Prediction of MYCN Amplification in Neuroblastoma Using Serum DNA and Real-Time Quantitative Polymerase Chain Reaction
http://www.100md.com
《临床肿瘤学》
the Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto
Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan
ABSTRACT
PURPOSE: MYCN amplification (MNA) indicates a poor prognosis in neuroblastoma (NB) and is routinely assayed for therapy stratification. We aimed to develop a diagnostic tool to predict MYCN status using serum DNA, which, in cancer patients, predominantly originates from tumor-released DNA.
PATIENTS AND METHODS: Using DNA-based real-time quantitative polymerase chain reaction, we simultaneously quantified MYCN (2p24) and a reference gene, NAGK (2p12), and evaluated MYCN copy number as an MYCN/NAGK (M/N) ratio in 87 NB patients whose MYCN status had been determined by Southern blotting. Of these patients, 17 had MYCN-amplified NB, and 70 had nonamplified NB.
RESULTS: The serum M/N ratio in the MNA group (median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26; Mann-Whitney U test). The sensitivity and specificity of the serum M/N ratio as a diagnostic test were both 100% when the serum M/N ratio cutoff was set at 10.0. Among six MNA patients whose clinical courses were followed, the serum ratios decreased to 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).
CONCLUSION: Measurement of the serum M/N ratio seems to be a promising method for accurately assessing MYCN status in NB, although a larger set of patients needs to be examined to confirm this result.
INTRODUCTION
Neuroblastoma (NB) is the most common extracranial solid tumor in children and is characterized by a wide range of clinical behaviors, from spontaneous regression to rapid progression with a fatal outcome. The clinical heterogeneity has been reported to be associated with a variety of biologic features of NB. One such aberration, MYCN amplification (MNA; ie, creation of multiple copies of the MYCN gene in the nuclei of tumor cells), is strongly associated with rapid tumor progression and a poor outcome. MNA is detected in 4% of patients in the early stages of NB, 8% of patients in stage 4S, and approximately 30% of patients in advanced stages. Currently, assessment of MYCN status is essential for determining therapy stratification in NB.1-6 Having rapid access to selected biologic data for each tumor has become increasingly important in routing patients to appropriate therapies. Several years ago, fluorescence in situ hybridization (FISH) replaced Southern blotting as the most accurate and timely way of evaluating tumors for MNA. Using FISH, the turnaround time for results was shortened from weeks to days, making its use in clinical trials realistic.
In this study, we describe a real-time polymerase chain reaction (PCR) method for evaluating MYCN status that shortens the turnaround for results to just a few hours. Furthermore, to facilitate the evaluation of MYCN status of tumors, we used serum DNA for the PCR template, which, in cancer patients, predominantly consists of tumor-released DNA.7 Quantification of serum DNA has also been proposed as a screening tool for early detection of lung cancer.8 Several groups were able to detect tumor-related aberrations, such as loss of heterozygosity and mutations in the p53 gene, using the serum DNA of patients with a malignant tumor.9-11
Recently, Combaret et al12 reported that high levels of MYCN DNA were present in the peripheral blood of patients with MYCN-amplified NB. However, they evaluated serum MYCN (2p24) dosage based on PCR without a reference gene, so their assay could be influenced by the quality of the template DNA or a numerical change of chromosome 2. To avoid these problems, we used DNA-based real-time quantitative PCR and a single copy reference gene, the N-acetylglucosamine kinase gene (NAGK; 2p12), so that MYCN copy number per chromosome 2 could be evaluated as the MYCN/NAGK (M/N) ratio. NAGK was chosen because it is on the same chromosome as MYCN but sufficiently distant from the region spanned by the MYCN amplicon (2p12 v 2p24)13 that a numerical change in chromosome 2 would not affect the M/N ratio. The diagnostic performance of the test was evaluated in patients with an NB whose MYCN status had been determined by Southern blotting.
PATIENTS AND METHODS
Subjects
Eighty-seven patients diagnosed with NB at the Hospital of the Kyoto Prefectural University of Medicine and Chiba Cancer Center Research Institute were enrolled onto this study with the informed consent of their parents. The studies were conducted under research protocols approved by each institutional review board. At the time of diagnosis, 44 patients were younger than 1 year, and 43 were between 1 and 13 years of age. Seventeen of the patients had MNA, and 70 patients did not have MNA, as determined by Southern blotting. According to the International Neuroblastoma Staging System,4 the 17 children with MNA included one patient each in stage 1 and 2B, two in stage 3, and 13 in stage 4, whereas the 70 children without MNA included 22 in stage 1, 18 in stage 2A and 2B, five in stage 4S, seven in stage 3, and 18 in stage 4.
Twelve of the 17 patients with MNA and 33 of the 70 nonamplified patients were also analyzed by dual-color FISH technique, as previously described,14 using an MYCN probe (pNb101) and a chromosome 2 centromere probe (D2Z). FISH results of these patients were consistent with the Southern blotting results, although three of the patients who were diagnosed as non-MNA by Southern blotting were found to have one to four extra copies of the MYCN gene relative to the chromosome 2 centromere number by FISH. This low level of amplification has been defined as MYCN gain, which is an intermediate stage between MNA and non-MNA.15 Because the prognostic significance of MYCN gain is still unclear, these patients were classified as non-MNA according to the Southern blotting results.
Sample Preparation
Tumor specimens were surgically resected and immediately stored at –80°C. Peripheral blood was obtained from each patient before any therapy and surgery. To avoid contamination of serum DNA by the DNA from WBCs, we prepared serum exclusively from the liquid fraction of clotted blood after centrifugation at 1,000 x g for 10 minutes and stored it at –20°C until DNA extraction.
DNA Isolation
DNA was extracted from tissues and serum samples by using the QIAmp tissue and blood kits (Qiagen, GmbH, Hilden, Germany), respectively, according to the manufacturer's protocols. For each patient, 200 μL of the stored serum was used for extraction of free DNA.
Real-Time Quantitative PCR
TaqMan PCR was performed using the ABI Prism 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR mixture contained TaqMan universal PCR master mix (Applied Biosystems), 200 nmol/L of each primer, and 100 nmol/L of fluorogenic probe. The principle of the TaqMan analysis has been described previously in detail.16-18 In addition to the MYCN sequence, NAGK (GenBank accession No. NM 017567) located at 2p12 was simultaneously measured as a single-copy reference gene. The sequence of primers and the TaqMan probe used for MYCN and NAGK are as follows: MYCN forward, 5'-GTGCTCTCCAATTCTCGCCT-3'; MYCN reverse, 5'-GATGGCCTAGAGGAGGGCT-3'; MYCN probe, 5'-FAM-CACTAAAGTTCCTTCCACCCTCTCCT-TAMRA-3'; NAGK forward, 5'-TGGGCAGACACATCGTAGCA-3'; NAGK reverse, 5'-CACCTTCACTCCCACCTCAAC-3'; and NAGK probe, 5'-VIC-TGTTGCCCGAGATTGACCCGGT-TAMRA-3'. All PCR reactions were performed with one cycle of 95°C for 5 minutes, followed by PCR amplification with 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. Standard curves were constructed in each PCR run with four-fold serial dilutions containing 20, 5, 1.25, 0.3125, and 0.078125 ng/μL of a healthy donor's DNA in addition to 20 ng/μL of salmon sperm DNA, and the dosages of the target genes in each sample were interpolated using these standard curves. The MYCN copy number of a sample of DNA was determined by the ratio of the MYCN dosage to the NAGK dosage (M/N ratio). Copy numbers were expressed as the average of two measurements.
Effect of WBC Contamination
To assess the effect of WBC contamination in serum samples on the serum M/N ratio, we measured the serum M/N ratio using DNA extracted from a series of WBC-contaminated serum samples. The samples were prepared by adding 0, 1 x 10, 1 x 102, 1 x 103, 1 x 104, and 1 x 105 of WBCs from a healthy donor to 200 μL of serum from a MYCN-amplified patient.
Statistical Methods
The difference in the serum M/N ratio between the MNA and non-MNA groups was assessed using the Mann-Whitney U test. P < .05 was judged as significant.
RESULTS
Serum M/N Ratio As a Predictor of MYCN Status of Tumor
Serum M/N ratios could be determined in approximately 4 hours by real-time quantitative PCR. Figure 1 shows the distribution of the serum M/N ratio in the MNA and non-MNA groups at the time of diagnosis. The serum M/N ratio in the MNA group (n = 17; median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (n = 70; median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26). In fact, there was no overlap between the two groups in the limited number of patients examined in this study. As a cutoff for the serum M/N ratio to distinguish between MNA and non-MNA patients, we empirically chose a value of 10, which was in the middle of the two ranges. With this value, the sensitivity and specificity of the serum M/N ratio as a diagnostic test to distinguish patients with MNA from those without MNA were both 100% for our limited number of patients. That is, the serum M/N ratio was in complete agreement with the Southern blotting results. The positive and negative predictive values were 100%. The serum M/N ratios were also consistent with results obtained by FISH for 45 of the patients (FISH analyses were performed in 12 of the 17 MNA patients and in 33 of the 70 nonamplified patients). Three of the patients who had one to four extra copies of the MYCN gene relative to chromosome 2 centromere number, as determined by FISH, also had slightly elevated serum M/N ratios (2.5, 3.3, and 4.6).
Change in Serum M/N Ratio Levels During Follow-Up
To evaluate whether an increase in the serum M/N ratio can be used as an indicator of relapse, we measured serum M/N ratios at several points in the clinical courses of six patients with MNA (Fig 2). In three patients who were in complete remission (patients 1, 2, and 3), the serum M/N ratios decreased to the normal range and were consistently low. In contrast, in one patient who failed to achieve remission (patient 4), the serum M/N ratio did not decrease to the normal range and remained at a high level until his death. In the other patients who experienced recurrence after remission (patients 5 and 6), the serum M/N ratio first decreased to the normal range and then increased beyond the cutoff value by the time of diagnosis.
Effect of WBC Contamination on Serum M/N Ratio
We found that a high serum M/N ratio could be masked by the presence of WBC. The M/N ratio of serum from an MYCN-amplified patient decreased with increasing WBC contamination (Fig 3). When 200 μL of serum was contaminated with 1 x 105 of WBC, corresponding to approximately one fortieth of the WBC concentration in normal whole blood, the serum M/N ratio decreased below the cutoff level.
DISCUSSION
Serum markers, such as ferritin,19 lactic dehydrogenase,20 and neuron-specific enolase,21 have been proposed as prognostic markers of NB, although they have shown little prognostic value. Recently, elevated levels of plasma midkine have been reported to correlate with a poor prognosis. However, the significance of this finding is controversial because plasma midkine levels are highest in stage 4S patients.22 Therefore, a noninvasive assay of tumor-related genetic aberrations using serum DNA is desirable for the assessment of prognosis and therapy stratification at the time of diagnosis. Among the tumor-related genetic aberrations detected in NB, MNA was of greatest interest to us because of its significant prognostic value.
By using DNA-based real-time quantitative PCR with a single-copy reference gene, we have demonstrated that the M/N ratio in serum DNA is a valuable diagnostic tool to discriminate MNA patients from non-MNA patients. The serum M/N ratio in the MNA group was significantly higher than the ratio in the non-MNA group, without an overlap. The highest sensitivity (100%), highest specificity (100%), highest positive predictive value (100%), and highest negative predictive value (100%) were obtained with a serum M/N ratio cutoff value of 10.0. Furthermore, we found an elevated level of the serum M/N ratio in a stage 1 patient and a stage 2B patient with MNA (188.7 and 901.6, respectively), even though the tumor was localized in these patients. This suggests that tumors could release a significant amount of genomic DNA into the systemic circulation even at an early stage. Furthermore, Sozzi et al23 reported that the concentration of plasma DNA in 84 lung cancer patients was higher than the concentration in 43 controls, regardless of the tumor stage, and suggested that circulating DNA in peripheral blood was an early event in lung carcinogenesis.
Another clinical benefit of the serum M/N assay is that it could be used as a marker to monitor therapeutic efficacy and recurrence after therapies. The serum M/N ratio decreased to the normal range in the patients in remission but remained at a high level in the patient who failed to achieve remission. Furthermore, in two patients with recurrence after remission, the serum M/N ratio initially decreased to the normal range but then increased beyond the cutoff value by the time of diagnosis. The serum M/N ratio did not increase to the initial level as long as the metastasis was localized in the brain, but it did increase to the initial level when the patient later developed a bone metastasis (patient 6). This is noteworthy because it suggests that a brain metastasis releases genomic DNA into the systemic circulation less easily than extracranial tumors. If this is confirmed by examination of additional patients, then it is possible that tumors localized in brain could be overlooked with diagnostic assays based on serum DNA.
A possible pitfall of our serum M/N assay is that a high serum M/N ratio could be reduced by WBC contamination (Fig 3). This could be a result of dilution of tumor DNA with the WBC DNA, which would be expected to have an M/N ratio of 1. Therefore, the importance of removing WBCs from serum should be addressed in diagnostic assays that use serum DNA. For the same reason, a predominance of any nontumor DNA in serum may lower an elevated M/N ratio of an MYCN-amplified patient. However, this assay can be accurate on the premise that, in cancer patients, serum DNA predominantly consists of tumor-released DNA.7 In addition, the use of serum DNA as a diagnostic tool in lung cancer patients has resulted in a diversity of findings, suggesting that these differences likely reflect variations in the manner in which the blood specimens were collected and handled and variations in the methods by which the assay were conducted.24 Therefore, it is necessary to standardize the serum collection procedure to ensure that different laboratories obtain the same result with a given blood sample. An additional high-speed centrifugation step (16,000 x g for 5 minutes) was found to eliminate cellular contamination even after thawing of stored samples.25 By using the appropriate centrifugation methods, we believe that WBC-free serum can be reliably achieved.
Although a large set of patients needs to be studied to verify the accuracy of this assay and to set an appropriate cutoff, our results are promising and need to be further tested. The advantages of this method are that it takes only 4 hours and much less effort than FISH and Southern blotting, which should make this assay an alternative to these other methods for determining MYCN status. A third advantage is that the serum M/N ratio seems to be a promising indicator of therapeutic efficacy and relapse in the follow-up of patients with MNA, although more patients need to be examined to confirm its reliability.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank two anonymous reviewers for helpful comments.
NOTES
Supported by Grants-in-Aid for Scientific Research grant Nos. 15659248, 15659249, and 14370250 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Brodeur GM, Seeger RC, Schwab M, et al: Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224: 1121-1124, 1984
Seeger RC, Brodeur GM, Sather H, et al: Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 313: 1111-1116, 1985
Brodeur GM, Maris JM, Yamashiro DJ, et al: Biology and genetics of human neuroblastomas. J Pediatr Hematol Oncol 19: 93-101, 1997
Brodeur GM, Pritchard J, Berthold F, et al: Revisions of the international criteria for neuroblastoma diagnosis, staging and response to treatment. J Clin Oncol 11: 1466-1477, 1993
Castleberry RP, Prichard J, Ambros P, et al: The International Neuroblastoma Risk Groups (INRG): A preliminary report. Eur J Cancer 33: 2113-2116, 1997
Shimada H, Ambros IM, Dehner LP, et al: The International Neuroblastoma Pathology Classification System (the Shimada system). Cancer 86: 364-372, 1999
Shapiro B, Chakrabarty M, Cohn EM, et al: Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease. Cancer 51: 2116-2120, 1983
Sozzi G, Conte D, Leon M, et al: Quantification of free circulating DNA as a diagnostic marker in lung cancer. J Clin Oncol 21: 3902-3908, 2003
Sozzi G, Musso K, Ratcliffe C, et al: Detection of microsatellite alterations in plasma DNA of non-small cell lung cancer patients: A prospect for early diagnosis. Clin Cancer Res 5: 2689-2692, 1999
Chen X, Bonnefoi H, Diebold-Berger S, et al: Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer. Clin Cancer Res 5: 2297-2303, 1999
Silva JM, Dominguez G, Garcia JM, et al: Presence of tumor DNA in plasma of breast cancer patients: Clinicopathological correlations. Cancer Res 59: 3251-3256, 1999
Combaret V, Audoynaud C, Iacono I, et al: Circulating MYCN DNA as a tumor-specific marker in neuroblastoma patients. Cancer Res 62: 3646-3648, 2002
Akiyama K, Kanda N, Yamada M, et al: Megabase-scale analysis of the origin of N-myc amplicons in human neuroblastoma. Nucleic Acids Res 22: 187-193, 1994
Gotoh T, Sugihara H, Matsumura T, et al: Human neuroblastoma demonstrating clonal evolution in vivo. Genes Chromosomes Cancer 22: 42-49, 1998
Spitz R, Hero B, Skowron M, et al: MYCN-status in neuroblastoma: Characteristics of tumours showing amplification, gain, and non-amplification. Eur J Cancer 40: 2753-2759, 2004
Gelmini S, Orlando C, Sestini R, et al: Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification. Clin Chem 43: 752-758, 1997
Claudia CR, Maria LB, Gian PT, et al: Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system. Clin Chem 45: 1918-1924, 1999
Chiang PW, Beer DG, Wei WL, et al: Detection of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res 5: 1381-1386, 1999
Hann HWL, Evans AE, Siegel SE, et al: Prognostic importance of serum ferritin in patients with stage III and IV neuroblastoma: The Children's Cancer Study Group experience. Cancer Res 45: 2843-2848, 1985
Quinn JJ, Altman AJ, Frantz CN, et al: Serum lactic dehydrogenase, an indicator of tumor activity in neuroblastoma. J Pediatr 97: 89-91, 1980
Massaron S, Seregni E, Luksch R, et al: Neuron-specific enolase evaluation in patients neuroblastoma. Tumour Biol 19: 261-268, 1998
Ikematsu S, Nakagawara A, Nakamura Y, et al: Correlation of elevated level of blood midkine with poor prognostic factors of human neuroblastomas. Br J Cancer 88: 1522-1526, 2003
Sozzi G, Conte D, Mariani L, et al: Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res 61: 4675-4678, 2001
Bunn PA: Early detection of lung cancer using serum RNA or DNA markers: Ready for "prime time" or for validation? J Clin Oncol 21: 3891-3893, 2003
Swinkels DW, Wiegerinck E, Steegers EAP, et al: Effect of blood-processing protocols on cell-free DNA quantification in plasma. Clin Chem 49: 525-526, 2003(Takahiro Gotoh, Hajime Ho)
Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan
ABSTRACT
PURPOSE: MYCN amplification (MNA) indicates a poor prognosis in neuroblastoma (NB) and is routinely assayed for therapy stratification. We aimed to develop a diagnostic tool to predict MYCN status using serum DNA, which, in cancer patients, predominantly originates from tumor-released DNA.
PATIENTS AND METHODS: Using DNA-based real-time quantitative polymerase chain reaction, we simultaneously quantified MYCN (2p24) and a reference gene, NAGK (2p12), and evaluated MYCN copy number as an MYCN/NAGK (M/N) ratio in 87 NB patients whose MYCN status had been determined by Southern blotting. Of these patients, 17 had MYCN-amplified NB, and 70 had nonamplified NB.
RESULTS: The serum M/N ratio in the MNA group (median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26; Mann-Whitney U test). The sensitivity and specificity of the serum M/N ratio as a diagnostic test were both 100% when the serum M/N ratio cutoff was set at 10.0. Among six MNA patients whose clinical courses were followed, the serum ratios decreased to 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).
CONCLUSION: Measurement of the serum M/N ratio seems to be a promising method for accurately assessing MYCN status in NB, although a larger set of patients needs to be examined to confirm this result.
INTRODUCTION
Neuroblastoma (NB) is the most common extracranial solid tumor in children and is characterized by a wide range of clinical behaviors, from spontaneous regression to rapid progression with a fatal outcome. The clinical heterogeneity has been reported to be associated with a variety of biologic features of NB. One such aberration, MYCN amplification (MNA; ie, creation of multiple copies of the MYCN gene in the nuclei of tumor cells), is strongly associated with rapid tumor progression and a poor outcome. MNA is detected in 4% of patients in the early stages of NB, 8% of patients in stage 4S, and approximately 30% of patients in advanced stages. Currently, assessment of MYCN status is essential for determining therapy stratification in NB.1-6 Having rapid access to selected biologic data for each tumor has become increasingly important in routing patients to appropriate therapies. Several years ago, fluorescence in situ hybridization (FISH) replaced Southern blotting as the most accurate and timely way of evaluating tumors for MNA. Using FISH, the turnaround time for results was shortened from weeks to days, making its use in clinical trials realistic.
In this study, we describe a real-time polymerase chain reaction (PCR) method for evaluating MYCN status that shortens the turnaround for results to just a few hours. Furthermore, to facilitate the evaluation of MYCN status of tumors, we used serum DNA for the PCR template, which, in cancer patients, predominantly consists of tumor-released DNA.7 Quantification of serum DNA has also been proposed as a screening tool for early detection of lung cancer.8 Several groups were able to detect tumor-related aberrations, such as loss of heterozygosity and mutations in the p53 gene, using the serum DNA of patients with a malignant tumor.9-11
Recently, Combaret et al12 reported that high levels of MYCN DNA were present in the peripheral blood of patients with MYCN-amplified NB. However, they evaluated serum MYCN (2p24) dosage based on PCR without a reference gene, so their assay could be influenced by the quality of the template DNA or a numerical change of chromosome 2. To avoid these problems, we used DNA-based real-time quantitative PCR and a single copy reference gene, the N-acetylglucosamine kinase gene (NAGK; 2p12), so that MYCN copy number per chromosome 2 could be evaluated as the MYCN/NAGK (M/N) ratio. NAGK was chosen because it is on the same chromosome as MYCN but sufficiently distant from the region spanned by the MYCN amplicon (2p12 v 2p24)13 that a numerical change in chromosome 2 would not affect the M/N ratio. The diagnostic performance of the test was evaluated in patients with an NB whose MYCN status had been determined by Southern blotting.
PATIENTS AND METHODS
Subjects
Eighty-seven patients diagnosed with NB at the Hospital of the Kyoto Prefectural University of Medicine and Chiba Cancer Center Research Institute were enrolled onto this study with the informed consent of their parents. The studies were conducted under research protocols approved by each institutional review board. At the time of diagnosis, 44 patients were younger than 1 year, and 43 were between 1 and 13 years of age. Seventeen of the patients had MNA, and 70 patients did not have MNA, as determined by Southern blotting. According to the International Neuroblastoma Staging System,4 the 17 children with MNA included one patient each in stage 1 and 2B, two in stage 3, and 13 in stage 4, whereas the 70 children without MNA included 22 in stage 1, 18 in stage 2A and 2B, five in stage 4S, seven in stage 3, and 18 in stage 4.
Twelve of the 17 patients with MNA and 33 of the 70 nonamplified patients were also analyzed by dual-color FISH technique, as previously described,14 using an MYCN probe (pNb101) and a chromosome 2 centromere probe (D2Z). FISH results of these patients were consistent with the Southern blotting results, although three of the patients who were diagnosed as non-MNA by Southern blotting were found to have one to four extra copies of the MYCN gene relative to the chromosome 2 centromere number by FISH. This low level of amplification has been defined as MYCN gain, which is an intermediate stage between MNA and non-MNA.15 Because the prognostic significance of MYCN gain is still unclear, these patients were classified as non-MNA according to the Southern blotting results.
Sample Preparation
Tumor specimens were surgically resected and immediately stored at –80°C. Peripheral blood was obtained from each patient before any therapy and surgery. To avoid contamination of serum DNA by the DNA from WBCs, we prepared serum exclusively from the liquid fraction of clotted blood after centrifugation at 1,000 x g for 10 minutes and stored it at –20°C until DNA extraction.
DNA Isolation
DNA was extracted from tissues and serum samples by using the QIAmp tissue and blood kits (Qiagen, GmbH, Hilden, Germany), respectively, according to the manufacturer's protocols. For each patient, 200 μL of the stored serum was used for extraction of free DNA.
Real-Time Quantitative PCR
TaqMan PCR was performed using the ABI Prism 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR mixture contained TaqMan universal PCR master mix (Applied Biosystems), 200 nmol/L of each primer, and 100 nmol/L of fluorogenic probe. The principle of the TaqMan analysis has been described previously in detail.16-18 In addition to the MYCN sequence, NAGK (GenBank accession No. NM 017567) located at 2p12 was simultaneously measured as a single-copy reference gene. The sequence of primers and the TaqMan probe used for MYCN and NAGK are as follows: MYCN forward, 5'-GTGCTCTCCAATTCTCGCCT-3'; MYCN reverse, 5'-GATGGCCTAGAGGAGGGCT-3'; MYCN probe, 5'-FAM-CACTAAAGTTCCTTCCACCCTCTCCT-TAMRA-3'; NAGK forward, 5'-TGGGCAGACACATCGTAGCA-3'; NAGK reverse, 5'-CACCTTCACTCCCACCTCAAC-3'; and NAGK probe, 5'-VIC-TGTTGCCCGAGATTGACCCGGT-TAMRA-3'. All PCR reactions were performed with one cycle of 95°C for 5 minutes, followed by PCR amplification with 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. Standard curves were constructed in each PCR run with four-fold serial dilutions containing 20, 5, 1.25, 0.3125, and 0.078125 ng/μL of a healthy donor's DNA in addition to 20 ng/μL of salmon sperm DNA, and the dosages of the target genes in each sample were interpolated using these standard curves. The MYCN copy number of a sample of DNA was determined by the ratio of the MYCN dosage to the NAGK dosage (M/N ratio). Copy numbers were expressed as the average of two measurements.
Effect of WBC Contamination
To assess the effect of WBC contamination in serum samples on the serum M/N ratio, we measured the serum M/N ratio using DNA extracted from a series of WBC-contaminated serum samples. The samples were prepared by adding 0, 1 x 10, 1 x 102, 1 x 103, 1 x 104, and 1 x 105 of WBCs from a healthy donor to 200 μL of serum from a MYCN-amplified patient.
Statistical Methods
The difference in the serum M/N ratio between the MNA and non-MNA groups was assessed using the Mann-Whitney U test. P < .05 was judged as significant.
RESULTS
Serum M/N Ratio As a Predictor of MYCN Status of Tumor
Serum M/N ratios could be determined in approximately 4 hours by real-time quantitative PCR. Figure 1 shows the distribution of the serum M/N ratio in the MNA and non-MNA groups at the time of diagnosis. The serum M/N ratio in the MNA group (n = 17; median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (n = 70; median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26). In fact, there was no overlap between the two groups in the limited number of patients examined in this study. As a cutoff for the serum M/N ratio to distinguish between MNA and non-MNA patients, we empirically chose a value of 10, which was in the middle of the two ranges. With this value, the sensitivity and specificity of the serum M/N ratio as a diagnostic test to distinguish patients with MNA from those without MNA were both 100% for our limited number of patients. That is, the serum M/N ratio was in complete agreement with the Southern blotting results. The positive and negative predictive values were 100%. The serum M/N ratios were also consistent with results obtained by FISH for 45 of the patients (FISH analyses were performed in 12 of the 17 MNA patients and in 33 of the 70 nonamplified patients). Three of the patients who had one to four extra copies of the MYCN gene relative to chromosome 2 centromere number, as determined by FISH, also had slightly elevated serum M/N ratios (2.5, 3.3, and 4.6).
Change in Serum M/N Ratio Levels During Follow-Up
To evaluate whether an increase in the serum M/N ratio can be used as an indicator of relapse, we measured serum M/N ratios at several points in the clinical courses of six patients with MNA (Fig 2). In three patients who were in complete remission (patients 1, 2, and 3), the serum M/N ratios decreased to the normal range and were consistently low. In contrast, in one patient who failed to achieve remission (patient 4), the serum M/N ratio did not decrease to the normal range and remained at a high level until his death. In the other patients who experienced recurrence after remission (patients 5 and 6), the serum M/N ratio first decreased to the normal range and then increased beyond the cutoff value by the time of diagnosis.
Effect of WBC Contamination on Serum M/N Ratio
We found that a high serum M/N ratio could be masked by the presence of WBC. The M/N ratio of serum from an MYCN-amplified patient decreased with increasing WBC contamination (Fig 3). When 200 μL of serum was contaminated with 1 x 105 of WBC, corresponding to approximately one fortieth of the WBC concentration in normal whole blood, the serum M/N ratio decreased below the cutoff level.
DISCUSSION
Serum markers, such as ferritin,19 lactic dehydrogenase,20 and neuron-specific enolase,21 have been proposed as prognostic markers of NB, although they have shown little prognostic value. Recently, elevated levels of plasma midkine have been reported to correlate with a poor prognosis. However, the significance of this finding is controversial because plasma midkine levels are highest in stage 4S patients.22 Therefore, a noninvasive assay of tumor-related genetic aberrations using serum DNA is desirable for the assessment of prognosis and therapy stratification at the time of diagnosis. Among the tumor-related genetic aberrations detected in NB, MNA was of greatest interest to us because of its significant prognostic value.
By using DNA-based real-time quantitative PCR with a single-copy reference gene, we have demonstrated that the M/N ratio in serum DNA is a valuable diagnostic tool to discriminate MNA patients from non-MNA patients. The serum M/N ratio in the MNA group was significantly higher than the ratio in the non-MNA group, without an overlap. The highest sensitivity (100%), highest specificity (100%), highest positive predictive value (100%), and highest negative predictive value (100%) were obtained with a serum M/N ratio cutoff value of 10.0. Furthermore, we found an elevated level of the serum M/N ratio in a stage 1 patient and a stage 2B patient with MNA (188.7 and 901.6, respectively), even though the tumor was localized in these patients. This suggests that tumors could release a significant amount of genomic DNA into the systemic circulation even at an early stage. Furthermore, Sozzi et al23 reported that the concentration of plasma DNA in 84 lung cancer patients was higher than the concentration in 43 controls, regardless of the tumor stage, and suggested that circulating DNA in peripheral blood was an early event in lung carcinogenesis.
Another clinical benefit of the serum M/N assay is that it could be used as a marker to monitor therapeutic efficacy and recurrence after therapies. The serum M/N ratio decreased to the normal range in the patients in remission but remained at a high level in the patient who failed to achieve remission. Furthermore, in two patients with recurrence after remission, the serum M/N ratio initially decreased to the normal range but then increased beyond the cutoff value by the time of diagnosis. The serum M/N ratio did not increase to the initial level as long as the metastasis was localized in the brain, but it did increase to the initial level when the patient later developed a bone metastasis (patient 6). This is noteworthy because it suggests that a brain metastasis releases genomic DNA into the systemic circulation less easily than extracranial tumors. If this is confirmed by examination of additional patients, then it is possible that tumors localized in brain could be overlooked with diagnostic assays based on serum DNA.
A possible pitfall of our serum M/N assay is that a high serum M/N ratio could be reduced by WBC contamination (Fig 3). This could be a result of dilution of tumor DNA with the WBC DNA, which would be expected to have an M/N ratio of 1. Therefore, the importance of removing WBCs from serum should be addressed in diagnostic assays that use serum DNA. For the same reason, a predominance of any nontumor DNA in serum may lower an elevated M/N ratio of an MYCN-amplified patient. However, this assay can be accurate on the premise that, in cancer patients, serum DNA predominantly consists of tumor-released DNA.7 In addition, the use of serum DNA as a diagnostic tool in lung cancer patients has resulted in a diversity of findings, suggesting that these differences likely reflect variations in the manner in which the blood specimens were collected and handled and variations in the methods by which the assay were conducted.24 Therefore, it is necessary to standardize the serum collection procedure to ensure that different laboratories obtain the same result with a given blood sample. An additional high-speed centrifugation step (16,000 x g for 5 minutes) was found to eliminate cellular contamination even after thawing of stored samples.25 By using the appropriate centrifugation methods, we believe that WBC-free serum can be reliably achieved.
Although a large set of patients needs to be studied to verify the accuracy of this assay and to set an appropriate cutoff, our results are promising and need to be further tested. The advantages of this method are that it takes only 4 hours and much less effort than FISH and Southern blotting, which should make this assay an alternative to these other methods for determining MYCN status. A third advantage is that the serum M/N ratio seems to be a promising indicator of therapeutic efficacy and relapse in the follow-up of patients with MNA, although more patients need to be examined to confirm its reliability.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank two anonymous reviewers for helpful comments.
NOTES
Supported by Grants-in-Aid for Scientific Research grant Nos. 15659248, 15659249, and 14370250 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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