Department of Clinical Laboratory, Taizhou People's Hospital (Postgraduate Training Base of Dalian Medical University), Taizhou 225300, Jiangsu Province, China.
Department of General Surgery, Taizhou People's Hospital (Postgraduate Training Base of Dalian Medical University), Taizhou 225300, Jiangsu Province, China.
World J Gastroenterol. 2022 Jul 7;28(25):2920-2936. doi: 10.3748/wjg.v28.i25.2920.
Colorectal cancer (CRC) is one of the most common malignancies worldwide. Given its insidious onset, the condition often already progresses to advanced stage when symptoms occur. Thus, early diagnosis is of great significance for timely clinical intervention, efficacy enhancement, and prognostic improvement. Featuring high throughput, fastness, and rich information, next generation sequencing (NGS) can greatly shorten the detection time, which is a widely used detection technique at present.
To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC, and to establish a technological platform for CRC screening, diagnosis, and efficacy monitoring through fecal DNA detection.
NGS was used to sequence the stool DNA of patients with CRC, which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease, as well as the tumor tissues of CRC patients. Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened, and their significances in diagnosing CRC and predicting patients' prognosis were comprehensively evaluated.
High mutation frequencies of , and were detected in the stools and tumor tissues of CRC patients prior to surgery. Contrastively, no pathogenic mutations of the above three genes were noted in the postoperative stools, the normal controls, or the benign intestinal disease group. This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients. The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent. Compared to the postoperative stools and the stools in the two control groups, the pathogenic mutation frequencies of and were significantly higher for the preoperative stools ( 7.328, < 0.05; 4.219, < 0.05), suggesting that fecal and genes can be used for CRC screening, diagnosis, and prognostic prediction. No significant difference in the pathogenic mutation frequency of the gene was found from the postoperative stools or the two control groups ( 0.878, > 0.05), so further analysis with larger sample size is required. Among CRC patients, the pathogenic mutation sites of occurred in 16 of 27 preoperative stools, with a true positive rate of 59.26%, while the pathogenic mutation sites of occurred in 10 stools, with a true positive rate of 37.04%. The sensitivity and negative predictive values of the combined genetic testing of and were 66.67% (18/27) and 68.97%, respectively, both of which were higher than those of or mutation detection alone, suggesting that the combined genetic testing can improve the CRC detection rate. The mutation sites exon 4 A84G and exon 20 I821T (mutation start and stop positions were both 7579436 for the former, while 55249164 for the latter) were found in the preoperative stools and tumor tissues. These "undetected" mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression, which needs to be confirmed through further research. Some mutations of "unknown clinical significance" were found in such genes as , , , , , and , whose clinical values is worthy of further exploration.
NGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis. Fecal and can be used as specific genes for the screening, diagnosis, prognostic prediction, and recurrence monitoring of CRC. Moreover, the combined testing of and genes can improve the CRC detection rate.
结直肠癌（CRC）是全球最常见的恶性肿瘤之一。由于其发病隐匿，当出现症状时，病情往往已经进展到晚期。因此，早期诊断对于及时的临床干预、疗效增强和预后改善具有重要意义。基于高通量、快速和丰富信息的下一代测序（NGS）可以大大缩短检测时间，是目前广泛使用的检测技术。
筛选粪便 DNA 中适合 CRC 诊断和预后预测的特定基因或基因组合，并通过粪便 DNA 检测建立 CRC 筛查、诊断和疗效监测的技术平台。
使用 NGS 对 CRC 患者的粪便 DNA 进行测序，然后将其与正常对照组和良性肠病患者的粪便样本以及 CRC 患者的肿瘤组织的基因检测结果进行比较。筛选出适合 CRC 诊断和预后预测的粪便 DNA 中的特定基因或基因组合，并综合评估其在诊断 CRC 和预测患者预后方面的意义。
在手术前，CRC 患者的粪便和肿瘤组织中检测到高突变频率的、和。相比之下，在术后粪便、正常对照组和良性肠病组中均未发现上述三个基因的致病性突变。这表明可以在 CRC 患者的术前粪便中检测到肿瘤特异性 DNA。术前粪便中肿瘤相关基因的表达可以在一定程度上反映肿瘤组织中的基因突变。与术后粪便和两个对照组相比，术前粪便中 和 的致病性突变频率明显更高（ 7.328，<0.05； 4.219，<0.05），提示粪便和 基因可用于 CRC 筛查、诊断和预后预测。未发现 基因的致病性突变频率在术后粪便或两个对照组中存在显著差异（ 0.878，>0.05），因此需要进一步进行更大样本量的分析。在 CRC 患者中，16 例术前粪便中出现了 基因的致病性突变位点，真阳性率为 59.26%，而 10 例粪便中出现了 基因的致病性突变位点，真阳性率为 37.04%。和联合基因检测的敏感性和阴性预测值分别为 66.67%（18/27）和 68.97%，均高于或单独突变检测，提示联合基因检测可以提高 CRC 的检出率。在术前粪便和肿瘤组织中发现了 外显子 4 A84G 和 外显子 20 I821T（前者的突变起始和终止位置均为 7579436，后者为 55249164）的“未检出”突变位点。这些“未知临床意义”的突变可能是 CRC 发生和发展过程中新出现的突变类型，需要通过进一步研究来证实。在 、、、、、和 等基因中发现了一些“未知临床意义”的突变，其临床价值值得进一步探索。
基于 NGS 的粪便基因检测可作为 CRC 诊断的补充技术。粪便和 可作为 CRC 筛查、诊断、预后预测和复发监测的特异性基因。此外，和 基因的联合检测可以提高 CRC 的检出率。
