Background
Peripheral neuroblastic tumors (NTs) including neuroblastoma(NBL), ganglioneuroblastoma(GNBL), and ganglioneuroma(GN) comprise one of the most common groups of neoplastic disease in infants and children. NBL and GNBL are considered malignant. In contrast, GNs are considered as benign tumors. In histology, NBL, GNBL, and GN can be conceptualized as three maturational manifestations of a common neoplasm[
1].
Amplification of
MYCN oncogene is an established marker indicating aggressive tumor progression of NBL[
2,
3]. Brodeur et al.[
4] were the first to show that
MYCN amplification occurs in a substantial subset of primary untreated NBLs and is highly correlated with advanced stage. Seeger et al.[
5,
6] then demonstrated a strong association with rapid disease progression and a poor prognosis. Analysis of
MYCN remains an essential component of disease evaluation for newly diagnosed NBL patients and serves as a paradigm for the utility of molecular biologic information in cancer treatment stratification[
7‐
9].
MYCN is vital for proliferation, migration and stem cell homeostasis while decreased levels are associated with terminal neuronal differentiation[
10]. On the other hand, downregulation of
MYCN leads to decreased proliferation and differentiation, emphasizing the importance of MYC signaling in NBL biology[
11,
12]. But copy number status of
MYCN gene in GNBL and GN is poorly described in the literature[
13].
In the study, we evaluated the copy number aberrations of MYCN gene in formalin-fixed, paraffin-embedded clinical samples of NBLs, GNBLs and GNs and analyzed their association with clinical outcome of the patients.
Discussion
The peripheral neuroblastic tumour group includes NBL, GNBL and GN. NBL is the most common extracranial solid tumour of childhood and the incidence of pediatric neuroblastoma are increasing[
17,
18].
MYCN gene amplification is a known molecular marker for aggressive progression of NBL[
4]. In the study, we evaluated the histological presentation and
MYCN gene copy number in 220 pediatric neuroblastic tumors, which include 178 NBLs, 32 GNBLs and 10 GNs and analyzed their association with clinical outcome of the patients. To our knowledge, this is the first article for
MYCN gene and chromosome 2 aneusomy analyses by using fluorescence in situ hybridization (FISH) method in chinese pediatric patients.
Our study reaffirmed the need for
MYCN copy number to be determined in light of chromosome 2 copy number.
MYCN copy number had been determined by southern blot analysis[
19]. After 1993, fluorescence in situ hybridization (FISH) was used to determine the presence of
MYCN amplification[
20,
21]. In these studies, the results of southern blotting and FISH analysis were prospectively compared and a
MYCN copy number of ≥ 10 was determined to be the optimal cutoff by FISH[
20], as the vast majority of amplified tumors have very large numbers of double minutes in each tumor cell. By southern blotting, any normal cells in the tissue were included in the measurement, whereas by FISH, each tumor nucleus was visualized directly and simultaneous cohybridization with a specific chromosome probe is of great value in predicting the prognosis of patients[
22]. FISH has a higher sensitivity because it detects the
MYCN copy number on the single-cell level and allows correlation of morphologic details. In our estimation, FISH is a practical, useful and reliable method for analysis of
MYCN copy number in neuroblastic tumors.
Our results showed that aberrant
MYCN copy number was detected in 153 (85.9%) of 178 NBLs, with amplification constituting 24 (13.5%), gain 129(72.4%). In contrast,
MYCN amplification is only observed in one GNBL case (1/32, 3.1%) and no GN cases (0/10, 0%). Moll A et al.[
23] also reported that no amplification of the
MYCN-oncogene was found in mixed hepatoblastoma and teratoma of the liver in a 3-year-old boy. Wan,T.S et al.[
24] investigated 12 NBL patients for
MYCN amplification by FISH and found that 16.7% cases had
MYCN amplification. Angelini,P et al.[
13] reported that only about 2% had
MYCN gene amplified tumours in 232 GNBL patients. Our results also showed that the frequency of MYCN gene gain was significantly higher in GNBL (78.1%, 25/32) and NBL (72.5%, 129/178) than in GN (40.0%, 4/10). Toraman,A.D et al.[
25] found that chromosomal gains displayed by chromosomes and chromosome loci were 2p25 approximately pter (60%) in five GNBL cases by comparative genomic hybridization. Truong LN et al.[
26] also detected
MYCN oncogenes in malignant brain tumors by using multiplex ligation dependent probe amplification (MLPA). Thus, higher frequency of
MYCN gene aberrations in undifferentiated or less differentiated tumors indicates an important function of
MYCN gene in tumor malignancy.
MYCN amplification is an established marker indicating aggressive tumor progression of NBL[
27]. Our data also showed that
MYCN amplification is correlated with decreased overall survival in NBL (P=0.017) (Figure
3A). More significantly, we demonstrated for the first time that the presence of extra copies of
MYCN gene is an independent prognostic factor for NBL in our case series. The patients with
MYCN gene gain had a significantly longer mean survival time than those with normal
MYCN gene copy number (P=0.012) (Figure
3B). In our data, NBL cases with
MYCN gene gain also showed gain of centromere 2, suggesting polyploidy in these cases. In 1991, look et al.[
28] found that NBL patietns treated with cyclophosphamide-doxorubicin, hyperdiploidy was closely associated with long-term disease-free survival (greater than 90% of cases), while diploidy invariably predicted early treatment failure (P <0.001). Recently, George et al. had also found that NB patients with hyperdiploidy plus no amplified
MYCN confers a favorable prognosis[
29], which is in line with our study. Furthermore, they also found that hyperdiploidy plus no amplified
MYCN NBL patients may respond well to contemporary chemotherapy, and could be spared intensive myeloablative therapy with stem-cell rescue[
29]. Thus, the classification of
MYCN gene status as three groups by FISH may provide more powerful prognostic indicator and better treatment options in NBL.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Miao Wang, Yong Li and Rongqin Cai carried out the FISH studies. Miao Wang drafted the manuscript. Miao Wang, Chunju Zhou and Liping Gong participated in the design of the study. Chunju Zhou participated in and coordinated specimen and clinical data retrieval and characterization. Liping Gong conceived and coordinated the study. All authors read and approved the final manuscript. This study was conducted with approval from Capital Medical University.