the University Children's Hospital, Division of Oncology
    University Hospital Zurich, Department of Pathology, Zurich, Switzerland
    University of Kiel, Department of Pathology, Kiel
    Olga Hospital, Department of Pediatric Oncology/Hematology, Stuttgart, Germany

    ABSTRACT

    PURPOSE: Rhabdomyosarcoma (RMS) is classified into two main subgroups: the embryonal (ERMS) and the alveolar (ARMS) form. The majority of the ARMSs are associated with specific chromosomal translocations (pARMS). Because ARMS is much more aggressive than ERMS, RMS subclassification has clinical relevance. However, diagnosis of RMS subgroups on the basis of histology or molecular biology can be difficult, and supplementing diagnostic methods would be desirable. The aim of this study was to establish a panel of markers for RMS subgroup classification by immunohistochemistry.

    MATERIALS AND METHODS: Gene expression data were used for selection of subgroup-specific markers. Single sections of RMS with available expression data were used for establishment of the immunohistochemistry. Evaluation of the sensitivity and specificity of the markers was carried out using a tissue array representing 252 RMSs. Kaplan-Meier survival curves were calculated for determination of differences in overall survival of the different staining subgroups.

    RESULTS: AP2 and P-cadherin were selected as markers for pARMS, and epidermal growth factor receptor (EGFR) and fibrillin-2 as markers for ERMS. EGFR + fibrillin-2 detected ERMS with a specificity of 90% and with a sensitivity of 60%. AP2 + P-cadherin detected pARMS with a specificity of 98% and a sensitivity of 64%, and allowed the detection of several misclassified tumors. The EGFR + fibrillin-2–positive group is associated with a favorable outcome, and the AP2 + P-cadherin–positive group is associated with an unfavorable outcome.

    CONCLUSION: The presented set of marker proteins detects RMS subgroups with high specificity and may be useful in routine subtype classification of RMS.

    INTRODUCTION

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, accounting for 5% to 10% of all pediatric malignancies.1 This tumor is thought to derive from myogenic precursor cells and belongs to the group of small round blue-cell tumors (SRBCTs). On the basis of histology, two main RMS subgroups are distinguished: the alveolar RMS (ARMS) and the embryonal RMS (ERMS).

    The two subtypes are associated with distinct clinical outcomes. Whereas improved chemo- and radiotherapeutic regimens have increased the 5-year event-free survival of ERMS patients to approximately 70%, the 5-year event free survival of patients with the alveolar form is still low at approximately 40%, mainly as a result of a greater frequency of disseminated metastases.2 Hence, harsher treatment regimens are used for ARMS, giving clinical relevance to accurate subclassification. Unfortunately, however, classification based solely on histology is not easy and does not always allow an unambiguous subclassification.

    Therefore, in addition to histology, different molecular biologic methods that make use of subtype-specific genetic aberrations have been applied for RMS subclassification. For example, approximately 80% of the ARMSs have specific translocations t(2;13) or t(1;13), leading to PAX3/FKHR or PAX7/FKHR fusion genes (reviewed in Barr 3), which are detectable on the DNA-level by cytogenetic analysis or fluorescence in situ hybridization, and on the RNA-level by specific reverse transcriptase polymerase chain reaction (RT-PCR). However the clinical adaptability of all these methods is complicated by the need for high-quality, most suitably fresh material, which often is not available, especially when microbiopsy samples are collected.

    An alternative to these methods is immunohistochemistry (IHC), which generally is widely accepted as a standard tool for tumor diagnosis. In the case of RMS, several immunohistochemical markers that allow discrimination of this tumor from other SRBCTs have been described, including desmin, myogenin and MyoD1.4,5 However, for RMS-specific subgroup classification, no good immunohistochemical marker proteins have been defined. The only subgroup marker proposed to date, myogenin, shows a certain association with ARMS based on a stronger positivity in this subtype,6,7 but the significant variability in the extent of myogenin expression among ERMSs casts doubts as to whether this marker allows an unambiguous distinction between ARMS and ERMS.8 Recent gene expression analysis of RMS biopsy samples generated lists of genes that discriminate with high specificity between the different RMS subgroups on the RNA level,9 and that are, therefore, excellent candidates for diagnostic immunohistochemical RMS markers.

    Here, we defined a panel of subgroup-specific marker proteins for immunohistochemical subgroup classification of RMS on the basis of gene expression data. Immunohistochemical stainings with antibodies against these markers were established on tumor sections with available expression data (real-time PCR and microarray). Specificity and sensitivity of the markers were evaluated on a RMS tissue array representing 252 RMSs. Generally, our marker panel allowed subgroup classification with high specificity, including identification of previously falsely classified samples.

    Our data, therefore, provide evidence for a simplified diagnostic classification of RMS on the level of immunohistochemistry, and suggests that further large-scale prospective studies using these markers are warranted.

    PATIENTS AND METHODS

    Tissue Array Construction

    Two hundred fifty-two paraffin-embedded RMSs were used for punching tissue cylinders, which were then transferred into a recipient block.

    RT Nested PCR Detection of Fusion Transcripts

    RNA extraction and cDNA synthesis was performed as described previously.10 For amplification of the PAX/FKHR fusion transcripts, PCR (30 seconds at 95°C, 30 seconds at 53°C, and 30 seconds 72°C; 60 cycles) with 5 μL cDNA template was performed. In the first round of PCR, the forward PAX3 (5'-AGACAGCTTTGTGCCTCCGTC-3') or PAX7 primer (5'-GACAGCTTCATGAATCCGG-3') was used in combination with the reverse FKHR primer (5'-TTCCCGCTCTTGCCACCCTCTGG-3'). Specificity was checked by nested PCR with internal primer pairs (forward PAX3: 5'-TCCAACCCCATGAACCCC-3', PAX7: 5'-CAACCACATGAACCCGGTC-3'; reverse FKHR: 5'-CTCTGGATTGAGCATCCACC-3'). For control of template quality, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment was amplified. Positive and negative controls were included in all steps of reverse transcription and PCR.

    Quantitative Real-Time PCR

    1 μg RNA from RMS biopsy samples or 2 μg RNA from paraffin-embedded tumors was used for cDNA synthesis with the SuperScript Kit (Invitrogen, Groningen, the Netherlands). Then, 5 to 10 ng (10 to 20 ng in case of paraffin material) cDNA was used as template for the PCR reaction under universal cycling parameters on an ABI7900 instrument using commercially available target probes ("Assay-On-Demand") and Mastermix (all from Applied Biosystems, Applera Europe BV, Rotkreuz, Switzerland). Cycle threshold (CT) values were normalized to GAPDH. Relative expression levels of the target genes among the different samples were calculated using the CT method.

    Immunohistochemistry

    Immunohistochemistry (IHC) for each antigen was performed with the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, AZ) on 2-mm paraffin sections. For antigen retrieval, slides were heated with cell conditioner 1 (mild protocol). The staining for fibrillin-2 and epidermal growth factor receptor (EGFR) required enzymatic predigestion with Ventana protease 1 for 8 minutes and 4 minutes, respectively. Primary antibodies against the following antigens were applied: AP2 (H-87, purified rabbit immunoglobulin fraction, 1:40; Santa Cruz Biotechnology, Santa Cruz, CA), P-cadherin (clone 56, 1:50; BD Transduction Labs, San Jose, CA), EGFR (clone 3C6, prediluted by Ventana), fibrillin-2 (mAb48, 1:200; described in Charbonneau, Dzamba, Ono, et al11), myogenin (clone LO26, prediluted by Ventana), and desmin (clone D33, 1:20; DakoCytomation, Glostrup, Denmark). Primary antibodies were revealed with the Ventana iVIEW DAB detection kit or the Ventana-Red enhanced detection kit (some EGFR stainings). For P-cadherin and fibrillin-2, the signal was enhanced with the Ventana amplification kit. Slides were counterstained with hematoxylin.

    IHC Assessment

    The staining results of our markers were scored and classified using the following five-point scoring system: 0, no staining; 1, weak staining of a minority of the cells; 2, weak staining of the majority of the cells; 3, strong staining of a minority of the cells; 4, strong staining of the majority of the cells.

    In the case of AP2 and P-cadherin score 3 and 4 stainings (nuclear localization of AB2) were interpreted as positive. In the case of the EGFR, fibrillin-2 and desmin score 1 to 4 stainings were interpreted as positive. As described,12 in case of myogenin stainings sections with > 50% positive cells were scored as ARMS, and sections with < 50% stained cells were scored as ERMS. Tumors represented by more than one core on the tissue array were interpreted as positive if one of the cores was positive. Desmin-negative tumors were excluded from the study.

    Sensitivity S and specificity Y of the stainings were calculated with the following formulas, where

    p is the number of positive samples in a subgroup, n is the number of samples in tested subgroup, f is the number of false-positive samples, and g is the number of samples in all subgroups.

    RESULTS

    Microarray and Quantitative Real-Time PCR Expression Levels Correlate With Protein Expression

    Recent RMS cDNA expression profiling analyses detected numerous potential RMS subgroup-specific marker genes potentially detectable on the protein level by IHC.9 On the basis of gene expression data, we selected a panel of four markers with available antibodies for immunohistochemical detection of RMS subgroups, namely AP2 and P-cadherin, as markers for the group of translocation-positive ARMS (pARMS), as well as EGFR and fibrillin-2 as markers for the group of ERMS (Fig 1). According to the gene expression data, the first two markers are expressed exclusively in pARMS, irrespective of the translocation involved, whereas the latter two markers are highly expressed in ERMS and at low levels also in translocation-negative ARMS (nARMS), but not in pARMS, allowing clear discrimination of the three RMS subgroups on the RNA level. For comparison, myogenin as an established RMS marker was included.

    To establish the immunohistochemical stainings with antibodies against these marker proteins, we used tissue sections of tumors that represent each RMS subclass (ERMS, pARMS and nARMS). Gene expression data was available for all the selected tumors and was further validated by quantitative PCR (qRT-PCR) analysis for the four selected markers. The data of the two methods are in agreement (Fig 1, bottom left insets). Therefore, for evaluation of the staining parameters for each antibody, tumors expected to be positive or negative were available. This allowed evaluation of both the background and positive stainings on the tissue sections.

    The AP2 antibody showed a strong nuclear staining in positive tumors (Fig 1) whereas in negative tumors, a mostly extranuclear granular background was detected sometimes (Fig 1, top right inset, arrow). On the basis of these findings, only strong nuclear staining of score 3 or 4 was defined as true positive for the AP2 antibody.

    The P-cadherin antibody displayed a strong staining at the cell periphery in positive cases, as expected for this classical member of the cadherin family (Fig 1). P-cadherin–negative tumors showed a slight, but detectable background (Fig 1). On the basis of these results, again only strong stainings of score 3 or 4 were defined as true positive.

    In the case of both the EGFR and fibrillin-2 antibodies, strong staining was found in ERMS tumors, for EGFR at a circumcellular localization (Fig 1B), and for fibrillin-2 often in a parallel filamentous distribution throughout the tissue, whereas pARMS tumors were free of any background staining (Fig 1). In nARMS tumors, a weak staining was found for these markers, corresponding exactly to the lower expression values found on the RNA level (Fig 1). Therefore, for these two antibodies, every detectable staining of score 1 to 4 was defined as positive.

    Subgroup classification on tissue microarrays. To evaluate specificity and selectivity of the selected markers in RMS subgroup detection, an RMS tissue array (TMA) representing 252 tumors was used for stainings applying the parameters established herein. The results of the stainings were compared to the initial subgroup classification of the samples, which was based on histologic analysis and determination of the translocation status of the ARMS samples by RT nested PCR using RNA from fixed and paraffin-embedded material (193 ERMS, 43 pARMS, and 16 nARMS), as well as to the myogenin staining. The clinical parameters of the tumors used for the TMA are summarized in Table 1. Subgroup distribution and overall survival in the different histologic subgroups on the TMA reflect the natural distribution found in RMS. The stainability of the samples on the TMA was evaluated by staining of the general RMS marker desmin. Desmin-negative samples were excluded from further analysis.

    Three tumors histologically classified as ERMS as well as four tumors classified as nARMS were found to be immunohistochemically double positive for AP2 and P-cadherin, as would be expected for pARMS samples. To further clarify the correct classification of these samples, RNA extracted from paraffin-embedded material of these seven tumors was used for RT-PCR detection of PAX3(7)/FKHR-translocations, as well as for detection of the expression of PAX3(7)/FKHR target genes such as AP2 (unpublished observation) and cannabinoid receptor 1 (CB1),13 which are specifically expressed in pARMS. This analysis led to the detection of PAX translocations in two of the cases classified as ERMS (Fig 2B). Furthermore, qRT-PCR detected expression of AP2 and CB1 in all three ERMS cases, as well as in one of two tested nARMS cases. We conclude from this analysis that these ERMS and nARMS samples had high likelihood originally erroneously classified, and therefore we reclassified these samples as pARMS for the statistical analysis (as indicated in Table 1, after validation). Furthermore, these data underscore the value of the antibody stainings.

    Statistical analysis of the immunostains on the TMA gave the following results: The AP2 antibody showed a sensitivity of 64% and a specificity of 92% for the detection of pARMS, whereas the P-cadherin antibody displayed a sensitivity of 76% and a specificity of 85% (Fig 2C). Combination of the two markers increased specificity to 98% with a sensitivity of 64%, demonstrating that the sensitivity is dominated by the AP2 staining.

    For the detection of ERMS, the EGFR antibody showed a sensitivity of 84% and a specificity of 81%, whereas the fibrillin-2 antibody showed a sensitivity of 68% and a specificity of 85%. Combination of the two markers decreased sensitivity slightly to 60% but increased specificity to 90%. In comparison to that, myogenin detected ARMS and ERMS with specificities of 82% and 76%, respectively (Fig 2C). This demonstrates that the four markers allow RMS subgroup-classification with very high specificity (Fig 2D).

    Clinical outcome of the staining subgroups. To test whether the established discrepancy in overall survival of the different histologic subgroups are also reflected in the classification on the basis of our staining results, Kaplan-Meier survival curves were calculated.

    As demonstrated in Figure 3A AP2 and P-cadherin double-positive samples showed a significantly worse outcome (P = .0044) compared with samples not double positive for these markers. In contrast, fibrillin-2 and EGFR double-positive samples were associated with a better outcome (P = .0254) than samples not double positive for these two markers. Therefore, the staining results are able to predict outcome.

    DISCUSSION

    Clinicopathologic behavior and resulting prognosis of RMS differ significantly among the different subgroups, necessitating subgroup-specific treatment regimens and making subgroup classification clinically relevant. Because of its feasibility and wide distribution, IHC is the method of choice for tumor diagnosis in general, and is in clinical use for discrimination of RMS from other types of SRBCTs. Therefore this method would also be preferable for RMS subclassification.

    We present here a set of markers that allow RMS subgroup classification by IHC, based on previously published gene expression data.9 The described subgroup-specific gene signatures allowed selection of markers with available antibodies specific for pARMS (AP2 and p-Cadherin) and ERMS (EGFR and fibrillin-2). The gene signature specific for the group of nARMS was much smaller, and no appropriate antibody for this group was available.

    According to the underlying microarray data, the two pARMS-specific markers are consistently expressed in all pARMS samples irrespective of the PAX fusion partner, suggesting that these might be potential target genes of the fusion proteins. Therefore, indirect detection of the PAX translocations in RMS by determining the expression levels of potential target genes (eg, by immunohistochemistry) allows identification of not only samples expressing the well-known fusion proteins PAX3(7)/FKHR, but also cases with novel variants such as PAX3/AFX14 or PAX3/NCOA-1.9 These latter variants would not have been detected by molecular analysis using specific RT-PCR. This observation, together with the fact that immunohistochemistry can be performed on paraffin-embedded material and is more widely available in clinical laboratories than elaborate PCR techniques, may qualify the two marker sets as useful alternatives to direct RT-PCR-detection of the translocation also in a routine clinical setting. This statement is further substantiated by the immunohistochemical detection of several falsely classified pARMS in our study. In fact, we could demonstrate that several tumors originally classified as ERMS or nARMS, but double-positive for AP2 and P-cadherin, were indeed originally falsely classified pARMS, as validated by RT-PCR detection of a PAX3(7) translocation or by measuring high expression levels of PAX3(7)/FKHR target genes by qRT-PCR. Interestingly, two of these cases were PCR negative for the known PAX3(7)/FKHR translocations, but nevertheless expressed high levels of the pARMS-specific genes AP2 and CB1. Hence, in these cases an alternative PAX translocation with known (AFX, NCOA1) or unknown partner proteins may be present. Alternatively, RT-PCR may be not sensitive enough to detect the PAX3(7)/FKHR-fusion product in cases with poor RNA quality. In fact, it has been shown recently that the group of nARMS is quite heterogeneous, and in addition to true translocation-negative samples, samples with cryptic translocations not detected by standard PCR may be assigned to this group.14

    The sensitivity that was achieved for the detection of both pARMS and ERMS was with 64% and 60%, respectively, somewhat lower than the sensitivities on the level of gene expression profiling. Especially in the case of the pARMS-specific markers, a very low theoretical rate of true negatives in IHC is expected, taking the consistency of expression among the pARMS samples in the underlying gene expression data into account. However, different factors may increase the rate of false negatives in practice. First of all, the stainability of the tissue samples may be influenced by the age of the tissues, which varied from 5 to 19 years, on the TMA, and also by the fixation protocol, which was not standardized among the different tissues used for the TMA. Secondly, although most tumors on the TMA were represented by several cores representing different areas of the tumor tissue, in some cases, positive areas may be missed. Furthermore, the scoring system applied for the pARMS-specific markers was optimized to maximize specificity by counting only strongly stained tissues. Borderline cases were therefore not counted, limiting sensitivity, but leading to a high specificity.

    The specificity values reached were comparable for both subgroups, with 85% to 92% for the single pARMS-markers and 81% to 85% for the single ERMS-markers. These values even could be improved to 98% (pARMS) and 90% (ERMS) by combination of the two markers. The specificities were therefore considerably higher than the ones found for myogenin here (82% ARMS, 76% ERMS) or in a recent study of 109 RMS (correct identification of approximately 70% of the ERMS).12 Furthermore, myogenin is not subtype-specifically expressed and therefore subgroup discrimination is based on the qualitative and quantitative differences of the stainings being exclusively nuclear in a majority of the cells in ARMS and typically focal in a minority of the cells in ERMS.6,7 Additionally, its variability of expression among ERMS has cast doubts about the usefulness of myogenin for an unambiguous distinction between ARMS and ERMS.8 In contrast, the markers identified in this study are subtype specific, and therefore should allow a simpler interpretation of the staining results.

    Additional evidence for the usefulness of our immunohistochemical classification was gathered by analyzing the clinical outcome of the different staining subgroups. A significantly worse outcome of the AP2 + P-cadherin double-positive cases and a significantly better outcome of the EGFR + fibrillin-2 double-positive samples could be demonstrated by Kaplan-Meier survival curve analysis. These results correlate with the survival data known for histology-based subgroups of RMS.

    In summary, our data suggest that an immunohistochemically-based subgroup classification of RMS with this marker panel could be a useful supplementation of the established routine histology. This suggests that extended prospective studies may be warranted to validate our data further in a routine clinical setting.

    Authors' Disclosures of Potential Conflicts of Interest

    The authors indicated no potential conflicts of interest.

    Author Contributions

    Conception and design: Marco Wachtel, Felix K. Niggli, Beat W. Schfer

    Financial support: Beat W. Schfer

    Provision of study materials or patients: Ivo Leuschner, Ewa Koscielniak, Jrn Treuner

    Collection and assembly of data: Tina Runge, Sabine Stegmaier, Bernhard Odermatt, Silvia Behnke, Beat W. Schfer

    Data analysis and interpretation: Marco Wachtel, Tina Runge, Sabine Stegmaier, Beat W. Schfer

    Manuscript writing: Marco Wachtel, Beat W. Schfer

    Final approval of manuscript: Beat W. Schfer

    Glossary

    Desmin: A member of the type III family of intermediate filaments, a class of cytoskeletal elements. Desmin expression is muscle specific and found in skeletal, cardiac, and heart muscles, but also in rhabdomyosarcoma, for which it serves as general marker.

    EGFR (epidermal growth factor receptor): Also known as HER-1, EGFR belongs to a family of receptors (HER-2, HER-3, HER-4 are other members of the family) and binds to the EGF, TGF-, and other related proteins, leading to the generation of proliferative and survival signals within the cell. It also belongs to the larger family of tyrosine kinase receptors and is generally overexpressed in several solid tumors of epithelial origin.

    FISH (fluorescence in situ hybridization): In situ hydridization is a sensitive method that is generally used to detect specific gene sequences in tissues sections or cell preparations by hybridizing the complimentary strand of a nucleotide probe to the sequence of interest. FISH uses a fluorescence probe to increase the sensitivity of in situ hybridization.

    Gene expression analysis: Microarray-based technique for the simultaneous quantification of the mRNA expression level of thousands of genes.

    Myogenin: A muscle-specific transcription factor that can induce myogenesis in a variety of cell types in tissue culture. Myogenin is a member of the basic helix-loop-helix gene family.

    qRT-PCR (quantitative real-time polymerase chain reaction): Also known as real-time PCR, qRT-PCR consists of detecting PCR products as they accumulate. It can be applied to gene expression quantification by reverse transcription of RNA into cDNA, thus receiving the name of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). In spite of its name—quantitative—results are usually normalized to an endogenous reference. Current devices allow the simultaneous assessment of many RNA sequences.

    Rhabdomyosarcoma: A malignant solid tumor arising from mesenchymal tissues, which normally differentiate to form striated muscle. It is divided into two main subtypes: the alveolar rhabdomyosarcoma, mainly arising in adolescents and young adults, and the embryonal rhabdomyosarcoma, predominantly affecting infants and children. It is one of the most frequently occurring soft tissue sarcomas and the most common in children younger than 15 years, accounting for 6% to 8% of all childhood cancers.

    Small round blue-cell tumors: A class of tumors united by the appearance of characteristic small, round, blue tumor cells. Besides rhabdomyosarcoma, desmoplastic small round blue-cell tumor, Ewing sarcoma, lymphoma, small-cell mesothelioma, neuroblastoma, primitive neuroectodermal tumor and Wilms tumor belong to this class.

    Tissue array: Used to analyze the expression of genes of interest simultaneously in multiple tissue samples, tissue microarrays consist of hundreds of individual tissue samples placed on slides ranging from 2 to 3 mm in diameter. Using conventional histochemical and molecular detection techniques, tissue microarrays are powerful tools to evaluate the expression of genes of interest in tissue samples. In cancer research, tissue microarrays are used to analyze the frequency of a molecular alteration in different tumor type to evaluate prognostic markers, and to test potential diagnostic markers.

    Translocation: A translocation of genetic material from one chromosome to another, chromosomal translocations occur during meiosis when chromosomal breaks occur. However, in translocations, fragments of one chromosome rejoin to other chromosomes.

    Acknowledgment

    We thank Lynn Y. Sakai, MD, for the anti–fibrillin-2 antibody and Norbert Wey for advice on microscopic analysis.

    NOTES

    Supported by a grant from the University Zurich, and by Oncosuisse (Grant No. 01473-02-2004), Krebsliga of the Kanton Zug, and by the Schweizer Forschungsstiftung Kind + Krebs.

    Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

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

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《临床肿瘤学医学期刊》2006年2月第24卷第2期