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Relationship Between MYCN Copy Number and Expression in Rhabdomyosarcomas and Correlation With Adverse Prognosis in the Alveolar Subtype

From the Molecular Cytogenetics, Section of Molecular Carcinogenesis and Section of Paediatric Oncology, The Institute of Cancer Research, Sutton, Surrey; Manchester Children's Hospital, Manchester; Department of Histopathology, the Royal Marsden National Health Service Trust; Unit of Molecular Haematology and Oncology, Institute of Child Health, London, United Kingdom; Department of Pathology, Catholic University of Leuven, Leuven, Belgium; and Institut fur Pathologie, Universitatsklinikum der Heinrich-Heine-Universitat, Dusseldorf, Germany

Address reprint requests to Janet Shipley, Molecular Cytogenetics, Male Urological Cancer Research Centre, Institute of Cancer Research, 15 Cotswold Rd, Sutton, Surrey SM2 5NG, United Kingdom; e-mail: janet.shipley{at}icr.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Amplification of the transcription factor MYCN is an important molecular diagnostic tool in stratifying treatment for neuroblastoma. Increased copy number and overexpression of MYCN in the pediatric cancer rhabdomyosarcoma has been described in a number of small studies with conflicting conclusions about its association with clinicopathologic characteristics. We aimed to study the phenomenon in the largest series to date.

PATIENTS AND METHODS: Using quantitative polymerase chain reaction, we measured MYCN copy number and expression levels in rhabdomyosarcoma samples from 113 and 92 individuals with a confirmed diagnosis of rhabdomyosarcoma, respectively.

RESULTS: Increased copy number of MYCN was found to be a feature of both the embryonal and alveolar subtypes. The copy number and expression levels were significantly greater in the alveolar subtype, although the range of expression in both subtypes spanned several orders of magnitude. MYCN copy number showed a significant correlation with expression in the alveolar subtype; this relationship between copy number and expression could be modeled as a logarithmic function. It is notable that relatively high expression frequently occurred in embryonal rhabdomyosarcoma without high copy number and that low expression was found in some cases with high copy number. In patients with alveolar rhabdomyosarcoma, overexpression (greater than median) or gain of genomic copies of MYCN were significantly associated with adverse outcome.

CONCLUSION: MYCN deregulation is a feature of rhabdomyosarcoma tumorigenesis, defines groups of patients with a poor prognosis, and is a potential target for novel therapies.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Rhabdomyosarcomas (RMS) are the most common pediatric soft-tissue sarcomas. RMS are broadly divided into two main subgroups on the basis of histology: alveolar (ARMS) and embryonal (ERMS). The ARMS are generally associated with a poorer prognosis than ERMS¹ and often contain either a t(2;13)(q35;q14) or t(1;13)(p36;q14) translocation that produces a PAX3/FOX01A or PAX7/FOX01A gene fusion, respectively.²

The gene MYCN is a member of the MYC family of highly conserved oncogenes that activate transcription from E-box promoters as part of a heterodimeric complex with MAX. The MYC protein can exert an effect on multiple pathways promoting cell-cycle progression and inhibiting cell differentiation; it is perhaps unsurprising then that MYC genes are both amplified and overexpressed in many cancers. The best characterized example of MYCN amplification is in neuroblastoma, where a core 130-kbp region including MYCN is amplified.³ MYCN amplification of up to 700-fold has been observed in neuroblastoma cell lines with concomitant overexpression and increased protein levels.⁴ MYCN amplification is a demonstrated adverse prognostic factor in neuroblastoma and is used clinically for treatment stratification of neuroblastomas.⁵ Amplification of MYCN has also been described in retinoblastoma, small-cell lung cancer, medulloblastoma, and small numbers of RMS.^6-8

A study of seven ARMS and six ERMS showed that amplification, as detected by Southern blot analysis, was present in 43% of ARMS but no ERMS and stated there was no significant difference in clinical outcome.⁹ A more recent study using semi-quantitative reverse transcriptase polymerase chain reaction (PCR) to study 19 primary samples suggested that there was no clear relationship between expression of MYCN and histology or clinical features.¹⁰ Analysis of 15 ARMS by fluorescence in situ hybridization (FISH) showed amplification of MYCN in nine (60%) of 15 ARMS and claimed a significant correlation with survival.¹¹ Our previous studies of RMS using comparative genomic hybridization and comparative expressed sequence hybridization indicate that the 2p24 region where MYCN is located is frequently amplified, and even more frequently, genes from this region are overexpressed.^12,13

In this study, we aimed to measure the expression and genomic copy number of MYCN in a series of well-characterized RMS and to identify any associations with histology and clinical characteristics. We used the TaqMan (Applied Biosystems, Foster City, CA) quantitative PCR method that has a much greater sensitivity and dynamic range than either Northern or Southern blots and semi-quantitative PCR. TaqMan analysis has previously been validated to detect single copy number changes in MYCN.¹⁴ Furthermore, by measuring quantitatively (rather than semi-quantitatively) expression and copy number from the same sample, we aimed to study the relationship between copy number and expression.

	PATIENTS AND METHODS

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Samples
This study had both local and national ethical approval (Local Research Ethics Committee protocol No. 1836 and Multi-Regional Research Ethics Committee/98/4/023, respectively), and, where appropriate, consent was obtained. Samples were collected from patients with a diagnosis of RMS from the Royal Marsden National Health Service Trust or participating United Kingdom Children's Cancer Study Group centers around the time of first diagnosis. In addition, 22 samples were collected at the University Hospital in Leuven, Belgium, and two samples were collected from University Hospital in Dusseldorf, Germany. Samples were snap-frozen, with the exception of seven samples, which were fixed in formalin and embedded in paraffin. Eighty-four of the 115 RMS patients available for this study were involved in Malignant Mesenchymal Tumour (MMT) studies and therefore had the pathology of their tumors centrally reviewed by the MMT panel (according to standard International Agency for Research on Cancer–WHO guidelines). Because it was important to maximize the size of our study, we also included a minority of samples that had not been registered for the MMT trial. To maintain parity with the MMT studies, the pathology of these samples were examined by the pathologist authors. In all cases, the diagnosis of ARMS applied the current histopathologic classification, whereby any alveolar focus is sufficient to result in ARMS classification. Subsequent to pathology review, cytogenetic and/or molecular biologic data for the presence of PAX/FOX01A fusion genes were available for 85 of 115 samples, most of which have been previously reported.¹⁴ The presence of PAX/FOX01A matched with the histopathologic diagnosis of ARMS in all but two cases. These two cases, classified as ERMS, were excluded from the study, as the presence of the fusion genes is widely considered consistent with ARMS. In accordance with the guidelines at the time of the pathology review, anaplasia was not a feature noted for RMS. However, it has been possible to review 44 of the cases for this feature (16 ARMS and 28 ERMS). Of these, two ERMS were identified to have focal anaplasia. Clinical data for the majority of tumors were obtained from the United Kingdom Children's Cancer Study Group data center (Leicester, United Kingdom); otherwise, data were collected directly from participating hospitals. The clinical details of the patients used in survival analysis are outlined in Table 1. The mean age of patients was 8.5 years, and only patients younger than 21 years were used in survival analysis. The majority of patients were treated using the International Society of Pediatric Oncology MMT89 protocol or the closely related MMT95 and MMT98 protocols. The remaining patients were treated using local treatment protocols that were comparable to the MMT protocols. All patients were treated with a combination of drugs, including vincristine, ifosfamide, and actinomycin. Higher-stage tumors and some alveolar tumors additionally received epirubicin, etoposide, and carboplatin. All patients were treated with intensive chemotherapy and surgery, with or without radiotherapy for local control. High-dose therapy with bone marrow or stem-cell rescue in first remission was limited to metastatic patients or patients with stage III disease and alveolar histology. In addition, tumor samples from eight individuals with a diagnosis of the rare adult pleomorphic form of RMS were available for study. The cell lines called RMS and SCMC-RM2 were derived from RMS and were cultured as previously described.^15,16 Blood and muscle samples from 10 and 11 normal individuals, respectively, were used as control tissues.

TaqMan Quantification of MYCN
Three sets of primers and probes were designed to measure the amount of genomic and mRNA copies of MYCN in RMS samples (Table 2). All primers and probes were designed in accordance with Applied Biosystems' TaqMan standard requirements. Using GenBank sequence Y00664, primers and a probe were designed within exon 3 of the MYCN gene to quantify genomic copies of MYCN. To correct for aneuploidy, the gene POLR2D was chosen as an endogenous control. POLR2D is a gene that is not believed to be involved in tumorigenesis and is located in a region of chromosome 2 (2q21) rarely altered in RMS other than through whole gain of chromosome 2.¹³ Using GenBank sequence NM_004805, the primers and a probe were designed within exon 4. To measure the amount of mature spliced mRNA copies of MYCN, a probe was designed across the exon 2–exon 3 boundary. Applied Biosystems' predeveloped glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control. Triplicate 25-µL multiplex PCR reactions using 2x Universal TaqMan Master Mix (Applied Biosystems; part No. 4304437), the concentration of primers and probes shown in Table 2 and 10 ng of DNA or cDNA were run under standard operating conditions on an ABI7700 SDS TaqMan Machine (Applied Biosystems). Limiting primer conditions were determined, and template titration showed that the reactions were equally efficient, and hence the comparative method was appropriate for both genomic and expression reactions (data not shown). The amount of MYCN was measured relative to either normal genomic DNA in the case of genomic measurements and relative to normal muscle pooled from 11 normal skeletal muscle samples for expression measurements.

Statistics
All statistics tests were performed using the SPSS 10.0 (SPSS Inc, Chicago, IL) package and tested to the 5% significance level. Failure-free survival was defined as the time from diagnosis to relapse, progression, death, or, if event free, to the date of last contact. Time to death was defined as the time from diagnosis to death or, if event free, to the date of last contact.

	RESULTS

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MYCN Genomic Copy Number
To determine the sensitivity of the TaqMan assay, we measured the copy number of MYCN in 10 healthy individuals and achieved a standard deviation of 0.2. We can thus be 95% confident that measurements above 1.4 (standard deviation = 1.96) represent true deviations from normal DNA copy number. Furthermore, a dilution series of normal DNA spiked with varying proportions of DNA from the cell line RMS, which has high-level MYCN amplification, produced an accurate and linear copy number response over the range measured in samples (data not shown).

Genomic copy number was measured in primary tumor samples taken from 113 individuals with a diagnosis of RMS, of which 48 were ARMS, 58 were ERMS, and seven were RMS not otherwise specified. The distribution of MYCN genomic copies relative to normal genomic DNA is shown in Figure 1. The definition of clinically relevant MYCN amplification in neuroblastoma is the presence of four times as many MYCN signals as chromosome 2 centromere signals by FISH.¹⁹ As an approximation to the situation in neuroblastoma, we have defined samples that give a TaqMan result of four times that of normal DNA as having high-level MYCN copy number changes. Using this definition, 23 (20.4%) of 113 RMS show high-level MYCN copy number changes; by subtype, 12 (25%) of 48 ARMS and nine (16%) of 58 ERMS show high-level MYCN copy number changes. Among these RMS, the genomic copy number of the ARMS is significantly greater than that of the ERMS (Mann-Whitney U = 13.00; n = 21; P = .002). Where data was available for the presence of the PAX/FOXO1A (nine of 12), each ARMS showing high-level MYCN copy number changes also tested positive for the presence of a PAX/FOXO1A fusion (8 x PAX3/FOX01A 1 x PAX7/FOX01A). Anaplasia was present in only two embryonal cases of 44 RMS reviewed for this feature, although neither of these samples had high-level MYCN copy number changes. It is noteworthy that low-level gains of MYCN were more frequent than high level, as defined earlier. Eighty-nine (79%) of 113 RMS samples give a relative measurement greater than 1.5 (the equivalent of three copies in a diploid cell; by subtype, 38 [79%] of 48 ARMS and 45 [78%] of 58 ERMS).

View larger version (39K): [in this window] [in a new window]   Fig 1. (Left) Histogram showing distribution of genomic copy number in (A) alveolar rhabdomyosarcoma (ARMS) and (C) embryonal rhabdomyosarcoma (ERMS); vertical line represents cutoff for definition of high-level genomic copy number gain. (Right) Histogram showing distribution of expression in (B) ARMS and (D) ERMS; vertical line represents the rhabdomyosarcomas median value.

View larger version (54K): [in this window] [in a new window]   Fig 2. Fluorescence in situ hybridization analysis supporting MYCN copy numbers. (A) comparative genomic hybridization analysis; (B) chromosomes; and (C) nuclei of SCMC-RM2. Nuclei from primary samples for (D) embryonal, no MYCN gain; (E) embryonal, 1.7-fold increase in MYCN indicated by TaqMan; (F) alveolar, 24-fold increase; and (G) embryonal, 4.4-fold increase. MYCN signals in red and 2 centromere in green.

Copy Number and Expression Correlation
There is significant correlation between genomic copy number and expression, where DNA and RNA were taken from the same sample within the ARMS but not the ERMS (ARMS, = 0.500, n = 37, P = .002; ERMS, = –0.119, n = 36, P = .313). The relationship between MYCN copy number and expression in the ARMS can be best modeled by a logarithmic regression with no constant: 22603.5Ln(Genomic) = expression. This function significantly matches the real data (analysis of variance between regression and residuals, F = 9.13, P = .005). This relationship is illustrated in Figure 3.

View larger version (15K): [in this window] [in a new window]   Fig 3. Scatterplot of genomic copy versus expression in embryonal rhabdomyosarcoma (A) and alveolar rhabdomyosarcoma (B). Vertical line marks cutoff for definition of high-level genomic copy number gain. Horizontal line marks the median expression value. Logarithmic curve of the form Ln (genomic copies) = expression fitted to the data.

View larger version (21K): [in this window] [in a new window]   Fig 4. Kaplan-Meier plot showing difference in failure-free survival between high (left column) and low (right column) expression of MYCN in alveolar rhabdomyosarcoma (ARMS; A), embryonal rhabdomyosarcoma (ERMS; C), and difference in survival between ARMS (B) and ERMS (D) with and without gain of MYCN genomic copies. Cum., cumulative.

Pleomorphic RMS
In addition to the ARMS/ERMS samples, seven samples of DNA and four samples of cDNA from the pleomorphic RMS were measured. One case of amplification (7.2-fold change relative to 2q) was observed, but overall expression was relatively low (median, 4.15 times greater than normal muscle).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
MYCN amplification is a clinically important prognostic marker in neuroblastoma. Here we have assessed the potential clinical importance of MYCN genomic gain and expression in the largest series of RMS to date. We have demonstrated TaqMan analysis with supporting FISH evidence that gain of genomic copies of the MYCN gene is a common feature of both ERMS and ARMS. The FISH data are consistent with amplification events, large rearrangements and duplications involving 2p material, and specific low copy increases of MYCN, all of which have been described in neuroblastomas.^20,21 There does, however, seem to be a biologic and clinical difference between the phenomenon of MYCN gain in the two subtypes of RMS. Of those samples that we determined to have high-level copy number changes, the ARMS have significantly more copies than the ERMS. Those ARMS with gain of copies of MYCN are less likely to survive and more likely to relapse than those with no gain. Curiously, this does not apply to ERMS, and there is even perhaps a trend toward the opposite (Fig 4).

As seen with the level of copy number change of MYCN, expression is significantly greater in ARMS compared with ERMS. Patients with high-expressing ARMS (greater than the median 490 times greater than normal skeletal muscle) are more likely to experience relapse and less likely to survive than patients with low-expressing ARMS. The clinical and biologic precedent for this adverse correlation has been set in neuroblastoma, albeit that the level at which gain is prognostic in RMS is different in neuroblastoma. The reason for this difference is unclear but may be due to a differing relationship between genomic copy number, rate of transcription, rate of translation, and/or biologic activity of MYCN between the two tumors. Overexpression of MYCN is likely to promote cell growth and repress differentiation in myoblastic progenitor cells of RMS as it does in the neuronal progenitors of neuroblastoma.²² Indeed, one study has already examined the biologic behavior of high MYCN-expressing RMS cell lines. This study demonstrated that high MYCN-expressing RMS cell lines had a more invasive growth pattern in xenografts compared with that of low MYCN expressing RMS cell lines, although growth rate in vitro was similar.²³

Expression of MYCN in RMS regardless of histologic subtype is frequently though not always greater than that of normal skeletal muscle, and the range of this expression spans several orders of magnitude. Overexpression is more common than high-level genomic gains, and relatively high expression can occur without similarly high genomic gain, particularly in ERMS. This is consistent with our previous CESH results showing overexpression from 2p24.¹³ Conversely, there are some ERMS that, despite high-level MYCN copy number gain, show relatively low expression. Consequently, ERMS do not show significant correlation between copy number and expression, indicating that genomic gain is not the primary method of MYCN deregulation in ERMS.

Increased copy number without overexpression is not wholly without precedent. Fan et al²⁴ quote unpublished results of medulloblastomas with MYCN amplification but with relatively low expression in their article on the amplified oncogene hTERT, a gene which also occasionally shows amplification without overexpression. Furthermore, Grotzer et al²⁵ found that only a subset of medulloblastoma showed C-MYC overexpression after amplification. This suggests either that only small increases in MYCN expression are required to produce a tumorigenic effect or that in some ERMS, MYCN is not the critical gene at 2p24.

Previous studies of the relationship between copy number of amplified oncogenes and their concomitant expression levels have used semi-quantitative techniques. One study of particular note showed a linear correlation between copy number of hTERT in embryonal CNS tumors and expression and used quantitative PCR to measure expression but a semi-quantitative technique to measure copy number.²⁴ The fact that our study uses the quantitative TaqMan method to measure both copy number and expression from the same sample, rather than semi-quantitative methods like reverse transcriptase PCR, Southern blot analysis, and Northern blot analysis, is unique among studies of amplified oncogenes. Examining those cases of ARMS where it was possible to measure both expression and copy number reveals a logarithmic relationship of the form Ln(Genomic copies) = expression (Fig 3). This phenomenon has not been previously described but suggests that expression of an oncogene is ultimately limited by factors other than the amount of genomic template (ie, availability of transcription factors). The implication of this relationship is a law of diminishing expression returns for increasing genomic gains. Potentially, these effects could act to limit the copy number of genes by ultimately limiting the selective clonal advantage produced by additional copy number gain. It is noteworthy that in culture, even after many passages, amplicons do not gain copies ad infinitum. Furthermore, neuroblastoma tend to be associated with higher copy numbers of MYCN than the RMS studied here. This is also true of neuroblastoma and RMS cell lines.⁴ The fact that the ERMS do not demonstrate the same relationship suggests that this may apply only under certain conditions in a particular cellular environment.

In conclusion, we have demonstrated that gain of genomic copies and overexpression of MYCN is a common phenomenon in both ARMS and ERMS, although the copy number gain and expression tend to be greater in ARMS. Furthermore, gain of genomic copies and overexpression of MYCN are associated with an adverse prognosis in patients with ARMS. An immunostain for the MYCN protein may also be of clinical relevance. Although the role of this gene in the different RMS subtypes requires further investigation, it is has been suggested that MYCN offers a target for novel therapies.²⁶ This study suggests that such therapies may be of relevance in RMS.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We thank Richard Carter, Andy Pearson, Annabel Foot, Richard Grundy, Ananth Shankar, Sue Picton, Jan Kohler, Syuiti Abe, Takayuki Nojima, Penny Flohr, and the United Kingdom Children's Cancer Study Group. We also thank Brenda Summersgill, Beata Grygalewicz, and Dyanne Rampling for their excellent technical assistance.

	NOTES

 
Supported by Cancer Research United Kingdom.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
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14. De Preter K, Speleman F, Combaret V, et al: Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay. Mod Pathol 15:159-166, 2002[CrossRef][Medline]

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Submitted November 13, 2003; accepted October 27, 2004.

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