Ji Won Lee and Joon Seol Bae contributed equally to this work.
We performed this study to determine whether the degree of neutropenia after the first chemotherapy cycle can be used as a surrogate marker of individual susceptibility to chemotherapeutic agents affecting treatment outcome in patients with neuroblastoma.
The study included 313 patients who received the first cycle chemotherapy with a CEDC (cisplatin+etoposide+doxorubicin+cyclophosphamide) regimen and had absolute neutrophil count (ANC) data available. The cumulative incidences of progression and treatment-related mortality (TRM) were estimated. To identify genetic variations associated with the ANC, a genome-wide association study (GWAS) was performed.
An ANC of 32.5/μL was determined as the cutoff point to categorize patients into the good and poor prognosis subgroups in terms of progression. Patients with a high nadir ANC had a higher cumulative incidence of progression than those with a low nadir ANC (p < 0.001). In multivariate analysis, high nadir ANC, age, bone marrow involvement, and unfavorable histology were poor prognostic factors. With regard to the TRM, patients with a low nadir ANC (ANC < 51.0/μL) had a higher cumulative incidence of TRM than those with a high nadir ANC (p=0.010). In GWAS, single-nucleotide polymorphisms of
In neuroblastoma patients, the degree of neutropenia after the first chemotherapy cycle can be used as a surrogate marker to predict an individual’s susceptibility to chemotherapeutic agents. Tailoring of treatment based on the degree of neutropenia needs to be considered.
Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for 6%–10% of all childhood cancers [
Individual variation in susceptibility to chemotherapeutic agents may occur because of differences in germline genomics, including single-nucleotide polymorphisms (SNPs). Many pharmacogenomic studies have shown that SNPs are useful predictive biomarkers for drug-induced adverse events and drug response [
In the present study, we hypothesized that the degree of neutropenia after the first chemotherapy cycle could be used as a surrogate marker of individual susceptibility to chemotherapeutic agents and that it affects treatment outcome. We analyzed whether the absolute neutrophil count (ANC) at the nadir after the first chemotherapy cycle is associated with treatment outcome in patients with NB. In addition, we determined whether germline genetic polymorphisms can affect the degree of neutropenia using a genome-wide association study (GWAS).
Patients who were diagnosed with NB and received chemotherapy between March 2000 and December 2018 were screened. Patients who underwent the first cycle chemotherapy with a cisplatin+etoposide+doxorubicin+cyclophosphamide (CEDC) regimen and had ANC data available from the first cycle chemotherapy were included in this study. In total, 313 patients were enrolled in the present study. The patients’ medical records were reviewed for detailed clinical and biological data, including clinical features at diagnosis, tumor biology (including
Patients were classified into three risk groups according to age at diagnosis, stage according to the International Neuroblastoma Staging System (INSS), and
Genomic DNA was extracted from the patients’ peripheral blood lymphocytes using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) according to the manufacturer’s protocol. Approximately 250 ng of genomic DNA was used to genotype each sample using Illumina’s Global Screening Array BeadChip (Illumina, San Diego, CA). The samples were then processed according to the Illumina Infinium assay manual. The quality of the sample was checked based on the sample call rate (> 95%). The clustering quality of each marker was measured based on the GenTrain score. Markers with a score of > 0.7 were used in the study. We performed imputation using the Michigan Imputation Server (
Maximally selected log-rank statistics were tested to obtain the optimal ANC cutoff values that categorized patients into the good and poor prognosis subgroups in terms of progression and treatment-related mortality (TRM) [
To investigate significant markers, numeric regression analyses were performed on the ANC with genotypes using HelixTree software (Golden Helix Inc., Bozeman, MT). To assess multiple corrections, the false discovery rate was used. The DAVID functional annotation tool (
Altogether, 313 patients were enrolled in the current study. The median age at diagnosis was 2.4 years (range, 0.0 to 19.2 years). One hundred ninety-one patients (61.0%) had metastatic disease and 110 (35.1%) patients had bone marrow metastasis. Tumor histology according to the INPC was favorable in 137 patients (43.8%), unfavorable in 154 patients (49.2%), and unknown in 22 patients (7.0%). Cytogenetic analysis was only performed in some patients; 68 of 310 patients (21.9%) had
During the study period, 59 patients showed progression and 25 experienced TRM; the 5-year EFS and OS rates were 73.1%±2.6% and 80.7%±2.3%, respectively. In high-risk patients, progression and TRM occurred in 51 and 21 patients, respectively, and the 5-year EFS and OS rates were 58.6%±3.9% and 69.0%±3.7%, respectively.
First, we analyzed whether the degree of neutropenia after the first cycle of chemotherapy was associated with the cumulative incidence of progression. Maximally selected log-rank statistics determined that an ANC of 32.5/μL was an optimal cutoff point to categorize patients into the good and poor prognosis subgroups in terms of progression. The patient characteristics in the nadir ANC subgroups are presented in
Because age was significantly different between the two groups and patients aged < 2 years received chemotherapy at a reduced dose, patients aged > 2 years were included in subgroup analysis. In subgroup analysis, patients with a low nadir ANC had a better outcome in terms of cumulative incidence of progression than those with a high nadir ANC (p=0.004), but the EFS and OS did not differ among the groups (p=0.051 and p=0.304, respectively) (
In patients with high-risk NB, the difference in the cumulative incidence of progression and EFS became more prominent (
The primary tumor volume was measured at diagnosis and at the first response evaluation after three cycles of induction chemotherapy. The percentage tumor volume at the first response evaluation compared with the initial tumor volume was calculated. Tumor volume was only evaluated in 164 patients who had undergone three-dimensional computed tomography or magnetic resonance imaging at diagnosis and had not undergone front-line surgery at diagnosis. Patients with a low nadir ANC showed greater tumor volume reduction than those with a high nadir ANC (median residual volume %: 17.5% vs. 47.8%, respectively; p=0.004) (
Subgroup analysis only including undifferentiated or poorly differentiated NB was performed because histological tumor differentiation, which can affect the tumor response, was different between the two groups (
TRM occurred in 25 patients—three patients died during induction chemotherapy, 11 patients showed acute TRM after HDCT, nine patients had late TRM after HDCT, and two patients died due to secondary malignancy. Maximally selected log-rank statistics showed that an ANC of 51.0/μL was the optimal cutoff point to categorize patients based on TRM. Patients with a low nadir ANC (≤ 51.0/μL) showed a higher cumulative incidence of TRM than those with a high nadir ANC (p=0.010). The 5-year cumulative incidences of TRM were 9.43%±0.04% and 1.25%±0.02% in the low and high nadir ANC groups, respectively (
A quantile–quantile plot of the association test using the ANC showed a significant deviation of measures at the tail (
In the present study, we investigated whether the degree of neutropenia after the first chemotherapy cycle could be used as a surrogate marker for determining an individual’s susceptibility to chemotherapeutic agents and its effect on treatment outcome in patients with NB. The final outcomes such as EFS and OS did not differ according to the nadir ANC group in all patients. However, this was because the ANC nadir group was inversely associated with the cumulative incidence of progression and TRM (even though the ANC cutoff points were different between progression and TRM). In other words, the patients in the high nadir ANC group showed a higher cumulative incidence of progression, but the cumulative incidence of TRM was higher in patients in the low nadir ANC group. These findings suggest that patients who are more susceptible to chemotherapeutic agents could have profound neutropenia after the first cycle of chemotherapy and have better outcomes in terms of progression. However, these patients are more vulnerable to toxicities. Collectively, our findings suggest that the degree of neutropenia could be used as a clinical marker to predict an individual’s susceptibility to chemotherapeutic agents, and tailoring of treatment based on the degree of neutropenia needs to be considered.
Several studies have shown an association between chemotherapy-induced neutropenia and survival outcome in adult patients with cancer [
TRM is an extreme form of treatment-related toxicity and is particularly common in children receiving intensive chemotherapy, which can result in infection, bleeding, or organ dysfunction [
The effects of the degree of neutropenia on treatment outcome were more prominent when the analysis was confined to high-risk patients who received very intensive treatment, including tandem HDCT. Conversely, the effects of the degree of neutropenia were not significant in low- or intermediate-risk patients, suggesting that treatment outcome is not significantly affected by the degree of neutropenia when treatment is less intensive, such as that in the low- and intermediate-risk patients in the present cohort. Therefore, in high-risk patients who receive intensive treatment, tailoring of treatment based on the degree of neutropenia needs to be considered. A prospective study is needed to investigate whether personalized treatment according to the degree of neutropenia can improve the final survival rate by reducing the cumulative incidence of progression and TRM in patients with high-risk NB.
We performed the GWAS to identify genetic factors responsible for the variable degree of neutropenia after the same chemotherapy; SNPs of
The number of patients in our cohort was lower than that in previous adult studies because NB is rare. Furthermore, patients in our cohort received treatment according to our own protocols, including intensive tandem HDCT in high-risk patients, which was uncommon globally during the study period. Hence, we could not validate the findings of the present study in another cohort. Therefore, our findings need to be confirmed in a larger cohort of children and adolescents with NB or other solid tumors.
In conclusion, the degree of neutropenia after the first chemotherapy cycle could be used as a surrogate marker to predict an individual’s susceptibility to chemotherapeutic agents, and thus, it can predict treatment outcomes in patients with NB. Further confirmatory studies on genetic markers are needed to explain the results of the present study. In addition, tailoring of treatment based on the degree of neutropenia needs to be considered, especially in high-risk patients.
Supplementary materials are available at Cancer Research and Treatment website (
Written informed consent was obtained from the parents or guardians of each patient. This study was approved by the Institutional Review Board (IRB) of Samsung Medical Center (IRB No. SMC 2015-06-068 and SMC 2020-12-164).
Conceived and designed the analysis: Lee JW, Sung KW.
Collected the data: Lee JW, Cho HW, Ju HY, Yoo KH, Koo HH.
Contributed data or analysis tools: Bae JS, Kim JH, Woo SY, Kim S.
Performed the analysis: Lee JW, Bae JS, Kim JH, Cho HW, Ju HY, Yoo KH, Koo HH, Woo SY, Kim S.
Wrote the paper: Lee JW, Bae JS, Sung KW.
Conflict of interest relevant to this article was not reported.
This work was supported by grants from the National Research Foundation of Korea (NRF), which is funded by the Korea government (NRF-2016R1A2B1012908 and NRF-2018R1A2B600325313), and by a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (No. 1520210). The biospecimens for this study were provided by Samsung Medical Center BioBank.
Survival outcomes according to the absolute neutrophil count (ANC) group. Cumulative incidence of progression/treatment-related mortality, event-free survival, and overall survival based on an ANC cutoff value of 32.5/μL in all patients (A), in patients aged ≥ 2 years (B), and in high-risk patients (C).
Tumor response according to the absolute neutrophil count (ANC) group. The percentage of residual tumor volume at the first response evaluation in all patients (A), in high-risk patients (B), and in patients with undifferentiated (UD) or poorly differentiated (PD) neuroblastoma (C).
Cumulative incidence of treatment-related mortality (TRM) according to the absolute neutrophil count (ANC) group. An ANC of 51.0/μL was selected as an optimal cutoff point for the cumulative incidence of TRM, and patients in the ANC > 51.0/μL group showed a lower 5-year cumulative incidence of TRM than those in the ANC ≤ 51.0/μL group (1.3%±0.02% vs. 9.4%±0.04%, respectively) in all patients (A) and in high-risk patients (B).
Manhattan plot of the genome-wide association study. (A) Results of the genome-wide association analyses of common single- nucleotide polymorphisms (SNPs) (minor allele frequency > 0.05) associated with the absolute neutrophil count represented as a Manhattan plot. The X-axis represents the SNP markers on each chromosome. (B) Regional association plots at the
Patient characteristics
ANC ≤ 32.5/μL (n=191) | ANC > 32.5/μL (n=122) | p-value | |
---|---|---|---|
Male | 111 (58.1) | 66 (54.1) | 0.560 |
Female | 80 (41.9) | 56 (45.9) | |
1.9 (0.0–18.2) | 3.4 (0.0–19.2) | < 0.001 | |
2,825 (530–13,900) | 3,115 (1,030–25,820) | 0.108 | |
Yes | 60 (31.4) | 49 (40.2) | 0.143 |
No | 131 (68.6) | 73 (59.8) | |
Localized | 71 (37.2) | 51 (41.8) | 0.484 |
Metastatic | 120 (62.8) | 71 (58.2) | |
Yes | 47 (25.0) | 21 (17.2) | 0.139 |
No | 141 (75.0) | 101 (82.8) | |
Non–high-risk | 85 (44.5) | 55 (45.1) | > 0.99 |
High-risk | 106 (55.5) | 67 (54.9) | |
Favorable | 82 (46.9) | 55 (47.4) | > 0.99 |
Unfavorable | 93 (53.1) | 61 (52.6) | |
Undifferentiated | 30 (16.8) | 6 (5.1) | 0.001 |
Poorly differentiated | 73 (40.8) | 57 (48.3) | |
Differentiating | 48 (26.8) | 21 (17.8) | |
Ganglioneuroblastoma | 28 (15.6) | 34 (28.8) | |
Abdomen | 143 (74.9) | 93 (76.2) | 0.902 |
Mediastinum | 47 (24.6) | 28 (23.0) | |
Other | 1 (0.5) | 1 (0.8) | |
881.0 (176.0–15,720.0) | 848.0 (257.0–12,160.0) | 0.064 | |
139.7 (7.6–3,283.7) | 141.2 (13.0–16,500.1) | 0.477 | |
59.0 (7.3–1,815.0) | 58.4 (7.4–865.4) | 0.428 | |
5.5 (0.2–205.0) | 10.9 (0.2–106.0) | 0.017 |
Values are presented as number (%) or median (range). ANC, absolute neutrophil count; BM, bone marrow; INPC, International Neuro-blastoma Pathology Classification; LDH, lactate dehydrogenase; NSE, neuron specific enolase; VMA, vanillylmandelic acid.
Multivariate analysis for the cumulative incidence of progression
All patients | Age > 2 years | High-risk patients | |||||||
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HR | 95% CI | p-value | HR | 95% CI | p-value | HR | 95% CI | p-value | |
ANC at nadir > 32.5/μL | 2.25 | 1.32–3.84 | 0.003 | 2.55 | 1.36–4.77 | 0.003 | 2.55 | 1.42–4.55 | 0.002 |
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Age | 1.01 | 1.00–1.01 | 0.021 | 1.00 | 1.00–1.01 | 0.280 | 1.00 | 0.99–1.01 | 0.590 |
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Stage IV | 1.59 | 0.62–4.06 | 0.340 | 1.88 | 0.54–6.54 | 0.320 | 1.37 | 0.25–7.50 | 0.720 |
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1.56 | 0.88–2.79 | 0.130 | 1.64 | 0.84–3.19 | 0.150 | 1.28 | 0.69–2.40 | 0.440 | |
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BM involvement | 2.12 | 1.12–4.02 | 0.022 | 1.69 | 0.83–3.46 | 0.150 | 1.95 | 0.99–3.80 | 0.051 |
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Unfavorable histology | 2.26 | 1.19–4.29 | 0.012 | 1.90 | 0.85–4.22 | 0.120 | 1.77 | 0.87–3.60 | 0.110 |
ANC, absolute neutrophil count; BM, bone marrow; CI, confidence interval; HR, hazard ratio.