Smilkou Stavroula, Kaklamanis Loukas, Balgouranidou Ioanna, Linardou Helena, Papatheodoridi Alkistis Maria, Zagouri Flora, Razis Evangelia, Kakolyris Stylianos, Psyrri Amanda, Papadimitriou Christos, Markou Athina, Lianidou Evi
Analysis of Circulating Tumor Cells, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece.
Department of Pathology, Onassis Cardiac Surgery Center, Athens, Greece.
Front Oncol. 2024 Dec 6;14:1435559. doi: 10.3389/fonc.2024.1435559. eCollection 2024.
Detection of mutations in primary tumors and liquid biopsy samples is of increasing importance for treatment decisions and therapy resistance in many types of cancer. The aim of the present study was to directly compare the efficacy of a relatively inexpensive ultrasensitive real-time PCR with the well-established and highly sensitive technology of ddPCR for the detection of the three most common hotspot mutations of , in exons 9 and 20, that are all of clinical importance in various types of cancer.
We analyzed 42 gDNAs from primary tumors (FFPEs), 29 plasma-cfDNA samples, and 29 paired CTC-derived gDNAs, all from patients with ER+ metastatic breast cancer, and plasma from 10 healthy donors. The same blood draws were used for CTC isolation using EpCAM beads for positive immunomagnetic enrichment. All FFPEs and plasma-cfDNA samples were analyzed in parallel for mutations by ultrasensitive real-time PCR assay and droplet digital PCR.
In gDNAs from FFPEs, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 22/42(52.4%), and the p.E542K and p.H1047R mutations were detected in 14/42(33.3%) and 16/42(38.1%), respectively. Using ddPCR, the p.E545K mutation was detected in 22/42(52.4%), p.E542K in 17/42(40.5%), and p.H1047R in 19/42(45.2%) samples, revealing a concordance between the two methodologies of 81%, 78.6% and 78.6% for each mutation respectively. In plasma-cfDNA, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 11/29(38%) and both p.E542K and p.H1047R mutations in 2/29(6.9%).In the same plasma-cfDNA samples using ddPCR, p.E545K was detected in 1/29(3.5%), p.E542K in 2/29(6.9%), and p.H1047R in 3/29(10.5%) samples, revealing a concordance of 65.5%,100% and 93.1% for each mutation respectively. In paired CTC-derived gDNAs p.E545K was detected in 11/29(38%), p.E542K in 3/29(10.3%), and p.H1047R in 7/29(24.1%) samples.
This low-cost, high-throughput and ultrasensitive real-time PCR assay provides accurate and specific detection of hotspot mutations in liquid biopsy samples and gives similar results to ddPCR. This assay can be performed in labs where digital PCR instrumentation is not available. In CTC-derived gDNA and paired plasma-cfDNA, mutations detected were not identical, revealing that CTC and plasma-cfDNA give complementary information.
在许多类型的癌症中，检测原发性肿瘤和液体活检样本中的突变对于治疗决策和治疗耐药性越来越重要。本研究的目的是直接比较相对便宜的超灵敏实时聚合酶链反应（PCR）与成熟且高度灵敏的数字滴式PCR（ddPCR）技术检测三个最常见热点突变（外显子9和20中的 ，这些突变在各种类型的癌症中均具有临床重要性）的效果。
我们分析了42份来自原发性肿瘤（福尔马林固定石蜡包埋组织，FFPEs）的基因组DNA（gDNA）、29份血浆游离DNA（cfDNA）样本以及29份配对的循环肿瘤细胞（CTC）来源的gDNA，所有样本均来自雌激素受体阳性（ER+）转移性乳腺癌患者，还分析了10名健康供者的血浆。使用EpCAM磁珠进行阳性免疫磁珠富集，从相同的血样中分离CTC。所有FFPEs和血浆cfDNA样本通过超灵敏实时PCR检测法和数字滴式PCR并行分析 突变情况。
在FFPEs的gDNA中，使用超灵敏实时PCR，p.E545K突变在22/42（52.4%）中被检测到，p.E542K和p.H1047R突变分别在14/42（33.3%）和16/42（38.1%）中被检测到。使用ddPCR时，p.E545K突变在22/42（52.4%）中被检测到，p.E542K在17/42（40.5%）中被检测到，p.H1047R在19/42（45.2%）样本中被检测到，两种方法对于每种突变的一致性分别为81%、78.6%和78.6%。在血浆cfDNA中，使用超灵敏实时PCR，p.E545K突变在11/29（38%）中被检测到，p.E542K和p.H1047R突变在2/29（6.9%）中均被检测到。在相同的血浆cfDNA样本中使用ddPCR时，p.E545K在1/29（3.5%）中被检测到，p.E542K在2/29（6.9%）中被检测到，p.H1047R在3/29（10.5%）样本中被检测到，每种突变的一致性分别为65.5%、100%和93.1%。在配对的CTC来源的gDNA中，p.E545K在11/29（38%）中被检测到，p.E542K在3/29（10.3%）中被检测到，p.H1047R在7/29（24.1%）样本中被检测到。
这种低成本、高通量且超灵敏的实时PCR检测法能准确、特异地检测液体活检样本中的 热点突变，且结果与ddPCR相似。该检测法可在没有数字PCR仪器的实验室中进行。在CTC来源的gDNA和配对的血浆cfDNA中，检测到的 突变并不相同，这表明CTC和血浆cfDNA提供互补信息。
