Open access peer-reviewed chapter

A Novel Diagnostic Tool for Therapy Stratification of Neuroblastoma: Preoperative Analysis of Tumor Biology Using Circulating Tumor-Released DNA in Serum

Written By

Shigeki Yagyu, Tomoko Iehara and Hajime Hosoi

Submitted: May 8th, 2012Reviewed: January 10th, 2013Published: May 29th, 2013

DOI: 10.5772/55793

Chapter metrics overview

1,379 Chapter Downloads

View Full Metrics

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 MYCNgene amplification (MNA) [4, 5]. Above all, the patterns of genetic changes can predict the subgroups of NB [6]. The low-risk, favorable NBs have mitotic disorders, and are characterized by near triploid karyotypes with whole chromosomal gains. On the other hand, the high-risk, unfavorable NBs exhibit genetic instability, and are characterized by chromosomal structural changes, including deletion of 1p or 11q, unbalanced gain of 17q, and/or MNA. MNA is the most powerful prognostic factor identified so far, and is useful regardless of the tumor stage. In the recent studies, the 5-year event free survival of patients who had NB tumors with MNA was 53% for localized NB [7] and 29% for all NBs [8], even with intensive, multimodal treatment. On the other hand, NB without MYCNamplification (non-MNA) falls into another two clinically distinct subgroups: a low-risk subgroup with overall survival rates of more than 95% without any intensive therapy, and a high-risk subgroup with overall survival rates of less than 40% despite the use of dose-intensive, multimodal therapy. In the high-risk non-MNA group, the development of NB depends on factors other than MNA, such as the expression profiles of other genes [9], aberrant hypermethylation of tumor suppressor genes [10], and chromosomal loss of heterozygosity (LOH) [11]. To determine the stratification of NB patients more precisely with regard to the treatment approach, a screen for these genetic aberrations should be performed before the initial treatment. Indeed, in the INRG staging system, routine assessments of MNA and 11q loss were required for the therapeutic stratification of NB [8]. Currently, genetic alterations in NB tumors are clinically evaluated by interphase dual-color fluorescence in situ hybridization (I-FISH), array comprehensive genomic hybridization (aCGH), PCR and multiple ligation probe amplification (MLPA) [12]. However, the evaluation of tumor-related genetic aberrations requires a fresh tumor sample, which is often difficult to obtain due to the frequent occurrence of life-threatening conditions in NB patients.

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 MYCNgene amplification, aberrant promoter hypermethylation, and chromosomal loss of 11q. These less invasive techniques are clinically useful for the preoperative risk stratification of NB patients, because genomic changes in the tumor correlate with the tumor behavior, survival outcome, and response to therapy of NB patients. In this paper, we provide an update on the novel serum-based system for evaluating genetic aberrations for the risk stratification of patients with NB, including our recent studies.

Advertisement

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 MYCNgene in the sera of NB patients

The MYCNoncogene can be detected in the serum of patients with MNA-NB. We previously established a system for quantitatively evaluating system of MYCNgene copy number using serum DNA of NB patients [17]. We simultaneously quantified the dosages of the MYCNgene located on 2p24 as a target, and the NAGKgene located on 2p12 as a reference by real-time PCR using DNA samples obtained from paired serum and tumor samples before the initial treatment. Also, we evaluated the MYCNgene copy number to obtain the MYCN/NAGKratio. The MYCK/NAGKratios of serum DNA and tumor DNA were strongly correlated, and the serum MYCN/NAGKratio in the MNA group was significantly higher than the ratio in the non-MNA group. Notably, the sensitivity and specificity of the serum MYCN/NAGKratio as a diagnostic test were both 100% when the serum MYCN/NAGKratio cutoff was set at 10.0 (Figure 1). We also reported that the serum-based MYCNstatus was an indicator of therapeutic efficacy. Among six MNA patients whose clinical courses were followed, the serum ratios decreased to within the normal range in the patients in remission (n=3), whereas the ratios increased to high levels in the patients who relapsed (n=2) or failed to achieve remission (n=1). These data strongly suggested that the serum-based quantitative MYCNstatus was a useful tool for preoperatively determining the stratification for therapy and for the evaluation of therapeutic efficacy during the course of treatment, when tumor cells were not available for a molecular analysis. Considering that it is often impossible to obtain tumor samples from patients with advanced NB for biological studies due to their life-threatening conditions, these data may have important clinical implications.

Figure 1.

A scatter plot of serumMYCN/NAGKratio according to tumorMYCNstatus. The serumMYCN/NAGKratio was significantly (p<0.001) higher in the MNA group (median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) than in the non-MNA group (median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26; Mann-WhitneyUtest). Adopted from Ref. [17]

Combaret et al., who first reported the existence of the MYCNgene in the sera of NB patients confirmed our data using a European cohort and the same method that we used. Also, they indicated that the sensitivity of serum-based MYCNstatus depends on the stage of NB and is lower for patients with locoregional NB [22]. Indeed, they analyzed 10 serum samples obtained at diagnosis from MNA-NB patients with INSS stages 1 and 2, and 16 serum samples from MNA-NB patients with INSS stage 3 disease, and revealed that only one of 10 patients with stage 1 and 2 disease, and 12 of the 16 INSS patients with stage 3 disease showed high levels of circulating MYCNDNA sequences (10% and 75% sensitivity, respectively). In contrast, significant levels of circulating MYCNDNA sequences were detected in the patients with stage 4 disease and MNA (85% sensitivity).

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 MYCN/NAGKratio would be a specific tool for the prediction of tumor MYCNamplification in patients with NB regardless of tumor stage, even though the sensitivity of serum MNA analysis showed a tendency to be low among the patients with INSS stages 1 and 2. Indeed, for most therapeutic regimens, primary radical resection is recommended for localized non-MNA NB. Moreover, up-front surgical resection is not indicated for advanced localized NB, including tumors with MNA. Therefore, knowing the preoperative serum-based MYCNstatus should result in better decision-making, especially for localized NB. As mentioned above, circulating tumor-derived cell-free DNA could be detected in the serum of cancer patients regardless of the tumor stage. Therefore, it was of interest to determine whether the serum-based MYCNstatus was also useful to determine the risk classification for localized NB. Being able to determine the MYCNstatus of NB patients from a blood sample would be very useful for cases who cannot provide tumor samples for a molecular analysis.

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 DCR2gene in serum is a potent prognostic factor especially in non-MNA NB [18]. DCR2(decoy receptor 2) is a tumor necrosis factor alpha receptor superfamily gene, and is negatively associated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, because it lacks an intracellular death domain [23]. In NB tumors, the methylation profile of DCR2has been found to be drastically different and independent of the MYCNstatus [10, 24, 25]. Moreover, DCR2methylation was found to be associated with rapidly progressing tumors and a reduced overall survival [10]. Using the methylation-specific real-time PCR analysis we established [18], the aberrant hypermethylation in the promoter region of DCR2gene could also be detected in the serum DNA, and strongly correlated with the expression in the tumor. The 5-year event-free survival and overall survival of NB patients with methylated DCR2, as detected by a serum-based assay, were significantly poorer than those of NB patients with unmethylated DCR2[18] (Figure 2). These observations indicated that the serum-based DCR2methylation status could help predict the prognosis of NB patients, especially those without MNA. Additionally, the serum-based DCR2methylation status can distinguish patients with a poor outcome within the non-MNA group, and may allow for a new type of risk stratification for patients with non-MNA NB in future trials.

Figure 2.

Event-free and overall survival for patients with neuroblastoma according to the serum-basedDCR2methylation status. A: Event-free survival in all patients with neuroblastoma: methylated (n = 24) and unmethylated (n = 62); P < 0.001. B: Event-free survival in patients with non-MNA neuroblastoma: methylated (n = 15) and unmethylated (n = 53); P < 0.001. C: Overall survival in all patients with neuroblastoma: methylated (n = 24) and unmethylated (n = 62); P = 0.008. D: Overall survival in patients with non-MNA neuroblastoma: methylated (n = 15) and unmethylated (n = 53); P < 0.001. Adopted from Ref. [18]

RASSF1Ais hypermethylated in 93-94% of NB primary tumors and in 100% of relapsed tumors [19, 26]. RASSF1Ahypermethylation in tumors appears to be a relatively early event in NB tumorigenesis, as it is usually detectable in early stage tumors. On the other hand, RASSF1Amethylation was detected in the serum from only 25% of NB patients, and methylated RASSF1Awas more frequently detectable in the serum of advanced NB patients than in those with early stage NB, although the RASSF1Amethylation status in NB tumors was not significantly different between patients with advanced and early stage NB. These discrepancies in the sensitivities between serum-based and tumor-based methylation assays could be explained by the smaller quantity of DNA released from a small tumor burden. Nevertheless, this serum-based RASSF1Amethylation assay should help to determine the appropriate risk classification.

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 MYCNstatus.

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.

Figure 3.

Representative case of 11q loss. The results of 11q microsatellite analysis were shown. This case was a neuroblastoma categorized into low-risk subgroup (INSS stage2,MYCNnon-amplified NB, Shimada: Favorable Histology). Complete resection after initial diagnosis was performed without any additional chemotherapy. Relapse at bone and bone marrow 1 year after resection was observed, and he died of disease 3year after relapse. Microsatellite analysis were performed using his serum, tumor and leucocyte DNA, and STS marker D11S4127 located on 11q 23.3. Non-tumor DNA purified from leukocytes has two fluorescent peaks (shown in blue), whereas in tumor and serum DNA, one of the peaks is reduced, suggesting the existence of 11q loss. Adopted from Ref. [21].

Advertisement

3. Discussion

In a large-scale randomized trial of children with high-risk NBs [28], the MYCNstatus was unknown in 27% of the children. In a clinical setting, some life-threatening cases with a huge mass or hepatomegaly (hepatic metastasis in stage 4s) received chemotherapy and/or radiotherapy based on elevated tumor markers and positive MIBG scintigraphy, prior to tumor biopsy without evaluation of the MYCNstatus. The serum-based assays described above will be most useful when a primary tumor biopsy is not possible and when genetic information will influence the risk grouping and treatment allocation of the NB patients.

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. 1.LondonW. BCastleberryR. PMatthayK. Ket alEvidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children’s Oncology Group.20052005236459
  2. 2.BrodeurG. MPritchardJBertholdFet alRevisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment.19931993111466
  3. 3.PeuchmaurMdAmoreE. SJoshiVV et al. Revision of the International Neuroblastoma Pathology Classification: confirmation of favorable and unfavorable prognostic subsets in ganglioneuroblastoma, nodular.20032003982274
  4. 4.BrodeurG. MSeegerR. CSchwabMet alAmplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage.198419842241121
  5. 5.SeegerR. CBrodeurG. MSatherHet alAssociation of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas.198519853131111
  6. 6.BrodeurG. MNeuroblastoma: biological insights into a clinical enigma.200320033203
  7. 7.BagatellRBeck-popovicMLondonW. Bet alSignificance of MYCN amplification in international neuroblastoma staging system stage 1 and 2 neuroblastoma: a report from the International Neuroblastoma Risk Group database.2009200927365
  8. 8.CohnS. LPearsonA. DLondonW. Bet alThe International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report.2009200927289
  9. 9.OhiraMObaSNakamuraYet alExpression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas.200520057337
  10. 10.BanelliBGelviIDi Vinci A et al. Distinct CpG methylation profiles characterize different clinical groups of neuroblastic tumors.20052005245619
  11. 11.AttiyehE. FLondonW. BMosseY. Pet alChromosome 1p and 11q deletions and outcome in neuroblastoma.200520053532243
  12. 12.AmbrosP. FAmbrosI. MBrodeurG. Met alInternational consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee.200920091001471
  13. 13.AnkerPLefortFVasioukhinVet alK-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer.199719971121114
  14. 14.KirkG. DLesiO. AMendyMet alser) TP53 mutation in plasma DNA, hepatitis B viral infection, and risk of hepatocellular carcinoma.20052005245858
  15. 15.NawrozHKochWAnkerPet alMicrosatellite alterations in serum DNA of head and neck cancer patients.1996199621035
  16. 16.CombaretVAudoynaudCIaconoIet alCirculating MYCN DNA as a tumor-specific marker in neuroblastoma patients.20022002623646
  17. 17.GotohTHosoiHIeharaTet alPrediction of MYCN amplification in neuroblastoma using serum DNA and real-time quantitative polymerase chain reaction.20052005235205
  18. 18.YagyuSGotohTIeharaTet alCirculating methylated-DCR2 gene in serum as an indicator of prognosis and therapeutic efficacy in patients with MYCN nonamplified neuroblastoma.20082008147011
  19. 19.MisawaATanakaSYagyuSet alRASSF1A hypermethylation in pretreatment serum DNA of neuroblastoma patients: a prognostic marker.20092009100399
  20. 20.CombaretVBrejonSIaconoIet alDetermination of 17q gain in patients with neuroblastoma by analysis of circulating DNA.2011
  21. 21.YagyuSIeharaTGotohTet alPreoperative analysis of 11q loss using circulating tumor-released DNA in serum: a novel diagnostic tool for therapy stratification of neuroblastoma.20112011309185
  22. 22.CombaretVHogartyM. DLondonW. Bet alInfluence of neuroblastoma stage on serum-based detection of MYCN amplification.2009
  23. 23.SrivastavaR. KTRAIL/Apo-2L: mechanisms and clinical applications in cancer.200120013535
  24. 24.YangQKiernanC. MTianYet alMethylation of CASP8, DCR2, and HIN-1 in neuroblastoma is associated with poor outcome.20072007133191
  25. 25.Van NoeselM. MVan BezouwSSalomonsG. Set alTumor-specific down-regulation of the tumor necrosis factor-related apoptosis-inducing ligand decoy receptors DcR1 and DcR2 is associated with dense promoter hypermethylation.20022002622157
  26. 26.MichalowskiM. BDe FraipontFPlantazDet alMethylation of tumor-suppressor genes in neuroblastoma: The RASSF1A gene is almost always methylated in primary tumors.200820085029
  27. 27.LastowskaMCotterillSPearsonA. Det alGain of chromosome arm 17q predicts unfavourable outcome in neuroblastoma patients. U.K. Children’s Cancer Study Group and the U.K. Cancer Cytogenetics Group.19971997331627
  28. 28.MatthayK. KVillablancaJ. GSeegerR. Cet alTreatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group.199919993411165
  29. 29.IeharaTHosoiHAkazawaKet alMYCN gene amplification is a powerful prognostic factor even in infantile neuroblastoma detected by mass screening.20062006941510
  30. 30.HeroBSimonTSpitzRet alLocalized infant neuroblastomas often show spontaneous regression: results of the prospective trials NB95-S and NB97.20082008261504

Written By

Shigeki Yagyu, Tomoko Iehara and Hajime Hosoi

Submitted: May 8th, 2012Reviewed: January 10th, 2013Published: May 29th, 2013