on day 3, and cisplatin 90 mg/m² on day 5) and from March 1991 to September 1993 consisted of regimen A₃ (cyclophosphamide 1,200 mg/m² on days 1 and 2, pirarubicin 40 mg/m² on day 3, etoposide 100 mg/m² on days 1 through 5, and continuous infusion cisplatin 25 mg/m² on days 1 through 5). Most of these patients underwent radical surgery to remove the original tumor and local metastases, irradiation, and supralethal preconditioning regimens, followed by blood stem-cell transplantation (SCT). Data on the patients were collected in December 1998, and the factors contributing to disease-free survival were analyzed.

RESULTS: During the study period, 66 patients with more than 10 copies of MYCN were treated. Five of nine patients with stage 3 disease, 13 of 55 with stage 4, and one of two with stage 4S survived for at least 66 months. It is interesting that all but one patient who survived for more than 66 months underwent SCT, in contrast with only five of 45 patients who died.

CONCLUSION: Not all patients with advanced neuroblastoma who have more than 10 copies of MYCN will die. The requisites for survival in such patients seem to be intensive induction chemotherapy, effective surgery, irradiation, and the use of SCT.

	INTRODUCTION

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IT IS WIDELY ACCEPTED that the most important prognostic factors in neuroblastoma are patient age, disease stage, and degree of MYCN amplification.^1-3 Early communications regarding MYCN amplification and prognosis reported that of 11 patients with stage 4 disease and amplified MYCN, all had progressive disease within 8 months after diagnosis,^1,2 and that most patients with amplified MYCN died of the disease within approximately 1 year, regardless of disease stage.^4-8

However, after receiving intensive chemotherapy and megatherapy with stem-cell rescue,^5,9-11 many patients can be expected to survive much longer. In this communication, we report the clinical details of long-term survivors among high-risk patients who were enrolled in a study conducted by the Study Group of Japan for Treatment of Advanced Neuroblastoma.

	PATIENTS AND METHODS

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Patients enrolled between January 1985 and September 1993 were analyzed. Clinical stage was classified according to the International Neuroblastoma Staging System (INSS).^12,13

Amplification of the genomic DNA sequence of MYCN was studied using the Southern blot technique at the Special Reference Laboratories, Tokyo, Japan. Investigators at all participating institutions (see Appendix) were requested to send samples of surgical specimens to the Special Reference Laboratories, on which Southern blot hybridization was carried out as follows: DNA (10 µg) was digested completely with EcoRI, electrophoresed in 0.8% agarose gel, and blotted onto a nylon filter membrane (Zeta-Probe, Bio-Rad, Hercules, CA) using the standard Southern blot transfer method. Hybridization was performed with an EcoRI fragment containing the first exon of the human MYCN gene. The probe was labeled with alpha phosphorus-32 dCTP.⁹

Induction chemotherapy regimens administered to neuroblastoma patients with 10 or more copies of MYCN are listed in Table 1. Between January 1985 and February 1991, regimen A₁ was used, which consisted of cyclophosphamide 1,200 mg/m² administered as an intravenous infusion over 6 hours on day 1, vincristine 1.5 mg/m² on day 1, pirarubicin 40 mg/m² on day 3, and cisplatin 90 mg/m² on day 5. However, from March 1991 to September 1993, the regimens designated as A₃ and new A₁ were used. Regimen A₃ consisted of cyclophosphamide 1,200 mg/m² administered as an intravenous infusion over 6 hours on days 1 and 2, pirarubicin 40 mg/m² on day 3, etoposide 100 mg/m² on days 1 through 5, and cisplatin 25 mg/m² administered as a continuous infusion on days 1 through 5; regimen new A₁ consisted of cyclophosphamide 1,200 mg/m² administered as an intravenous infusion over 6 hours on day 1, pirarubicin 40 mg/m² on day 3, etoposide 100 mg/m² on days 1 through 5, and cisplatin 90 mg/m² on day 5. The office of the Study Group of Japan for Treatment of Advanced Neuroblastoma requested all of the institutions to perform biopsies before treatment and to treat patients with one cycle of regimen new A₁ while waiting for the results of the Southern blot analyses. If the copy number of the MYCN DNA sequence was found to be less than 10, then the patients continued to receive further courses of regimen new A₁ until a total of six cycles was reached. However, if the copy number of the MYCN DNA sequence was found to be 10, then the patients received five courses of the much more intensive regimen, A₃, until a total of six cycles was reached. In regimen A₃, the cyclophosphamide dose is double that used in regimen A_1.⁹ However, some institutions preferred to use regimen new A₁, which is similar in intensity to regimen A₁. Patients who received regimen new A₁ after March 1991 were also included in the present analysis.

The treatment of patients with 10 copies of MYCN is indicated in the lower portion of Table 1. First, the patients received induction chemotherapy, and between the third and sixth cycles of chemotherapy, surgery and local irradiation were carried out. After the sixth cycle of either regimen A₁, new A₁, or A₃, patients were randomized by institutional choice between the two arms of continuation chemotherapy and autologous bone marrow transplantation (ABMT)/peripheral-blood stem-cell transplantation (PBSCT), as described elsewhere.⁹

Statistical analysis of clinical results was performed using the ² test, the Cochran-Armitage trend test, and Fisher's exact test.

	RESULTS

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From January 1985 to September 1993, 66 patients with 10 copies of MYCN were treated at the institutions comprising our group. Figure 1 shows the age distribution of 66 patients with MYCN-amplified neuroblastoma in stage 3, 4, or 4S among 186 registered cases. The 186 cases consisted of patients who were 12 months of age or older with stage 3 or 4 disease; a small number of patients younger than 12 months who had stage 4 disease and bone cortex, distant lymph node, and/or remote organ metastases;¹⁴ and a small number of patients with stage 4S disease with amplified MYCN. The incidence of MYCN-amplified neuroblastoma was highest in the group of patients who were 1 year of age at diagnosis (24 of 41 patients, or 59%), and it declined with age in older patients aged 2 to 4 years (27 of 86 patients, or 31%).

View larger version (18K): [in this window] [in a new window]   Fig 1. Distribution of MYCN amplification in stage 3, 4, and 4S neuroblastoma patients according to age at diagnosis.

No patients with stage 3 disease and more than 10 copies of MYCN had any gross total resection of the main tumor and regional lymph nodes at diagnosis. Of 55 patients with stage 4 disease and more than 10 copies of MYCN, 39 (70.9%) had extensive bone marrow involvement. Three of the 55 patients were classified as stage 4 on the basis of distant nodal disease in the absence of bone marrow metastases. The remaining 13 patients had either bone cortex metastases, remote organ metastases, or both, in the absence of bone marrow metastases.

The patients were followed-up until death or for at least 66 months if they remained alive. Among the 66 patients, 21 survived for more than 50 months; two of them, however, died afterward (disease, one patient; accident, one patient). The remaining 19 survived for more than 66 months. Except for patient no. 1, who experienced recurrence but achieved a second complete remission (CR), the remaining 18 patients were in CR and event-free survival continuously. Their follow-up period averaged 94 months (range, 66 to 155 months). Patient no. 1 developed recurrence in the skull at 57 months after diagnosis. However, after local and systemic therapy was administered, this patient remained relapse-free for the next 98 months and remains as such at the present time. MYCN amplification was not present in the recurrent tumor.

Table 2 lists the characteristics of 19 long-term survivors. Among the nine patients with stage 3 disease and amplified MYCN, five (56%) survived and four died. Among the 55 stage 4 patients with amplified MYCN, 13 (24%) were long-term survivors with no evidence of disease and 42 eventually died. It seems that the results of treatment were better in stage 3 disease, but there were no statistical differences in survival rate between stages 3 and 4. One of the two stage 4S patients survived.

Clinical outcomes of patients with amplified MYCN were analyzed according to the therapeutic modality. Among the 23 patients treated with the A₁ induction chemotherapy regimen, six (26%) were long-term survivors and 17 died. Among the 34 patients treated with the A₃ regimen, 11 (32%) were long-term survivors and 23 died (Fig 2). Results of treatment with the A₃ induction chemotherapy regimen seemed better than those achieved with the A₁ regimen, but there were no statistical differences in survival between patients treated with regimens A₁ and A_3, nor between those with the new A₁ and A₃ regimens (Fig 2).

View larger version (13K): [in this window] [in a new window]   Fig 2. Overall survival curve for MYCN-amplified neuroblastoma patients relative to induction chemotherapy.

Among the 25 patients who underwent ABMT or PBSCT, 18 (72%) were long-term survivors and seven died, whereas among 41 patients who did not undergo ABMT/PBSCT or failed to achieve CR to undergo ABMT/PBSCT, all but one died (Fig 3).

View larger version (12K): [in this window] [in a new window]   Fig 3. Overall survival curve for MYCN-amplified neuroblastoma patients relative to treatment after induction chemotherapy. A, patients who underwent ABMT/PBSCT; B, patients who did not undergo ABMT/PBSCT.

	DISCUSSION

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We previously reported our results of treatment of advanced neuroblastoma relative to MYCN amplification⁴ and relative to treatment with bone marrow transplantation.¹⁰ In the present analysis, it seems that the results of ABMT in patients with advanced neuroblastoma and amplified MYCN are surprisingly promising, but these results should be evaluated carefully, because our group recommended that ABMT/PBSCT be performed only when patients are in CR.^10,14

We also noted that the results of treatment with the A₃ regimen were much better than those with the A₁ regimen in a combined series of patients with MYCN-amplified and -unamplified tumors.⁹ According to a previous report,⁹ 70% of MYCN-amplified stage 4 neuroblastoma patients achieved CR with the A₃ regimen, whereas only 33% of the same group of patients obtained CR with the A₁ regimen. In the present analysis, 11 (32%) of 34 patients treated with the A₃ induction chemotherapy regimen were long-term survivors, whereas six (26%) of 23 patients treated with the A₁ regimen were long-term survivors. It is expected that further study will clarify how regimen A₃ increases the number of patients who achieve CR and are able to undergo ABMT/PBSCT. The percentage of MYCN-amplified patients was higher in the group of patients who were 1 year of age than in the group of patients who were 2 to 4 years of age, but the survival rates in both MYCN-amplified groups were 37.5% (nine of 24) and 27% (seven of 26), respectively, with no statistical difference (P > .05).

It is generally accepted that patients who undergo ABMT/PBSCT during CR do better than those who do during partial remission (PR).^5,10,11,15 Therefore, intensified induction chemotherapy such as regimen A₃ seems essential to treating high-risk neuroblastoma patients and achieving CR. All but one of the 19 long-term survivors underwent either ABMT or PBSCT. This would indicate, although not prove, the important role of megatherapy and ABMT/PBSCT in the treatment of high-risk neuroblastoma patients with amplified MYCN. It is not possible at present to prove the definitive role of the megatherapy and ABMT/PBSCT alone in treating such high-risk patients with amplified MYCN, because our study was not designed to randomize patients between treatment with and without ABMT/PBSCT. Stram et al⁵ recently reported that among six stage 4 patients with MYCN amplification who achieved CR and underwent ABMT, four were surviving, whereas seven other patients with MYCN amplification who achieved CR and received continued chemotherapy all died. In a series conducted from 1985 to 1994 of 23 stage 4 neuroblastoma patients with MYCN amplification, Stram et al⁵ found that only four patients (17%) survived for more than 28 months. The disease-free survival rate of MYCN-amplified stage 4 neuroblastoma patients would seem higher in the present series (24%) than in the series of Stram et al.⁵ Patients were followed-up much longer in the present series (66 months or longer) than in their series (28 months or longer).⁵

The results of treatment of neuroblastoma patients with MYCN amplification have been improving gradually.^4-8 After reviewing the clinical results in all patients, we believe that to achieve long-term survival in MYCN-amplified neuroblastoma patients, it is essential that they undergo intensive induction chemotherapy, radical surgery, effective irradiation, and ABMT/PBSCT without any major delay in the time sequence. Stage 3 rather than 4 disease, age of 1 year rather than 2 to 4 years, and induction chemotherapy with regimen A₃ rather than A₁ seem to be factors that contribute to the long-term survival of MYCN-amplified neuroblastoma patients, although this is not conclusive because no statistically significant differences were found in the present analysis. Review of the clinical courses of some patients with amplified MYCN who failed to survive clearly shows that the disease in these patients was truly progressive, and if the patients were treated inappropriately, recurrence resulted.

We would emphasize the need to treat without any major delay in the time sequence of multimodal treatment. However, six patients in Table 2 actually underwent transplantation at 12 or more months after diagnosis. One possibility is that patients with highly chemotherapy-sensitive tumors will do well, regardless of whether or not they receive maximally intensive treatment, whereas patients with tumors that are relatively insensitive to chemotherapy will have progressive disease early in the treatment.

It is of interest to compare results in the MYCN-amplified stage 4 (> 1 year old) patients with results in the non–MYCN-amplified stage 4 (> 1 year old) patients, because it might be that MYCN amplification is not a prognostic factor in children with stage 4 disease. On this point, we found in 1994 that the survival rate for stage 4 patients without MYCN amplification was better than that of stage 4 patients with MYCN amplification, the difference being statistically significant (P = .0038).⁴ Even now, we find that there is a significant difference between stage 4 patients (> 1 year old) with and without MYCN amplification, even though the survival curves for these two groups of patients have become closer in recent years, according to the authors' observations.¹⁶ The 2-year overall survival rate was 74.1% for stage 4 patients (> 1 year old) without MYCN amplification and 45.5% for stage 4 patients (> 1 year old) with MYCN amplification, and this difference was statistically significant (P = .0385).¹⁶

In conclusion, we clarified that not all patients with MYCN-amplified neuroblastoma will die. Nineteen (29%) of the 66 patients in the present study were long-term disease-free survivors for more than 66 months. There have been very few reports⁵ to date on the detailed characteristics of long-term survivors.

	APPENDIX: Participating Institutions and Main Investigators

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Gunma Children's Medical Center, Gunma (Y. Tsuchida); University of Tsukuba, Tsukuba (M. Kaneko); Hokkaido University (F. Sasaki) and Sapporo National Hospital (T. Takeda), Sapporo; Niigata University, Niigata (M. Iwafuchi); Ibaraki Children's Hospital, Mito (H. Kenmotsu); Jichi University, Tochigi (S. Makino); Saitama Children's Medical Center, Iwatsuki (K. Yamamoto); Chiba University (N. Ohnuma) and Chiba Children's Hospital (T. Etoh), Chiba; National Cancer Center (M. Ohhira), National Children's Hospital (T. Honna), Nihon University (H. Mugishima), University of Tokyo (F. Bessho), Keio Univesity (J. Yokoyama), Juntendo University (K. Ishimoto), and Jikei University (J. Yoshizawa), Tokyo; Metropolitan Children's Hospital, Kiyose (A. Hayashi); Kanagawa Children's Medical Center, Yokohama (H. Nishihira); St. Marianna University, Kawasaki (K. Nakada); Tohkai University, Isehara (S. Yokoyama); Kitasato University, Sagamihara (K. Ishida); Yamanashi Medical College, Yamanashi (K. Takano); Shizuoka Children's Hospital, Shizuoka (J. Mimaya); Kanazawa Medical University, Ishikawa (Y. Takahara); Nagoya City University, Nagoya (S. Kondo); Mie University, Tsu (M. Sakurai); Kyoto Prefectural University of Medicine, Kyoto (T. Sawada); Nara Medical University, Kashiwara (A. Yoshioka); Osaka City Children's Medical Center (N. Nagahara), Osaka City University (K. Yamato), and Osaka University (A. Okada), Osaka; Osaka Medical Center for Maternal and Child Health, Izumi (K. Kawa); Kinki University, Osaka-Sayama (H. Miyata); Hyogo University, Nishinomiya (A. Toyosaka); Hiroshima University, Hiroshima (E. Hiyama); Kyushu University (S. Suita) and National Kyushu Cancer Center (J. Okamura), Fukuoka; Kumamoto University, Kumamoto (Y. Sera); and Kagoshima University, Kagoshima (H. Takamatsu).

	ACKNOWLEDGMENTS

 
Supported in part by a grant-in-aid for cancer research (no. 9-14) from the Ministry of Health and Welfare of the Government of Japan.

We thank C. Yenches for editorial assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION APPENDIX: Participating... REFERENCES

 
1. Brodeur GM, Seeger RC, Schwab M, et al: Amplification of N-myc in untreated human neuroblastoma correlates with advanced stage disease. Science244:1121-1124, 1984

2. Seeger RC, Brodeur GM, Sather H, et al: Association of multiple copies of N-myc oncogene with rapid progression of neuroblastoma. N Engl J Med313:1111-1116, 1985[Abstract]

3. Tsuchida Y, Hemmi H, Inoue A, et al: Genetic clinical markers of human neuroblastoma with special reference to N-myc oncogene: Amplified or not amplified? Tumour Biol17:65-74, 1996[Medline]

4. Suita S, Zaizen Y, Kaneko M, et al: What is the benefit of aggressive chemotherapy for advanced neuroblastoma with N-myc amplification? A report from the Japanese Study Group for Treatment of Advanced Neuroblastoma. J Pediatr Surg20:746-750, 1994

5. Stram DO, Matthay KK, O'Leary M, et al: Consolidation chemoradiotherapy and autologous bone marrow transplantation versus continued chemotherapy for metastatic neuroblastoma: A report of two concurrent Children's Cancer Group studies. J Clin Oncol14:2417-2426, 1996[Abstract]

6. Rubie H, Hartmann O, Michon J, et al: N-myc gene amplification is a major factor in localized neuroblastoma: Results of the French NBL 90 Study. J Clin Oncol15:1171-1182, 1997[Abstract/Free Full Text]

7. Bowman LC, Castleberry RP, Cantor A, et al: Genetic staging of unresectable or metastatic neuroblastoma in infants: A Pediatric Oncology Group Study. J Natl Cancer Inst89:373-380, 1997[Abstract/Free Full Text]

8. Katzenstein HM, Bowman LC, Brodeur GM, et al: Prognostic significance of age, MYCN oncogene amplification, tumor cell ploidy, and histology in 110 infants with stage D(S) neuroblastoma: The Pediatric Oncology Group experience—A Pediatric Oncology Group Study. J Clin Oncol16:2007-2017, 1998[Abstract]

9. Kaneko M, Nishihira H, Mugishima H, et al: Stratification of treatment of stage 4 neuroblastoma patients based on N-myc amplification status. Med Pediatr Oncol31:1-7, 1998[Medline]

10. Ohnuma N, Takahashi H, Kaneko M, et al: Treatment combined with bone marrow transplantation for advanced neuroblastoma: An analysis of patients who were pretreated intensively with the protocol of the Study Group of Japan. Med Pediatr Oncol24:181-187, 1995[Medline]

11. Garaventa A, Rondelli R, Lanino E, et al: Myeloablative therapy and bone marrow rescue in advanced neuroblastoma: Report from the Italian Bone Marrow Transplant Registry. Bone Marrow Transplant18:125-130, 1996[Medline]

12. Brodeur GM, Seeger RC, Barrett A, et al: International criteria for diagnosis, staging and response to treatment in patients with neuroblastoma. J Clin Oncol6:1874-1881, 1988[Abstract]

13. Brodeur GM, Pritchard J, Berthold F, et al: Revisions of the international criteria for neuroblastoma diagnosis, staging and response to treatment. J Clin Oncol11:1466-1477, 1993[Abstract/Free Full Text]

14. Sawaguchi S, Kaneko M, Uchino J, et al: Treatment of advanced neuroblastoma with emphasis on intensive induction chemotherapy: A report from the Study Group of Japan. Cancer66:1879-1887, 1990[Medline]

15. 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 Oncol16:953-965, 1998[Abstract]

16. Kaneko M, Tsuchida Y, Uchino J, et al: Treatment results of advanced neuroblastoma with the First Japanese Study Group Protocol: Study Group of Japan for Treatment of Advanced Neuroblastoma. J Pediatr Hematol Oncol21:190-197, 1999[Medline]

Submitted February 9, 1999; accepted June 18, 1999.

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