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Journal of Clinical Oncology, Vol 23, No 3 (January 20), 2005: pp. 548-558 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.02.081
Ewing Sarcomas With p53 Mutation or p16/p14ARF Homozygous Deletion: A Highly Lethal Subset Associated With Poor ChemoresponseFrom the Departments of Pathology, Pediatrics, Surgery (Orthopaedics), and Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY Address reprint requests to Marc Ladanyi, MD, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021; e-mail: ladanyim{at}mskcc.org
PURPOSE: EWS-FLI1 fusion type, p53 mutation, and homozygous deletion of p16/p14ARF have each been shown to be prognostically significant in Ewing sarcoma (ES). We provide the first combined prognostic analysis of these three molecular parameters in ES. PATIENTS AND METHODS: We studied 60 patients with ES (stage: localized in 54, metastatic in six). All cases were confirmed to contain the EWS-FLI1 (29 type 1, 12 type 2, 14 other types) or EWS-ERG fusions (five cases). Homozygous deletion of p16/p14ARF, and p53 mutations were determined by fluorescent in situ hybridization and Affymetrix (Santa Clara, CA) p53 GeneChip microarray hybridization, respectively. RESULTS: Eight cases (13.3%) contained point mutations of p53, and eight cases (13.3%) showed p16/p14ARF deletion, including one case with both alterations. Among 32 cases with data on histologic chemoresponse, all 10 with alterations in p53 or p16/p14ARF showed a poor chemoresponse (P = .03). Variables predicting poorer overall survival included p53 mutation alone (P < .001), either p53 or p16/p14ARF alteration (P < .001), and stage (P < .01). In multivariate analysis, alterations of p53 and/or p16/p14ARF as a single variable, was the most adverse prognostic factor (P < .001), followed by stage (P = .04). In a multivariate analysis with alterations of p53 and p16/p14ARF as separate variables, both were significant (P < .001 and P = .03, respectively). Six cases with p16/p14ARF deletion were also studied for co-deletion of the contiguous methylthioadenosine phosphorylase gene, and this was detected in four cases. CONCLUSION: Alterations in p53 or p16/p14ARF are found in a fourth of ES cases and define a subset with highly aggressive behavior and poor chemoresponse.
Significant progress has been made in the treatment of Ewing sarcoma (ES), sometimes also termed peripheral neuroectodermal tumor (PNET). At least two thirds of patients with localized ES/PNET achieve long-term survival with a combination of intensive chemotherapy and local control by surgery and/or radiation. Nevertheless, among patients with localized tumor at diagnosis, 20% relapse within 4 years and die of the disease despite the most aggressive current protocols.1,2 Therefore, in addition to conventional factors such as age, tumor volume, serum lactate dehydrogenase level, and chemotherapy-induced necrosis, new molecular prognostic parameters are needed to risk-stratify patients with localized ES/PNET. Furthermore, up to 15% to 25% of patients first present with overt metastases, the strongest adverse conventional prognostic factor, and these patients fare poorly regardless of therapy.1,3 At the molecular level, ES/PNET is characterized by chromosomal translocations that fuse EWS, located at 22q12, and a gene of the ETS family of transcription factors. In more than 95% of cases, the gene fusion is EWS-FLI1 (90% to 95%) due to the classic t(11;22)(q24;q12), or EWS-ERG (5% to 10%) due to a variant translocation with the ERG gene at 21q22.4-6 These gene fusions are presumed to be the initiating oncogenic event in ES/PNET, and seem to play a critical role in the proliferation and tumorigenesis of ES/PNET cells.7,8 Very rare cases of ES/PNET show fusions of EWS to other ETS family genes (such as ETV1, E1AF, and FEV), or TLS-ERG fusion.9 At least some of the clinical heterogeneity in ES/PNET may correlate with EWS-FLI1 fusion transcript structure.5 We and others have found that the survival of patients whose tumor contains the most common type of EWS-FLI1 fusion (type 1: EWS exon 7 fused to FLI1 exon 6) is significantly better than that of patients with other EWS-FLI1 fusion types.10-12 Although the differences in EWS-FLI1 fusion structure are paralleled by differences in transactivation and proliferative rate,13,14 the clinical differences in retrospective series have been moderate. The variant EWS-ERG fusion has been found to be associated with clinical phenotypes indistinguishable from EWS-FLI1–positive ES/PNET.15 ES/PNET is also heterogeneous for the occurrence of genetic alterations involving certain critical regulators of cell cycle progression and apoptosis, in particular p16/p14ARF and p53. As reviewed in Table 1, homozygous deletion of p16/p14ARF and p53 alteration have been detected in approximately 20% and 10% of ES/PNET tumor samples, respectively. Although these are small subsets, both alterations are prognostically unfavorable, as reported by our group and others.23-25,27 However, these studies have had several limitations—many of them were small, few studies looked at both alterations, and some did not use optimal methods for the detection of these alterations.
The cellular effects of inactivating alterations of p16/p14ARF and p53 may overlap because of the roles of these proteins in the response to inappropriate mitogenic signals and the regulation of G1/S phase progression by the RB protein. The p53 protein mediates G1/S phase arrest or apoptosis (or senescence) in response to DNA damage or inappropriate mitogenic signals.28 P53, a transcription factor, blocks G1/S progression primarily through its downstream target gene, p21WAF. P53 itself is negatively modulated by binding to MDM2. The p16 and p14ARF proteins are encoded by the CDKN2A gene, located at 9p21 and closely flanked by the MTAP gene. MTAP, a gene 100 kb telomeric to p16/p14ARF at 9p21, encodes methylthioadenosine phosphorylase, an enzyme essential in the salvage of adenine and methionine. The CDKN2A gene encodes p16 and p14ARF by overlapping transcripts that use different reading frames.29 In the present article, we use the term p16/p14ARF gene synonymously with CDKN2A to clearly reflect this dual biology. The p16 protein inhibits cyclin D1-dependent kinases CDK4 and CDK6 and thus prevents phosphorylation of the RB protein, thereby blocking G1/S progression. In contrast to p16, p14ARF acts mainly through the p53 pathway by inhibiting the p53 antagonist MDM2. To clarify the interrelationships between fusion gene structure, p53 mutation, and p16/p14ARF deletion and their relative prognostic impact, we characterized these genetic alterations in 60 patients with ES/PNET and correlated molecular data with clinical parameters and overall survival. In addition, the subset of tumors with homozygous deletion of p16/p14ARF was examined for co-deletion of the adjacent MTAP gene, frequently co-deleted with p16/p14ARF in other cancers30-32 because the loss of this gene renders tumors susceptible to novel MTAP-directed agents.33
Clinical Material and Demographic Data The study group of 60 patients was drawn from 67 patients diagnosed with ES/PNET between 1991 and 2001 (except for one patient diagnosed in 1986). Inclusion requirements were as follows: (1) presence of either EWS-FLI1 or EWS-ERG fusion transcripts, (2) available material for p16/p14ARF and p53 studies, and (3) adequate clinical follow-up information. Seven cases had to be excluded because they did not meet the second or third requirement. Of the final study group of 60 patients, 57 were operated on at Memorial Sloan-Kettering Cancer Center (MSKCC), and three were operated on elsewhere, but frozen tumor material was submitted to MSKCC for fusion transcript analysis. Procurement of frozen tissues and retrospective clinical data collection was approved by the MSKCC institutional review board. There were 30 males and 30 females. Mean age at diagnosis was 23.7 years (median, 17 years; range, 1 to 72 years). The primary tumors were skeletal in origin in 38 patients and extraskeletal in 22. The higher proportion of extraskeletal primaries and cases in older adults may reflect the impact of confirmatory molecular diagnostic data in establishing this diagnosis in nonclassic settings.34,35 The primary tumor locations were axial in 32 patients and peripheral in 28 (lesions arising in limb girdles were considered peripheral). All patients had a chest computed tomography and radionuclide bone scan as part of their pretreatment extent of disease evaluation. At presentation, the tumor was localized in 54 patients, whereas six (10%) had distant metastases. The source of tissue used for molecular analyses was metastatic in 16 cases and nonmetastatic (primary or local recurrence) in 44 cases. The procurement of metastatic samples did not bias the molecular data compared with nonmetastatic samples (see Results), but may be responsible for poorer than expected chemoresponse and overall survival figures in the present series (see Results).
Patients With Repeat or Serial Analyses
Treatment and Response to Chemotherapy
Fusion Transcript Detection
FISH
p53 Mutation Analysis
Statistical Analysis
Fusion Transcript Data The distribution of fusion transcripts among the 60 cases, as summarized in Table 2, was similar to that of previous studies.
P16/P14ARF Deletion and MTAP Co-Deletion Homozygous and hemizygous deletions of the p16/p14ARF gene were detected in eight (13.3%; Table 1; Fig 1A through D) and six (10%) cases, respectively, and the former group consisted of one metastatic and seven localized cases. FISH analysis showed no deletion of p16/p14ARF in the remaining 46 (76.7%) cases. Because the survival of patients with tumors containing loss of only one copy of p16/p14ARF paralleled that of cases lacking any evidence of deletion (see Univariate Survival Analysis subsection), cases with hemizygous deletion of p16/p14ARF were grouped with nondeleted cases for all subsequent analyses. Of the eight cases with homozygous deletion of the p16/p14ARF gene,six were also studied for co-deletion of MTAP. Homozygous or hemizygous MTAP deletions were observed in four and one cases, respectively, as detected by probe P1-1069 (Fig 1E and F).
p53 Mutation By IHC, 17 cases showed p53 immunoreactivity in  10% tumor nuclei. These cases, together with another 11 cases lacking IHC data, were subjected to Affymetrix p53 GeneChip analysis to detect point mutations. This approach identified p53 point mutations in eight (13.3%) of 60 cases (Table 1), including six patients with localized tumors and two with metastatic disease. Affymetrix p53 GeneChip scores for specific point mutations ranged from 14 to 37 in six cases (Table 3). In two cases with borderline scores (ie, 7 and 10), direct sequencing confirmed the base change detected by chip analysis (results not shown). Among the eight cases showing p53 mutation, there was only one case (case 2 in Table 3) that also displayed homozygous deletion of p16/p14ARF. Taken together, the total number of cases with either p53 missense mutation or p16/p14ARF homozygous deletion was 15 (25%; Table 1). Table 3 summarizes the clinical and molecular data on these 15 cases.
Correlations Between Parameters Among the 32 cases available for evaluation of histological response to chemotherapy, there were nine good, and 23 poor responders. Good chemotherapeutic effect was correlated with the absence of either p53 or p16/p14ARF alterations (P = .03, Fisher's exact test; Table 4).
There was also a trend for p53 mutation alone (P = .14) and the combination of either p53 or p16/p14ARF alteration (P = .13) to show a relationship to stage, reflecting the finding that three of six tumors from patients with metastatic disease at diagnosis contained one of these two alterations, compared with only 11 of the remaining 54 patients. The association of p16/p14ARF status alone and stage did not approach statistical significance. There were no significant associations among the other variables. Specifically, there was no significant association between EWS-FLI1 fusion type (type 1 v others) and alterations in p53 and p16/p14ARF in the 55 EWS-FLI1 cases. EWS-FLI1 fusion type was also not related to chemotherapy response. The seemingly mutually exclusive relationship between p53 mutation and p16/p14ARF deletion was also not statistically significant. In addition, neither the EWS-FLI1 type nor the status of the p16/p14ARF gene was associated with stage or whether the tumor was axial or peripheral, or skeletal or extraskeletal. Because the source of the tissues used for molecular analyses was metastatic in 16 cases, we examined the possibility of a relationship between sample source and abnormalities of p53 or p16/p14ARF, but there was none: five of 16 metastatic and 10 of 44 nonmetastatic samples showed p53 or p16/p14ARF alteration (P = .52). This supports the notion that these genetic changes generally occur in the primary tumor at a preclinical time point.
Univariate Survival Analyses
By univariate analysis (Table 5), stage (Fig 2A, P < .001; risk ratio [RR] = 2.2; 95% CI, 1.4 to 3.7), p53 mutation (Fig 2B, P < .0001; RR = 3.7; 95% CI, 2.1 to 4.6), and either or both of a p53 or p16 alteration (Fig 2C, P < .0001; RR = 2.5; 95% CI, 1.6 to 3.8), were strong negative predictors of overall survival, whereas p16/p14ARF deletion alone (Fig 2D, P = .06; RR = 1.6; 95% CI, 0.96 to 2.6) only reached marginal statistical significance. Since the six cases with evidence of hemizygous deletion of p16/p14ARF showed no difference in survival compared with the nondeleted cases (not shown), we combined these two groups for the survival analysis.
No other factors were associated with survival, including age, sex, location, and origin of the primary tumor. As in previous studies,10,11 among cases with EWS-FLI1 (n = 55), patients with type 1 fusion showed slightly better survival, but this did not reach statistical significance (Fig 2E, P = .4; RR = 1.5; 95% CI, 0.6 to 3.4).
Multivariate Survival Analyses
We found p53 mutation in eight patients (13.3%)—a proportion similar to previous reports (Table 1). The difference in overall survival between cases with and without p53 mutation was remarkable, with all eight patients whose tumors had p53 mutations dying within 2 years. By multivariate analysis, p53 mutation was the most important independent prognostic factor. The dramatic negative impact of p53 alteration on survival in ES/PNET is consistent with several previous smaller studies, most of which used IHC as a surrogate marker for p53 point mutations.20,23 In the present study, all eight cases with p53 point mutations showed more than 20% immunoreactive tumor cells, and only two cases with this IHC finding were negative for p53 mutations, supporting this cutoff for p53 immunoreactivity as a reasonable surrogate marker for this genetic alteration in ES/PNET. Homozygous deletion of p16/p14ARF (n = 8, 13%) was as frequent as p53 mutation in this series, a somewhat lower prevalence than most previous studies (Table 1). Possible reasons for this difference may include variability in methodologies or in criteria to define positive cases, and small sample sizes in previous studies. By univariate analysis, p16/p14ARF deletion alone had only marginal value as a negative factor. However, in the multivariate analysis, p16/p14ARF homozygous deletion emerged as the second most significant factor after p53 mutation. The lack of significance of stage in the latter multivariate analysis should be interpreted with caution given the low number of metastatic samples (n = 6) in the present study. Finally, when p16/p14ARF deletion and p53 mutation were combined as a single factor, it was the most crucial determinant of overall survival followed by stage in both univariate and multivariate analysis. The negative impact of p16/p14ARF loss on survival in ES/PNET is consistent with several previous smaller studies.26,27,42 Some groups have found an association between p16/p14ARF or p53 alterations and advanced stage,23,42 while others have not.24,25 In the current series, the presence of p53 mutation or p16/p14ARF deletion was weakly, but not significantly, related to distant metastases at presentation, but the small number of metastatic cases (n = 6; 10%) limited the power of this analysis. Regardless of this possible association, it is of more practical clinical importance that most p53 mutations or p16/p14ARF deletions are detected in patients with localized disease at diagnosis—22% in the present series. Our finding that the six cases with hemizygous deletion of p16/p14ARF showed no difference in survival compared with the nondeleted cases suggests that loss of one copy of p16/p14ARF combined with inactivation of the remaining copy by mutation or epigenetic mechanisms is unlikely to be significant in ES/PNET. Indeed, Kovar et al17 found only one tumor in 27 to show mutation of p16/p14ARF with loss of heterozygosity. In another study, the presence of p16 promoter hypermethylation had no impact on survival of ES/PNET patients.27 Finally, further evidence indicating that p16 promoter hypermethylation is rare in ES/PNET is that the prevalence of loss of p16 protein expression in both tumors and cell lines closely approximates that of p16/p14ARF homozygous deletion.42,43 In aggregate, these data support homozygous deletion as the principal mode of p16/p14ARF inactivation in ES/PNET. The finding of combined p16/p14ARF deletion and p53 mutation in only a single case of a total of 15 with either or both alterations might suggest that alterations in these two genes are mutually exclusive. However, this apparent reciprocal relationship p16/p14ARF deletion and p53 mutation did not approach statistical significance. This, along with the report of another ES/PNET case with both alterations26 and of several ES/PNET cell lines with both p16/p14ARF deletion and p53 mutation,17 reinforces the impression that the infrequent coincidence of these two alterations in primary ES/PNET stems mainly from their low prevalence. Other modes of inactivation of the p53 and Rb pathways, through MDM2, CCND1, or Rb alterations, are rare in ES/PNET.16,44 The striking impact of p53 and p16/p14ARF alterations on prognosis points to profound biologic differences inconsistent with a functionally equivalent perturbation of these pathways in the remaining 75% of cases. The strong clinical impact of these alterations in ES/PNET is reminiscent of the marked impact of impaired apoptosis due to p14ARF or p53 loss on lymphomagenesis in Em-myc transgenic mice, a model for P53 pathway alterations in human tumors with translocation oncogenes.45,46 Oncogenic or inappropriate proliferative stimuli are known to elicit an apoptotic or senescence response dependent on the P53/p14ARF pathway.47,48 By analogy, it is tempting to hypothesize that the oncogenicity of EWS-FLI1 may be modulated or tempered by the apoptotic function of an intact P53/p14ARF pathway. Indeed, data from three groups support an important relationship between EWS-FLI1 and the P53 pathway. Kovar et al found that reintroduction of normal p53 in p53-null ES/PNET cell lines triggers extensive apoptosis.49,50 It should be noted that essentially all ES/PNET cell lines contain p53 alterations or p16/p14ARF deletions, suggesting selection pressure for these genetic alterations to permit in vitro growth.8,51 These data suggest that reintroduction of functional p53 in ES/PNET cell lines promptly leads EWS-FLI1 to trigger a p53-dependent apoptotic program because ES/PNET cells cannot overcome an apoptotic response to in vitro adherent culture conditions unless the p53 pathway is nonfunctional. Deneen et al showed that EWS-FLI1 induces apoptosis and growth arrest in normal mouse embryonic fibroblasts (MEF).52 However, in p16/p14ARF–null or p53-deficient MEFs, apoptosis and growth arrest in response to EWS-FLI1 were reduced.52 Finally, Lessnick et al53 found that in primary human fibroblasts immortalized with telomerase cDNA, expression of EWS-FLI triggered growth arrest associated with transcriptional upregulation of p53. While these data suggest that specific response mechanisms to EWS-FLI1 may differ in different experimental settings, taken together, they support a central role for the p53 pathway in the cellular response to EWS-FLI1. A similar pattern of infrequent but highly clinically significant p53 or p16/p14ARF alterations has been reported in several other developmental and/or translocation-associated cancers.54 For instance, the outcome of Wilms' tumors,55,56 neuroblastomas,57 follicular lymphomas,58,59 and myxoid liposarcomas60 is dramatically worsened by p53/p14ARF pathway alterations. This is in contrast to most carcinomas and most sarcomas lacking specific translocations, where p53 alterations usually have only moderate or marginal clinical significance.61-63 MTAP is co-deleted in 75% to 90% of tumors, with p16/p14ARF homozygous deletion32 reflecting the close genetic linkage between these two genes. Indeed, four of our six p16/p14ARF–deleted cases (66%) had also lost both copies of MTAP. Codeletion of the MTAP gene with p16/p14ARF has been observed in a variety of cancers.30,32,64-66 Because cancer cells that lack MTAP are unable to salvage adenine from methylthioadenosine, they become dependent on the de novo synthesis pathway of purine metabolism. Thus, MTAP-deleted cancer cells are highly and specifically sensitive to inhibition of the de novo synthesis pathway (eg, by L-alanosine, also known as SDX-102), presenting an opportunity for tailored chemotherapy.33,64 Although only a small proportion of patients with ES/PNET would be candidates for such MTAP-directed chemotherapeutic approaches, it is a subset of ES/PNET that includes approximately a third of the most lethal cases. We and others have previously shown that the survival of patients with localized disease whose ES/PNET bear the type 1 EWS-FLI1 fusion is better than those with tumors containing other EWS-FLI1 fusion types.10-12 These series have ranged from 55 to 83 informative cases.12 In the present series, among the 55 EWS-FLI1 cases (including six metastatic), EWS-FLI1 fusion type was not a statistically significant prognostic factor, though the survival trends did parallel the findings in prior larger series in that patients with the type 1 fusion seemed to have better outcomes (Fig 2E). The prognostic impact of EWS-FLI1 fusion type appears lesser than that of p53 or p16/p14ARF alterations and may not be demonstrable unless a larger sample size is studied. A poor histologic response to preoperative chemotherapy has been found to be a highly significant adverse factor in studies of 74 and 118 patients with ES/PNET, respectively.36,67 The limited number of cases in the present study with available chemotherapy response data (n = 32) made the series unsuitable for re-evaluation of this question. However, a significant correlation was observed between good chemotherapeutic effect and the absence of genetic alterations in p53 or p16/p14ARF. Previous studies relating p53 alterations (by IHC) to chemoresponse in ES/PNET reached conflicting conclusions.23,24 P53 mutations have been shown to be associated with resistance to chemotherapy and radiotherapy in diverse tumor types, including sarcomas.68,69 The drug resistance caused by p53 dysfunction can be exerted through several underlying mechanisms.68,69 In addition, p14ARF deletions promote chemoresistance in murine Eµ-myc lymphomas by disabling p53 function with consequent apoptotic defects,45 further emphasizing the links between p16/p14ARF and p53 in oncogenic transformation and chemoresistance. There had so far been no strong prognostic factor available for prechemotherapy risk stratification of patients presenting with localized ES/PNET. The present retrospective study suggests that alterations in p53 and p16/p14ARF, found in approximately a fourth of all ES/PNET, are strong negative predictors of overall survival, thereby providing for the first time a robust molecular marker of clinical outcome in ES/PNET that could be used to assign certain patients with localized disease to high-risk regimens before initial chemotherapy. Definitive confirmation of these results will require a prospective analysis of these molecular prognostic markers in a larger series of patients with ES/PNET, with longer follow-up.
The authors indicated no potential conflicts of interest.
We thank the following for providing clinical follow-up data: Ronald Jaffe, MD, Children's Hospital of Pittsburgh; Alex Aledo, MD, New York Presbyterian Hospital; Charles F. Timmons, MD, PhD, Children's Medical Center of Dallas.
Supported by American Cancer Society Project Grant 99-216 (M.L.) and the Ewing Sarcoma Research Fund (M.L.). H.-Y.H. was a visiting fellow sponsored by Chang Gung Memorial Hospital, Kaohsiung Medical Center, Kaohsiung, Taiwan. H.-Y.H. and P.B.I. contributed equally to this work. P.B.I. is presently in the Department of Pathology, New York University Medical Center, 560 First Ave, New York, NY 10016. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
50%; grade 2 = 51% to 90% tumor necrosis; grade 3 = only scattered foci of viable tumor cells [91% to 99% tumor necrosis]; grade 4 = complete tumor necrosis [100%]).
