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.     

Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer

A new method for detecting circulating tumor cells that is based on magnetic-activated cell separation (MACS) and nested mutant allele-specific amplification (nested MASA) was evaluated in patients with colorectal cancer using the p53 and K-ras genes as genetic markers. By negative selection with anti-CD45 monoclonal antibody-conjugated supermagnetic microbeads, the proportion of tumor cells was enriched 9-fold. By the combination of MACS and nested MASA, 10 tumor cells in 10(7) normal peripheral blood mononuclear cells could be detected without false-positives. Using this method, we examined blood taken from the tumor drainage veins of 23 patients with colorectal cancer. Eighty-seven percent (20/23) of primary tumor tissues showed p53 and/or K-ras gene mutations. Forty-five percent (9/20) of patients with p53 and/or K-ras mutations in the primary tumor showed the same mutated genes in the blood samples. There was a significant association between the presence of p53 and K-ras gene mutation in the blood and tumor size, depth of invasion, and venous invasion. Blood gene mutation was detected in 80% (4/5) of samples from patients with synchronous liver metastases. Sixty percent (3/5) of patients with mutant genes in the blood developed asynchronous liver metastases after surgery. The overall survival of patients with p53 and/or K-ras gene mutation-positive findings in blood was significantly shorter than that of patients testing negative on Kaplan-Meier analysis. Our results suggest that the method may be useful for reliable detection of tumor cells circulating in the blood and may help to identify patients at high risk for relapse.     

Evaluation of p53 alterations in occult lymph node metastases

BACKGROUND AND OBJECTIVES: This study was designed to evaluate p53 alterations in occult lymph node metastases. METHODS: We examined 41 patients with stage I non-small-cell lung cancer. We investigated p53 gene mutation by polymerase chain reaction and single-strand conformation polymorphism analysis of exons 5-8, p53 protein accumulation by immunostaining with monoclonal antibody DO-7, and detection of tumor cells in lymph nodes by immunohistochemistry with monoclonal antibodies to cytokeratin (CK). RESULTS: p53 gene mutation was detected in 34% of tumors and nuclear p53 accumulation in 46%. CK-positive cells in the hilar and mediastinal region lymph nodes were detected in 43.9% of patients and 29.3%, respectively. Of the 14 cases with p53 mutation and the 19 cases with p53 accumulation, 12 and 15 had micrometastases in the hilar or mediastinal lymph nodes, respectively. However, p53 alterations were not significantly associated with occult lymph node metastases. In cases with occult lymph node metastases, the 5-year survival was 81. 9% for the p53 wild-type group and 45.8% for the p53 mutation group. CONCLUSIONS: p53 alterations are not correlated with occult lymph node metastases, while p53 gene mutation is considered to be an unfavorable prognostic marker in patients with occult lymph node metastases. J. Surg. Oncol. 2000;73:143-147.     

Association of K-ras mutations with liver metastases from colorectal carcinoma

BACKGROUND: Colorectal carcinomas were studied regarding early proof of liver metastases through determination of K-ras mutations. PATIENTS AND METHODS: Seventy-seven colorectal carcinomas were investigated for the presence of point mutations in codon 12 and 13 of the K-ras gene, using single-strand conformation polymorphism (SSCP) analysis and direct sequencing. RESULTS: Twenty-six carcinomas were positive for K-ras mutations, of which 21 had codon 12 and 5 had codon 13 mutations. Twenty patients with K-ras-positive tumor (20 out of 26: 77%) developed liver metastases, of which 13 had simultaneous metastases and 7 had metachronous metastases. There was a significant association between K-ras mutations and liver metastases (p=0.03). A multivariate logistic regression model demonstrated that the involvement of lymph node (p<0.01) and K-ras mutations (p=0.02) were predictive factors for liver metastases from colorectal carcinoma. Sequencing in carcinomas without liver metastases showed the base change in the first position of codon 12, whereas with liver metastases it showed significantly frequent base change in the second position of codon 12 (p<0.01). CONCLUSION: It is suggested that the presence of K-ras mutation, especially base change in the second position of codon 12, may predict liver metastases from colorectal carcinoma.     

Quantitative detection of mutant alleles of the K-ras gene with minor groove binder-conjugated fluorogenic DNA probes

Tumor-specific point mutations are stable biomarkers compared with tumor-specific mRNA expression, and are therefore useful to detect occult tumor cells. These mutations have never been used for real-time quantitative polymerase chain reaction (RQ-PCR) assays, because the ability of conventional probes to discriminate between wild-type and mutant alleles is poor. Recently, DNA probes with conjugated minor groove binder (MGB) have been developed. Because of their high melting temperature, these probes achieve high performance in detecting single nucleotide mismatches. Using the MGB technology, we developed a new RQ-PCR system for detecting occult tumor cells in patients with colorectal cancer (CRC), targeting K-ras point mutations. Sixteen MGB-conjugated DNA probes were designed for all previously reported K-ras mutations. The performance of these probes was examined with plasmid DNAs into which K-ras point mutations had been inserted, 32 cancer cell lines and 338 lymph nodes obtained from 15 CRC patients. Fifteen of the 16 MGB probes designed were useful for accurate quantitative assessment, and achieved high sensitivity (1/10(4)-10(5) background cells) and high reproducibility (coefficients of variation <10%). Performance in discriminating single nucleotide mismatches was superior for MGB probes compared with non-MGB probes. We detected a micrometastasis (5.85/10(4) cells equivalent) in one (0.9%) of 110 lymph nodes obtained from 6 patients with K-ras mutations. There was no true false-positive result in 209 lymph nodes obtained from 9 patients without K-ras mutations. The MGB RQ-PCR assay targeting K-ras mutations is an accurate quantitative method for detecting occult tumor cells in CRC.     

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.     

Simultaneous mutations in K-ras and TP53 are indicative of poor prognosis in sporadic colorectal cancer

Despite the fact that the mutations in K-ras codon 12 and TP53 are common abnormalities in colorectal cancer, the determination of K-ras mutation combined with TP53 gene mutation, with diagnostic and prognostic purposes is still controversial. We have analyzed K-ras and TP53 mutations in 77 sporadic colorectal adenocarcinomas by means of polymerase chain reaction and sequencing. We observed a negative correlation between both K-ras and TP53 mutations. Patients with mutations in K-ras but not in TP53 exhibited worse survival rates than those with mutations in TP53 and not in K-ras. Moreover, we found the worst outcome in patients with mutations in both K-ras and TP53. These results may relate to the previously published data about primary human and rodent cells, in which transformation by Ras require either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. In conclusion, simultaneous mutations in K-ras and TP53 are indicative of a worse prognosis in sporadic colorectal cancer.     

Assessment of K-ras, Smad4 and p53 gene alterations in colorectal metastases and their role in the metastatic process

To date there is no genetic marker that gives accurate information on the prognostic impact for patients with colorectal cancer. A particular clone, not detected in the tumor, could be responsible for the metastatic process. To overcome this problem, genetic alterations were analyzed in metastatic tissues from 58 patients who developed metastases after curative surgery for colorectal cancer. K-ras, p53 and Smad4 alterations were observed in respectively 38, 60 and 27% of the metastases. These frequencies are similar to the ones reported in primary colorectal tumors. Thus, these genetic alterations cannot be used as prognostic biomarkers in patients with colorectal cancer. The metastases were stratified into 3 groups, according to the metastatic localization. K-ras mutations were detected in respectively 75, 26 and 11% of the distant, peritoneal and liver metastases. Loss of Smad4 expression was observed more frequently in the liver (62%) than in other metastases (13%). These results suggest that the genetic changes of the tumor cells indicate the location of the metastases and thus, the route of metastatic spread.     

Sensitive detection of K-ras mutations augments diagnosis of colorectal cancer metastases in the liver.

Postoperative survival of colorectal cancer patients is often delineated by metastases spreading to the liver. Current clinical diagnostic procedures are unable to discover micrometastases in this organ. Our aim was to develop a diagnostic tool for detecting micrometastases that are present at the time of surgery. Therefore, a PCR-RFLP assay was set up tracking point mutations of the K-ras oncogene at codons 12 and 13, based on mismatch primers and restriction enzymes BstXI and XcmI. The detection limit of this assay was one mutant in one million wild-type cells. One hundred forty-two patients with colorectal carcinoma were screened for these mutations in tissue samples from their tumor, proximally adjacent mucosa, and liver. Of these, 67 patients (46%) were positive for a K-ras mutation, of which 58 had codon 12 and 9 had codon 13 mutations. No patient without a K-ras-positive tumor showed a mutation in mucosa, but 11 patients with a K-ras-positive tumor (11 of 58; 19%) were found to bear a K-ras mutation in their mucosa, and in 21 patients (21 of 64; 33%), a K-ras mutation was detected in liver tissue. Sequencing of all mutated samples revealed a 92% confirmation of PCR-RFLP results. In summary, the assay is a useful tool for detecting K-ras codon 12 and 13 mutations and allows early proof of molecular determinants of liver metastases. Such knowledge will improve the staging of colorectal cancer patients and could beneficially influence their prognosis if followed by an effective therapy.     

P53 mutation analysis of colorectal liver metastases: relation to actual survival, angiogenic status, and p53 overexpression.

PURPOSE: To correlate TP53 mutations with angiogenic status of the tumor and prognosis after liver surgery in patients with colorectal liver metastases and to correlate immunohistochemical staining of p53 protein with TP53 gene mutations. EXPERIMENTAL DESIGN: Tumors of 44 patients with surgically treated colorectal liver metastases were analyzed for (a) TP53 mutations using denaturing gradient gel electrophoresis followed by sequencing, (b) microvessel density using the hot spot overlap technique, (c) apoptotic rate in tumor cells and endothelial cells of tumor microvessels using double immunostaining for anti-cleaved caspase 3 and anti-CD34, and (d) expression of p53 protein using immunohistochemistry. RESULTS: TP53 mutations were detected in 36% of the metastases and occurred more frequently in liver metastases from left-sided colon tumors than from right-sided colon tumors (P = 0.04). In metastases with TP53 mutations, microvessel density was higher compared with tumors with wild-type p53. Endothelial cell apoptosis was not different in tumor microvessels from TP53-mutated versus nonmutated tumors. The 5-year actual survival was not influenced by TP53 mutational status, microvessel density, or endothelial cell apoptotic rate of the tumors. Based on immunohistochemical p53 overexpression, the positive and negative predictive values of TP53 mutations were 61% and 82%. CONCLUSIONS: In patients with surgically treated colorectal liver metastases, TP53 mutations and angiogenic status did not influence prognosis. Immunohistochemistry is not a reliable technique for detecting TP53 mutations.     

Multiple detection of genetic alterations in tumors and stool.

Detection of genetic alterations in exfoliated intestinal cells in stool could represent an alternative, noninvasive tool for the screening of colorectal tumors. To verify this, we analyzed p53 and K-ras mutations and microsatellite instability on 46 cases of colorectal cancer and compared the presence of molecular alterations in tumor tissue and stool samples from individual patients. p53 exons 5-8 and K-ras exons 1-2 were analyzed by denaturing gradient gel electrophoresis. For the microsatellite instability, a set of 5 microsatellite markers (D2S123, D5S346, D17S250, BAT25, and BAT26) was evaluated. In the 18 healthy individuals, no genetic alterations in either tissue or stool were detected. p53 mutations were detected in 17 (37%), K-ras alterations in 15 (33%), and microsatellite instabilities in 5 (11%) of the 46 tumors analyzed. In a side study, we analyzed the correlation in genetic alteration profiles between tumors and macroscopically normal or healthy tissue from the same patient. The presence of at least one molecular alteration in tumor was observed in 31 (67%) of the cases. p53, K-ras mutations, and microsatellite instabilities were detected in stool samples in 18, 40, and 60% of patients with tumors harboring the same alterations. Due to the largely complementary presence of p53 and K-ras mutations in tumors, the use of highly sensitive procedures for stool analysis could offer a means competitive with colonoscopy and the fecal occult blood test.     

Determination of microsatellite instability, p53 and K-RAS mutations in hepatic metastases from patients with colorectal cancer: relationship with response to 5-fluorouracil and survival

In vitro and clinical studies have suggested that high-frequency microsatellite instability (MSI-H) phenotype, p53 and K-ras mutations might influence the response to chemotherapy in a variety of tumors, including primary colorectal cancers (CRC). Unresectable hepatic metastases from CRC are commonly treated with 5-fluorouracil (5FU) and folinic acid. Since several new active drugs are now used for treating CRC, molecular determinants predictive to response to 5FU would thus be crucial for optimizing indications of chemotherapy to those patients. MSI-H phenotype, p53 and K-ras status were characterized in a prospective study of 56 patients with CRC metastatic to the liver and treated with 5FU-based chemotherapy. The objective response rate after a 3-month treatment was 32.1%. The prevalence of p53 mutations, K-ras mutations and MSI-H phenotype was 62.5%, 30.3% and 1.8%, respectively. No significant association was found between response to chemotherapy and p53 mutations (78% mutated tumors in responders vs. 55% in nonresponders; p = 0.10) and K-ras mutations (39% mutated tumors in responders vs. 26% in nonresponders; p = 0.34). Survival was longer for patients with p53-mutated metastases than for patients with unresected wild-type p53 metastases (median survival 15 months vs. 17 months; p = 0.06). The determination of the MSI-H phenotype, p53 and K-ras status in hepatic metastases from CRC does not discriminate a group of patients that should preferentially benefit from 5FU-based chemotherapy. The prognosis of patients with treated liver metastases is better when p53 is mutated.     

P53 mutations in primary tumors and subsequent liver metastases are related to survival in patients with colorectal carcinoma who undergo liver resection

BACKGROUND: The appearance of p53 mutations in colorectal carcinoma was determined, independent of differentiation and tumor stage of the primary tumors, in relation to the survival of patients who were scheduled to undergo liver resection. METHODS: Tumor material was analyzed for p53 mutations in primary colorectal tumors and subsequent liver metastases from 41 consecutive patients who were scheduled to undergo surgical liver resection. DNA sequencing and immunohistochemical staining of p53 protein within tumor nuclei were performed. RESULTS: Primary tumors displayed p53 mutations within exons 5-9 in 41% of patients. No mutations were found in exons 4, 10, or 11. Forty-one percent of metastatic lesions had the same single mutation that was found in the primary tumor, whereas 11% of metastatic lesions had one additional mutation within exons 5-9; 22% had mutations only in their liver metastases, whereas corresponding primary tumors displayed wild-type p53. None of the patients had mutated p53 in their primary tumor and wild type in their metastases. Survival after undergoing liver resection was correlated negatively (P < 0.05-0.01) with Duke Stages A-D classification of the primary tumors, tumor differentiation, and radicality (> 0.7-0.8 mm) of resected liver metastases. CONCLUSIONS: The presence of p53 mutations in patients with metastatic lesions was related significantly (P < 0.003) to better survival after the patients underwent liver resection compared with patients with wild type p53 in their metastatic lesions. This finding was not related to covariates, such as Duke classification, tumor differentiation, type of liver metastasis, or metastatic radicality during resections. Explanations for this unexpected finding remain unclear, although the authors speculate that occult tumor cells with p53 mutations may be less responsive to growth factor(s) exposure during hepatic regeneration after resection.     

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。     

K-ras codon 12 and 13 mutations are correlated with differential patterns of tumor cell dissemination in colorectal cancer patients

The aim of this prospective study was to relate the incidences of cytokeratin 20 (CK20) and guanylylcyclase C (GCC) in lymph node, liver, and bone marrow specimens of 245 colorectal cancer (CRC) patients with the K-ras oncogene status of the corresponding primary tumor. Qualitative RT-PCR detection of CK20 and GCC mRNA was used as marker of circulating epithelial cells (CEC). Samples were considered positive for CEC only when both markers were detected concomitantly. For the detection of K-ras mutations, a PCR-RFLP assay was used. In the group with K-ras mutated primary carcinomas (n=92), CEC were detected in 62% of lymph node-, 43% of liver-, and 2% of bone marrow samples. No statistical significance was found when comparing these results with those from patients with K-ras wild-type carcinoma (59%, 46%, and 0%, respectively). In contrast to this combined evaluation, separate analysis of K-ras codons 12 (n=75, 82%) and 13 (n=17, 18%) revealed significantly differing CEC incidences. Lymph node specimens from corresponding K-ras codon 13 mutated carcinomas showed a significantly higher CEC incidence (82%) than the groups with codon 12 mutation (57%, p<0.05) or K-ras wild-type sequence (59%, p<0.05). Unlike these findings in lymph nodes, liver biopsies from corresponding carcinomas with K-ras codon 12 mutation or wild-type sequence were significantly more often positive for CEC (31% and 29%) than specimens from K-ras codon 13 mutated primary CRC (12%, p<0.04, respectively). In conclusion, colorectal carcinomas with K-ras codon 12 mutation showed the same pattern of tumor cell dissemination as their K-ras wild-type counterparts. Since K-ras codon 12 mutations prevailed 4-fold over codon 13 mutations, combined analysis of the two codons showed the same result. However, sub-analysis of patients with K-ras codon 13 mutation revealed that the respective CEC incidence was significantly increased in lymph nodes, but decreased in liver biopsies.     

Molecular staging individualizing cancer management

Although the most important prognostic and predictive marker in colorectal cancer is tumor cells in lymph nodes, approximately 30% of patients who are node-negative die from occult metastases. Molecular staging employing specific markers and sensitive detection technologies has emerged as a powerful platform to assess prognosis in node-negative colon cancer. Integrating molecular staging into algorithms that individualize patient management will require validation and the definition of relationships between occult tumor cells, prognosis, and responses to chemotherapy.     

Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis.

Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.     

Detection of tumor DNA in serum of colorectal cancer patients.

We previously found p16 promoter methylation in DNA in the sera of 13 colorectal cancer patients out of 44 (30%) whose tumor DNA exhibited the methylation, using methylation-specific PCR (MSP). To examine whether the cancer detection rate could be improved by using a different tumor marker, we examined the K-ras status in 90 colorectal cancer patients using a mismatch ligation assay. Among the 31 patients showing K-ras gene mutations in their tumors, the same mutations were observed in serum DNA of 11 patients (35%). Among the 90 patients, 63 showed tumors positive for K-ras mutation or p16 promoter methylation, or both, and 22 had serum DNA positive for one or both. K-ras mutation was found even in serum DNA of patients with Dukes A cancer, suggesting that colorectal cancer might be detected even in patients without symptoms by using this ligation assay.     

Detection of mutated K12-ras in histologically negative lymph nodes as an indicator of poor prognosis in stage II colorectal cancer

Stage II colorectal carcinoma is characterized by negative lymph node pathology as determined by conventional microscopic examination. These patients generally do not receive adjuvant therapy although 20%-30% will die from metastatic disease. To determine whether K-ras mutations at codon 12 could be used as a sensitive indicator of occult lymph node metastasis in stage II colon carcinoma, a retrospective study was performed using restriction endonuclease-mediated selective polymerase chain reaction (REMS-PCR) amplification. Of 106 colonic tumors analyzed, 46 were identified as positive for a K12-ras mutation in the primary tumor. Multiple lymph node samples from 38 of these 46 patients were examined by a sensitive nested PCR protocol for the presence of a K12-ras mutation. Of these 38 patients, 14 had 1 or more positive lymph nodes by PCR (37%) and 24 were negative for the mutation (63%). Of the 14 patients with a K12-ras mutation detected in lymph nodes, 8 died of the disease within 5 years (57%) compared to only 4 of the 24 patients with ras-negative lymph nodes (17%). The difference in time to death from disease, stratified using K12-ras status of lymph nodes, was statistically significant (P = 0.036; log-rank test). These results suggest K-ras mutation status of lymph nodes in patients with stage II colon cancer might identify a subgroup of patients who are more likely to develop recurrent and/or metastatic disease and benefit from adjuvant therapy. Larger studies are indicated to determine whether detection of K-ras mutation positivity in histologically negative lymph nodes portends a poor prognosis and to determine whether more aggressive use of adjuvant therapy is warranted.     

Frequent detection of K-ras mutation in stool samples of colorectal carcinoma patients after improved DNA extraction: comparison with tissue samples

Fecal occult blood testing is widely used in the clinical screening of colorectal tumors. However, this method has so frequent false-positive results that more accurate screening-strategy should be established. Although the molecular screening using K-ras gene mutation in stools has been attempted to improve the results, the low rate of DNA extraction from stools leaves this measurement under utility value. In this study, we investigated whether or not our applied DNA extraction method from stools could produce enough DNA for the molecular screening of colorectal tumors by K-ras gene mutations in stools. We applied cetyltrimethylammonium bromide (CTAB) solution to improve human DNA extraction from stools and a mutant-allele-sensitive amplification (MASA) method to detect K-ras mutation within codon 12. We were able to confirm the stool DNA by identifying K-ras fragments in all the 20 patients. Tissue K-ras mutation was identified in 4 (2 cancers and 2 adenomas) of 20 patients. Stool K-ras mutations were found in 6 patients, 3 tissue K-ras mutation positive patients (2 cancers and an adenoma) and 3 tissue K-ras mutation negative patients. These results indicate that it is possible to extract enough DNA from human stool samples of all patients with colorectal tumors for K-ras mutation studies. K-ras mutations are more frequently detected in stools than in resected colorectal tumors. This study indicates that K-ras mutation screening in stools for colorectal cancer may include not only a primary colorectal cancer but also precancerous lesions in all parts of a gastrointestinal tract.