目的：利用微滴数字PCR(droplet digital PCR, DD-PCR)技术进行CpG岛甲基化表型(CpG island methylation phenotype, CIMP)结直肠癌分型, 并探讨以血浆游离DNA进行CIMP分型的可行性。方法：收集2008到2012年在长海医院行结直肠癌手术的患者216例，抽提肿瘤组织DNA和血浆游离DNA, 重亚硫酸盐处理后，利用DD-PCR进行CIMP分型。CIMP分型选用CACNA1G、IGF2、NEUROG1、RUNX3、SOCS1五个基因的甲基化位点，利用免疫组织化学技术（IHC）检测P53突变情况, 以巢式PCR测序方法检测K-RAS 和BRAF突变。结果：216例肿瘤中17例(7.9%)存在BRAF突变, 96例(444%) 存在K-RAS 突变和112例 (51.9%) P53-IHC(+)。31.5%(68/216)的结直肠癌为CIMP（+）(5个基因位点中高甲基化位点数目≥3)，823%(14/17)的BRAF突变属于CIMP(+), 而只有9.4% (9/96)的K-RAS 突变和7.1% (8/112) 的P53突变属于CIMP(+)。除了K-RAS ，BRAF和P53突变外，同CIMP（-）结直肠癌相比，CIMP（+）肿瘤还具有独特的临床病理特征：肿瘤更易发生在结直肠的近端位置，黏液性癌比例偏高，分化程度较高，CIMP(+) 患者生存时间显著短于CIMP (-)患者。利用血浆游离DNA进行分型和利用肿瘤组织进行分型的一致性为93.4%、灵敏度为872%、特异性为100%。结论：CIMP（+）结直肠癌具有独特的临床病理特征, 且预后较差；在缺乏肿瘤组织的情况下, 利用DD-PCR对血浆游离DNA进行CIMP分型是可行的。

Objective:To investigate the clinicopathologic features of colorectal cancers with CpG island methylation phenotype (CIMP) by using droplet digital PCR, and to evaluate the feasibility of determining the CIMP status using plasma free DNA. Methods: Two hundred sixteen tumor and paired plasma samples were collected from patients with colorectal cancer enrolled into Changhai Hospital from 2008 to 2012. DNA was extracted from tumor tissues and plasmas and underwent bisulfate conversion. CIMP was determined by using digital PCR for the five genes CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1. P53 mutation was detected by immunohistochemical analysis. K-RAS and BRAF mutations were assessed by nest PCR. Results: Among the 216 colorectal cancers, 17 (7.9%) had BRAF mutations, 96 (44.4%) contained K-RAS mutations, 112 (51.9%) were P53- IHC (+), and 68 (31.5%) were CIMP positive (≧3 gene loci were hypermethylated). Most of the cancers with BRAF mutation (82.3%, 14/17) were CIMP(+), whereas only 94% (9/96) of the cancer with K-RAS mutations and 7.1% (8/112) of cancers harboring P53 mutations (+) were CIMP(+). Compared to CIMP(-) tumors, the unique clinical pathological features of the CIMP(+) tumors included: (1) more likely to happen in the proximal locations; (2) more likely to be mucinous carcinoma; (3) higher degree of differentiation. The overall survival period of colorectal patients with CIMP(+) was significantly shorter than that with CIMP(-). When plasma free DNA from these patients was used for CIMP analysis, the consistency, sensitivity and specificity were 93.4%, 87.2% and 100% respectively compared with the assay using tumor DNA.Conclusion: Patients with CIMP(+) colorectal cancer have unique clinical pathological characteristics and poor prognosis. When tumor tissues are not available, CIMP analysis with plasma free DNA using digital PCR is a feasible alternative.

国家自然科学基金资助项目（No.81471605）