Assessment of Predictive Biomarkers of the Response to Pazopanib Based on an Integrative Analysis of High-grade Soft-tissue Sarcomas: Analysis of a Tumor Sample from a Responder and Patients with Other Soft-tissue Sarcomas
Assessment of Predictive Biomarkers of the Response to Pazopanib Based on an Integrative Analysis of High-grade Soft-tissue Sarcomas: Analysis of a Tumor Sample from a Responder and Patients with Other Soft-tissue Sarcomas
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DOI：
10.1097/corr.0000000000001322
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发表时间：
2020-11-01
影响因子：
4.2
通讯作者：
Mano, Hiroyuki
中科院分区：
文献类型：
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作者：
Suehara, Yoshiyuki;Kohsaka, Shinji;Mano, Hiroyuki
背景软组织肉瘤是一组罕见的恶性肿瘤，通常采用手术切除和放射治疗，但最近，帕唑帕尼，口服酪氨酸激酶抑制剂，已被用于转移患者谁不响应标准化疗方案。基于既往接受过化疗的晚期软组织肉瘤患者，几项临床研究报告了使用帕唑帕尼治疗软组织肉瘤患者的生存率和敏感性（约5%至10%敏感）。最近，下一代测序（NGS）技术已被用于提供广泛的遗传信息，并在癌症治疗中开发个性化药物。然而，有很少的报告，没有基因分析的软组织肉瘤患者谁有一个完全响应（CR）帕唑帕尼。问题/目的我们描述了一个罕见的，先进的软组织肉瘤谁实现了CR帕唑帕尼治疗患者的临床病理特征。此外，使用NGS和阵列进行综合分析，以阐明特征性改变，包括与帕唑帕尼应答相关的基因突变、拷贝数变化和蛋白质表达。此外，还进行了由体外和体内测定组成的功能分析，以阐明所鉴定的改变是否与致癌能力和药物反应相关。方法在一个70岁的晚期软组织肉瘤的女性患者的样本中，每天口服帕唑帕尼800 mg治疗1个月，CT扫描显示治疗CR。据我们所知，在帕唑帕尼的几项临床试验中，没有软组织肉瘤患者达到CR，因此，我们的患者被认为是非常罕见的。我们进行了一项综合分析，包括全外显子组测序，转录组测序和磷酸化分析的受体酪氨酸激酶（RTK）使用肿瘤样本与正常样本匹配的CR患者。从这里开始，我们将该患者称为CR，尽管短期高级别部分缓解可能更准确。这些分析使用NGS和磷酸受体酪氨酸激酶（磷酸-RTK）阵列进行。作为验证研究，我们还使用来自长期稳定疾病患者的三个样本和来自对帕唑帕尼治疗有反应的进行性疾病患者的两个样本进行了靶向测序。此外，通过定量实时聚合酶链反应验证了1例CR患者、3例长期稳定疾病患者和27例具有不同组织学亚型和对帕唑帕尼不同反应的高级别软组织肉瘤患者中根据对帕唑帕尼的反应确定的特征性基因改变。我们进行了一个焦点形成试验，以评估这些基因组改变的转化活性。结果在对帕唑帕尼有CR的患者中，我们鉴定了几种体细胞突变，包括Fms相关受体酪氨酸激酶1（FLT 1）p.G38S、血小板衍生生长因子受体α（PDGFRA）p.T83S和血小板衍生生长因子受体β（PDGFRB）外显子13跳跃。在染色体12 q13 -14处也检测到扩增，包括GLI家族锌指1（GLI 1）和细胞周期蛋白依赖性激酶4（CDK 4）。在5名患者的靶序列分析中，3名长期稳定疾病的患者中有1名具有12 q13 -14扩增。GLI 1、CDK 4和帕唑帕尼靶点的mRNA表达包括PDGFRA、PDGFRB、血管内皮生长因子受体（VEGFR）1-3、和干细胞因子受体（KIT）的样本中的CR患者，和27例高级别软组织肉瘤患者进行了验证。相对于其他软组织肉瘤患者，GLI 1的表达在CR患者和长期稳定疾病患者中特征性增加。过表达GLI 1在3 T3细胞中表现出较强的转化潜力。此外，GLI 1的过表达上调PDGFRB蛋白的表达并促进磷酸化，而帕唑帕尼以剂量依赖性方式抑制磷酸化。然而，在病灶形成试验中，帕唑帕尼对GLI 1诱导转化的抑制作用有限;因此，PDGFRB活化以外的机制可能有助于转化。结论：我们发现了一些基因改变，这些基因改变可能与接受帕唑帕尼治疗晚期软组织肉瘤的患者的CR和长期稳定的疾病有关。因此，我们认为，这种独特的分子特征值得进一步研究，以确定对帕唑帕尼的反应的预测生物标志物。
Background Soft-tissue sarcomas are a rare group of malignant tumors that usually are treated with surgical excision and radiation therapy, but recently, pazopanib, an oral tyrosine kinase inhibitor, has been used in patients with metastases who do not respond to standard chemotherapy regimens. Based on patients with advanced soft-tissue sarcomas who had received prior chemotherapy, several clinical studies have reported the survival and sensitivity (approximately 5% to 10% sensitive) of patients with soft-tissue sarcomas treated with pazopanib. Recently, next-generation sequencing (NGS) technologies have been used to provide a wide genetic information and to develop personalized medicine in cancer treatment. However, there are few reports and no genetic analyses of patients with soft-tissue sarcomas who had a complete response (CR) to pazopanib. Questions/purposes We described the clinicopathologic features of a patient with a rare, advanced soft-tissue sarcoma who achieved a CR to pazopanib treatment. Furthermore, integrative analyses using NGS and arrays were performed to elucidate characteristic alterations, including gene mutations, copy number changes, and protein expression that were associated with response to pazopanib. Additionally, functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified alterations were associated with oncogenic abilities and drug responses. Methods In a sample from a 70-year-old woman with an advanced soft-tissue sarcoma treated for 1 month with 800 mg of oral pazopanib daily, CT scans demonstrated a CR to treatment. To our knowledge, there have been no patients with soft-tissue sarcomas among several clinical trials of pazopanib that have achieved a CR and therefore, our patient is considered to be extremely rare. We performed an integrative analysis including whole-exome sequencing, transcriptome sequencing, and phosphorylation profiling of receptor tyrosine kinases (RTK) using tumor samples from a patient with a CR matched to normal samples. From here on we will refer to this patient as having a CR, although a short term high-grade partial response may be more accurate. These analyses were performed using NGS and the phosphoreceptor tyrosine kinase (phospho-RTK) array. As a validation study, we also performed target sequencing using three samples from patients with long-term stable disease and two samples from patients with progressive disease who responded to pazopanib treatment. In addition, characteristic gene alterations that were identified according to the response to pazopanib in one patient with a CR, in three patients with long-term stable disease, and in 27 patients with high-grade soft-tissue sarcomas with different histologic subtypes and different responses to pazopanib were verified by quantitative real-time polymerase chain reaction. We conducted a focus formation assay to evaluate the transforming activities of these genomic alterations. Results In the patient with a CR to pazopanib, we identified several somatic mutations including Fms related receptor tyrosine kinase 1 (FLT1) p.G38S, platelet-derived growth factor receptor alpha (PDGFRA) p.T83S, and platelet-derived growth factor receptor beta (PDGFRB) exon 13 skipping. Amplification at chromosome 12q13-14 encompassing GLI family zinc finger 1 (GLI1) and cyclin-dependent kinase-4 (CDK4) was also detected. Furthermore, an elevated PDGFRB phosphorylation level was observed in the tumor.In target sequencing analyses in five patients, one of three patients with long-term stable disease had 12q13-14 amplification.The mRNA expression of GLI1, CDK4, and pazopanib targets including PDGFRA, PDGFRB, vascular endothelial growth factor receptor (VEGFR)1-3, and stem cell factor receptor (KIT) in samples from the patient with a CR, and 27 patients with high-grade soft-tissue sarcomas was verified. The expression of GLI1 was characteristically increased in the patient with a CR and in those with long-term stable disease relative to other patients with soft-tissue sarcomas. Overexpression of GLI1 showed strong transforming potential in 3T3 cells. Moreover, the overexpression of GLI1 upregulated the expression of the PDGFRB protein and promoted phosphorylation, which was dose-dependently inhibited by pazopanib. However, inhibition of GLI1-induced transformation by pazopanib was limited in the focus formation assay; therefore, mechanisms other than PDGFRB activation may contribute to transformation. Conclusions We identified several gene alterations that might be associated with a CR and long-term stable disease in patients who received pazopanib for advanced soft-tissue sarcomas. We therefore believe that this distinct molecular profile warrants further investigation to identify predictive biomarkers of the response to pazopanib.