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TP53 Mutations and Outcome in Osteosarcoma: A Prospective, Multicenter Study

From the Samuel Lunenfeld Research Institute, and the University Musculoskeletal Oncology Unit, Mount Sinai Hospital; Departments of Surgery, Public Health Sciences, Medical Genetics and Microbiology, Pathology and Laboratory Medicine, University of Toronto; Division of Hematology-Oncology, Hospital for Sick Children, Toronto, Ontario; University of British Columbia, Vancouver, British Columbia, Canada; University of Washington Medical Center, Seattle, WA; Royal Orthopaedic Hospital, Birmingham, England; Memorial Sloan-Kettering Cancer Center, NY, New York; and Mayo Clinic, Rochester, MN

Address reprint requests to Jay S. Wunder, MD, 476E-600 University Ave, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; e-mail: wunder{at}mshri.on.ca

	ABSTRACT

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

 
PURPOSE: Mutations of the TP53 gene have been associated with resistance to chemotherapy as well as poor prognosis in many different malignancies. This is the first prospective study of the prognostic value of somatic TP53 mutations in patients with newly diagnosed extremity osteosarcoma.

PATIENTS AND METHODS: One hundred ninety-six patients with high-grade, nonmetastatic osteosarcoma of the extremities were enrolled from seven tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 44 months). All patients received neoadjuvant or adjuvant chemotherapy and surgery. Tumors were analyzed for the presence of TP53 mutations by polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing. The association of the status of the TP53 gene with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors.

RESULTS: Patient age was the only factor that varied with TP53 gene status (P = .05). No relationship was identified between TP53 status and systemic relapse (relative risk, 1.24; P = .41). Analyses based on missense or nonsense mutations gave similar results (P > .10). In multivariate analysis, large (> 9 cm) tumor size (relative risk, 1.9; P = .006) and poor histologic response ( 90% necrosis) to chemotherapy (relative risk, 2.14; P = .02) were the only significant independent predictors of systemic outcome.

CONCLUSION: We found no evidence that TP53 mutations predict for development of metastases in patients with high-grade osteosarcoma. Identification of other genes that influence chemotherapy response and clinical outcome in osteosarcoma is needed to facilitate further improvements in patient outcomes.

	INTRODUCTION

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

 
Recent advances in the treatment of osteosarcoma have led to significant improvements in patient outcome. Because of better radiographic imaging combined with the benefits of preoperative chemotherapy, few patients require amputation.^1–3 Introduction of intensive multiagent chemotherapy has also contributed to major improvements in patient survival. Long-term cure rates for patients with high-grade osteosarcoma presenting without metastases have been reported at between 50% and 75%.^4–7 However, 25% to 50% of patients will relapse, and only a minority of these will be salvaged.

One of the major difficulties in treating patients with osteosarcoma is the lack of accurate prognostic factors. For patients who present with a resectable tumor and without metastases, large tumor size predicts for a poor outcome.^4,8–10 Poor response to preoperative chemotherapy is also a negative prognostic indicator; however, this information does not become available until the primary tumor is resected, which is well into the patient's treatment protocol.^4–6 Neither of these factors alone, or in combination, can accurately predict outcome for an individual patient.

As a result, there has been a growing expectation that molecular genetic alterations may be better predictors of outcome for patients with osteosarcoma. The tumor suppressor gene TP53 is a candidate prognostic marker for osteosarcoma. Patients with Li-Fraumeni syndrome who have germline TP53 mutations are at risk for multiple cancers, including osteosarcoma.^11,12 A variety of tumors, including osteosarcoma, also develop in TP53-deficient and transgenic mice.^13–15 TP53 is one of the most frequently mutated genes in human cancers, and we previously identified TP53 mutations in 60 (22%) of 272 sporadic osteosarcomas.¹⁶ For a variety of malignancies, TP53 mutations predict for a more aggressive tumor phenotype and worse clinical outcome.^17–22 The status of the TP53 gene has also been identified as a determinant of chemotherapy response both in vivo and in vitro, likely as a result of its roles in cell cycle arrest and apoptosis.^{19,20,23–27} This could be of critical importance in osteosarcoma since the effectiveness of chemotherapy is the prime determinant of patient outcome. In a previous prospective study aimed at identifying a prognostic marker for chemotherapy response and outcome in osteosarcoma, we examined expression of the MDR1 gene. No correlation with disease progression was identified,⁸ suggesting that alterations of other genes, such as TP53, may play a more important role in this disease.

This study is the first prospective assessment of the prognostic value of TP53 gene status in osteosarcoma. In this study, we evaluated the effect of TP53 status and standard prognostic variables on clinical outcome in 196 newly diagnosed patients with conventional high-grade nonmetastatic osteosarcoma of the extremities.

	PATIENTS AND METHODS

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Patient Eligibility, Treatment, and Clinical Follow-Up
Two hundred forty-five patients with high grade, nonmetastatic osteosarcoma of the extremities were prospectively enrolled from seven tertiary care institutions between 1989 and 1998. Patients were not consecutively treated, and therefore represent a subgroup of the eligible patients from each institution. All patients had newly diagnosed, biopsy-proven osteosarcoma without prior treatment. Systemic staging studies included total-body technetium bone scans and computed tomography scans of the chest. All patients received pre- and postoperative chemotherapy or postoperative chemotherapy alone, which included doxorubicin and was based on one of three protocols incorporating the following major agents: (1) doxorubicin and cisplatin⁷; (2) high-dose methotrexate, doxorubicin, and cisplatin⁶; and (3) ifosfamide, high-dose methotrexate, doxorubicin, and cisplatin.²⁸ The decision of which chemotherapy protocol to administer was not based on the presenting patient or tumor characteristics, but on the protocol in use at each participating institution. All patients were seen in regular follow-up for a minimum of 24 months from the time of diagnosis, or until systemic relapse. No patients were lost to follow-up. Each eligible patient provided a signed consent form before study entry, as approved by each participating institution's research ethics board.

Patients with pre-existing Paget's disease, radiation-induced tumors, or previous malignancies (excluding nonmelanoma skin cancer or cervical carcinoma in situ) were not eligible for this study. Similarly, patients with concurrent nonmalignant disease that would preclude the use of one of the standard chemotherapy protocols were ineligible. Of 245 patients entered into this study, those with high-grade parosteal tumors were excluded (n = 14) because they may have had a different natural history and clinical course following treatment compared with conventional high-grade intramedullary osteosarcoma.^29–32 The occurrence of local tumor relapse was taken to be an indication of inadequate surgical resection, and these patients were also excluded (n = 19). In 16 of the remaining cases, it was not possible to obtain the TP53 mutation status, owing to the absence of available tumor tissue (n = 15) or poor quality DNA (n = 1). This left 196 patients with high-grade intramedullary osteosarcoma in whom to study the relevance of TP53 mutations to the risk of systemic relapse.

Molecular Analyses
For each patient, a tumor sample was chosen by a pathologist, with the aid of frozen histologic analysis before and/or following chemotherapy, and was immediately snap frozen and stored at –70°C. Specimens were frozen on dry ice and transported by overnight courier to a single laboratory where all molecular analyses were performed. DNA was extracted by conventional techniques.³³ Exons 4 to 10 of the TP53 gene were screened for sequence alterations using single-strand conformation polymorphism analysis (SSCP). Genomic DNA was used as a template for polymerase chain reaction amplification of fragments containing an exon and its adjacent intron boundaries, as previously described.^16,34 Quantitative analysis of SSCP band shift patterns was used to determine the possibility of allelic loss or deletion. Sequence alterations were detected as electrophoretic mobility shifts on SSCP gels and were confirmed and characterized independently by direct sequencing using the same SSCP primers. Southern blot analysis for the TP53 gene was performed as previously described.³⁸

Statistical Analysis
A total of 196 patients with 83 systemic recurrences met the eligibility criteria and are the focus of this study, which investigated the relevance of TP53 mutations, as determined by SSCP and sequencing, to the risk of systemic relapse. The original study protocol anticipated 200 patients with 70 recurrences in order to detect a relative risk of 3.0 with a power of 80% and an overall level of .05. In 16 additional cases, it was not possible to determine the TP53 mutation status. To assess generalizability, characteristics of patients with and without TP53 mutation information were compared. However, patients lacking TP53 data were not considered for the primary outcome analysis.

Patients with wild-type TP53 were designated as the referent category and were compared with all patients with (1) any TP53 mutation, (2) nonsense mutations, and (3) missense mutations. To examine the association of TP53 mutations with the risk of systemic relapse in osteosarcoma, TP53 was considered alone and then in combination with other prognostic factors, which included: tumor size ( 9 cm or > 9 cm) based on the median of the maximum dimension determined from pretreatment radiographs; chemotherapy-induced tumor necrosis ( 90%, > 90%, or missing [Tables 1 and 2]) based on pathology review; tumor site in the extremity (proximal or distal to the elbow or knee joint); age at diagnosis (< 14 years, 14 to 19 years, or > 19 years); and chemotherapy protocol (doxorubicin/cisplatin v others). Categorical codings for these prognostic factors were selected before the analysis, based on previous studies or clinical convention. Preliminary analysis compared the frequency distributions of the prognostic factors among TP53 mutation subgroups using the Wilcoxon-Mann-Whitney test.

	RESULTS

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Patient and Tumor Characteristics
This study assessed 196 patients with classical high-grade intramedullary osteosarcoma who presented with a localized tumor, had no metastatic disease, and did not develop a local tumor relapse. Definitive surgical treatment of the primary tumor was by en bloc resection (n = 156) or amputation (n = 40). All patients were observed for a minimum of 24 months or until systemic relapse. After an average of 4.3 years of follow-up (median, 3.7 years; range, 0.1 to 12.0 years), there were 83 systemic recurrences (42%), while 112 patients remained continuously alive and disease free, and one patient died with no tumor recurrence.

Table 1 summarizes patient and tumor characteristics for those with and without TP53 mutations. Of the clinicopathologic features studied, only patient age varied with TP53 status (P = .05). Overall, patients with TP53 mutations were older than those who had a wild-type gene. Patients with nonsense TP53 mutations were slightly younger than those with a wild-type gene (median, 14.4 v 16.6 years; P = .31), while the missense group was considerably older (median, 21.8 v 16.6 years; P = .008). There was no evidence of any association between TP53 status and the other prognostic factors.

Characteristics of TP53 Mutations
In the 196 tumors analyzed for TP53 status by SSCP and sequencing, 38 mutations (19.4%) were identified. There were 23 missense mutations and 15 other alterations, including 11 nonsense mutations, three splice site changes, and one in-frame insertion.¹⁶ Sixteen (42%) of 38 cases with mutations had evidence of loss of heterozygosity indicating allelic loss or deletion. For the purpose of the following analyses, we considered all mutations that were not missense to be nonsense alterations.

Prognostic Value of TP53 and Other Factors
There was no evidence of any association between TP53 mutation status (regardless of whether missense or nonsense mutations were considered individually, or all mutations combined) and the risk of systemic relapse. Considering TP53 status alone (Table 2, "Univariate Model"), the estimated HR of any mutation relative to wild type was 1.24, with a 95% CI of 0.74 to 2.10. Similar HRs were estimated for missense (HR = 1.01; 95% CI, 0.5 to 2.03) and nonsense (HR = 1.6; 95% CI, 0.64 to 4.04) mutations relative to wild type. Consistent with the HR estimates, the Kaplan-Meier curve for patients with TP53 mutations shows an only slightly worse prognosis than for individuals with the wild-type gene (Fig 1; P = .41, log-rank test). Neither nonsense (P = .17) nor missense (P = .99) mutations showed statistically significant differences in systemic recurrences using the log-rank test, as compared with the wild type.

View larger version (16K): [in this window] [in a new window]   Fig 1. Kaplan-Meier metastasis-free survival curves stratified by TP53 mutation status as determined by single-strand conformation polymorphism analysis and sequencing (N = 196).

In univariate analysis of the other prognostic factors, there was evidence that larger tumor size (> 9 cm v 9 cm) and lower degree of necrosis ( 90% v > 90%) were associated with an increased risk of systemic relapse (Figs 2 and 3). Each of these factors retained their statistical significance when considered together in the multivariate model (Table 2, "Multivariate Model"). The lack of significance of TP53 in the multivariate model was not affected by alternative specifications of tumor size and necrosis, including the use of cubic splines (data not shown). There was no indication that the use of the Cox regression model was inappropriate. The P value of the global test of departure from the proportional hazards assumption in the multivariate Cox regression model was .78, and was not less than .30 for any of the individual model parameters.³⁶

View larger version (16K): [in this window] [in a new window]   Fig 2. Kaplan-Meier metastasis-free survival curves stratified by tumor size (N = 196).

View larger version (15K): [in this window] [in a new window]   Fig 3. Kaplan-Meier metastasis-free survival curves stratified by chemotherapy-induced tumor necrosis (n = 171).

Missing TP53 Results
When the 16 patients without TP53 results were compared with the main study group of 196 patients, there was no significant difference in the occurrence of metastases (P = .17, log-rank test). There were fewer patients in the doxorubicin/cisplatin group missing TP53 results (five [4.3%] of 115) compared with the other chemotherapy protocols (11 [11.3%] of 97; P = .052), and there was a higher proportion of patients with greater than 90% necrosis (eight [14.3%] of 56) who were missing TP53 results as compared with patients with 90% necrosis (eight [6.1%] of 131; P = .064). Otherwise, there were no notable differences between the two groups.

A sensitivity analysis was carried out to assess the possible impact of the missing TP53 data on the association between TP53 mutation status and systemic recurrence. The estimated HR in the univariate model increased to 1.50 (P = .11) in the extreme scenario in which cases without TP53 results were assumed to be mutated if a recurrence had occurred, or wild type if there was no recurrence. The estimated HR decreased to 0.87 (P = .60) when cases without TP53 results were assumed to be wild type if a recurrence had occurred, or mutated otherwise. In all scenarios, there was no significant effect of TP53 mutation status on outcome. This demonstrates that a significant effect was not masked by the missing TP53 results in these 16 patients.

Southern Blot Analysis of TP53
To investigate the possibility of rearrangements or large deletions that may not have been detected by SSCP, we performed Southern blot analysis on 27 tumors that were TP53 wild type by SSCP analysis. These tumors were from 14 patients who remained disease-free at follow-up, and from 13 who relapsed. Three gene rearrangements and one deletion (14.8%) were identified. Two of these large structural gene alterations occurred in the group of patients who remained disease-free at follow-up, and two were in the group of those who developed metastases.

	DISCUSSION

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

 
In this prospective study, we found no evidence that TP53 mutations predict outcome in patients presenting with high-grade intramedullary osteosarcoma. Based on the 95% CIs estimated in this study, it is unlikely that the true relative risk of metastasis in a patient with any TP53 mutation could be more than double that of a patient with wild-type TP53. A similar result was found when TP53 mutations were considered missense or nonsense alterations. These results are consistent with our previous single-institution study, which found a similar proportion of TP53 mutations in patients presenting with localized osteosarcoma (55 [22.3%] of 247), many of whom will be cured, compared with those with metastatic disease at diagnosis (five [20%] of 25) who are only rarely curable.¹⁶ Two small retrospective studies reached a similar conclusion, though they were based on only 17 patients³⁷ and 36 patients.³⁸ Together, these results suggest that TP53 status cannot be used as a prognostic indicator for high-grade intramedullary osteosarcoma.

TP53 mutations are likely to be early events in high-grade osteosarcoma, with a more critical role in tumorigenesis than metastasis. We previously found that the status of the TP53 gene was concordant in all of 21 metastases from patients with osteosarcoma as compared with their matched primary tumors, by DNA sequencing.¹⁶ Using immunohistochemistry, Wadayama et al³⁹ found no difference in TP53 protein expression between primary tumors and their matched metastases from nine patients with osteosarcoma. These results are consistent with the investigations of other cancers, which identified only rare cases in which TP53 mutations were present in metastases but not in matched primary tumors.^40–45 TP53 mutations have also been identified in three (15.8%) of 19 patients with low-grade osteosarcomas, which are associated with a good prognosis following surgical resection alone.⁴⁶ It seems that for a wide range of tumor manifestations, the TP53 mutation rate does not differ substantially from the 19.4% (38 of 196 patients) observed in this study of high-grade nonmetastatic intramedullary tumors, further supporting the idea that TP53 mutations are an early event unrelated to metastasis.

Overexpression of MDM2, generally as a result of gene amplification, may be particularly relevant in high-grade osteosarcoma since the MDM2 protein binds and inactivates TP53.⁴⁷ As a result, MDM2 overexpression may provide an alternate mechanism for inactivation of the TP53 pathway. Although some studies have found MDM2 amplification and overexpression in osteosarcoma,^37,48,49 our group and others have not found this to be a common occurrence in high-grade intramedullary cases.^50–53

Tumor size and histologic response to preoperative chemotherapy were both found to be independent predictors of outcome in this study. Each of these factors has previously been identified as a strong prognostic indicator, with histologic response typically being the best prognostic factor in osteosarcoma.^4–6,8–10 TP53 status was not associated with either of these factors. This was particularly surprising since TP53 has been shown to play a critical role in chemotherapy response to adriamycin and cisplatin, and both of these agents were part of each chemotherapy protocol used in this study.^{19,20,24,25,54,55} In the present investigation, only patient age varied with TP53 status. Patients with TP53 missense mutations were significantly older than those with nonsense alterations or a wild-type gene. We made the same observation in a previous single-institution study of 272 osteosarcomas.¹⁶ A similar association between TP53 status and age was also identified by Lonardo et al in an immunohistochemical study of osteosarcoma.⁵¹ This could be clinically relevant because older patients with osteosarcoma may have a worse clinical outcome,^5,51,56,57 and missense mutations of the TP53 gene frequently produce gain-of-function effects that increase oncogenicity.^21,58–60 However, neither older age nor TP53 missense mutations were found to be predictors of patient outcome in this study. In fact, the lack of TP53 status correlation with clinical outcome or histologic response to chemotherapy suggests that TP53 mutations are likely just one of a number of genetic alterations that occur during the development of this disease.^61–63

In this study, the status of the TP53 gene was analyzed directly using SSCP and sequencing in order to determine the exact mutation type and codon location for each tumor. This conforms with recent recommendations for analyzing TP53 in cancer studies that suggested that only molecular analyses should be employed.⁶⁶ This approach has advantages over immunohistochemical analysis, which only identifies TP53 protein overexpression thought to be indicative of missense mutations. Immunohistochemistry generally does not detect frameshift or nonsense mutations that result in deletion or truncation of the TP53 protein, leading to frequent discrepancies between the two techniques. In a review of 84 studies, Greenblatt et al⁶⁴ found that the sensitivity of immunohistochemistry for identification of TP53 alterations was only 75%, with frequent discrepancies as compared with molecular results. In a previous study comparing TP53 alterations in bone and soft tissue sarcomas by molecular and immunohistochemical methods, we found concordant results in 64 (93%) of 69 cases.⁶⁵ However, most TP53 alterations in that study were missense mutations. In this larger study, 15 (39.5%) of 38 TP53 mutations in osteosarcoma were nonsense changes, suggesting that a high proportion of TP53 alterations would likely be missed by immunohistochemical detection methods.

Some investigations of the TP53 gene in osteosarcoma have identified large DNA abnormalities based on Southern blot analysis.^{37–39,48,53} To address this further, we analyzed a subgroup of tumors without TP53 mutations, by SSCP analysis, using the Southern blot technique. Four (14.8%) of 27 tumors had large detectable TP53 alterations, including three gene rearrangements and one deletion. However, these additional TP53 mutations were evenly divided between the group of patients who relapsed with systemic disease (two of 14 patients) and those who remained disease-free (two of 13 patients). Therefore, identification of these large TP53 alterations did not change the results relating to a lack of correlation with clinical outcome. However, since SSCP and DNA sequencing may miss some types of mutations, this should be taken into account when evaluating the precise mutation spectrum.

Despite accurate identification of TP53 alterations in this study (by DNA sequencing), neither the presence of any mutation nor the specific type of mutation correlated with patient outcome. Additionally, TP53 status did not correlate with chemotherapy-induced necrosis, which is considered the strongest prognostic factor for osteosarcoma. TP53 mutations likely play an important role in the development of osteosarcoma, but not metastasis. Although the outcome of patients with osteosarcoma has significantly improved throughout the last two decades, there have been no recent major advances. The continued search for clinically relevant genes that might encode novel therapeutic targets in this disease will likely lead to the next phase of clinical advances.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	NOTES

 
Supported by grants from the National Cancer Institute of Canada and Canadian Institutes of Health Research (J.S.W., A.M.D., D.M., R.S.B., I.L.A.).

J.S.W. and N.G. contributed equally to this work.

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

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Submitted April 8, 2003; accepted November 29, 2004.

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