Galbiati Silvia, Damin Francesco, Brambilla Dario, Ferraro Lucia, Soriani Nadia, Ferretti Anna M, Burgio Valentina, Ronzoni Monica, Vago Riccardo, Sola Laura, Chiari Marcella
Complications of Diabetes Units, Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" SCITEC CNR, 20131 Milan, Italy.
Pharmaceuticals (Basel). 2021 Feb 6;14(2):128. doi: 10.3390/ph14020128.
It is widely accepted that assessing circular tumor DNA (ctDNA) in the plasma of cancer patients is a promising practice to evaluate somatic mutations from solid tumors noninvasively. Recently, it was reported that isolation of extracellular vesicles improves the detection of mutant DNA from plasma in metastatic patients; however, no consensus on the presence of dsDNA in exosomes has been reached yet. We analyzed small extracellular vesicle (sEV)-associated DNA of eleven metastatic colorectal cancer (mCRC) patients and compared the results obtained by microarray and droplet digital PCR (ddPCR) to those reported on the ctDNA fraction. We detected the same mutations found in tissue biopsies and ctDNA in all samples but, unexpectedly, in one sample, we found a mutation that was not identified either in ctDNA or tissue biopsy. Furthermore, to assess the exact location of sEV-associated DNA (outside or inside the vesicle), we treated with DNase I sEVs isolated with three different methodologies. We found that the DNA inside the vesicles is only a small fraction of that surrounding the vesicles. Its amount seems to correlate with the total amount of circulating tumor DNA. The results obtained in our experimental setting suggest that integrating ctDNA and sEV-associated DNA in mCRC patient management could provide a complete real-time assessment of the cancer mutation status.
人们普遍认为，评估癌症患者血浆中的循环肿瘤DNA（ctDNA）是一种很有前景的做法，可用于无创评估实体瘤中的体细胞突变。最近有报道称，分离细胞外囊泡可提高转移性患者血浆中突变DNA的检测率；然而，关于外泌体中双链DNA的存在尚未达成共识。我们分析了11例转移性结直肠癌（mCRC）患者的小细胞外囊泡（sEV）相关DNA，并将微阵列和液滴数字PCR（ddPCR）获得的结果与ctDNA部分报道的结果进行了比较。我们在所有样本中都检测到了在组织活检和ctDNA中发现的相同突变，但出乎意料的是，在一个样本中，我们发现了一个在ctDNA或组织活检中均未鉴定出的突变。此外，为了评估sEV相关DNA的确切位置（在囊泡外部还是内部），我们用三种不同方法分离的sEV用DNase I进行了处理。我们发现囊泡内部的DNA只是囊泡周围DNA的一小部分。其数量似乎与循环肿瘤DNA的总量相关。我们在实验环境中获得的结果表明，将ctDNA和sEV相关DNA整合到mCRC患者管理中可以提供癌症突变状态的完整实时评估。
