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Clinical Chemistry 45: 1918-1924, 1999;
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(Clinical Chemistry. 1999;45:1918-1924.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Real-Time Quantitative PCR for the Measurement of MYCN Amplification in Human Neuroblastoma with the TaqMan Detection System

Claudia Casini Raggi1, Maria Letizia Bagnoni1, Gian Paolo Tonini3, Mario Maggi2, Giovanna Vona1, Pamela Pinzani1, Katia Mazzocco3, Bruno De Bernardi4, Mario Pazzagli1 and Claudio Orlando1,a

1 Clinical Biochemistry and
2 Andrology Units, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy.

3 Unit of Solid Tumor Biology, Advanced Biotechnology Centre,
4 Giannina Gaslini Children's Hospital, 16132 Genoa, Italy.
a Address correspondence to this author at: Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. Fax 39-55-4377290; e-mail c.orlando{at}dfc.unifi.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients.

Methods: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqManTM reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, ß-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA.

Results: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0.987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (<9; P = 0.02) and high (>=9; P = 0.03) oncogene amplification.

Conclusion: The assay is rapid and reproducible and does not require any post-PCR analytical procedure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neuroblastoma is the most common extracranial solid malignancy in children under 5 years of age (1), accounting for a large proportion of cancer deaths in this population. The prognosis is negative in many cases, in part because of the extent of disease at diagnosis as well as resistance of the tumors to conventional therapies (2). However, this tumor can also exhibit favorable characteristics, including the possibility of evolving into a more benign form or regressing spontaneously (2). The clinical evaluation of the disease (3), including in vivo imaging and bone marrow examinations, and the age of the child at diagnosis (4) allow definition of a prognosis in most cases. Unfortunately, some patients in which a favorable prognosis is expected on the basis of these clinical considerations can relapse, and some of them will die of the disease (5).

Progress in the management of neuroblastoma requires a more precise evaluation based on the characterization of some biochemical and molecular abnormalities. Serum ferritin (6), neuron-specific enolase (7), lactate dehydrogenase (8), and catecholamine metabolites in urine (9) have shown limited prognostic value.

The molecular characterization of neuroblastoma seems a more appropriate tool in the diagnosis of tumor aggressiveness and progression. Several molecular markers have been identified, variously related to patient survival: deletion or allelic loss of the short arm of chromosome 1 (10); DNA ploidy (11); the expression of nerve growth factor receptor, encoded by the TRKA gene (12); and the expression of genes involved in multidrug resistance (MDR1 and MRP) (13)(14) as well as of genes related to tumor invasion and metastasis (nm23 and CD44) (15)(16). More recently, additional insights have been derived from the detection of telomerase activity (17) and from the measurement of type 2 somatostatin receptor (18). However, in clinical practice, the measurement of MYCN amplification remains the cornerstone molecular marker routinely determined at diagnosis (2)(5)(19)(20)(21)(22)(23)(24)(25). This oncogene seems to play a pivotal role in the biological features of neuroblastoma. MYCN amplification correlates with both advanced disease stage (19) and rapid tumor progression (20). In localized neuroblastomas, MYCN amplification is the major prognostic factor (26), identifying patients who do not require aggressive therapy (27).

Oncogene amplification is a common DNA alteration in cancer, causing an increase of encoded protein synthesis (28). Several methods have been proposed to detect oncogene amplification, based mainly on Southern or dot blot (29)(30), on quantitative PCR (31)(32), and on fluorescence in situ hybridization techniques (33). More recently, we proposed the measurement of c-erbB-2 oncogene amplification by a PCR-based homogeneous assay that uses fluorogenic probes (34). This procedure was based on the endpoint measurement of fluorescence generated by the cleavage of the fluorogenic probe via the 5' -> 3' exonuclease activity of Taq polymerase according the TaqManTM reaction. However, even under the best controlled experimental conditions, the measurement of endpoint PCR products represents a difficult instrument for quantitative PCR.

Here we present an evolution of the TaqMan reaction for measurement of MYCN amplification in DNA from neuroblastoma tumors, based on the real-time detection of PCR kinetics. This technique allows the measurement of PCR products in the first cycles of amplification, without any influence deriving from the complex reactions that take place as the PCR reaction approaches the plateau phase. The results obtained with the proposed method were compared with those of a previously assessed competitive PCR method based on the use of a multiple synthetic competitor for oncogene amplification (32).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
real-time TaqMan ASSAY PRINCIPLE
The principle of the TaqMan reaction has been described previously in detail (34)(35). Briefly, the reaction is based on the use of fluorogenic probes designed to hybridize to the gene target sequence of the two PCR primers. Each probe contains a fluorescent reporter dye and a quencher dye at the 5' and 3' ends, respectively. In the intact probe, the presence of the quencher inhibits reporter emission by quenching energy emission. During the extension phase of PCR cycling, the annealed probe is cleaved by the 5' -> 3' exonuclease activity of Taq polymerase. The cleavage produces an increase of fluorescence emission of reporter dye. This event occurs in each PCR cycle only if probe is annealed to the target sequence, which leads to an increase of fluorescence proportional to the concentration of target sequences in the initial sample. The real-time fluorescence detection is performed with the ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystem). In this instrument, a 96-well thermal cycler is connected by fiber optic cables to a CCD camera detector. Laser excitation (488 nm) and fluorescence detection (between 520 and 660 nm) are performed every 7 s during the entire PCR cycling. The signal attributable to the 5' nuclease reaction is expressed as {Delta}Rn values, which represents the reporter signal normalized against the emission of passive reference (ROX) minus the baseline signal established in first cycles of PCR (conventionally from cycles 3 to 15). This range can also be increased up to the cycle immediately prior the appearance of fluorescent signal of the more concentrated template, according to the different kinetics of amplification. This value increases during PCR because the amplicon copy number increases until the reaction approaches a plateau. At the same time, the algorithm determines the threshold cycle (CT), which represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected. The sequence detection software generates a calibration curve of CT vs the quantity of reference DNA and then determines unknowns by interpolation.

tumor samples
DNA was extracted from neuroblastomas with a conventional phenol-chloroform extraction (36). MYCN amplification was measured in 49 neuroblastomas, in which the presence of oncogene amplification had previously been tested by a competitive PCR assay based on the use of a synthetic internal standard, as previously described (18)(32). The group of patients included 33 males and 16 females. At diagnosis, 26 patients were <2 years of age, whereas 23 were >=2 years. Patients were staged according the International Neuroblastoma Staging System (INSS) (37) as follows: 5 patients (10%) were stage 1; 7 (15%) were stage 2A; 5 (10%) were stage 2B; 7 (14%) were stage 3; 20 (41%) were stage 4, and 5 (10%) were stage 4S. Patients had a maximum follow-up of 99 months (median value, 39 months).

external calibration curve
DNA for external calibrator preparation was obtained from 50 mL of blood from five healthy volunteers (age range, 30–36 years) by a conventional phenol-chloroform extraction (36). The DNA concentration was determined by spectrophotometric measurement and then accurately assessed by the measurement of the ß-globin single-copy reference gene with a competitive PCR analysis, as described previously (31)(32). The DNA concentration was expressed as number of ß-globin molecules per microliter.

For each TaqMan assay, we prepared a reference calibration curve containing a DNA quantity corresponding to 1 x 103, 2 x 103, 5 x 103, 1 x 104, and 2 x 104 molecules of reference ß-globin DNA (and, presumably, the same number of molecules of the other single-copy gene, ß-actin).

One of the most important tools to evaluate the efficiency and reproducibility among different assays with the TaqMan method is the control of calibration curve parameters. We tested the reproducibility of the external calibration curves for ß-actin and MYCN gene determination, evaluating the slope and the correlation coefficient of experimental fitting of the calibration curve in each experiment. In addition, the variability of CT values for each calibrator concentration was also evaluated. All data are reported as the mean and coefficient of variation of data obtained in eight consecutive assays.

pcr conditions
All PCR reactions were performed in the ABI PRISM 7700 Sequence Detector in a 50-µL final volume. The PCR mixture contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 10 mmol/L EDTA, 60 nmol/L passive reference dye ROX, 3.5 mmol MgCl2, 0.2 mmol/L each dNTP, 300 nmol/L each primer, 200 nmol/L each probe, 0.5 U of AmpliTaq Gold, and 1 U of AmpErase UNG. All reagents were from Perkin-Elmer Cetus.

The primers for ß-actin gene amplification were 5' TCACCCACACTGTGCCCATCTACGA-3' (forward primer,positions 2141–2165) and 5'-CAGCGGAACCGCTCATTGCCAATGG-3' (reverse primer, positions 2411–2435). The sequence of the TaqMan fluorogenic probe for the ß-actin gene was 5'-ATGCCCTCCCCCATGCCATCCTGCGT-3' (positions 2171–2196). For the MYCN oncogene, the primers and probe were 5'-CCCCTGGGTCTGCCCCGTTT-3' (forward primer, positions 1456–1475), 5'-GCCGAAGTAGAAGTCATCTT-3' (reverse primer, positions 1720–1739), and 5'-CCCACCCTCTCCGGTGTGTCTGTCGGTT-3' (fluorogenic probe, positions 1477–1501). Both genes were amplified by a first step of 120 s at 95 °C, followed by 45 cycles of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C.

mycn amplification measurement in neuroblastoma with TaqMan REACTION
Conventionally, the measurement of oncogene amplification is a comparative assay in which the degree of amplification is derived from the ratio of the target gene and a single-copy reference gene. In our proposed method, the concentrations of the target gene and the reference single-copy gene were estimated, in the same assay, in terms of the number of molecules of the gene per microliter, by using the same external reference calibrator DNA for the assay of MYCN and the ß-actin reference gene. We assayed 5-ng samples of DNA simultaneously and in duplicate for both genes. In the case of high values of MYCN amplification (>9) that were not readable in competitive or TaqMan assays, the quantity of starting DNA was reduced accordingly and the measurement was repeated. The concentration of each gene was then calculated based on the respective calibration curve (i.e., for MYCN and ß-actin) generated with the same calibrator DNA and amplified with the appropriate primers and probes (i.e., MYCN primers and probe for the MYCN curve). The CT value of each unknown was used to estimate the target concentration by extrapolation from the calibration curve. The degree of amplification of each sample was derived from the ratio of the number of molecules of MYCN to the number of molecules of ß-actin. In each assay, we included two positive controls with different MYCN amplifications and two noncancer DNA samples as nonamplified controls. Only samples in which the MYCN/ß-actin ratio was more than two were considered as amplified for this oncogene.

The measurements of MYCN amplification with competitive PCR and TaqMan assays were performed by two different researchers in a blinded fashion.

statistical analysis
Overall survival was evaluated from the date of diagnosis to the date of last follow-up or until death occurred. Estimates of the progression-free survival of various subgroups of children with neuroblastoma were calculated using the method of Kaplan and Meier (38). Curves were compared using the log-rank test.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
calibration curves
The mean slopes of the calibration curves for the two genes were similar: -3.628 for ß-actin and -3.234 for MYCN, with CVs of 17% and 19%, respectively. The mean correlation coefficients of curve fitting were 0.988 (CV = 3.1%) and 0.984 (CV = 3.7%), respectively. The variability of CT values for each calibrator concentration was always <10% (range, 4.0–6.5% for ß-actin and 4.9–8.8% for MYCN).

Figs. 1 and 2 show two examples of amplification plots of real-time detection of ß-actin and MYCN in the TaqMan assay and the corresponding calibration curves, which were generated by plotting the CT value for each calibration point against the concentration of reference DNA.



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  		Figure 1. Amplification plots of real-time detection of the ß-actin gene by TaqMan reaction with a variable number of starting DNA molecules (A), and calibration curve for ß-actin (B).

(A), starting DNA molecule concentrations of 1 x 103, 2 x 103, 5 x 103, 1 x 104, and 2 x 104. Cycle number reported on x-axis and plotted vs the logarithm of change in normalized reporter signal ({Delta}Rn). {Delta}Rn is the intensity of reporter dye emission of each sample minus the fluorescence intensity of control tubes without templates. (— · — · —), experimental threshold of the mean baseline signal plus 10 SD, established in the first 20 cycles. For each sample, the algorithm calculated the PCR cycle (CT value) at which an increase in reporter fluorescence above the baseline signal can first be detected. Data are reported as the mean of three replicates for each dilution. (B), calibration curve for ß-actin was generated by plotting the value of CT (on the y-axis) vs the log concentration of reference DNA (from 1 x 103 to 2 x 104 DNA molecules; x-axis). The data from all three replicates at each dilution of the calibrator are plotted. The unknowns were calculated by interpolation of respective CT values on the calibration curve.

assay performance
To test the precision of the TaqMan real-time detection system and its ability to discriminate between amplified and nonamplified samples, we measured in the same assay 10 replicates of two neuroblastoma samples (the first carrying a threefold amplified MYCN oncogene and the second a nonamplified MYCN oncogene) for both ß-actin and MYCN. The intraassay CV for the first sample was 9.7% for ß-actin and 13% for MYCN. In the second sample, the intraassay CV was 7.4% and 11%, respectively. Similarly, interassay precision, evaluated in the same samples in seven different analytical runs, was 14% for ß-actin and 18% for MYCN for the first sample and 12% and 17% in the second sample.

mycn amplification measurement in neuroblastomas
The amplification of MYCN was determined with TaqMan assay in 49 neuroblastomas. In 26 (53%) of these patients, we found a variable degree of MYCN amplification, ranging from 2 to 29. Table 1 shows the relationship between the magnitude of MYCN amplification and the clinical stage of our patients. In the same samples, the amplification of this oncogene has already been documented by a competitive PCR assay (18). We found a good relationship between the two PCR-based techniques (r = 0.987; Fig. 3 ).


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  		Table 1. Relationship between MYCN amplification and clinical stage of 49 neuroblastomas.



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  		Figure 3. Linear relationship of MYCN amplification in 49 neuroblastomas as measured by the real-time quantitative PCR (y-axis) with TaqMan reaction and by competitive PCR (x-axis).

Oncogene amplification reported in logarithmic scales.

We analyzed cumulative survival by classifying our patients in three groups according to the extent of MYCN amplification. Group A (n = 23) included patients without MYCN amplification; group B (n = 22) included patients with low amplification (between 2 and 8); group C (n = 4) included patients with high amplification (>=9). The analysis of cumulative survival by Kaplan-Meier curves showed that patients with MYCN amplification had a significantly worse prognosis compared with patients in whom the oncogene was not amplified (log-rank test; group A vs B, P = 0.03; group A vs C, P = 0.02; Fig. 4 ). The lower survival of group C in comparison with group B was not statistically significant in this small study.



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  		Figure 4. Kaplan-Meier survival curves for 49 patients with neuroblastoma classified according to different degrees of MYCN amplification.

Group A (n = 23) included patients with no MYCN amplification; group B (n = 22) included patients with low amplification (between 2 and 8); and group C (n = 4) included patients with high amplification (>=9). Differences in survival times between the groups were significant on the basis of the log-rank test (group A vs B, P = 0.03; group A vs C, P = 0.02; group B vs C, not significant).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Quantification of nucleic acids has become an essential tool in molecular diagnostics. In the last few years, many PCR-based methods have been proposed for measurement of both DNA and mRNA [for a review, see Ref. (35)]. Many of these approaches have also been applied to the measurement of oncogene amplification in human tumors. Most of the methods that do not adopt an internal standard for quantitative PCR show serious limitations because of the inaccuracy of the assay. At the same time, methods based on the introduction of internal standards (competitive PCR), require long and complex phases for the preparation of the reagents and the assessment of the method. Furthermore, whatever the strategy followed for nucleic acid quantification, the post-PCR steps are time-consuming; require amplicon manipulation, which is a possible source of carryover contamination; and add further uncontrolled variables via the analytical procedures used to identify and measure the PCR products.

Recently we proposed a novel procedure for the measurement of c-erbB-2 oncogene amplification based on the TaqMan technique for quantitative PCR (34). In this method, fluorescence was generated from a continuous succession of annealing and cleavage of a fluorogenic probe during PCR cycling (39). The homogeneous signal produced by this cycling reaction was then recorded with a conventional fluorometer as the endpoint measurement of fluorescence. We demonstrated that in very well-controlled experimental conditions, the endpoint measurement of fluorescence can also provide accurate quantitative information on target concentration. However, this approach is quite complex, is not easily standardizable, and is affected by unpredictable variables that interfere with PCR yield.

More recently, an important evolution of this technique was introduced that combined the high sensitivity and specificity of 5' nuclease assay with an instrument (7700 Sequence Detector; Perkin-Elmer Applied Biosystem) that can monitor fluorescence continuously as a homogeneous real-time signal generated during PCR cycling. This instrument combines a conventional 96-well thermal cycler with a laser for the excitation of fluorescent dyes and a CCD camera for the detection of specific fluorescence released during the reaction. The homogeneous signal generated from the cyclic cleavage of the fluorogenic probe is specifically associated with the amplified target and quantitatively related to the amount of PCR product (40).

Compared with the conventional methods for the detection of oncogene amplification in cancers, based either on hybridization techniques or on quantitative PCR (competitive or differential PCR), this method allows rapid and accurate determinations without post-PCR steps. Compared with our previous method, which was based on the endpoint measurement of the TaqMan reaction (34) and allowed only a relative estimation of gene abundance, the real-time measurement allows accurate evaluation of the absolute concentration of target and reference genes in analyzed DNA.

Results obtained with this assay were superimposable to those obtained in the same samples with a competitive PCR procedure (18), the only other PCR technique that can provide the absolute measurement of DNA (or RNA) targets (35) The introduction of an internal standard (competitor) in the assay protocols, even if it represents an important tool in ensuring the accuracy of the assay, introduces a series of complicated steps, limiting the possibility of a large assay throughput. On the other hand, real-time method represents a promising technique for the measurement of oncogene amplification with a semi-automated procedure. Furthermore, the use of the same reference DNA calibrator could allow, for the first time, the comparison of data obtained in different laboratories, one of the main limitations for the rationale clinical applications of oncogene amplification in cancer.

The measurement of MYCN gene amplification in neuroblastoma is one of the most relevant clinical applications of oncogene amplification detection in oncology. Both prognosis and treatment of this tumor are deeply influenced by this molecular marker (2)(5)(19)(20)(21)(22)(23)(24)(25)(26)(27). Any improvement of accuracy and reliability of the determination of MYCN amplification could represent an important tool in the management of this cancer. Our data demonstrated that the measurement of MYCN amplification with the TaqMan reaction is easy, rapid, precise, and accurate. Our results confirmed the high incidence of MYCN amplification in neuroblastoma (2)(5)(19)(20)(21)(22)(23)(24)(25)(26)(27). In our 49 patients, we found oncogene amplification in ~53% of tumors, with a major incidence in advanced tumor stages (20). Most amplified cases were found in stage 4 neuroblastomas (12 of 26, 46%), whereas few amplified cases were found in the 4S group (3 of 26, 11%), which included patients with spontaneous remission. Most cases (22 of 26, 84.5%) showed low MYCN amplification (>2 and <9), whereas the incidence of high MYCN amplification (>=9) was limited to patients in advanced clinical stage. However, the clinical significance of low-level amplification of the MYCN oncogene remains to be defined, in part because the number of patients examined in this study was low. It is also important to remember that aneuploidy is a quite common finding in neuroblastoma and that, at least in some cases, the MYCN/ß-actin ratio could be partially affected in aneuploid cells without genomic amplification.

In conclusion, the prognostic value of MYCN amplification in neuroblastoma is confirmed from our data. The analysis of cumulative survival curves confirmed that the prognosis is worse in patients with MYCN amplification and that survival probability is particularly reduced in patients with a high degree MYCN amplification (>=9). These data confirm the importance of a correct measurement of oncogene amplification in the clinical evaluation of neuroblastomas to direct more aggressive therapies in patients with higher risk of cancer progression. However, to have clear evidence of the possible clinical impact of TaqMan assay for MYCN amplification measurement, larger groups of patients must be studied.



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  		Figure 2. Amplification plots of real-time detection of MYCN gene by TaqMan reaction with a variable number of starting DNA molecules (A), and calibration curve for MYCN (B).

(A), reference DNA, dilutions, and result calculations are the same as in the legend of Fig. 1Up . (B), calibration curve for MYCN was generated by plotting the value of CT (y-axis) vs the log concentration of reference DNA (from 1 x 103 to 2 x 104 DNA molecules; x-axis). The data from all three replicates at each dilution are plotted. The unknowns were calculated by interpolation of respective CT values on a calibration curve.


   Acknowledgments
 
This work was supported by a grant from AIRC (Associazione Italiana per la Ricerca sul Cancro) and by a grant from the Associazione Italiana Neuroblastoma, Genoa, Italy


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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