Background
Neuroblastoma (NB) is a malignant solid tumor of children originating in the adrenal medulla and sympathetic nervous system [
1]. It is the widely diagnosed in children, with 8–10.2 cases of NB occurring per million children under 15 years of age [
2]. It has high heterogeneity, hidden onset, and poor prognosis, with a 5-year survival rate of less than 40% in high-risk NB [
3]. Only 1–2% of NB varieties have genetic predisposition, and most cases are sporadic [
4]. However, the mechanism of occurrence, proliferation, and metastasis of NB is not clear. With the development of chromosome karyotype analysis, whole-genome sequencing, and proteomics, the relationship between chromosome abnormalities and apoptosis, differentiation, spontaneous regression, proliferation, and metastasis of NB tumor cells has gradually been revealed [
5].
Chromosome 10 is one of 23 pairs of autosomes in humans, and contains about 135 million base pairs. It is possible that important genes related to the development of NB exist on chromosome 10. One of the tumor suppressor genes, PTEN (phosphatase and tensin homolog), is located at 10q23.3. It can influence the development of NB through the PI3K/ AKT/ mTOR pathway [
6]. Children with high-risk NB at Stage IV, without MYCN gene amplification, and with a whole chromosome aneuploidies (WCAS) factor of less than 2 have a poorer prognosis than those with a WCAS factor of greater than 2. This phenomenon is most significant on chromosome 10 (
P = 0.002) [
7]. A previous clinical study demonstrated that 50% of the children with metastatic NB had chromosomal abnormalities, and 70% of them have concurrent abnormalities related to chromosome number and structure. Among them, abnormalities in number occurred frequently on chromosomes 21, 10, and 11, with abnormalities on chromosome 10 being the most frequent.
This study aims to summarize the clinical characteristics of children with NB with abnormal chromosome 10 in a single treatment center. The relationship between the abnormal number and structure of chromosome 10 and the occurrence, development and prognosis of NB will be explored in order to to provide a new argument for chromosome genetics of neuroblastomas.
Methods
Patients samples
This study analyzed a total of 150 children with NB who had been diagnosed with bone marrow metastases by routine bone marrow cytology or bone marrow biopsy from May 2015 to December 2018 in the Medical Oncology Department of the Beijing Children’s Hospital. All patients were staged according to the International Neuroblastoma Stage System (INSS) [
8]. Risk stratification was conducted according to the Children’s Oncology Group (COG) [
9]. Bone marrow specimens were tested by chromosome G-banding when the patients were first hospitalized. All patients were regularly treated and followed up with at the center until November 30, 2019.
Complete medical records were collected for each participant. Clinical data included age at diagnosis, staging, sex, MYCN gene, chromosome report, and outcome. Based on detailed karyotype analysis, it was found that 13 patients had abnormal chromosome 10 and shared similar clinical characteristics. A focus was placed on their clinical features, such as primary tumor location, tumor markers at initial diagnosis, the largest diameter of the tumor, metastatic site, and so on. The study was approved by the Ethics Committee of the Beijing Children’s Hospital of Capital Medical University (2019-k-390). Informed consent was acquired from all participants and their parents prior to the collection of samples and information.
Therapeutic regimen and follow-up
All patients were treated according to NB protocols of the Beijing Children’s Hospital (BCH-NB-2007), which was developed based on Hong Kong NB protocol 7 and the European low- and intermediate-risk (IR) NB protocol [
10‐
12]. For the low-risk (LR) and IR groups with favorable pathological NB, therapies were CBVP (carboplatin, etoposide) and CADO (cyclophosphamide, doxorubicin, vincristine) alternately for 4–6 courses, and surgery. For the IR group with unfavorable pathological NB, therapy included 6–8 courses of chemotherapy combined with surgery, radiotherapy, and 6 courses of cis-retinoic acid. For the high-risk (HR) group, the chemotherapy regimen was CAV (cyclophosphamide, doxorubicin, vincristine) and CVP (etoposide, cisplatin), administered sequentially, which was combined with surgery, autologous hematopoietic stem cell transplantation, local radiotherapy, and 13-cis-retinoid acid. Patients were regularly followed up with every 3 months in the first year, every 4 months in the second year, and every 6 months in year 3 and 4.
Chromosome examination
Chromosome karyotype analysis of bone marrow cells was performed on G-banded preparations. All methods were performed in accordance with the experiment guidelines and ethical permission. First, bone marrow specimens were anticoagulated with 2 ml heparin and cultured in 1640 culture media at 37 °C and 5.0% CO
2 for 24 h. After adding 0.075 mol/L KCL hypotonic solution, 1.5 ml fixing solution (3,1 mixture of methanol and acetic acid) was used to mix and pre-fix two times [
13]. Each specimen was divided into four pieces and baked at 80 °C overnight. Before the examination, each piece was digested with trypsin and stained with Giemsa. Each split phase was observed and analyzed using high resolution chromosome analysis system (SRL, Tokyo, Japan). Twenty metaphase mitotic phases were analyzed in each specimen.
The karyotypes were defined based on the International System for Human Cytogenomic Nomenclature (ISCN 2016) [
14]. In tumor cells, it was considered a meaningful clone if 2 or more cells showed the same gain or structural abnormality of chromosomes, and 3 or more cells showed the same loss.
Statistical analysis
Descriptive statistics were conducted using Statistical Package for Social Scientists (SPSS) version 22. Overall survival (OS) was defined as the time from enrollment to disease-caused death or final follow-up. Event-free survival (EFS) was defined as the time to first occurrence of any event, such as disease progression, or death from any cause. The survival curves for OS or EFS were generated via the Kaplan-Meier method, and the difference between the two groups was evaluated with a log-rank test. Correlation between chromosome 10 and MYCN gene was analyzed by continuous correction chi-square test, where p < 0.05 was considered statistically significant.
Discussion
As a heterogeneous and occult tumor, most children with NB have chromosomal abnormalities when they are first diagnosed [
15]. According to the Mitelman database, more than 60% of NB is aneuploidy [
16]. In WCAS, NB is prone to co-occur with trisomy of chromosomes 6–9, 12, 13, 17, 18, 20, and 21, and monosomy of chromosomes 3, 4, 9–11, 15, 17, 19, 22 and X [
7]. NB in stages I, II, and 4S is mostly triploid, with relatively good prognosis. Patients often have chromosome 6, 7, and 17 gains and chromosomes 3, 4, 11, and 14 losses [
17]. Parodi et al. came to a preliminary conclusion that the prognosis of children with NB and whole X-chromosome-loss is relatively poor, which can be used as a new prognostic indicator, and patients with this chromosomal abnormality should be treated in the IR group [
18]. Marked by the centromere, each chromosome is divided into a long arm (q) and short arm (p). High-risk NB children without MYCN gene amplification at stage IV often show an increase in the number of chromosomes 7, 12 and 17, lost of 11q and 3p alleles, and 17q gains [
19]. However, no specific association between chromosome 10 and neuroblastoma has been reported. As can be seen from Fig.
1, in accordance with previous studies, the most common gain is seen in chromosome 7, and the most common structural abnormalities are seen in chromosomes 1 and 11. Chromosome 10 is the most frequent loss.
Previous studies have shown that there are tumor suppressor genes such as PTEN, DBMT, and LGI1 on the long arm of chromosome 10, and IDI1, AKR1C3, DDH1, NET1A, PRKCQ, and GATA-binding protein 3 on the short arm [
20,
21]. Among them, PTEN is the second largest deletion/mutation gene in human tumors, with a mutation rate of 50% [
22]. Li et al. [
23] have confirmed that GDNF family receptor alpha 2(GFRA2) promotes proliferation of NB cells by activating the PTEN/PI3K/AKT pathway. The allele imbalance of 10p 11.23–15.1 and 8q 21.3 appears to be specific to stage 4 tumors with MCYN amplification [
24]. The complete loss of chromosome 10 is common in tumors of the brain, lungs, ovaries, and skin [
25]. Although there are no prior studies of chromosome 10 and NB, it has been reported that genetic changes in chromosome 10q are common in other neurological tumors [
26]. For example, members of the cysteine-rich scavenger receptor family, DMBT1(10q25.3–26.1), are heterozygously absent in oligodendrogliomas, medulloblastoma, gastrointestinal cancer, and lung cancer [
27,
28]. Park et al. reported that in the CpG island methylator phenotype (G-CIMP) subtype of glioma, children with 10q loss have a poor prognosis [
29]. MGMT, located at 10q26.1, encodes a protein associated with DNA repair that can remove proto-mutant alkyl from O. It affects the development of glioblastoma by the oncogene TP53 [
30]. Fibroblast growth factor receptor 2 (FGFR2) at 10q26 is associated with cell proliferation, differentiation, migration, and inhibition of apoptosis. It is overexpressed in breast cancer [
31], and downregulated in prostate cancer [
32].
This study showed that the OS rate of NB children with abnormal chromosome 10 was significantly lower than that of children with normal chromosome 10, including children with no chromosomal abnormalities and children with abnormal chromosomes but normal chromosome 10. The OS and EFS of the group with all normal chromosomes were significantly higher than those in the abnormal chromosome 10 group. 10q22 was found to be the sites of all structural abnormalities on chromosome 10, which indicates that 10q22 may have tumor suppressor or oncogenic genes. Other non-statistically significant results may be due to the number of chromosome 10 abnormalities still being insufficient.
In addition, the individual effects of chromosome 10 abnormalities cannot be evaluated because many abnormal chromosome karyotypes in children with NB contain complex quantitative or structural abnormalities. There may be other chromosomes associated with neuroblastoma in the group of chromosomal abnormalities affecting prognosis.
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