Ewing Sarcoma: A tough road to clinically relevant biomarkers

I n this issue of Pediatric Blood & Cancer, Lerman et al. present results from a prospective study interrogating TP53 and CDKN2A alterations in tumor specimens from patients with localized Ewing Sarcoma (ES) who were enrolled onto the recently completed Children’s Oncology Group (COG) treatment protocol AEWS0031 [1]. Their analysis reveals TP53 mutations and CDKN2A deletions in 8.6% and 11.2%, respectively, of evaluable samples. The prevalence of identified alterations is consistent with previously performed retrospective studies of these two genes in ES samples. Survival comparisons between patients with mutationpositive and mutation-negative tumors did not reveal a statistically significant difference in event-free survival (EFS). The primary conclusionmade by the authors is that TP53mutation andCDKN2A deletion status of diagnostic tumor specimens do not serve as reliable prognostic biomarkers for patients with localized ES. The findings of this study highlight some important issues including: 1) the importance of prospective experimental design and specimen banking for biomarker studies of rare diseases such as ES and 2) the utility of data emerging from next-generation sequencing (NGS) techniques applied to ES. For over a decade, numerous retrospective studies have been published evaluating cell-cycle pathway alterations in ES which have made statistically significant correlations between TP53 and/ or CDKN2A mutations and inferior patient outcomes [2–5]. However, these studies had limitations such as relatively small sample sizes, variability in disease characteristics (i.e., metastatic vs. localized disease), and significant variability in treatment modalities employed for patients. This study by Lerman et al. examines the question of whether or not TP53 and/or CDKN2A mutation status corresponds to the clinical outcomes of a relatively large, uniform group of patients (i.e., only localized disease, same treatment protocol) with prospectively collected tumor specimens. These criteria provide a stronger level of evidence than predecessor studies [6]. An analogous circumstance recently arose in ES biomarker research when two separate and large prospective studies failed to show prognostic significance of EWSR1 fusion type on clinical outcomes, despite multiple smaller retrospective, previous studies reporting the contrary [7,8]. However, we must be careful about prematurely eliminating TP53 and CDKN2A as potentially relevant biomarkers. Although the number of tumor specimens evaluated by Lerman et al. was larger than in previous studies, the overall numbers of mutation positive tumors were still modest; 8/93 with TP53 mutations and 12/107 withCDKN2Amutations. Therefore, even though this study provides a stronger level of evidence than any previous individual study, it would be helpful to have a larger cohort of tumors to definitively show a lack of correlation between mutation status and outcomes, especially since the majority of previous studies have demonstrated such correlation. One large retrospective study of 107 specimens, which was discussed in the report by Lerman et al., also failed to show a correlation between mutation status and survival. However in this study, immunohistochemistry and fluorescent in situ hybridization were utilized to evaluate TP53 and CDKN2A status, making it more difficult to compare to studies in which mutational analyses were performed [9]. Three separate and extensive NGS studies of ES have been published in the past six months [10–12]. All three studies reported very low overall mutation rates as compared to othermalignancies; an observation which appears to be consistent among translocationassociated sarcomas [13]. Only three genes appear to be recurrently altered at a significant frequency; TP53, CDKN2A, and STAG2. In their NGS study of ES, Tirode et al. made an intriguing observation that tumors harboring both TP53 and STAG2mutations are associated with particularly poor clinical outcomes. Similar to other studies, the double-mutant subset analyzed was small with only eight patients included in the overall survival analysis. However, the results raise an intriguing possibility that the poor outcomes seen in previous studies of TP53mutant ES cases aremore specifically associatedwith those tumors harboring an accompanying STAG2mutation. It will be crucial to ascertain whether or not this finding maintains statistical significance in larger, prospective cohorts of patients. Finally, the study by Lerman et al. underscores the importance of prospective specimen collection and the responsibility of investigators to design and perform prospective studies, despite the challenges encountered conducting such research in relatively rare diseases. An essential component to successfully conduct this type of study in rare diseases like ES is thorough specimen collection and storage from all patients who consent to participate in banking protocols. In the study by Lerman et al., less than 20% of patients (112 out of 558) enrolled onto AEWS0031 had tumor specimens of sufficient quantity and quality to perform their mutational evaluation, thereby limiting the robustness of the results of this highly important prospective analysis. The COG Ewing Sarcoma Biology Committee recently published a report on the banking efforts and acquisition rates over the past decade [14]. Their analysis revealed that only 18% of all ES cases diagnosed in the United States, and 8% of patients >20 years old at diagnosis, had tumor samples banked on COG banking studies despite the fact that 80% of patients were diagnosed at COG affiliated centers.