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PAX3-FKHR and PAX7-FKHR Gene Fusions Are Prognostic Indicators in Alveolar Rhabdomyosarcoma: A Report From the Children’s Oncology Group

From the Departments of Pathology and Pediatrics, Children’s and Women’s Hospital of British Columbia, Vancouver, British Columbia, Canada; Intergroup Rhabdomyosarcoma Study Group Statistical Center, Office of the Chancellor, and Department of Microbiology/Pathology, University of Nebraska Medical Center, Omaha, NE; Department of Laboratory Medicine, Columbus Children’s Hospital, Columbus, OH; Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles, Los Angeles, CA; Office of the Dean, School of Medicine, University of Missouri at Columbia, Columbia, MO; and Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA.

Address reprint requests to Poul H.B. Sorensen, Operations Office, Children’s Oncology Group, 440 East Huntington Dr, Suite 300, PO Box 60012, Arcadia, CA 91066-6012; email: psor{at}interchange.ubc.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Alveolar rhabdomyosarcoma (ARMS) is an aggressive soft tissue malignancy of children and adolescents. Most ARMS patients express PAX3-FKHR or PAX7-FKHR gene fusions resulting from t(2;13) or t(1;13) translocations, respectively. We wished to confirm the diagnostic specificity of gene fusion detection in a large cohort of RMS patients and to evaluate whether these alterations influence clinical outcome in ARMS.

PATIENTS AND METHODS: We determined PAX3-FKHR or PAX7-FKHR fusion status in 171 childhood rhabdomyosarcoma (RMS) patients entered onto the Intergroup Rhabdomyosarcoma Study IV, including 78 ARMS patients, using established reverse transcriptase polymerase chain reaction assays. All patients received central pathologic review and were treated using uniform protocols, allowing for meaningful outcome analysis. We examined the relationship between gene fusion status and clinical outcome in the ARMS cohort.

RESULTS: PAX3-FKHR and PAX7-FKHR fusion transcripts were detected in 55% and 22% of ARMS patients, respectively; 23% were fusion-negative. All other RMS patients lacked transcripts, confirming the specificity of these alterations for ARMS. Fusion status was not associated with outcome differences in patients with locoregional ARMS. However, in patients presenting with metastatic disease, there was a striking difference in outcome between PAX7-FKHR and PAX3-FKHR patient groups (estimated 4-year overall survival rate of 75% for PAX7-FKHR v 8% for PAX3-FKHR; P = .0015). Multivariate analysis demonstrated a significantly increased risk of failure (P = .025) and death (P = .019) in patients with metastatic disease if their tumors expressed PAX3-FKHR. Among metastatic ARMS, bone marrow involvement was significantly higher in PAX3-FKHR–positive patients.

CONCLUSION: Not only are PAX-FKHR fusion transcripts specific for ARMS, but expression of PAX3-FKHR and PAX7-FKHR identifies a very high-risk subgroup and a favorable outcome subgroup, respectively, among patients presenting with metastatic ARMS.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
RHABDOMYOSARCOMAS (RMS) are a family of soft tissue sarcomas that demonstrate morphologic, immunophenotypic, or ultrastructural evidence of skeletal muscle differentiation.¹ These malignancies are the most common soft tissue sarcomas in children.^2,3 The considerable variation in outcome in RMS has led to an intense search for prognostic indicators useful for therapeutic stratification. Currently, the most useful prognostic parameters are extent of disease at presentation, primary tumor site, histology, and type of therapy used.⁴

Pathologic classification schemes have historically divided RMS into embryonal RMS (ERMS) and alveolar RMS (ARMS) subtypes. More recently, RMS classification was expanded to include so-called botryoid and spindle cell morphologic variants of ERMS and a rare primitive tumor known as undifferentiated sarcoma.⁵ Clinical group classification is determined on the basis of local and distant spread and the adequacy of surgical removal of local disease, whereas the primary tumor sites are roughly subdivided into favorable versus less favorable sites (reviewed in Wexler and Helman⁶). These predictors are combined to establish risk categories with differing outcomes. Lesions in the "low-risk" category (30% of RMS patients, 95% long-term survival) tend to be localized ERMS at favorable sites, whereas "high-risk" lesions (10% of RMS, 25% long-term survival) tend to be metastatic ARMS. The remaining 60% of cases are "intermediate-risk" (50% to 65% survival) and consist of a clinically heterogeneous group of ERMS and ARMS. The identification of clinicopathologic predictors that more reliably distinguish these risk groups from each other and further subdivide these categories would be highly desirable for therapeutic stratification at diagnosis.

Analysis of recurrent cytogenetic alterations in solid tumors reveals an association of sarcomas with reciprocal chromosomal translocations. Cloning studies have demonstrated in-frame fusion of coding sequences from each rearranged gene, giving rise to fusion transcripts encoding chimeric proteins with oncogenic activity.^7,8 The consistent association of these gene fusions with specific sarcoma subtypes has facilitated the development of molecular diagnostic assays.^9-13 Chromosomal analyses have demonstrated two translocations associated with ARMS, t(2;13)(q35;q14) and t(1;13)(p36;q14). Physical mapping and cloning studies revealed that these translocations fuse the FKHR locus on chromosome 13 to either PAX3 on chromosome 2¹⁴ or the chromosome 1 PAX7 gene.¹⁵ The resulting gene fusions encode PAX3-FKHR and PAX7-FKHR chimeric proteins that combine transcriptional domains from the corresponding wild-type proteins and thereby acquire oncogenic activity.¹⁶

The association of these gene fusions with ARMS led to the establishment of reverse transcriptase polymerase chain reaction (RT-PCR) assays to detect fusion transcripts in primary tumor tissue.^10,17 Initial studies detected these gene fusions in more than 85% of ARMS,^10,17 with PAX3-FKHR being 4.5-fold more prevalent than PAX7-FKHR.¹⁰ These gene fusions were also identified in a small proportion of ERMS cases but were not found in other "small round cell tumors" of childhood, such as Ewing tumor, neuroblastoma, and desmoplastic small round cell tumor (reviewed in Triche³), making such assays useful in the diagnostic work-up of ARMS. Although both ARMS-associated gene fusions are reliable diagnostic markers, several observations suggest that there may be clinical and biologic differences between PAX3-FKHR– and PAX7-FKHR–expressing tumors. This hypothesis was first indicated by the finding of genomic amplification of the PAX7-FKHR locus in most t(1;13)-containing ARMS, whereas PAX3-FKHR amplification in t(2;13)-positive tumors is rare.^18,19 A potential clinical difference was found in an analysis of a small series of PAX3-FKHR– and PAX7-FKHR–positive ARMS tumors.²⁰ This study found that the PAX7-FKHR cohort was younger, presented more often with an extremity lesion, and appeared to have improved overall survival. These results provided the first evidence that PAX3-FKHR and PAX7-FKHR gene fusions are associated with distinct clinical phenotypes and may be useful as prognostic markers. In the present study, we wished to definitively test the clinical impact of fusion status in ARMS and therefore analyzed a large series of uniformly treated RMS cases from the recently completed Intergroup Rhabdomyosarcoma Study IV (IRS-IV) (1991 to 1997).²¹

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
IRS-IV Primary Tumor Specimens
Primary tumor samples stored in liquid nitrogen were retrieved from the Intergroup Rhabdomyosarcoma Study Group (IRSG) Tissue Bank/Pathology Center in Columbus, OH. All IRS-IV cases received central pathologic review and patients were treated using uniform sets of protocols.²¹ The inclusion criteria for this study were an IRSG review panel diagnosis of RMS (including each of the RMS subtypes), confirmation of registration on IRS-IV, and the availability of frozen tissue in the IRSG tumor bank. On the basis of these criteria, we identified 200 RMS cases of the total of 1,319 IRS-IV RMS cases for possible inclusion. We then excluded specimens lacking viable tumor tissue by histologic evaluation of frozen sections or cytologic touch preparations, and those lacking amplifiable RNA by RT-PCR screening of total RNA for the ubiquitously expressed MIC2 or beta-actin genes.¹² These exclusions left 144 RMS cases (including 51 ARMS cases) suitable for molecular studies, which were then subjected to double-blinded rereview by the IRSG pathology committee using established criteria.^5,21 Because one of the goals of this study was to correlate gene fusion status with outcome in ARMS, we increased the ARMS sample size using two approaches. First, we cross-referenced all ARMS cases registered on IRS-IV from two of our institutions (Children’s and Women’s Hospital of British Columbia and University of Pennsylvania) to identify additional IRS-IV ARMS cases that were not originally included on our study but which had institutionally banked frozen tissue specimens that had not been submitted to the IRSG tumor bank. Second, we searched the IRSG database for ARMS cases with frozen tissue that had been registered on IRS-IV after the initiation of our study. These approaches allowed us to add 27 more IRS-IV ARMS cases to the original study cohort of 144 cases, for a total of 171 RMS cases (all of which were registered on IRS-IV). All histologic diagnoses were assigned without any knowledge of the molecular findings. The histopathologic subtypes of the cases studied are listed in Table 1.

RNA Extraction and RT-PCR Analysis
RNA extraction from frozen primary tumors using the acid guanidinium-phenol-chloroform method was performed as previously described.^9,10 Molecular analysis of ARMS-associated gene fusions was carried out using previously described RT-PCR assays for PAX3-FKHR and PAX7-FKHR.^10,22 Amplified products were identified by agarose gel electrophoresis, and then transferred to nylon filters. Blots were sequentially probed for PAX3-FKHR and PAX7-FKHR gene fusions as previously described²² using phosphorus-32–labeled oligonucleotide probes for FKHR, PAX3, and PAX7 followed by autoradiography. Representative PCR products were purified and subjected to sequence analysis to confirm the identity of amplification products. Gene fusion status was evaluated in three independent laboratories blinded to the histopathologic subtypes, with each RT-PCR result requiring corroboration in at least two separate experiments.

Statistical Analysis
Clinical characteristics and outcome of IRSG cases were retrieved from the IRSG Biostatistical Center database. Failure-free survival was measured from the time of diagnosis until progression, relapse after response, or death (if occurring before relapse or progression). Patients whose treatment did not fail were censored at their date of last contact. Survival was measured from the time of diagnosis until death, or until the date of last contact, if the patient was alive at last report. All deaths were counted as failures, whether or not they were disease-related. Estimates of the time-to-event distributions were calculated using the Kaplan-Meier method.²³ Comparisons of outcome among patient subsets were made using the log-rank test,²⁴ and the Cox proportional hazards regression model was used for multivariate analysis.²⁵ Comparisons of demographic and disease characteristics among patient subsets were made using the ² test for association or, where appropriate because of sparseness, Fisher’s exact test.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PAX3-FKHR and PAX7-FKHR Gene Fusions Are Specific for ARMS
We assessed the utility of PAX3-FKHR and PAX7-FKHR gene fusion detection in the diagnostic and prognostic stratification of childhood RMS. The IRS-IV RMS cohort was particularly suitable for this study, as all patients registered on IRS-IV received central pathologic review and were treated using uniform sets of protocols,^21,26 thus allowing for meaningful outcome analysis. The first aim of this study was to correlate the fusion status with RMS histopathologic subtypes to confirm the utility of these gene fusions as specific tumor markers for ARMS. As outlined in the Patients and Methods section, we identified 171 IRS-IV cases that had a review diagnosis of RMS and were suitable for molecular analysis. Gene fusion status was evaluated using RT-PCR assays for PAX3-FKHR and PAX7-FKHR fusion transcripts and was then compared to histopathologic subtyping as summarized in Table 1. All histopathologic diagnoses were arrived at without any knowledge of the molecular findings. In this series, 55% of ARMS cases expressed PAX3-FKHR gene fusions, 22% expressed PAX7-FKHR, and 23% were fusion-negative. In contrast, all cases of ERMS (including botryoid and spindle cell variants) and undifferentiated sarcoma were fusion-negative, confirming that these fusions are specific for tumors with ARMS morphology.

Fusion Status Correlates With Clinical Outcome in ARMS
We next correlated gene fusion status with clinical characteristics and outcome in the 78 IRS-IV ARMS cases. The age range of the cohort was 0 to 21 years of age, and the male/female ratio was 55:45. The median follow-up of surviving patients was 3.9 years. Three fusion categories were used in subsequent analyses: PAX3-FKHR–positive ARMS, PAX7-FKHR–positive ARMS, and fusion-negative ARMS. We found no association of fusion status with sex, race, tumor site or size, stage, or clinical group (data not shown). A similar fraction of PAX3-FKHR, PAX7-FKHR, and fusion-negative patients presented with metastatic disease (30%, 35%, and 28%, respectively). However, PAX3-FKHR expression was associated with older age (P = .04) and higher tumor invasiveness (T2 v T1; P = .007) compared with the other groups (data not shown).

In our evaluation of clinical outcome, we found a trend toward better overall survival in PAX7-FKHR–positive compared with PAX3-FKHR–positive ARMS; the estimated 4-year overall survival was 77% for PAX7-FKHR versus 52% for PAX3-FKHR (P = .08), whereas fusion-negative patients had an intermediate outcome (Fig 1A). To further assess this trend, we subdivided patients into those presenting with either locoregional (including regional lymph nodes) or metastatic disease. Among fusion-positive patients, fusion status was not associated with an outcome difference in patients with locoregional disease at diagnosis (Fig 1B). However, when fusion status was compared in patients presenting with metastatic disease, there was a striking difference in outcome between PAX7-FKHR– and PAX3-FKHR–positive patients; the estimated 4-year overall survival rate was 75% for PAX7-FKHR versus 8% for PAX3-FKHR (P = .002) (Fig 1C). Notably, treatment regimens were balanced across the cohorts analyzed (data not shown). We then analyzed the PAX7-FKHR patients alone and found little difference in outcome whether patients presented with locoregional or metastatic disease (Fig 1D). In contrast, the difference between these categories was dramatic in the PAX3-FKHR cohort (Fig 1E).

View larger version (26K): [in this window] [in a new window]   Fig 1. Kaplan-Meier plots of overall survival for RMS patients registered on IRS-IV. P values were based on the log-rank test. (A) All ARMS patients: PAX3-FKHR (n = 43), PAX7-FKHR (n = 17), or fusion-negative (n = 18). (B) ARMS patients presenting with locoregional disease: PAX3-FKHR (n = 30) or PAX7-FKHR (n = 11). (C) ARMS patients presenting with metastatic disease: PAX3-FKHR (n = 13) or PAX7-FKHR (n = 6). (D) All PAX7-FKHR–positive cases: locoregional (n = 11) or metastatic (n = 6). (E) All PAX3-FKHR–positive cases: locoregional (n = 30) or metastatic (n = 13).

PAX3-FKHR Fusion Is a Marker of Poor Prognosis in Multivariate Analysis
To better understand how fusion status might influence the prognosis of ARMS in the context of known or suspected risk factors in this disease, the Cox proportional hazards model was used for the multivariate analysis of risk of failure or death in patients with ERMS or ARMS.⁴ Among patients with locoregional disease, there was increased relative risk of failure or death for each of the ARMS categories compared with locoregional ERMS, although only the presence of the PAX3-FKHR gene fusion reached statistical significance (P = .033 for failure or death) (Table 2). However, these findings may be due to the low numbers of events and correspondingly wide confidence intervals, and therefore larger cohorts are required to compare locoregional ARMS with locoregional ERMS. Similar analyses were performed for cohorts presenting with metastatic disease. Among ERMS cases, there was an increased risk of failure and death for patients with metastatic disease as expected from previous studies⁴ (Table 2). Among metastatic ARMS cases, the PAX3-FKHR–positive and fusion-negative groups also had increased risks for failure and death. In marked contrast, the presence of metastatic disease was not associated with worse outcome in the PAX7-FKHR group (Table 2). In fact, we found that among fusion-positive ARMS patients with metastatic disease, there was a significantly increased risk of failure (P = .025) and death (P = .019) in patients if their tumors expressed the PAX3-FKHR but not the PAX7-FKHR gene fusion (Table 2). The PAX3-FKHR gene fusion is therefore an independent indicator of adverse outcome, whereas PAX7-FKHR expression is an independent indicator of favorable outcome in metastatic ARMS. After adjustment for extent of disease, fusion status, and histology, the parameters of age, sex, race, primary tumor site, nodal involvement, tumor size, and tumor invasion were not significantly related to outcome (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

As a group, fusion-negative ARMS cases demonstrated outcomes that were intermediate to those of the PAX7-FKHR and PAX3-FKHR cohorts (Fig 1A and Table 2). Fusion-negative ARMS seems to represent a genetically heterogeneous subgroup, and we are currently exploring this subgroup (Barr et al, manuscript in preparation). Some of these cases may have variant translocations involving related members of the PAX and/or FKHR gene families, or involving different genes altogether. Alternatively, some cases may have other types of genetic abnormalities that lead to activation of the same oncogenic pathways as PAX-FKHR proteins. Larger studies of fusion-negative cases will be required before the genetic basis and prognostic significance of this category of ARMS can be rigorously determined.

The predictive power of fusion status on outcome in this study was most evident in patients presenting with metastatic disease. This observation may simply be due to the small numbers of patients within the locoregional subgroups: it is well known that pairwise comparisons of favorable-risk categories, such as patients with limited extent of disease, require larger patient numbers to demonstrate outcome differences than do comparisons of high-risk categories where outcome is poor. However, the findings in ARMS clearly contrast with similar studies of other solid tumors of children and adolescents. Almost all Ewing tumors express either EWS-FLI1 or EWS-ERG gene fusions resulting from 11;22 or 21;22 translocations, respectively.^27,28 Further diversity of these rearrangements is conferred by different combinations of exons from EWS and its partner genes giving rise to variably sized fusion products.²⁹ Although no difference in outcome was observed when EWS-FLI1– and EWS-ERG–positive tumors were recently compared,³⁰ an analysis limited to EWS-FLI1–positive cases revealed a significantly better prognosis in patients with the more common type 1 gene fusion (EWS exon 7 fused to FLI1 exon 6) in comparison with cases with less common fusion types.³¹ Notably, however, this difference was only seen among cases with localized disease. In synovial sarcomas, which express t(X;18)-associated SYT-SSX1 or SYT-SSX2 fusion transcripts,^32,33 a better outcome in patients with the SYT-SSX2 gene fusion was observed only in cases with locoregional disease.^34,35 In our study, there was a marked difference in overall survival between the PAX3-FKHR and PAX7-FKHR cohorts with metastatic disease. Because the incidence of metastatic disease was similar in both cohorts, the observed difference is not due to PAX3-FKHR expression predisposing to metastasis. It is tempting to speculate that the PAX3-FKHR oncoprotein may confer increased survival or resistance to chemotherapy of metastatic cells, and that PAX7-FKHR lacks this protective effect. Interestingly, we also found marked differences in the pattern of metastatic disease in the two cohorts. Over 50% of the PAX3-FKHR metastatic cases had bone marrow involvement, whereas none of the metastatic PAX7-FKHR cases involved this site (P = .044). Bone marrow metastatic disease has previously been shown to predict poor outcome in RMS.^36,37 However, we failed to demonstrate outcome differences in bone marrow positive versus negative PAX3-FKHR metastatic ARMS. This finding may be due to the small numbers analyzed, or alternatively because other mechanisms underlie the aggressive nature of PAX3-FKHR metastatic ARMS. Further studies are required to determine the biologic basis of the observed clinical differences in ARMS fusion subclasses and the relationship of these fusions to the metastatic process in general.

This study provides strong evidence that fusion status is essential for prognostic stratification in ARMS. We propose that gene fusion assessment be considered as a major end point in the diagnostic and prognostic work-up of RMS, and that clinical trials should be initiated to rigorously evaluate whether fusion status should dictate treatment assignment in ARMS. Also, given the poor outcome in PAX3-FKHR–positive ARMS patients with metastatic disease at diagnosis, it is hoped that our findings will prompt further efforts to focus on the PAX3-FKHR oncoprotein as a therapeutic target in this patient cohort.

	ACKNOWLEDGMENTS

 
Supported in part by grant nos. CA24507, CA64202, CA72989, CA81659, and CA89461 from the National Institutes of Health, National Cancer Institute, Bethesda, MD.

We thank Joan Mathers, Heather Wildgrove, Michelle Macris, and Xian Fang for expert technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted March 28, 2001; accepted March 1, 2002.

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