Objective: To analyze the difference of somatic mutation of DNA mismatch repair (MMR) protein deletion (dMMR) /integrity (pMMR) in colorectal cancer (CRC). Methods: A total of 93 cases of paraffin pathological tissue derived from CRC patients underwent surgical treatment and postoperative routine immunohistochemical diagnosed as dMMR in the Second Affiliated Hospital of Zhejiang University Medical College from January 2015 to January 2017 were collected and conducted the second-generation sequencing test. The expressions of 4 MMR proteins (MLH1, MSH2, MSH6 and PMS2) in CRC tissue were detected by immunohistochemistry method, and the immunohistochemistry results were re-interpreted according to the American Association of Pathologists (CAP) standard. Second-generation sequencing technology was used to detect somatic mutations of 41 genes in 93 cases of paraffin pathological CRC tissue, and Fisher's exact test was used to analyze the gene mutation differences between groups. Results: After re-evaluation according to CAP standard, 31 cases were divided into pMMR group and 62 cases in dMMR group among the 93 CRC patients. The medium number of gene mutations in the dMMR group was 9.5, higher than 3.0 of the pMMR group (P<0.001). Somatic mutation differences were found in 17 genes between the dMMR and pMMR groups, including breast cancer susceptibility genes 1 (BRCA1), BRCA2, MLH1, PDGFRA, PIK3CA, APC, ATM, KIT, MET, PMS2, MSH6, POLE, MSH2, PTCH1, epidermal growth factor receptors (EGFR), TP53 and ERBB2 genes. The pathogenic somatic mutation rates of BRAF, MLH1, MSH2 and MSH6 in the dMMR group were higher than those in the pMMR group [21.0% (13/62) vs 9.7% (3/31), 9.7% (6/62) vs 0 (0/31), 21.0% (13/62) vs 0 (0/31), 22.6% (14/62) vs 0 (0/31), P<0.05]. The mutation rate differences of BLM N515fs, BRAF V600E, PTCH1 R1308fs and KRAS G13D sites were statistically different between the dMMR group and the pMMR group [22.6% (14/62) vs 0 (0/31), 19.4% (12/62) vs 3.2% (1/31), 11.3% (7/62) vs 0 (0/31), 16.1% (10/62) vs 3.2% (1/31), P<0.05]. The mutation rates of 3 uncommon sites including BLM N515fs, MSH6 F1088fs and PTCH1 R1308fs were 28.2% (11/39), 15.4% (6/39) and 15.4% (6/39) in patients with dMMR who were missing MLH1 and PMS2 together, statistically different from all of 0 (0/31) in pMMR patients (P<0.05). Conclusions: CRC Patients with dMMR have more related gene somatic mutations. The BRAF V600E mutation is closely related to dMMR. KRAS G13D, BLM N515fs and PTCH1 R1308fs mutation sites are also associated with the expression of MMR proteins.
目的： 分析DNA错配修复(MMR)蛋白缺陷(dMMR)和完整(pMMR)的结直肠癌(CRC)患者相关基因体细胞突变差异。 方法： 收集2015年1月至2017年1月于浙江大学医学院附属第二医院行手术治疗、术后常规免疫组化检测报告为dMMR的93例CRC患者石蜡病理组织行二代测序技术检测。采用免疫组化法检测93例CRC患者肿瘤组织中4种MMR蛋白(MLH1、MSH2、MSH6和PMS2)的表达情况，依据美国病理学家协会(CAP)标准对免疫组化检测结果进行重新判读。采用二代测序技术检测93例CRC患者石蜡病理组织中41个基因的体细胞突变情况，采用Fisher精确检验分析组间基因突变差异。 结果： 依据CAP标准重新判读后，93例CRC患者中pMMR组31例，dMMR组62例。dMMR组基因突变中位数为9.5个，高于pMMR组(3.0个，P<0.001)。dMMR组和pMMR组中17个基因存在体细胞突变差异，分别为乳腺癌易感基因1(BRCA1)、BRCA2、MLH1、PDGFRA、PIK3CA、APC、ATM、KIT、MET、PMS2、MSH6、POLE、MSH2、PTCH1、表皮生长因子受体(EGFR)、TP53和ERBB2基因。dMMR组致病体细胞BRAF、MLH1、MSH2和MSH6基因突变率高于pMMR组[分别为21.0%(13/62)和9.7%(3/31)，9.7%(6/62)和0(0/31)，21.0%(13/62)和0(0/31)，22.6%(14/62)和0(0/31)，均P<0.05]。BLM N515fs、BRAF V600E、PTCH1 R1308fs和KRAS G13D位点突变率在dMMR组和pMMR组CRC中差异均有统计学意义[分别为22.6%(14/62)和0(0/31)，19.4%(12/62)和3.2%(1/31)，11.3%(7/62)和0(0/31)，16.1%(10/62)和3.2%(1/31)，均P<0.05]。在MLH1和PMS2共同缺失的dMMR患者中3个不常见位点BLM N515fs、MSH6 F1088fs和PTCH1 R1308fs的突变率分别为28.2%(11/39)、15.4%(6/39)和15.4%(6/39)，与pMMR患者[均为0(0/31)]比较，差异均有统计学意义(均P<0.05)。 结论： dMMR结直肠癌患者有更多的相关基因发生体细胞突变。BRAF V600E突变与dMMR密切相关。KRAS G13D、BLM N515fs和PTCH1 R1308fs突变位点也与MMR蛋白的表达有关。.
Keywords: Colorectal neoplasms; DNA mismatch repair; Next generation sequencing; Somatic mutation.