Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health (NIH), 9000 Rockville Pike, Building 10, CRC, Bethesda, MD 20892, USA.
Computational and Systems Biology Branch, Biometric Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Rockville, Maryland 20850, USA.
Biomolecules. 2023 Oct 10;13(10):1499. doi: 10.3390/biom13101499.
Glioblastoma (GBM) is the most common brain tumor with an overall survival (OS) of less than 30% at two years. Valproic acid (VPA) demonstrated survival benefits documented in retrospective and prospective trials, when used in combination with chemo-radiotherapy (CRT).
The primary goal of this study was to examine if the differential alteration in proteomic expression pre vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA as compared to standard-of-care CRT. The second goal was to explore the associations between the proteomic alterations in response to VPA/RT/TMZ correlated to patient outcomes. The third goal was to use the proteomic profile to determine the mechanism of action of VPA in this setting.
Serum obtained pre- and post-CRT was analyzed using an aptamer-based SOMAScan proteomic assay. Twenty-nine patients received CRT plus VPA, and 53 patients received CRT alone. Clinical data were obtained via a database and chart review. Tests for differences in protein expression changes between radiation therapy (RT) with or without VPA were conducted for individual proteins using two-sided -tests, considering -values of <0.05 as significant. Adjustment for age, sex, and other clinical covariates and hierarchical clustering of significant differentially expressed proteins was carried out, and Gene Set Enrichment analyses were performed using the Hallmark gene sets. Univariate Cox proportional hazards models were used to test the individual protein expression changes for an association with survival. The lasso Cox regression method and 10-fold cross-validation were employed to test the combinations of expression changes of proteins that could predict survival. Predictiveness curves were plotted for significant proteins for VPA response (-value < 0.005) to show the survival probability vs. the protein expression percentiles.
A total of 124 proteins were identified pre- vs. post-CRT that were differentially expressed between the cohorts who received CRT plus VPA and those who received CRT alone. Clinical factors did not confound the results, and distinct proteomic clustering in the VPA-treated population was identified. Time-dependent ROC curves for OS and PFS for landmark times of 20 months and 6 months, respectively, revealed AUC of 0.531, 0.756, 0.774 for OS and 0.535, 0.723, 0.806 for PFS for protein expression, clinical factors, and the combination of protein expression and clinical factors, respectively, indicating that the proteome can provide additional survival risk discrimination to that already provided by the standard clinical factors with a greater impact on PFS. Several proteins of interest were identified. Alterations in GALNT14 (increased) and CCL17 (decreased) ( = 0.003 and 0.003, respectively, FDR 0.198 for both) were associated with an improvement in both OS and PFS. The pre-CRT protein expression revealed 480 proteins predictive for OS and 212 for PFS ( < 0.05), of which 112 overlapped between OS and PFS. However, FDR-adjusted values were high, with OS (the smallest p value of 0.586) and PFS (the smallest value of 0.998). The protein PLCD3 had the lowest -value ( = 0.002 and 0.0004 for OS and PFS, respectively), and its elevation prior to CRT predicted superior OS and PFS with VPA administration. Cancer hallmark genesets associated with proteomic alteration observed with the administration of VPA aligned with known signal transduction pathways of this agent in malignancy and non-malignancy settings, and GBM signaling, and included epithelial-mesenchymal transition, hedgehog signaling, Il6/JAK/STAT3, coagulation, NOTCH, apical junction, xenobiotic metabolism, and complement signaling.
Differential alteration in proteomic expression pre- vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA. Using pre- vs. post-data, prognostic proteins emerged in the analysis. Using pre-CRT data, potentially predictive proteins were identified. The protein signals and hallmark gene sets associated with the alteration in the proteome identified between patients who received VPA and those who did not, align with known biological mechanisms of action of VPA and may allow for the identification of novel biomarkers associated with outcomes that can help advance the study of VPA in future prospective trials.
胶质母细胞瘤（GBM）是最常见的脑肿瘤，其两年总体生存率（OS）不到 30%。在联合化疗-放疗（CRT）的基础上加用丙戊酸（VPA）的回顾性和前瞻性研究中，均显示出生存获益。
本研究的主要目的是检验在完成同期化疗-放疗（CRT）前后，与标准 CRT 相比，加用 VPA 是否存在蛋白质组表达差异的改变。次要目的是探索 VPA/RT/TMZ 治疗反应的蛋白质组改变与患者结局之间的相关性。第三个目的是利用蛋白质组谱来确定 VPA 在这种情况下的作用机制。
使用基于适配体的 SOMAScan 蛋白质组分析检测 CRT 前后获得的血清。29 例患者接受 CRT 加 VPA，53 例患者接受 CRT 单独治疗。通过数据库和图表回顾获得临床数据。使用双侧检验比较接受 RT 加或不加 VPA 的患者的蛋白质表达变化，当 P 值 < 0.05 时认为差异有统计学意义。进行了年龄、性别和其他临床协变量的调整，对差异表达的蛋白质进行了分层聚类，并使用标志性基因集进行了基因集富集分析。采用单因素 Cox 比例风险模型检测个体蛋白表达变化与生存的关系。采用 Lasso Cox 回归方法和 10 倍交叉验证检测能够预测生存的蛋白表达变化组合。对于 VPA 反应（P 值 < 0.005）的显著蛋白，绘制预测性曲线，以显示生存概率与蛋白表达百分位数的关系。
共鉴定出 124 种蛋白质，这些蛋白质在接受 CRT 加 VPA 和 CRT 单独治疗的患者中，在 CRT 前后存在差异表达。临床因素不影响结果，并且在 VPA 治疗组中确定了独特的蛋白质聚类。以 20 个月和 6 个月为时间标记点的 OS 和 PFS 的时间依赖性 ROC 曲线显示，蛋白表达、临床因素和蛋白表达与临床因素的组合的 AUC 分别为 0.531、0.756、0.774 和 0.535、0.723、0.806，表明蛋白质组可以为已经由标准临床因素提供的生存风险提供额外的区分，对 PFS 的影响更大。确定了一些感兴趣的蛋白质。GALNT14（增加）和 CCL17（减少）的改变（分别为 P 值 = 0.003 和 0.003，FDR 分别为 0.198）与 OS 和 PFS 均有改善相关。在 CRT 前的蛋白质表达中，发现了 480 个与 OS 相关的蛋白和 212 个与 PFS 相关的蛋白（P < 0.05），其中 OS 和 PFS 之间有 112 个重叠。然而，FDR 调整后的 P 值较高，OS（最小 P 值为 0.586）和 PFS（最小 P 值为 0.998）。PLCD3 蛋白的 P 值最低（OS 和 PFS 的 P 值分别为 0.002 和 0.0004），其在 CRT 前升高预示着接受 VPA 治疗时 OS 和 PFS 的预后更好。与 VPA 给药相关的蛋白质改变相关的癌症标志性基因集与这种药物在恶性和非恶性环境中的已知信号转导途径以及 GBM 信号转导相吻合，包括上皮-间充质转化、 hedgehog 信号转导、Il6/JAK/STAT3、凝血、NOTCH、顶端连接、外来代谢物和补体信号转导。
在加用 VPA 的同期化疗-放疗（CRT）前后，蛋白质组表达存在差异。利用前后数据，在分析中出现了预后蛋白。利用 CRT 前的数据，鉴定出了潜在的预测蛋白。与患者接受或不接受 VPA 之间的蛋白质组改变相关的蛋白信号和标志性基因集与 VPA 的已知作用机制相吻合，可能有助于确定与结局相关的新型生物标志物，从而有助于在未来的前瞻性试验中研究 VPA。
