结直肠癌原发灶和对应肝转移灶K-ras基因突变的对比研究

目的:观察结直肠癌肝转移的原发灶和对应肝转移灶中K-ras基因的突变情况,比较两者的一致性.方法:从75例结直肠癌肝转移患者的原发灶和对应肝转移灶病理标本中提取肿瘤组织DNA,经聚合酶链反应(PCR)扩增K-ras基因,采用DNA直接测序法检测K-ras基因的突变情况.结果:有3例因DNA提取质量较差出组.72例中,有24例(33.3%)结直肠癌原发组织中K-ras基因为突变型;23例(31.9%)肝转移灶癌组织中K-ras基因为突变型.24例原发灶K-ras基因为突变型者中,有21倒(87.5%)对应的肝转移灶K-ras基因为突变型;46例两者均为野生型.原发灶及对应肝转移灶两者的一致率为93.1%.两者不一致的情况有5例(6.9%).结论:结直肠癌肝转移的原发灶和对应肝转移灶中K-ras基因的突变情况较为一致,可作为选用分子靶向药物的依据.     

结直肠癌原发灶与转移灶K-ras基因突变的比较分析

背景与目的：K-ras基因突变是抗表皮生长因子受体(epidermal growth factor receptor,EGFR)靶向治疗的重要负性预测因子。本研究拟对结直肠癌原发灶与转移灶中K-ras基因状态的一致性进行比较,以探讨目前临床K-ras检测的科学性与严谨性。方法：收集复旦大学附属肿瘤医院手术切除的结直肠癌原发灶及转移灶石蜡包埋组织76对,提取DNA,经过PCR扩增后,对产物进行基因序列分析,检测结直肠癌中K-ras基因外显子2基因序列。结果：76例患者中有15例结直肠癌原发灶与转移灶的K-ras基因突变情况不一致。76例结直肠癌原发灶有31例发生突变,突变率为40.8%,其中第13号密码子突变16例,第12号密码子突变15例;76例结直肠癌转移灶有31例发生突变,突变率为40.8%,其中第13号密码子突变15例,第12号密码子突变16例。结论：结直肠癌原发灶和转移灶中K-ras基因状态并不一致,且存在19.7%的表达差异率,提示通过检测原发灶K-ras基因表达状态来确定针对转移灶的西妥昔单克隆抗体药物选择存在不严谨性,需要进一步完善。     

结直肠癌APC、K-ras、p53基因突变检测

目的:探讨结直肠癌中APC、K-ras、p53基因突变模式。方法:应用酚/氯仿法提取48例结直肠癌组织及其相应正常黏膜组织的DNA,用聚合酶链反应(PCR)、单链构象多态性分析(SSCP)和DNA测序等方法检测APC基因第15外显子突变密集区(mutation cluster region,MCR)区段、K-ras和p53基因的突变。结果:APC、K-ras和p53基因的突变率分别为37.5%(18/48)、43.8%(21/48)和35.4%(17/48)。48例结直肠癌组织中,有42例发生APC、K-ras或p53基因突变,突变率高达87.5%(42/48),其中仅有APC、K-ras或p53 1种基因发生突变的发生率分别为16.7%(8/48)、25.0%(12/48)和20.8%(10/48)。单独1种基因发生突变的总发生率为62.5%(30/48)。APC和p53,APC和K-ras或p53和K-ras 2种基因均有突变的发生率分别为6.3%(3/48)、10.4%(5/48)和4.2%(2/48)。APC、K-ras和p53 3种基因均发生突变的发生率为4.2%(2/48)。2种和3种基因均发生突变的总发生率为25%(12/48)。结论:结直肠癌的发生、发展并不完全遵循由正常结直肠黏膜上皮细胞向腺瘤和侵袭性癌转化的过程中,及依次发生“APC→K-ras→p53→DCC”突变累积这一经典的结直肠癌发生发展模式,可能存在其他结直肠癌发病机制。     

结直肠癌原发灶、相应肝转移灶K-ras基因状态的研究

目的 观察结直肠癌患者原发肿瘤组织及相应肝转移灶K-ras基因突变情况,分析二者的一致性.探讨结直肠癌K-ras基因状态与肝转移关系.方法 实时荧光定量PCR技术和基因测序技术检测76例结直肠癌患者原发肿瘤组织及其中22例肝转移灶K-ras基因突变,结合其临床资料分析.结果 76例结直肠癌组织中25例(32.9%)发现K-ras基因突变;22例肝转移灶中11例(50%)发现K.ras基因突变,其中10例对应原发癌组织也发现K-ras基因突变.1例对应原发癌组织未发现突变.原发癌组织与肝转移灶K-ras基因状态一致率为90.9%.肝转移患者原发癌组织K-ras基因突变率明显高于无肝转移患者(P<0.05).原发肿瘤组织和7例同时性、2例异时性肝转移灶的K-ras基因突变类型基本一致(即K-ras基因12密码子GGT突变为GAT或GTr).1例异时性和1例同时性肝转移灶K-ras基因突变类型为13密码子GGC突变为GAC.结论 结直肠癌的原发癌组织与肝转移灶的K-ras基因状态较为一致,原发癌组织有K-ras基因的突变,预示着肿瘤有肝脏转移倾向.     

干扰素诱导蛋白16在结直肠癌中的表达及其与K-ras基因突变的相关性研究

背景与目的:结直肠腺瘤-癌序列是目前公认的结直肠癌演变过程,该过程伴随一系列基因突变及蛋白的异常表达和积累.本研究旨在探讨结直肠癌发生、发展过程中干扰素诱导蛋白16(interferon induced 16,IFI16)表达及其与K-ras基因突变之间的相关性.方法:收集云南省第一人民医院病理科2008-2009年门诊及手术的结直肠正常黏膜组织、腺瘤和腺癌各发展阶段标本,应用免疫组化SP法检测IFI16表达情况;应用PCR-RFLP法对结直肠癌标本K-ras基因12密码子进行检测,并与IFI16进行相关性分析.结果:IFI16在结直肠腺癌与腺瘤、腺瘤与正常结直肠黏膜组织巾蛋白表达差异均有统计学意义(49.2% vs 25%,P＜0.05;25% vs 0%,P＜0.05).结直肠癌患者K-ras基因突变率为38.3%;K-ras基因变异与IFI16表达无相关性(55.2%vs 44.4%,P＞0.05),但与IFI16异位表达差异有统计学意义(13.8% vs 72.2%,P＜0.05),两者之间呈显著相关(r=0.635,P＜0.01).结论:IFI16参与了结直肠痛的发生、发展过程,K-ras基因突变与IFI16异位在结直肠癌的发生、发展过程中起重要作用.     

K-ras点突变与非小细胞肺癌经典HLA-Ⅰ类抗原表达下调相关性的研究

目的:探讨癌基因K-ras点突变与非小细胞肺癌(NSCLC)原发灶及淋巴结癌转移灶经典HLA-Ⅰ类抗原表达下调的关系.方法:应用流式细胞术检测65例NSCLC原发灶及31例淋巴结转移灶经典HLA-Ⅰ类抗原的表达;应用PCR-RFLP技术检测K-ras基因第12位密码子点突变(正常肺组织及淋巴结组织作对照);根据不同的变量类型及目的,采用t检验、Spearman相关分析和x2检验等进行统计分析.结果:淋巴结转移灶经典HLA-Ⅰ类抗原的表达率为(15.35±6.24)％,明显低于NSCLC原发灶的(39.68±12.46)％,t=2.06,P=0.034;且与NSCLC原发灶经典HLA-Ⅰ类抗原表达下调明显呈正相关,rs=0.487,P=0.008.经典HLA-Ⅰ类抗原表达阳性的NSCLC原发灶中未检测到K-ras基因突变,经典HLA-Ⅰ类抗原阴性表达者的K-ras基因突变率为54.5％(6/11),低表达者的K-ras基因突变率为20.0％(9/45),差异有统计学意义,x2 =5.38,P=0.042.结论:经典HLA-Ⅰ类抗原表达下调是NSCLC淋巴结转移的机制之一,癌基因K-ras突变与NSCLC原发灶及淋巴结转移灶经典HLA-Ⅰ类抗原低表达有关.     

结直肠癌中K-ras基因突变研究

目的研究K-ras基因突变表达与结直肠癌的相关性。方法采用实时荧光定量PCR方法检测89例患者结直肠癌组织中K-ras基因2号外显子12与13编码子突变情况,并结合病理资料进行分析。结果89例患者结直肠癌组织中K-ras基因突变者为37例,突变率为41．6％。其中12编码子突变为29例,突变率为32．6％。13编码子突变为8例,突变率为9．O％。K-ras基因突变与肿瘤位置、分化程度无明显相关性（P〉0．05）,与浸润深度、淋巴结转移、肝转移有相关性（P〈0．05）。淋巴结转移越多K-ras基因突变率越高,有肝转移者K-ras基因突变率高。结论K-ras基因突变在结直肠癌的发生、发展中起重要作用,与浸润深度、淋巴结转移和肝转移密切相关。     

Kiss-1基因启动子甲基化与结直肠癌的关系

目的探讨结直肠癌组织中Kiss-1基因启动子甲基化状态与临床病理学特征的关系及临床意义。方法采用甲基化特异性PCR检测142例正常结直肠组织、23例结直肠腺瘤组织、73例结直肠癌组织和相应癌旁组织中Kiss-1基因启动子甲基化状态。结果结直肠癌组织中Kiss-1基因启动子甲基化阳性率为82.2%,癌旁组织为30.1%,结直肠腺瘤组织为21.7%,正常结直肠组织为6.3%,总体比较差异有统计学意义(P<0.05)。Kiss-1基因启动子甲基化阳性率与结直肠癌的分化程度、浸润深度、淋巴结转移及远处转移有关(P<0.05)。结论 Kiss-1基因启动子甲基化可能促进结直肠癌的发生、发展与转移,Kiss-1基因启动子甲基化水平有可能成为评估结直肠癌转移风险及预后的指标之一。     

大肠癌Kai-1/CD82基因外显子9区域突变的研究

背景与目的:探讨Kai-1/CD82基因外显子9区域的突变在结直肠癌演进与转移中的意义.材料与方法:提取40例结直肠癌患者的肿瘤组织(24例无淋巴结转移,16例有淋巴结转移)DNA和RNA,PCR扩增DNA,测序判断Kai-1/CD82基因中外显子9区域的突变情况;RT-PCR后经测序Kai-1/CD82基因外显子9缺失情况.结果:40例组织标本中18例出现Kai-1/CD82基因突变;在有淋巴结转移的结直肠癌组织中,突变的频率明显高于无淋巴结转移的结直肠癌组织(P＜0.05),在大肠癌中晚期(Dukes C/D期)明显高于早期(Dukes A/B期)(P＜0.05),而Kai-1/CD82基因的突变频率与结直肠癌患者的年龄,性别,组织学类型以及分化程度无关(P＞0.05).结论:Kai-1/CD82基因突变可能与大肠癌演进、转移有关,检测其突变,可以为判断预后、指导临床治疗提供实验依据.     

非小细胞肺癌原发灶和转移淋巴结EGFR、KRAS和MET基因状态比较

目的:比较配对的原发肺癌灶和转移淋巴结EGFR、KRAS和MET基因状态,探讨NSCLC原发灶和转移淋巴结基因变化规律并指导临床实践。方法:22例手术切除的Ⅲa期非小细胞肺癌,术前未经靶向和化学治疗,获取配对的原发灶和N2站转移淋巴结。采用直接测序法检测EGFR外显子19-21,KRAS密码子12和13突变,实时定量PCR检测MET基因拷贝数。结果:原发灶和N2转移淋巴结中EGFR基因突变率分别为7/22例(31.82%)和6/22例(27.27%),EGFR基因型一致率达95.45%。KRAS基因突变率分别为2/22例(9.09%)和1/22例(4.55%)。转移淋巴结MET基因拷贝数(1.54±0.71)显著高于原发灶(1.19±0.41),P=0.038。EGFR 19和21基因突变与原发灶(P=0.24)、转移灶(P=0.97)的MET基因拷贝数以及原发灶和转移灶MET基因拷贝数变化(P=0.69)之间都无相关性,P>0.05。不同的EGFR基因状态和MET基因拷贝数其1年无病生存无显著差异(P>0.05)。结论:肺癌原发灶和相应转移淋巴结中EGFR基因突变较稳定;EGFR敏感基因突变与MET基因拷贝数可能无关;而未经EGFR-TKI治疗的肺癌患者其MET基因拷贝数在淋巴结转移时即已开始出现明显增高。     

Study on the Expression and Significance of TGF-β1, p-ERK1/2and K-ras in Colorectal Cancer Using Tissue Microarray Technique

OBJECTIVE This study aimed to explore the expression and significance of transforming growth factor β1(TGF-β1),extracellular signal-regulated kinases 1/2 (ERK1/2), and K-ras in colorectal cancer (CRC) using tissue microarray technology.METHODS The expressions of TGF-β1, ERK1/2, and K-ras in colon cancer cells taken from the specimens of 92 CRC patients (stage Ⅰ: 16 cases, stage Ⅱ: 28 cases, stage Ⅲ: 24 cases, and stage Ⅳ:24 cases) were analyzed using tissue microarray technology and immunohistochemistry, and compared with those of 20 normal colon tissue samples.RESULTS High immunoreactive scores (IRS) of TGF-β1,p-ERK1/2, and K-ras protein in CRC were obtained, which were 66.3% (61/92), 59.8% (55/92), and 48.9% (45/92), respectively, and those in normal epithelial cells of colon were 10% (2/20), 20% (4/20), and 30% (6/20), respectively (P < 0.05). The expressions of TGF-β1 and ERK1/2 in CRC at stage Ⅰwere 37.5% and 31.3%,respectively, and those in CRC at stage Ⅳ were 83.3% and79.3%, respectively, with statistically significant differences. No significant relationship was found between K-ras expression and tumor stages (P>0.05).CONCLUSION High level expressions of TGF-β1 and ERK1/2 are closely related to the clinical stages of colon cancer and crosstalk may exist between the 2 signal pathways.     

Epigenetic events in normal colonic mucosa surrounding colorectal cancer lesions

Gene inactivation by promoter hypermethylation has been demonstrated in the colonic mucosa of colorectal cancer (CRC) patients. However, current data do not prove direct involvement of this epigenetic modification in the early stages of CRC. Promoter methylation profiles of E-cadherin, hMLH1, MGMT, p16(INK4a), p15(INK4b) and p14(ARF); mutations of K-ras, B-raf and TP53 and microsatellite instability (MSI) were examined in normal and cancerous colonic mucosal tissue in 82 CRC patients using methylation-specific PCR assays. Methylation of hMLH1 and MGMT in normal mucosa correlated significantly with MSI and K-ras activation in neighbouring cancerous mucosal tissues. Similarly, poorly differentiated tumours were associated with methylated p16(INK4a) and E-cadherin in neighbouring normal colonic tissues (NCTs). Our results indicate that epigenetic changes in mucosa surrounding colorectal neoplastic lesions may describe a 'field cancerisation' phenomenon that may occur previous to genetic alterations in early stages of carcinogenesis.     

Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.     

K-ras oncogene mutation in cancer and precancerous lesions of the gallbladder

BACKGROUND AND OBJECTIVES: To determine whether K-ras mutation plays any role in the development and progression of gallbladder cancer, or has any clinical or pathological significance in gallbladder cancer patients, we investigated the presence and incidence of this mutation in the normal mucosa, and precancerous and cancerous lesions of the gallbladder. METHODS: DNA was obtained from normal mucosa, dysplastic mucosa, primary cancer tissues, and metastatic lymph nodes that were identified and microdissected from the paraffin blocks of 20 gallbladder cancer cases. K-ras codon 12 mutations were investigated using a modified two-step polymerase chain reaction and the restriction fragment length polymorphism method, and by direct sequencing with an automated sequencer. RESULTS: K-ras mutations were detected in the tissues of 10 out of the 20 patients. A mutation was present in the dysplastic epithelium associated with the primary carcinoma in 3 out of 12 specimens, in metastatic carcinoma in 1 out of 5 patients, and in primary carcinoma in 8 out of 20 patients. Mutation was found only once in the dysplastic, noncancerous epithelium, and only once in a metastatic tumor although not detectable in the primary cancer. Direct sequencing showed that the mutations were G to C substitutions (GGT-->CGT) at the first site of codon 12, except in two cases (GGT-->TGT). There were no correlations between K-ras mutations and clinicopathological factors. CONCLUSIONS: K-ras mutations were detected in half of the gallbladder cancer cases. We suggest that K-ras mutation may play a role in the development of premalignant lesions or early carcinogenesis in some gallbladder cancers. We were unable to find any evidence that K-ras mutation plays any role in tumor progression or metastasis, or that it has any clinicopathological significance.     

Ras mutational status is a biomarker for resistance to EGFR inhibitors in colorectal carcinoma

The epidermal growth factor receptor (EGFR) has been validated as a therapeutic target in several human tumours, including colorectal cancer (CRC). Although EGFR expression is used for patient selection, clinical experience shows that levels of EGFR expression (measured by immunohistochemistry) do not predict clinical benefit. Ras mutations in codons 12, 13 and 61 (found in 40-45% of CRC cases) result in inhibition of GTPase activity, thus leading to the constitutive activation of the ras proteins, which may render tumour cells independent of EGFR signalling and thereby, resistant to cetuximab, panitumumab and EGFR TKIs. Data from several recently published studies, as reviewed in this article, in patients with metastatic CRC (OPUS, CRYSTAL) clearly indicated that benefit from cetuximab, when added to chemotherapy, was only restricted to patients with wild-type K-ras tumours. These results showed that K-ras mutations predict the lack of clinical benefit from cetuximab and panitumumab therapies in CRC and indicate that K-ras status should be considered when selecting CRC patients as candidates for these antibodies. Moreover, the results from these studies should also trigger retrospective analyses of K-ras mutations from all available trials in CRC (as well as non-small cell lung cancer and pancreatic cancer). These studies may enable further establishment of the correlation between K-ras mutations and resistance to cetuximab and panitumumab in CRC patients.     

A novel high-specificity approach for colorectal neoplasia: Detection of K-ras2 oncogene mutation in normal mucosa

There is an important need for a high-specificity approach to colorectal cancer. Approximately 50% of colorectal tumors contain K-ras gene mutations, which occur as an early step in carcinogenesis. K-ras mutations were detectable not only in tumors but also in microscopically normal colorectal mucosa close to carcinomas in some patients with colorectal cancer. This is the first systematic analysis of K-ras mutations in normal colonic mucosa at multiple consistently-selected locations. A total of 480 normal colonic mucosal samples were obtained from 80 subjects, including 65 patients with sporadic colorectal cancer and 15 controls in whom a colorectal neoplasm was ruled out endoscopically. Normal mucosal samples were obtained at multiple consistently-selected locations using biopsy forceps during colonoscopy. Mutant allele-specific amplification (MASA)-PCR was performed; this could detect a K-ras mutation in normal colonic mucosa even though it was only sparsely present. The K-ras mutation was found in histologically normal mucosa from colorectal cancer patients (20 of 65 cases; 41 of 390 loci) by MASA-PCR, especially frequent (51%; 19 of 37 cases) when the tumor showed a K-ras mutation. In contrast, no mutation was found in normal mucosa from 15 controls (90 loci). K-ras mutation in normal mucosa showed a significant association with the presence of colorectal cancer (p = 0.008). The specificity of the MASA-PCR method for colorectal neoplasms was thus 100%. We conclude that detection of K-ras mutations in normal colonic mucosa might serve as a high-specificity approach to colorectal cancer.     

Clinico-Pathological Patterns and Survival Outcome of Colorectal Cancer in Young Patients: Western Saudi Arabia Experience

Background: The prognosis of young colorectal cancer (CRC) patients has been addressed by several studies but with contradictory results. The aim of the present study was to evaluate the clinico-pathological features of young Saudi patients with CRC in addition to displaying their survival outcome. Materials and Methods: In this retrospective study, young CRC patients (≤ 40 years) diagnosed between 2007 and 2011 from 4 centres in western Saudi Arabia, were included. Clinico-pathological features, tumor markers, dates of disease relapse and death were collected. Survival parameters were compared with those of older Saudi patients, reported in previous studies. Results: One hundred and sixteen young patients with CRC were identified (32.2% rectal, 67.8% colon). Some 44% were metastatic while 32.7% had stage III at diagnosis. Patients with grade 3 tumorsmade up 29.4% of the total while 49.5% had positive lymphovascular invasion (LVI), 56% had a lymph node (LN) ratio ≥ 0.2 and 40.2% were K-ras mutant. Median disease-free survival (DFS) and overall survival (OS) in non-metastatic cases were 22.8 and 49.6 months respectively with better median DFS in K-ras wild compared to mutant patients (28.5 vs 20.9 months, p=0.005). In metastatic cases, median OS was 19.5 months. These survival outcomes are inferior compared to those of older Saudi patients reported in prior studies. Conclusions: Young CRC patients present more commonly with advanced stage and a high incidence of adverse prognostic factors such as LVI and high LN ratio. Young CRC patients seem to have worse survival compared to older Saudi patients.     

基于GEPIA数据库分析SLC52A3在结直肠癌组织中的表达及临床病理意义

目的:通过研究SLC52A3在结直肠癌(CRC)组织中的表达及其与临床病理学参数的相关性,阐明其在CRC预后中的重要作用。方法:运用GEPIA大数据分析SLC52A3在正常结直肠组织和结直肠癌组织中的表达差异和其对胃癌预后的影响。收集2018年6月-2020年6月经手术切除的CRC患者石蜡标本84例。应用免疫组化方法检测84例CRC患者手术切除石蜡标本中SLC52A3表达情况和细胞定位。分析SLC52A3的表达与CRC临床病理指标的相关性。结果:数据库分析结果表明,与正常的结直肠组织相比,SLC52A3在结直肠癌组织中的表达显著提高,免疫组化结果显示,SLC52A3蛋白阳性信号定位于细胞膜。SLC52A3蛋白在84例CRC组织中低表达26例,高表达58例;在对应的84例CRC癌旁组织中9例呈高表达,75例为低表达,SLC52A3蛋白在CRC组织中的表达显著高于癌旁组织(P<0.01)。SLC52A3表达水平与CRC的肿瘤部位、肿瘤病理分化程度、浸润深度、TNM分期都明显相关,差异具有统计学意义(P<0.01)。在生存预后方面：SLC52A3高表达组的结直肠癌患者的生存率较高...     

Disseminated colorectal tumor cells in organs prone to metastasis detected by new double enriched nested-PCR in comparison with recognized assays

Hypothetically, K-ras mutations can be used as a marker of disseminated tumor cells (DTCs) in patients with K-ras mutated primary carcinoma. This study focused on the development of a useful assay for detecting low numbers of DTCs in potential target tissues of metastatic K-ras codon 12 mutated colorectal cancer. Tumor, liver, lymph node and bone marrow tissues from 46 colorectal carcinoma patients were examined for K-ras codon 12 mutations with a new double enriched nested (DEN)-PCR and the incidence of mutations was compared to those obtained from three established assays. DEN-PCR followed by sequencing found one mutated cell within 107 K-ras codon 12 wild-type cells and was more sensitive than other methods (1:106-1:102). Colon carcinomas (26/46) and adenomas (1/3) harbored mutations in K-ras codon 12. Sixteen of these 27 mutated tumors were found with all assays, two with three methods and one with the two most sensitive assays. In 8 cases, only DEN-PCR identified the K-ras mutation, and thus prevailed over the other methods used (p<0.002). Eight of 26 patients with K-ras mutated colorectal carcinoma also harbored K-ras mutated DTCs in liver and lymph nodes, respectively, and 4 in bone marrow. For liver and lymph node samples, DTC-mutations were identical to those in the primary carcinoma but those in bone marrow differed from the respective mutation in the primary carcinoma. In conclusion, DEN-PCR is a highly sensitive method for detecting K-ras mutations as marker of early and late tumor cell dissemination in tissues potentially harboring colorectal carcinoma metastases.     

Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes

Inaccurate staging of colorectal cancer (CRC) has been attributed to the failure to detect lymph node metastases by conventional pathology. We have previously reported the use of lymphatic mapping to accurately identify those lymph nodes most likely to harbor micrometastatic disease and permit focused pathologic examination. Mutation of K-ras allele at codons 12 or 13 occurs frequently in early stages of CRC development. The purpose of our study was to assess sentinel lymph nodes (SLN) for occult CRC micrometastases using a unique peptide nucleic acid (PNA) clamp PCR assay specific for K-ras mutations. Seventy-two paraffin-embedded primary CRC and paired SLN were evaluated by PNA clamp PCR for K-ras mutations. Thirty primary tumors (42%) were positive for K-ras mutations, and in 5 of these cases the SLN were positive for metastases by Hematoxylin and Eosin staining. PNA clamp PCR identified occult metastases in an additional 6 patients, upstaging 24% of K-ras positive primary CRCs (p = 0.014). No K-ras mutations were detected among the 20 noncancer lymph nodes assessed. This study demonstrates the utility, specificity and sensitivity of PNA clamp PCR assay in identifying occult micrometastases in the SLN of CRC patients by single-base mutation analysis.