1. Introduction
Neuroblastoma (NB) patients fall into two clinically distinct subgroups; a low-risk subgroup and a high-risk subgroup. These subgroups are correlated with the age of onset [1], the extent of the disease (International Neuroblastoma Staging System) [2], pathological findings (International Neuroblastoma Pathological Criteria) [3], and genomic changes in NB tumors as represented by
Recent improvements in molecular techniques such as PCR have made it possible to detect small amounts of cell-free DNA in the serum and plasma of patients with various diseases, including cancers. In particular, tumor-derived cell-free DNA has attracted attention as a novel genetic marker for cancer. Other improvements in molecular techniques have enabled the evaluation of tumor-related genetic aberrations using small amounts of cell-free DNA in the serum. NB has been detected with several tumor-related genetic alterations, such as RAS mutations [13], or TP53 mutations [14], microsatellite instability [15], gene amplification [16, 17], aberrant promoter hypermethylation [18, 19], and allelic gain and loss of oncogenes [15, 20, 21].
We have been attempting to establish a preoperative risk stratification system for NB based on a serum-based assay system for
2. Methods and results
Serum samples were collected from the patients with NB before initial treatment. Cellular components were immediately removed from the collected sera immediately by centrifugation or filtration. DNA fragments were purified from 200μl of serum according to manufacturer’s protocol and were used for further genetic analysis as mentioned below.
2.1. Detection of amplified MYCN gene in the sera of NB patients
The
Combaret et al., who first reported the existence of the
We also confirmed the sensitivity and specificity of serum MNA analysis using 148 samples obtained from NB patients with various INSS stages in two Japanese cohorts other than ours and a cohort from the Children Oncology Group. A sub-group analysis according to INSS stages revealed that the sensitivities and specificities were not statistically different (67% and 95%, respectively, in stages 1 and 2, 92% and 86% in stage 3, and 87% and 97% in stage 4), although the number of each group was not statistically sufficient (p=0.48 for sensitivity, p=0.68 for specificity, chi-squared test) (our unpublished data). Accordingly, the serum
2.2. Detection of methylated DNA fragments in the sera of NB patients
It is important to have additional biomarkers with prognostic value for the management of non-MNA cases of NB because some cases without MNA also have a poor prognosis. Recent studies have revealed that epigenetic alterations, such as silencing of tumor suppressor genes by aberrant hypermethylation of the promoter, often play important roles in the pathogenesis and progression of NB. A positive correlation has been found between the hypermethylation of the promoters of these genes and a poor prognosis, thus suggesting that hypermethylation influences the phenotype of neuroblastoma.
We previously revealed that the aberrant hypermethylation in the promoter region of the
2.3. Detection of chromosomal loss and gain in the serum DNA of NB patients
Chromosomal gain and/or loss are frequently observed in NB as mentioned above. Among the various unbalanced chromosomal aberrations, 17q gain is the most frequent chromosomal aberration, and correlates with a poor outcome [27]. The loss of 11q is also a strong prognostic factor that can be used in addition to MNA [11], and routine assessment of the 11q status, as well as MNA, is required for therapeutic stratification of NB in the INRG staging system [8].
Some groups, including ours, have developed serum-based assays to detect chromosomal gain and/or loss in NB using various techniques. Combaret et al. reported a serum-based detection system for 17q gain [20]. They simultaneously quantified the gene dose of MPO (17q.23.1) and survivin (17q25) as targets, and p53 as a reference, by quantitative real-time PCR using 142 serum samples. They revealed that the serum-based determination of 17q gain had good specificity (94.4%) and sensitivity (58.8%) in patients who were less than 18 months old (p<0.001), while this approach showed moderate specificity (71.4%) and sensitivity (51.2%) in patients over 18 months of age. In a subset analysis according to the stage of NB, the sensitivity of serum-based 17q gain determination tended to increase with the stage of the disease. On the other hand, for metastatic NB, the sensitivity of the test never exceeded 60%, which is lower than the results achieved by the analysis of the serum-based
We developed an assay for detecting chromosomal aberration of 11q, using a different method that doesn’t use quantitative real-time PCR. Previous studies have revealed the presence of a smallest region of overlap (SRO), which is a common region of deletion in all NB cases with 11q loss. By targeting some polymorphic markers in the SRO of 11q (most of which are located in 11q23), allelic loss could be detected using serum DNA as well as tumor DNA of NB patients [21] (Figure 3). Using this technique, the sensitivity and specificity of the results between the serum- and tumor-based 11q loss analyses were both 100%, although a further study is needed to confirm of these findings because of the limited number of cases that were analyzed.
3. Discussion
In a large-scale randomized trial of children with high-risk NBs [28], the
In Japan, infantile NB cases were formerly subclinically detected by mass-screening. Most of these cases showed a good prognosis and were recommended to undergo a reduced regimen, including the “wait-and-see” approach. However, we and others demonstrated that MNA was strongly correlated with a poor prognosis even in infantile, localized NB [7, 29]. The serum-based MNA status has considerable prognostic value for infantile NB cases. Indeed, the serum-based MNA status of NB patients has considerable prognostic value, especially in cases less than 18 months of age (our unpublished data).
On the other hand, most of the infantile, localized NB patients did not have MNA. In the Cooperative German Neuroblastoma NB95 and NB97 trials, some localized patients without MNA did not receive chemotherapy after biopsy and showed spontaneous regression [30]. Considering the clinical behavior of non-MNA NB, an early and non-invasive system for detecting genetic alterations besides MNA is needed to help select the appropriate therapy. In other words, combined preoperative assessments of MNA and 11q loss using serum DNA will make it possible to safely perform risk-adapted therapy according to the INRG staging system [8]. Particularly, preoperative serum-based MNA and 11q loss detection can be useful for cases that are in INRG stages L2 and MS, which have a wide range of clinical outcomes and potential therapeutic strategies depending on the existence of MNA and 11q loss. Further, we may be able to select infants with NB who truly need to receive treatment including surgical treatment, intensive chemotherapy, and even radiotherapy from infants with NB who do not need to require any treatment by using our technique.
In conclusion, serum-based, less invasive molecular analysis can provide much better clinical information to determine the optimal therapeutic strategy for NB patients. Prospective validation in a large cohort will be needed to confirm the utility of these tools for assessing biological risk. Serum-based, surgery-free, rapid, sensitive, and specific genetic assessments have great potential to provide a personalized, risk-adapted therapy for patients with NB.
References
- 1.
Evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children’s Oncology Group.London W. B Castleberry R. P Matthay K. K et al 2005 2005 23 6459 - 2.
Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment.Brodeur G. M Pritchard J Berthold F et al 1993 1993 11 1466 - 3.
VV et al. Revision of the International Neuroblastoma Pathology Classification: confirmation of favorable and unfavorable prognostic subsets in ganglioneuroblastoma, nodular.Peuchmaur M d Amore E. S Joshi 2003 2003 98 2274 - 4.
Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage.Brodeur G. M Seeger R. C Schwab M et al 1984 1984 224 1121 - 5.
Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas.Seeger R. C Brodeur G. M Sather H et al 1985 1985 313 1111 - 6.
Neuroblastoma: biological insights into a clinical enigma.Brodeur G. M 2003 2003 3 203 - 7.
Significance of MYCN amplification in international neuroblastoma staging system stage 1 and 2 neuroblastoma: a report from the International Neuroblastoma Risk Group database.Bagatell R Beck-popovic M London W. B et al 2009 2009 27 365 - 8.
The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report.Cohn S. L Pearson A. D London W. B et al 2009 2009 27 289 - 9.
Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas.Ohira M Oba S Nakamura Y et al 2005 2005 7 337 - 10.
Di Vinci A et al. Distinct CpG methylation profiles characterize different clinical groups of neuroblastic tumors.Banelli B Gelvi I 2005 2005 24 5619 - 11.
Chromosome 1p and 11q deletions and outcome in neuroblastoma.Attiyeh E. F London W. B Mosse Y. P et al 2005 2005 353 2243 - 12.
International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee.Ambros P. F Ambros I. M Brodeur G. M et al 2009 2009 100 1471 - 13.
K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer.Anker P Lefort F Vasioukhin V et al 1997 1997 112 1114 - 14.
ser) TP53 mutation in plasma DNA, hepatitis B viral infection, and risk of hepatocellular carcinoma.Kirk G. D Lesi O. A Mendy M et al 2005 2005 24 5858 - 15.
Microsatellite alterations in serum DNA of head and neck cancer patients.Nawroz H Koch W Anker P et al 1996 1996 2 1035 - 16.
Circulating MYCN DNA as a tumor-specific marker in neuroblastoma patients.Combaret V Audoynaud C Iacono I et al 2002 2002 62 3646 - 17.
Prediction of MYCN amplification in neuroblastoma using serum DNA and real-time quantitative polymerase chain reaction.Gotoh T Hosoi H Iehara T et al 2005 2005 23 5205 - 18.
Circulating methylated-DCR2 gene in serum as an indicator of prognosis and therapeutic efficacy in patients with MYCN nonamplified neuroblastoma.Yagyu S Gotoh T Iehara T et al 2008 2008 14 7011 - 19.
RASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker.Misawa A Tanaka S Yagyu S et al 2009 2009 100 399 - 20.
Determination of 17q gain in patients with neuroblastoma by analysis of circulating DNA.Combaret V Brejon S Iacono I et al 2011 - 21.
Preoperative analysis of 11q loss using circulating tumor-released DNA in serum: a novel diagnostic tool for therapy stratification of neuroblastoma.Yagyu S Iehara T Gotoh T et al 2011 2011 309 185 - 22.
Influence of neuroblastoma stage on serum-based detection of MYCN amplification.Combaret V Hogarty M. D London W. B et al 2009 - 23.
TRAIL/Apo-2L: mechanisms and clinical applications in cancer.Srivastava R. K 2001 2001 3 535 - 24.
Methylation of CASP8, DCR2, and HIN-1 in neuroblastoma is associated with poor outcome.Yang Q Kiernan C. M Tian Y et al 2007 2007 13 3191 - 25.
Tumor-specific down-regulation of the tumor necrosis factor-related apoptosis-inducing ligand decoy receptors DcR1 and DcR2 is associated with dense promoter hypermethylation.Van Noesel M. M Van Bezouw S Salomons G. S et al 2002 2002 62 2157 - 26.
Methylation of tumor-suppressor genes in neuroblastoma: The RASSF1A gene is almost always methylated in primary tumors.Michalowski M. B De Fraipont F Plantaz D et al 2008 2008 50 29 - 27.
Gain of chromosome arm 17q predicts unfavourable outcome in neuroblastoma patients. U.K. Children’s Cancer Study Group and the U.K. Cancer Cytogenetics Group.Lastowska M Cotterill S Pearson A. D et al 1997 1997 33 1627 - 28.
Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group.Matthay K. K Villablanca J. G Seeger R. C et al 1999 1999 341 1165 - 29.
MYCN gene amplification is a powerful prognostic factor even in infantile neuroblastoma detected by mass screening.Iehara T Hosoi H Akazawa K et al 2006 2006 94 1510 - 30.
Localized infant neuroblastomas often show spontaneous regression: results of the prospective trials NB95-S and NB97.Hero B Simon T Spitz R et al 2008 2008 26 1504