结直肠癌组织基因突变、微卫星不稳定与患者临床病理特征的相关性

目的 探讨结直肠癌组织KRAS、NRAS、BRAF基因突变及微卫星不稳定（MSI）的发生情况,及其与患者临床病理特征的相关性。 方法 回顾性分析山西省肿瘤医院2020年10月至2021年5月诊治的473例结直肠癌患者临床病理资料。应用扩增阻滞突变系统（ARMS）法检测石蜡包埋组织中KRAS、NRAS、BRAF基因突变情况,应用聚合酶链反应-毛细管电泳法分析MSI状态,并分析患者临床病理特征与基因突变及MSI状态的相关性。 结果 473例结直肠癌组织中KRAS、NRAS和BRAF基因突变率分别为45.03%（213/473）、2.96%（14/473）和5.50%（26/473）,未检出两种基因共突变患者。高/中分化腺癌中KRAS基因突变率高于低分化腺癌[47.4%（175/369）比36.5%（38/104）,χ²＝3.89,P＝0.049]。女性NRAS基因突变率高于男性[5.0%（10/202）比1.5%（4/271）,χ²＝4.86,P＝0.027];肿瘤长径≤3 cm者NRAS基因突变率高于肿瘤长径>3 cm者[7.1%（7/98）比1.9%（7/375）,P＝0.013]。病变位于结肠BRAF基因突变率高于直肠[11.7%（20/171）比2.0%（6/302）,χ²＝19.81,P<0.001];低分化者BRAF基因突变率高于高/中分化者[10.6%（11/104）比4.1%（15/369）,χ²＝6.62,P＝0.010];伴有黏液成分者BRAF基因突变率高于无黏液者[10.9%（11/101）比4.0%（15/372）,χ²＝7.19,P＝0.007];有淋巴结转移者BRAF基因突变率高于无淋巴结转移者[8.2%（15/182）比3.8%（11/291）,χ²＝4.29,P＝0.038]。473例结直肠癌组织中高度微卫星不稳定（MSI-H）的发生率为7.19%（34/473）,MSI-H在结肠中发生率高于直肠[14.0%（24/171）比3.3%（10/302）,χ²＝18.82,P<0.001];在低分化程度者中发生率高于高/中分化程度者[17.3%（18/104）比4.3%（16/369）,χ²＝20.46,P<0.001];伴有黏液成分者的MSI-H发生率高于无黏液成分者[11.9%（12/101）比5.9%（22/372）,χ²＝4.24,P＝0.039];无淋巴结转移者中MSI-H发生率高于有淋巴结转移者[10.0%（29/291）比2.7%（5/182）,χ²＝8.75,P＝0.003]。同时MSI-H在BRAF突变患者中的发生率增高（P<0.001）。 结论 结直肠癌中KRAS、NRAS、BRAF基因突变及MSI状态与患者临床病理特征存在相关性,MSI-H与BRAF突变相关。     

Multiple Primary Cancers with Microsatellite Instability: Report of a Case

We report here a patient who developed a variety of tumors both synchronously and metachronously over a 2-year period. The involved organs were the uterus, ureter, and small and large intestines. The patient underwent open surgery 3 times and polypcctomies 6 times. Postoperative histopathologic analysis showed 2 adenomas and 8 carcinomas. Genetic analysis revealed microsatellite instabilities at the tested loci in all 10 tumors, indicating that replication errors played an essential role in the tumorigenesis. Early identification of microsatellite instability could be useful for predicting development of additional primary cancers.     

Combined analysis of specific KRAS mutation,BRAF and microsatellite instability identifies prognostic subgroups of sporadic and hereditary colorectal cancer

Confounding effects of specific KRAS gene alterations on colorectal cancer (CRC) prognosis stratified by microsatellite instability (MSI) and BRAF^V600E have not yet been investigated. The aim of our study was to evaluate the combined effects of MSI, BRAF^V600E and specific KRAS mutation (Gly → Asp; G12D, Gly → Asp, G13D; Gly → Val; G12V) on prognosis in 404 sporadic and 94 hereditary CRC patients. MSI status was determined according to the Bethesda guidelines. Mutational status of KRAS and BRAF^V600E was assessed by direct DNA sequencing. In sporadic CRC, KRAS G12D mutations had a negative prognostic effect compared to G13D and wild‐type cancers (p = 0.038). With MSI, specific KRAS and BRAF^V600E mutations, 3 distinct prognostic subgroups were observed in univariate (p = 0.006) and multivariable (p = 0.051) analysis: patients with (i) KRAS mutation G12D, G12V or BRAF^V600E mutation, (ii) KRAS/BRAF^V600E wild‐type or KRAS G13D mutations in MSS/MSI‐L and (iii) MSI‐H and KRAS G13D mutations. Moreover, none of the sporadic MSI‐H or hereditary patients with KRAS G13 mutations had a fatal outcome. Specific KRAS mutation is an informative prognostic factor in both sporadic and hereditary CRC and applied in an algorithm with BRAF^V600E and MSI may identify sporadic CRC patients with poor clinical outcome.     

Wild-type KRAS and BRAF could predict response to Cetuximab in Chinese colorectal cancer patients

Objective  

To analyze the relationship between KRAS, BRAF mutations and the response to Cetuximab in Chinese colorectal cancer patients.     

Differential colorectal carcinogenesis: Molecular basis and clinical relevance

Colorectal cancer (CCR) is one of the most frequent cancers in developed countries. It poses a major public health problem and there is renewed interest in understanding the basic principles of the molecular biology of colorectal cancer. It has been established that sporadic CCRs can arise from at least two different carcinogenic pathways. The traditional pathway, also called the suppressor or chromosomal instability pathway, follows the Fearon and Vogelstein model and shows mutation in classical oncogenes and tumour suppressor genes, such as K-ras, adenomatous polyposis coli, deleted in colorectal cancer, or p53. Alterations in the Wnt pathway are also very common in this type of tumour. The second main colorectal carcinogenesis pathway is the mutator pathway. This pathway is present in nearly 15% of all cases of sporadic colorectal cancer. It is characterized by the presence of mutations in the microsatellite sequences caused by a defect in the DNA mismatch repair genes, mostly in hMLH1 or hMSH2. These two pathways have clear molecular differences, which will be reviewed in this article, but they also present distinct histopathological features. More strikingly, their clinical behaviours are completely different, having the "mutator" tumours a better outcome than the "suppressor" tumours.     

大肠癌微卫星不稳定性及其临床意义

目的 了解大肠癌微卫星不稳定性（ＭＩ）情况。方法 用６个微卫星位点检测ＰＣＲ扩增所选位点ＰＣＲ产物用８％变性聚丙烯酰胺凝胶电泳、银染。结果 ６０例大肠癌中,２０例表现ＭＩ,其中１１例为复制错误（ＲＥＲ）阳性。ＲＥＲ阳性与ＲＥＲ阴性大肠癌患者相比,发病年龄较年轻,一级亲属有恶性肿瘤病史者明显高于ＲＥＲ阴性,倾向位于结肠,呈浸润性生长,Ⅲ￣Ⅳ期比例高。５例大肠癌伴大肠腺瘤病例中,４例腺瘤有ＭＩ。结论     

KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers

Background:

Mutations in the Kirsten Ras (KRAS) oncogene are common in colorectal cancer (CRC). The role of KRAS-mutation status as a prognostic factor, however, is unclear. We evaluated the relationship between KRAS-mutation status and CRC survival, considering heterogeneity in this association by tumour and patient characteristics.

Methods:

The population-based study included individuals diagnosed with CRC between 1998–2007 in Western Washington State. Tumour specimens were tested for KRAS exon 2 mutations, the BRAF p.V600E mutation, and microsatellite instability (MSI). We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between KRAS-mutation status and disease-specific and overall survival. Stratified analyses were conducted by age, sex, tumour site, stage, and MSI. We conducted additional analyses combining KRAS-mutation, BRAF-mutation, and MSI status.

Results:

Among 1989 cases, 31% had KRAS-mutated CRC. Kirsten Ras (KRAS)-mutated CRC was associated with poorer disease-specific survival (HR=1.37, 95% CI: 1.13–1.66). This association was not evident in cases who presented with distant-stage CRC. Cases with KRAS-wild-type/BRAF-wild-type/MSI-high CRC had the most favourable prognosis; those with CRC exhibiting a KRAS- or BRAF-mutation and no MSI had the poorest prognosis. Patterns were similar for overall survival.

Conclusion:

Kirsten Ras (KRAS)-mutated CRC was associated with statistically significantly poorer survival after diagnosis than KRAS-wild-type CRC.     

不同错配修复状态结直肠癌患者临床病理特征及与KRAS/NRAS/BRAF基因突变的相关性

目的探究不同错配修复（MMR）状态下结直肠癌患者的临床病理特征及与KRAS/NRAF/BRAF（KNB）基因突变的相关性。方法收集477例结直肠癌患者临床病理资料,采用免疫组织化学染色（IHC）、PCR-毛细管电泳法、二代测序技术（NGS）分别检测MMR、微卫星不稳定性（MSI）及KNB状态,分析不同MMR状态分组下患者临床病理特征及与KNB突变的相关性。结果与pMMR组相比,经典dMMR组患者年龄小、女性多、右半结肠常见、多为黏液腺癌且分化差（均P<0.05）;非经典dMMR组患者右半结肠常见,且易见特殊组织学类型（均P<0.05）。经典/非经典dMMR组患者均常见MLH1-PMS2共缺失、BRAF突变和KRAS G13密码子突变（均P<0.05）,且MMR IHC与MSI PCR结果高度一致（分别为100%和99.1%）。经典dMMR组KRAS突变的患者年龄小、男性多、易见特殊组织学类型,但远处转移少见（均P<0.05）,而非经典dMMR组KRAS突变的患者淋巴结转移少见（P=0.005）。非经典dMMR组患者中MSH6基因突变率较高（P=0.002）,均为MLH1-PMS2完全缺失合并MSH6非经典表达（100%）。5例含髓样癌成分的患者均存在MLH1-PMS2完全缺失,其中3例合并MSH2/MSH6非经典表达,这3例中有2例携带MSH6基因c.3261位点突变。结论经典/非经典dMMR结直肠癌患者具有不同于pMMR患者的临床病理特征,MMR IHC检测可很好预测MSI状态。KRAS突变的经典/非经典dMMR结直肠癌患者之间临床病理特征不同,但均常见MLH1-PMS2共缺失、BRAF突变和KRAS G13密码子突变。非经典dMMR患者常见MLH1-PMS2完全缺失合并MSH6非经典表达模式。MSH6基因突变可在部分含髓样癌成分的结直肠癌发生中起关键作用。     

微卫星不稳定在结直肠癌预后及治疗指导中的研究进展

微卫星不稳定是基因组不稳定的表现之一,其在结直肠癌、乳癌、肺癌以及淋巴瘤等多种恶性肿瘤中均有存在,并且对肿瘤的发生、发展以及预后转归起到关键作用。近年来,国内外学者对微卫星不稳定的相关研究取得了显著成果,尤其在结直肠癌领域,其对结直肠癌免疫治疗和化学治疗的指导性意见已被纳入诊疗指南,并且成为该病治疗以及预后指导上的重要分子指标。本文就此总结国内外相关研究进展,以期为后续研究工作提供指导。     

Incorporation of somatic <Emphasis Type="Italic">BRAF</Emphasis> mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.     

散发性结直肠癌中微卫星不稳定性研究

目的：检测微卫星不稳定性（Microsatellte Instability,MIN)在国内散发性结直肠癌中的发性情况并探讨其与散发性结直肠癌临床病理参数之关系。方法：彩和PCR,变性聚丙烯酰胺凝胶电泳,银染色显色等技术,选用位于4条染色体上的6个不同的微卫星位点,对60例散发性结直肠癌病例进行了微卫星不稳定性检测。结果：51．67％（31／60）的病例在1个或1个以上位点出现MIN,33．33％（20／60）的病例在2个或2个以上位点出现MIN,即RER＋（Replication ERRORS pOSITIVE)。各位点在散发性结肠癌中的检出率不同,其顺序为P53（1）,D18S363,NF1,D18S46,ANK1,APC（1）。在临病理参数上,RER＋组和RER－组相比在肿瘤发生部位,组织学分化、浸润深度,肿瘤大小等方面具有不同的趋势,但没有显著性差异;而在局部淋巴结转移上具有显著性差异（P＜0．05）。结论：散发性结直肠癌中广泛存在着MIN;RER＋结直肠癌和RER－结直肠癌在临床病理参数上有着不同的趋势。