大多数NTRK1、NTRK2和NTRK3重排导致相关基因的激酶部分的表达增加,这是由于其与主动转录的基因配偶体融合。因此,对5'/3'末端表达失衡的分析可能能够检测NTRK基因融合的整个光谱。从8075例患者中获得的存档肿瘤标本进行了肿瘤细胞的手动解剖,DNA/RNA分离,和cDNA合成。通过实时PCR分析了NTRK基因的5'/3'端表达失衡。通过变体特异性PCR对44种常见的NTRK融合体进行基因重排的进一步鉴定,and,只要有必要,通过基于RNA的下一代测序(NGS)。在7424/8075(91.9%)肿瘤中获得足够质量的cDNA。在7/6436(0.1%)肺癌中检测到NTRK重排,11/137(8.0%)儿科肿瘤,和13/851(1.5%)成人非肺部恶性肿瘤。在小儿肉瘤中观察到NTRK易位的最高发生率(7/39,17.9%)。在微卫星不稳定的结直肠肿瘤中观察到NTRK融合的频率增加(6/48,12.5%),唾液腺癌(5/93,5.4%),和肉瘤(7/143,4.9%)。1293例EGFR/ALK/ROS1/RET/MET癌基因驱动改变的肺癌均未出现NTRK5'/3'端表达失衡。对744个5'/3'-末端表达率正常的肿瘤进行变异特异性PCR:172个EGFR/ALK/ROS1/RET/MET阴性肺癌和125个儿科肿瘤没有重排,而在2/447(0.5%)非肺成人恶性肿瘤中检测到NTRK3融合。总之,本研究描述了一种诊断管道,可作为传统NTRK1-3分析方法的一种经济有效的替代方法.
The majority of NTRK1, NTRK2, and
NTRK3 rearrangements result in increased expression of the kinase portion of the involved gene due to its fusion to an actively transcribed gene partner. Consequently, the analysis of 5\'/3\'-end expression imbalances is potentially capable of detecting the entire spectrum of NTRK gene fusions. Archival tumor specimens obtained from 8075 patients were subjected to manual dissection of tumor cells, DNA/RNA isolation, and cDNA synthesis. The 5\'/3\'-end expression imbalances in NTRK genes were analyzed by real-time PCR. Further identification of gene rearrangements was performed by variant-specific PCR for 44 common NTRK fusions, and, whenever necessary, by RNA-based next-generation sequencing (NGS). cDNA of sufficient quality was obtained in 7424/8075 (91.9%) tumors. NTRK rearrangements were detected in 7/6436 (0.1%) lung carcinomas, 11/137 (8.0%) pediatric tumors, and 13/851 (1.5%) adult non-lung malignancies. The highest incidence of NTRK translocations was observed in pediatric sarcomas (7/39, 17.9%). Increased frequency of NTRK fusions was seen in microsatellite-unstable colorectal tumors (6/48, 12.5%), salivary gland carcinomas (5/93, 5.4%), and sarcomas (7/143, 4.9%). None of the 1293 lung carcinomas with driver alterations in EGFR/ALK/ROS1/RET/MET oncogenes had NTRK 5\'/3\'-end expression imbalances. Variant-specific PCR was performed for 744 tumors with a normal 5\'/3\'-end expression ratio: there were no rearrangements in 172 EGFR/ALK/ROS1/RET/MET-negative lung cancers and 125 pediatric tumors, while
NTRK3 fusions were detected in 2/447 (0.5%) non-lung adult malignancies. In conclusion, this study describes a diagnostic pipeline that can be used as a cost-efficient alternative to conventional methods of NTRK1-3 analysis.