Results of the Neuroblastoma-2015 Protocol for Treating Neuroblastoma in China: Experience from Multiple Centers

ABSTRACT Background: The collaborative efforts of the Chinese Children’s Cancer Group (CCCG) have led to advances in our understanding of NB biology, a standardized classification, and stratified strategies, resulting in improved outcomes in China. We aimed to assess the NB-2015 protocol formulated by the CCCG in 2015 and evaluate the extent of the impact of autologous peripheral blood stem cell transplantation (APBSCT) . Methods: This study enrolled patients with NB between 2015 and 2018 from seven medical centers in China. The clinical characteristics of one hundred sixty-one patients were reviewed, and the therapeutic effect was evaluated as the impact on survival rate. Results: The 3-year event-free survival (EFS) rates of the low-, intermediate-, and high-risk groups were 95.5%, 91.6%, 51.1%, respectively. In total, 3 out of 7 (42.8%) patients with stage 2B disease, none of whom had MYCN amplifications, relapsed. A total of 47 of the 78 patients in the high-risk group underwent external-beam radiation to the primary tumor bed (dose ranging from 18 to 36 Gy). The therapeutic effect was significantly different between patients who received radiation therapy and those who did not (3-year OS P =0.041). Within the high-risk group, The 3-year EFS rates of the patients with and without APBSCT were 72.6% and 37.1%, respectively ( P =0.008). Conclusions: This study showed that using the NB-2015 protocol achieved satisfactory results without the occurrence of fatal complications. For patients in the stage 2B group, chemotherapy should be intensified. APBSCT can effectively improve the survival rate of patients with NB, especially high-risk patients. radiotherapy, and maintenance treatment with cis-retinoic acid based on the different INSS risk groups. The present article reports the results of the first retrospective study conducted by the various CCCG medical centers on NB treatment. This retrospective analysis of patients with NB who followed our NB-2015 protocol from 2015 to 2018 was performed to demonstrate the clinical features


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Introduction Neuroblastoma (NB) is the third most common cancer in children after leukemia and brain cancer [1].
NB is a complex heterogeneous disease that arises from the embryonic cells that form the primitive neural crest, with a natural history ranging from a benign course to a terminal illness and from spontaneous regression without intervention to highly fatal chemoresistant disease. NB accounts for 7-10% of all pediatric malignancies but is responsible for nearly 15% of pediatric cancer deaths because of the aggressive nature of the disease and the high likelihood of metastatic disease at diagnosis [2][3]. More than half of pediatric NB patients present with disseminated metastases at initial diagnosis, and most of these patients have advanced stage disease with poor prognoses. Patients can be divided into low-, intermediate-, and high-risk groups based on the International Neuroblastoma Staging System (INSS) criteria, which consider biological and clinical features such as age at diagnosis;, disease stage; tumor histology, including grade of neuroblastic differentiation and mitosiskaryorrhexis index (MKI); and molecular markers such as MYCN oncogene amplification. Most patients with INSS stage 1 and 2 NB (usually in the low-risk group) can be cured by surgery alone and have excellent prognoses, which is the consensus around the world [4][5]. However, the treatment for patients with stage 3 and 4 NB, especially those in the high-risk group, is still a challenge, and the outcomes are poor despite modern treatment strategies. Most of the studies show that the survival rate of patients with high-risk NB is approximately 50% with combination treatment that includes intensive chemotherapy, surgery, autologous stem cell transplantation, radiotherapy, immunotherapy and so on [6][7][8][9]. Although modern therapeutic treatments have improved the survival outcomes, these strategies still fail to eradicate the tumor in some patients with high-risk NB.
Racial and geographical disparities also exist in the risk status and survival of children with NB [10], and these disparities were thought to be due to genetic and economic differences. For decades, no relatively unified treatment protocol for NB existed in China; delayed diagnosis, lack of access to accurate staging, risk stratification, and optimal treatment, and abandonment led to survival rates much lower than those in high-income countries. Any of the abovementioned factors could lead to a  (Table 1). All patients were confirmed to have NB and adequate clinical data. All patients followed a treatment regimen based on the NB-2015 protocol that included neoadjuvant chemotherapy, surgery, adjuvant chemotherapy, APBSCT and radiotherapy according to the different risk groups determined by a professional team of pediatric oncologists.
Information regarding patient characteristics was retrospectively reviewed ( Table 2). The treatment protocol for each group is shown in Figs. 1. Local irradiation was administered to patients in the high-risk group.
positive High-risk Disease status and response to treatment were categorized as complete remission, very good partial remission (VGPR), partial remission (PR), stable disease (SD), or progressive disease (PD) according to the published International Neuroblastoma Response Criteria [12]. Complete response (CR) was defined as no evidence of the primary tumor and no evidence of metastases on imaging. Very good partial remission (VGPR) was defined as a decrease in tumor size of more than 90%. Partial response (PR) was defined volume reduction of more than 50% in the primary tumor and all measurable metastatic sites. Stable disease (SD) was defined as no new lesions with < 50% reduction but a < 25% size increase in any lesion; progressive disease (PD) was defined as a greater than 25% size increase in any pre-existing lesion or any new lesion. If surgical resection was attempted, the result was recorded according to the International Neuroblastoma Response Criteria: surgery was considered complete, near complete (leaving minimal residual disease, less than 5%), incomplete (between 5% and 50% disease remaining), or as a biopsy only (more than 50% remaining).   year EFS rates of the patients with and without transplantation were 72.6% and 37.1%, respectively (P = 0.008) (Fig. 2,3). The median survival time (MST) was 26 months (range 7-65 months). In the transplant group, no adverse effects specifically related to transplantation were observed up to 3 years after treatment. A total of 139 patients were still alive without recurrence, and 32 patients relapsed. Twenty-two patients died of disease progression after recurrence. The other 10 patients were still alive, including 3 with stage 2B disease, 1 with stage 3 disease, and 6 with stage 4 disease.
Among these 10 patients, 5 (50%) regained CR after further treatment. Although the patients experienced therapy-induced complications, nearly all patients could tolerate the intensive treatment.
None of the patients died of complications related to transplantation. In total, 47 of the 78 patients in the high-risk group underwent external-beam radiation to the primary tumor bed, and a dose ranging from 18 to 36 Gy was applied. The 3-year EFS rate was not significantly different between patients who received radiation therapy and those who did not (P = 0.354). However, the 3-year OS rate was significantly different between the two groups (P = 0.041). Among the patients who underwent total resection, the 3-year OS rate was also significantly different between patients with and without radiation therapy (P = 0.044). (Fig. 4)

Discussion
Patient age at the time of NB diagnosis is a known important independent prognostic factor. Among the 161 patients in this study, 51 patients were < 18 months old and the other 110 patients were ≥ 18 months old at diagnosis. The OS and EFS rates were significantly different between the two groups (3-year OS P = 0.004; 3-year EFS P = 0.000). These results are consistent with those of a previously published study [13].  Fig. 2, the EFS of patients with stage 2B disease was much lower than that of patients with stage 3 disease, which may be due to low chemotherapy intensity for patients in this group or the different genotypes associated with poor prognosis, resulting in poor therapeutic effects. Molecular genetic techniques should be used to stratify patients and to optimize treatment.
Local radiotherapy has been documented to achieve local control in patients with stage 4 or high-risk stage 3 NB through induction chemotherapy and tandem stem cell transplants [15]. According to our protocol, all patients belonging to the high-risk group are required to receive radiotherapy to the primary site and persistent metastatic foci. In our retrospective analysis, we found that only 47 of the 78 patients in the high-risk group underwent external-beam radiation to the primary tumor bed, where a dose ranging from 18 to 36 Gy was applied. On the one hand, the reason for the poor compliance with our treatment may be that radiotherapy is more challenging to administer for children than for adults, considering factors such as poor coordination and the need for sedation. On the other hand, this difference can be partially attributed to the lack of professional pediatric radiologists in some medical centers. There were no significant differences in the 3-year EFS rates between patients who received radiation therapy and those who did not (P = 0.354). However, the 3- year OS was significantly different between these two patient groups (P = 0.041). Particularly for patients who underwent total resection, the 3-year OS rate was significantly different between patients who received radiation and those who did not (P = 0.044). Although patients with gross or microscopic residual disease after surgery did not receive local radiation to the tumor bed, high-intensity chemotherapy played a role. Our study showed that for high-risk patients with NB, even after complete excision, local radiation was also necessary.
The incorporation of three distinct phases of therapy has improved outcomes for high-risk NB: intensive induction treatment, myeloablative chemotherapy with autologous hematopoietic stem cell rescue, and treatment of minimal residual disease (MRD). The survival outcomes of high-risk NB patients have continually improved in recent years, which is attributed to the intensification of therapeutic approaches [16][17][18]. Even after such therapies, the relapse rate remains higher than 40%, which has led to MRD-positive patients using post-transplant treatment such as isotretinoin and monoclonal anti-GD2 antibodies [19]. China has a lack of monoclonal anti-GD2 antibodies; thus, in our  [23]. Therefore, in our therapeutic protocol, we recommended tandem APBSCT with MM and CEC and observed very good effects. In our study, among the 34 patients who underwent APBSCT, 18 patients successfully received tandem transplantation. The other 16 patients failed to receive a second APBSCT after undergoing the first procedure, which was due to nonmedical reasons (parent preference, insurance limitations). In addition, we found that the tandem transplantation was well tolerated with no unbearable complications related to APBSCT, even including hepatic veno-occlusive disease (HVOD) or sinusoidal obstructive syndrome (SOS). Toxicities were as expected, and the time to engraftment was rapid for both cycles. Most importantly, APBSCT obviously improved the survival of patients in the high-risk group. The 3-year OS rate of the transplantation group was 79.0%, and the 3-year EFS rate of the transplantation group was 72.6%.
The survival outcomes were not significantly different between patients who received tandem transplantation and those who received single transplantation, which may be attributed to the small sample size and short follow-up period.

Conclusion
This study showed that the NB-2015 protocol obtained satisfactory results without the occurrence of fatal complications. Patients in the stage 2B group need further genotype testing, and the chemotherapy regimen should be intensified according to the different genotypes. Multidisciplinary treatment was essential, especially for high-risk patients, and APBSCT can effectively improve the survival rate. A longer follow-up period is needed to survey the survival rate and incidence of complications for Chinese NB patients who receive chemotherapy, radiotherapy and APBSCT. The outcomes for patients with NB have improved; however, the field continues to expand with efforts in developing more targeted therapies for high-risk patients.

Competing interests
The authors have no conflicts of interest.        Supplementary Files This is a list of supplementary files associated with this preprint. Click to download.
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