2.2.1 Demographics
In this cohort, there were 131 boys (61.8%) and 81 girls (38.2%), and the male-to-female ratio was 1.61:1. Sixty-three children (29.7%) were less than 18 months old, and 249 children (70.3%) were 18 months and above. The median age at initial diagnosis was 32 months (range from 1.2 to 156.3 months). Fifty-seven cases (26.9%) were found in the physical examination without any symptoms. Forty-nine cases (23.1%) were palpated by their parents. The chief complaint of 59 cases (27.8%) was digestive system symptoms such as jaundice, vomiting, and diarrhea. The initial symptom of 21 cases (9.9%) was asthenia, muscle strength and muscle tension changes, or other motor symptoms. Cough, hemoptysis, dyspnea, and other respiratory symptoms were chief complaints in 10 patients (4.7%). Fourteen patients (6.6%) only presented with fever (6.6%). MYCN genetic testing had been carried out since 2012 in this center, so 170 out of 212 patients had been detected, including 125 (58.9%) non-amplified patients and 45 (21.2%) amplified patients. RFS rate in 1-year, 3-year, 5-year, and 10-year was 0.811, 0.662, 0.639, and 0.604, respectively. The demographic and clinical characteristics of 212 NB patients were listed in Table 1.
The primary sites of neuroblastoma were mainly retroperitoneal/paranephros (166 cases, 78.3%) and mediastinum (33 cases,15.6%). Bone and bone marrow were the main metastasis sites. There were 69 patients (32.5%) with bone and bone marrow metastasis simultaneously. Eighty-nine children (42%) had bone metastases, including 10 cases (4.7%) with skull metastases, 20 cases (9.4%) with limb bone metastases, 17 cases (8%) with trunk bones metastases, and 42 cases (19.8%) with multiple bone metastases.
Eighty-two children (38.7%) had bone marrow metastasis at the initial onset. They accepted bone marrow biopsy before the therapy. The minimal residual disease (MRD) in their bone marrow was positive (MRD%≥0.01%) through flow cytometers. So, they were asked to have a bone marrow puncture to monitor MRD every two cycles of chemotherapy. After two cycles, MRD turned negative (<0.01%) in 54 cases (25.5%), while MRD in 24 cases (11.3%) and 4 patients (1.9%) converted negative after four and six cycles, respectively. Univariate analysis of RFS was performed on the above three groups of patients (2, 4, and 6 cycles). The median RFS of the three groups was 25.75 months, 12.07 months, and 39.80 months, respectively (P=0.023). Given the small number of patients whose MRD turned negative after 6 cycles of chemotherapy, the above comparison might be biased, so we compared the RFS of patients with negative MRD after 2 and 4 cycles by the Kaplan-Meier curve. (Figure 1).
2.2.2 Recurrent neuroblastoma
Seventy-one patients (33.5%) developed recurrent NB including 47 boys (66.2%) and 24 girls (33.8%). The ratio of male-to-female was 1.95:1. The median interval of recurrence from CR or VGPR was 10.27 months (range from 1.53 to 64.83months). While the median follow-up time was 70.80 months (range from 13.30 to 161.06months). Forty cases (56%) relapsed within one year. Sixty-two cases (87.3%) were older than 18 months at the initial diagnosis. Two cases (2.8%) were in stage INSS 2; 9 cases (12.7%) were in INSS stage 3; while 60 patients (84.5%) were in INSS stage 4. Eight (11.2%) were divided into the intermediate-risk group, while 63 cases (88.8%) were divided into the high-risk group (COG). The pathological type of 40 patients was undifferentiated neuroblastoma. Forty-six (64.8%) of them had bone marrow metastases.
2.2.3 Univariate analysis of RFS
The Kaplan-Meier curve was used to conduct a univariate analysis of the correlation between clinical data and recurrent NB. The univariate analysis was performed on the relevant laboratory tests of the newly diagnosed children. Children with increased NSE values, LDH>1000U/L, and increased VMA values had lower RFS (P=0.048, P=0.002, P<0.001, respectively, Log-rank test, figures 2A, B, C). Children with MYCN amplification also had lower RFS (median: 34.03 months vs. 42.23 months, P = 0.017, Log-rank test, figure 2D). Age and RFS showed a negative correlation. The risk of recurrence was increased in older children (median:62.30 months vs. 59.77 months vs. 36.33months, P corrected=0.0002, Log-rank test, figure 2E). For the pathological subtypes, the median RFS was the longest (57.47 months) in children with GNBi and the shortest (32.60 months) in children with GNBn (P = 0.023, Log-rank test, figure 2F). RFS was correlated with tumor localization. The median RFS of stages 1, 2, and 4s was 90.67 months, the median RFS of stage 3 was 52.46 months, and it was only 36.33 months in stage 4 (P corrected<0.0001, Log-rank test, figure 2G). The integrity of the tumor capsule was also correlated with RFS. Children with intact tumor capsules had a higher RFS (median: 62.60 months vs. 30.83 months, P <0.0001, Log-rank test, figure 2H). RFS was related to whether there was bone marrow metastasis at the initial onset. Children with bone marrow metastases had lower RFS (median: 21.05 months vs. 60.28 months, P<0.0001, Log-rank test, figure 2I). In summary, according to the univariate analysis, it can be concluded that the older onset age, with distant metastasis, bone marrow involvement, MYCN amplification, without intact tumor capsule, increased VMA and NSE value, and LDH>1000U/L were unfavorable factors for recurrence of NB. The univariate analysis data related to RFS was shown in Table 2.
2.2.4 Association Between MYCN and Relevant Laboratory Tests of NB
The study demonstrated the correlation between the MYCN gene and the data from the relevant laboratory tests. Children without MYCN amplification had a median initial VMA value of 2.03(0.48-35.89) mg/24h, and children with MYCN amplification had a median initial VMA value of 3.29(0.50-184.70) mg/24h (P=0.038). The median value of initial NSE in children without MYCN amplification was 74.55(12.79-1519.00) ug/l, and the median value of initial NSE with MYCN amplification was 244.00(12.90-4534.00) ug/l (P =0.031). It demonstrated that the MYCN gene was correlated with NSE and 24-hour urine VMA value at the initial onset. The correlation between the MYCN gene and other clinical features was listed in Table 3.
2.2.5 Multivariate Cox regression and nomogram
The significant variables in the univariate analysis included age at diagnosis, tumor localization, histologic category, bone marrow metastasis, MYCN state, the integrity of tumor capsule, VMA, NSE, and LDH value. The results from multivariate Cox regression analysis of RFS showed that onset age, tumor localization, MYCN state, histologic category, and integrity of tumor capsule were independent risk factors for RFS (all P values <0.05). The data of multivariate analysis were shown in Table 4.
Based on the multivariate analysis of RFS, all variables in the multivariate Cox regression were applied to build the nomogram model diagram for predicting RFS in the 1st-year, 5th-year, and 10th-year, as shown in Figure 3. Nomogram could assign points to each risk factor for recurrence, and finally, predict the RFS based on the total score individually. The corresponding meaning and description of the Arabic numbers in each risk factor in Figure 3 were shown in Table 5. For example, if there were a 3-year-old patient with INSS Stage 4 at diagnosis, normal VMA and NES, LDH≤1000U/L, MYCN non-amplification, bone marrow MRD-negative, histologic category classified as undifferentiated neuroblastoma, and fracted tumor capsule, The total score was calculated as follows, 0 + 0 + 23 + 51 + 5 + 29 + 5 + 19 + 0 = 132 (scores), and the patient's 1st-year, 5th-year, and 10th-year recurrence-free probability was approximately 87%, 62%, and 45% referring to Figure 3, respectively.
Two methods were taken to verify the model: (1) used the Bootstrap method and repeated the sample 1000 times for internal verification. It showed that the recurrence prediction model had a C-index of 0.824 (P=0.023). (2) used the graph calibration method to verify the RFS of the 1st-year, 5th-year, and 10th-year RFS. See Figure 4 for details. It showed that the prediction curve of 1-year, 5-year, and 10-year was entangled with the standard curve, indicating that the model had good predictive potential.