¹Pediatrics Department, Institut Gustave Roussy, Villejuif, France

²Biostatistics Department, Institut Gustave Roussy, Villejuif, France

³Hematology Unit, Institut Gustave Roussy, Villejuif, France

⁴Radiology Department, Institut Gustave Roussy, Villejuif, France

⁵Nuclear Medicine Department Institut Gustave Roussy, Villejuif, France

Correspondence to: Dr D Valteau-Couanet, Pediatrics Department, Institut Gustave Roussy, Rue Camille Desmoulins, 94800 Villejuif, France

Although infants with stage 4 neuroblastoma (NB) usually have a good prognosis, metastatic relapses after 1 year of age and amplification of the N-myc oncogene are established poor prognostic factors. In order to improve the survival of patients with such high-risk factors, we performed consolidation with a busulfan (600 mg/m²)-melphalan (140 mg/m²)-containing regimen followed by autologous stem cell transplantation (SCT). From 1986 to 1998, 12 patients were treated according to this strategy. Their median age at diagnosis was 9 months (1-11). Consolidation was performed after a metastatic relapse in five children, because of persistent bone metastases in one and as first-line consolidation in six patients whose tumor exhibited N-myc amplification. The 5-year EFS rate is 64.5% (36-85%) with a median follow-up of 92 months (20-126). One toxicity-related death occurred in a very heavily pretreated patient. Hepatic veno-occlusive disease was the major side-effect that occurred in nine of 12 children. This busulfan-melphalan combination appears to dramatically improve the prognosis of these high-risk infants with metastatic NB. Given its high toxicity, indications for this consolidation must be restricted to high-risk infants and a lower dose of busulfan (480 mg/m²) is recommended in children weighing less than 10 kg. Bone Marrow Transplantation (2000) 25, 937-942.

neuroblastoma; infant; SCT; busulfan

Neuroblastoma (NB) is the most common solid tumor affecting children under 1 year of age. Twenty percent of these children have stage 4s disease according to the definition proposed by Evans et al in 1971¹ and 10% have stage 4 disease with, as stipulated in the revised International Neuroblastoma Staging System,² either bone marrow involvement exceeding 10% or radiological evidence of bone metastases.³

Patients with stage 4 NB diagnosed under 1 year of age usually have a good prognosis^2,4,5 although they receive less aggressive treatment than older patients. However, not all of these good prognosis cases fare well. For a long time, the subgroup of children with a poor prognosis could only be identified once relapses had occurred. As N-myc amplification (NMA) has recently been demonstrated to be a significant indicator of a poor outcome, these less favorable cases may now be detected and treated more rapidly.^6,7,8,9,10

Since the mid-1980s we have been developing an intensification strategy in infants with metastatic NB. Administered either after recurrent disease or when N-myc amplification is demonstrated in their tumor, this intensive consolidation therapy consists of a busulfan (Bu)-melphalan (Mel)-containing regimen followed by autologous stem cell transplantation (SCT) and is similar to the conditioning regimens used in older patients with stage 4 NB.

Here we present the results of this aggressive consolidation strategy applied to very young children.

Patients and methods

Patients

From 1986 to 1998, 73 children with metastatic NB diagnosed before 1 year of age were treated in the Pediatrics Department of the Institut Gustave Roussy. Among them, 12 received consolidation with a busulfan-melphalan (Bu-Mel)-containing high-dose chemotherapy (HDC) regimen followed by SCT either as a consolidation after treatment of a metastatic relapse or as a first-line consolidation in patients with an NMA tumor. Their median age at the time of the diagnosis and HDC was 9 months (1-11) and 17.5 (12-35) respectively. There were six boys and six girls. Their clinical status at diagnosis is detailed in Table 1. Urinary catecholamines were elevated in all patients. They all received conventional chemotherapy either at diagnosis or at relapse or both, according to the on-going French Society of Paediatric Oncology (SFOP) protocols.^5,11,12 Combinations of the following drugs were administered: cyclophosphamide, doxorubicin, vincristine, cisplatinum, etoposide and carboplatin (Table 2). One patient (No. 10) received two courses of high-dose thiotepa (600 mg/m²) followed by peripheral blood stem transplantation (PBSCT) before Bu-Mel. In another patient (No. 2), initial treatment consisted of radiation therapy to the liver with 1.5 Gy being delivered in three consecutive sessions. One patient (No. 7) received radiotherapy to the orbit after two courses of CADO because of disease progression with sight-threatening lesions.

All patients underwent surgical excision of their primary tumor. Response to therapy was assessed according to INSS criteria.¹

Since 1990 N-myc oncogene evaluation has been planned for all tumors. Results were considered reliable only if tumor material contained over 10% of neuroblasts. N-myc genomic content was determined using Southern blotting with second exon probes. N-myc amplification (NMA) was defined as >10 copies per haploid genome.

HDC was administered as consolidation therapy, after treatment of a relapse in five patients whose N-myc status was either unknown (4) or not amplified (1). The bone was the site of recurrent disease in all of these patients and lesions were associated with bone marrow involvement in four. Their median age at the time of relapse was 22 months (range 21-30). For the seven remaining patients, HDC was given as consolidation therapy immediately after first-line chemotherapy to six patients with an NMA tumor and to one patient with persistent bone metastases in spite of eight courses of conventional chemotherapy.

Treatment regimens

The HDC regimen consisted of either a combination of busulfan, cyclophosphamide and melphalan or of busulfan and melphalan. Busulfan was administered orally (37.5 mg/m² every 6 h) over 4 days. Then cyclophosphamide (2.2 g/m²/day) was infused intravenously over 2 consecutive days. On the last day of the HDC regimen, melphalan (140 mg/m²) was administered as an i.v. bolus through a central venous line.

In five patients (Nos 5, 6, 7, 11 and 12) who weighed less than 10 kg, the dose of busulfan was reduced to 120 mg/m²/day (total dose: 480 mg/m²) and cyclophosphamide was administered at a maximum dose of 200 mg/kg in patients 5 to 7.

Transplantation procedures

Hematopoietic stem cell transplantation was performed within 48 h after the end of the melphalan infusion. Autologous bone marrow was grafted in nine patients and peripheral stem cells obtained by leukapheresis in three. Five of the 12 patients received G-CSF after SCT.

Supportive care

All children were isolated in single laminar air-flow rooms. Standard supportive care was provided, as previously described.¹³ During HDC, patients were hydrated with 5% dextrose and the usual concentrations of electrolytes (3 l/m²/day).

Prophylaxis against busulfan-related seizures was provided with continuous intravenous clonazepam from the first day of HDC to the day on which the SCT was performed.¹⁴

Evaluation of toxicity

Neutrophil recovery was defined as a blood neutrophil count above 0.5 ´ 10⁹/l and platelet recovery as a blood platelet count above 50 ´ 10⁹/l without transfusion. Visceral toxicity was evaluated according to the criteria defined by Bearman et al.¹⁵ Hepatic veno-occlusive disease was defined clinically according to McDonald's criteria.¹⁶

Post HDC treatment

Radiation therapy was delivered to the primary tumor in five patients (1, 8, 9, 11, 12) and also to residual metastatic disease in patient No. 1. Patient No. 2 received interleukin-2 after HDC according to a schedule described elsewhere.¹⁷

Statistical methods

Survival probabilities were estimated by the Kaplan-Meier method.¹⁸ Survival curves carry Rothman's 95% confidence intervals.¹⁹ Survival curves were compared by the log rank test.²⁰

Results

Survival

In May 1999, eight patients were alive in continuous CR. Their median follow-up post diagnosis was 108 months (20-144) and post HDC and SCT was 92 months (14-126).

Four patients died in total. Three of them relapsed 7, 11 and 13 months post SCT. Two had a metastatic relapse and one, a recurrent primary and metastatic lesions. Of the three patients, one initially had stage 4S disease and an unknown N-myc status and HDC was administered as consolidation therapy after a metastatic relapse. The other two had stage 4 disease and HDC was administered as consolidation therapy while they were in first CR because of NMA. One toxicity-related death occurred. Overall, event-free survival at 5 years is 64% (36-85%) (Figure1).

Toxicity

All patients experienced profound myelosuppression. The median duration of neutropenia (<0.5 ´ 10⁹/l) and thrombocytopenia (<50 ´ 10⁹/l) was 26 days (range 7-42) and 46 days (range 36-657), respectively. Patients were given a median of five RBC transfusions (range 2-50) and a median of 21 platelet transfusions (range 6-99) to counteract anemia and thrombocytopenia.

All but one patient experienced fever for a median duration of 8 days (range 1-146). Three patients developed septicemia related to Staphylococcus epidermidis in two cases and to Candida tropicalis in one case. Infection resolved easily in the former two cases and completely under amphotericin B therapy in the latter case.

Two patients experienced a viral infection: pneumonia due to respiratory syncithial virus during aplasia (1) and varicella (1) within the 6 months post-BMT. Both cases resolved under antiviral therapy.

Three cases of severe hemorrhagic cystitis were observed among the first patients treated. Although severe and requiring multiple blood transfusions, all recovered without late effects. As soon as effective prophylaxis was prescribed (hourly voiding of the bladder under forced diuresis with furosemide) during cyclophosphamide administration, this complication ceased to occur in consecutive patients.

Gastro-intestinal toxicity was frequent and often severe. Half of the patients had moderate or severe vomiting. Mucositis requiring narcotics ( grade 2) occurred in seven patients (four grade 3) and grade 2 diarrhea in seven (one grade 3). Two cases of gastro-intestinal hemorrhage were observed that resolved rapidly without sequelae. Liver toxicity was a major concern since nine of 12 experienced hepatic veno-occlusive disease (HVOD). It was mild in intensity in three, moderate in three and severe in three. All cases resolved completely except for one in whom it was associated with multi-organ failure. That patient, who had previously received two courses of high-dose thiotepa followed by SCT, died of multi-organ failure.

Discussion

During the last decade, HDC followed by autologous bone marrow transplantation has been used extensively as consolidation therapy in stage 4 NB. A randomized study recently demonstrated that it significantly improved the prognosis of NB patients over 1 year of age.²¹ In contrast, most patients with stage 4 NB under 1 year of age and stage 4S NB are usually cured with minimal treatment. The 5-year EFS of infants with stage 4 or 4S neuroblastoma treated with conventional chemotherapy varies between 60 and 85% according to published series.^6,8,10,22 Paul et al,²² who obtained a 75% 5-year EFS rate in 24 patients did not advocate using HDC in these patients. N-myc amplification has however been found to be correlated with a poor prognosis in NB^23,24,25 and was demonstrated to be the most relevant adverse prognostic indicator in localized NB.²⁶ In Bowman's series,⁶ patients with diploid tumors characterized by an amplified N-myc locus represented a particularly unfavorable risk group. In Hartmann's series,⁸ the presence of bone lesions and of NMA were the two major poor prognostic factors. Sixteen of the 17 patients with NMA in Lampert's series died during the study period.⁷ These three authors concluded that as NMA appeared to be a major indicator of a poor outcome, alternative therapies were warranted. In Katzenstein's series, the 3-year survival rate of patients with NMA was 33% (+/- 27%) vs 91% ( +/- 9%) in patients without NMA (P = 0.005).¹⁰ The authors concluded that these patients ought to receive myeloablative therapy with stem cell rescue as consolidation therapy after intensive induction chemotherapy. In Paul's series²² only one out of the seven tumors tested showed NMA, and it was the patient with this tumor who relapsed and died.

Of the 12 patients in the present study, five were treated with HDC after a metastatic relapse that occurred after 1 year of age, and six, with an NMA tumor, were consolidated while in first CR. The last patient had persistent bone metastases after eight courses of conventional chemotherapy. All these patients had a poor prognosis either because of their clinical outcome or because of their biological status (NMA). They all received a busulfan-melphalan-containing regimen followed by SCT. Their 5-year EFS rate is 64% (36-85%) and compares favorably with all published data. In Bowman's study, the 3-year DFS of patients with an NMA tumor was 20% (0-44.8%); all but one of the patients with an NMA tumor died in Lampert's series.

The benefits of consolidation with HDC appear greater in these young children than in older patients¹⁸ even though the conditioning regimens were similar.²⁷ That busulfan plays a major role in the treatment of NB has been demonstrated in xenograft models²⁸ as well as in clinical studies.^27,29,30 Hartmann et al³⁰ showed that a combination of three alkylating agents including busulfan improved the prognosis of poorly responding stage 4 NB in patients over 1 year of age. In addition, a multivariate analysis of 218 patients with stage 4 NB over 1 year of age demonstrated that being younger than 2 years at diagnosis and the presence of a busulfan-melphalan combination in the high-dose conditioning regimen were the two main independent significant prognostic factors. The present study shows that such conditioning regimens can indeed improve the survival of infants with poor prognosis NB. That NB carries a better prognosis in children under 2 years of age has already been demonstrated by the analysis of the EBMT registry,³¹ the CCG studies³² and by the series reported by Hartmann et al.²⁷ Although the number of patients in this latter series is low, the very high DFS emphasizes the prognostic significance of a young age at diagnosis. In poor risk patients, consolidation appears to improve the survival rate all the more when patients are young. No relapses have been observed after 2 years post SCT, which is not usually the case in children over 1 year of age at diagnosis, since survivors at 5 years have a DFS rate of only 80%.³³

The toxicity of the regimen used in these young children was very high. Myelotoxicity can now be attenuated by using peripheral stem cell transplantation.³⁴ Liver toxicity and especially HVOD was the major side-effect. We have shown that high doses of busulfan and the combination of three alkylating agents were the major risk factors in the occurrence of HVOD.³⁵ No patient died of this toxicity and all recovered completely. The incidence and the severity of this complication could probably be decreased by prophylaxis with ursodiol.³⁶

After a busulfan-cyclophosphamide regimen, the incidence of severe HVOD has been related to a high systemic exposure to busulfan³⁷ and dose adjustment is currently investigated by several teams, mainly in the hematological setting, in order to reduce drug-related toxicity. In our institution, we did not embark on such a prospective evaluation of pharmacokinetically guided dose adjustment of high-dose busulfan for two major reasons. First, we did not find a significant correlation between a high systemic exposure to busulfan and severe liver toxicity in the high-dose chemotherapy regimens we have developed for children with solid tumors.³⁸ Second, the wide inter- and intra-patient variability of busulfan disposition following oral dosing may be responsible, in part, for the absence of pharmacokinetic/pharmacodynamic correlation and will greatly jeopardize the accuracy and effectiveness of dose adjustment.³⁹ We strongly believe that the use of i.v. busulfan^40,41 will avoid the consequences of oral absorption and may allow accurate targetting of a maximum tolerated systemic exposure. Appropriate randomized studies will then evaluate the benefit of individual dose adjustment vs the administration of a fixed dose (corrected for body surface area) of i.v. busulfan, especially in infants with stage 4 neuroblastoma.

The only toxicity-related death occurred in a very heavily pretreated patient who had received two courses of high-dose thiotepa before busulfan and melphalan consolidation. We have recently observed that although rapidly sequenced courses of high-dose alkylating agents induced high response rates in poor risk patients, they were complicated by unacceptable visceral toxicity.⁴²

Although the results of this high-dose consolidation strategy are very encouraging, indications must strictly be restricted to infants with demonstrated poor prognostic factors, namely, NMA, metastatic relapses (consolidation in 2nd CR or 2nd good PR) whatever the biological status of the tumor. Toxicity-related complications must be avoided in patients who can be cured with conventional treatment. In order to reduce toxicity in these young children, we recommend administering a combination of busulfan 480 mg/m² and melphalan 140 mg/m² in patients weighing less than 10 kg. In the future, other biological factors such as 17q+ or del 1p, whose prognostic significance has recently been identified,⁴³ should be evaluated prospectively in order to single out infants with a poor prognosis at diagnosis, even in the absence of NMA.

Acknowledgements

We are grateful to L Saint Ange for editing the manuscript, and to Severine Faure for her secretarial assistance.

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Figure 1 Post transplantation EFS of the 12 patients consolidated with a busulfan-containing regimen.

Table 1  Tumor status at diagnosis

Table 2  Treatment before Bu-Mel-containing high-dose chemotherapy

Table 3  Conditioning regimen/tumor status before and after transplantation and current status