Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, St.-Petersburg, Russia.
Department of Medical Genetics, St.-Petersburg Pediatric Medical University, St.-Petersburg, Russia.
Cancer Med. 2022 Sep;11(17):3226-3237. doi: 10.1002/cam4.4686. Epub 2022 Mar 23.
Despite the progress in the development of next-generation sequencing (NGS), diagnostic PCR assays remain to be utilized in clinical routine due to their simplicity and low cost. Tests for 5'-/3'-end mRNA unbalanced expression can be used for variant-independent detection of translocations, however, many technical aspects of this methodology require additional investigations.
Known ALK/ROS1 fusions and 5'-/3'-end unbalanced expression were analyzed in 2009 EGFR mutation-negative non-small cell lung cancer (NSCLC) samples with RT-PCR tests, which were optimized for the use with FFPE-derived RNA.
Variant-specific PCR tests for 4 common ALK and 15 common ROS1 translocations detected 115 (5.7%) and 44 (2.2%) rearrangements, respectively. Virtually all samples with common ALK fusions demonstrated some level of 5'/3' mRNA ends unbalanced expression, and 8 additional NSCLCs with rare ALK fusions were further identified by PCR or NGS among 48 cases selected based on ALK expression measurements. Interestingly, NSCLCs with unbalanced 5'-/3'-end ALK expression but without identified ALK translocations had elevated frequency of RAS mutations (21/40, 53%) suggesting the role of RAS activation in the alternative splicing of ALK gene. In contrast to ALK, only a minority of ROS1 translocation-positive cases demonstrated unbalanced gene expression, with both 5'- and 3'-end mRNA expression being elevated in most of the samples with translocations. Surprisingly, high ROS1 expression level was also found to be characteristic for NSCLCs with activating mutations in other tyrosine kinases such as EGFR, ALK, or MET.
Comprehensive ALK analysis can be performed by the test for 5'-/3'-end unbalanced expression with minimal risk of missing an ALK rearrangement. In contrast, the use of the test for 5'-/3'-end unbalanced expression for the detection of ROS1 fusions is complicated; hence, the utilization of variant-specific PCR assays for ROS1 testing is preferable.
尽管下一代测序（NGS）技术取得了进展，但由于其简单性和低成本，诊断 PCR 检测仍在临床常规中得到应用。5'-/3'-端 mRNA 不平衡表达的检测可用于非依赖性检测易位，但该方法的许多技术方面仍需要进一步研究。
采用 RT-PCR 检测对 2009 年 EGFR 突变阴性非小细胞肺癌（NSCLC）样本中的已知 ALK/ROS1 融合和 5'-/3'-端不平衡表达进行分析，该方法经过优化，可用于 FFPE 衍生 RNA。
针对 4 种常见 ALK 和 15 种常见 ROS1 易位的特异性 PCR 检测分别检测到 115（5.7%）和 44（2.2%）重排。几乎所有常见 ALK 融合的样本均表现出某种程度的 5'/3'mRNA 末端不平衡表达，在根据 ALK 表达测量选择的 48 例样本中，通过 PCR 或 NGS 进一步鉴定了 8 例罕见 ALK 融合的 NSCLC。有趣的是，具有不平衡 5'-/3'-端 ALK 表达但未发现 ALK 易位的 NSCLC 中 RAS 突变的频率较高（21/40，53%），提示 RAS 激活在 ALK 基因的选择性剪接中的作用。与 ALK 相反，只有少数 ROS1 易位阳性病例表现出基因表达不平衡，大多数易位样本中均同时升高 5'-和 3'-端 mRNA 表达。令人惊讶的是，高水平的 ROS1 表达也被发现是具有其他酪氨酸激酶（如 EGFR、ALK 或 MET）激活突变的 NSCLC 的特征。
通过 5'-/3'-端不平衡表达的检测可以进行全面的 ALK 分析，并且具有最小的漏检 ALK 重排风险。相比之下，使用 5'-/3'-端不平衡表达的检测来检测 ROS1 融合较为复杂；因此，优选使用针对 ROS1 检测的变体特异性 PCR 检测。
