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Połeć A, Ekstrøm PO, Fougner C, Sørlie T, Norum JH. Rapid assessment of 3-dimensional intra-tumor heterogeneity through cycling temperature capillary electrophoresis. BMC Res Notes 2023; 16:167. [PMID: 37568187 PMCID: PMC10416412 DOI: 10.1186/s13104-023-06437-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2022] [Accepted: 07/20/2023] [Indexed: 08/13/2023] Open
Abstract
OBJECTIVE Tumors are heterogeneous three-dimensional masses populated by numerous cell types, including distinct sub-clones of cancerous cells. Various sub-clones within the same tumor mass may respond differently to cancer treatment, and intra-tumor heterogeneity contributes to acquired therapeutic resistance. Thus, one tissue biopsy will in most cases not be representative of the entire genetic landscape of a tumor mass. In this study, we aimed to establish an easily accessible, low cost method to address intra-tumor heterogeneity in three dimensions, for a limited number of DNA alterations. RESULTS This study includes analyses of the three-dimensional (3D) distribution of DNA mutations in human colon cancer and mouse mammary gland tumor tissue samples. We used laser capture microdissection for the unbiased collection of tissue in several XY-planes throughout the tumor masses. Cycling temperature capillary electrophoresis was used to determine mutant allele frequency. High-resolution distribution maps of KRAS and Trp53 mutations were generated for each XY-plane in human and mouse tumor samples, respectively. To provide a holistic interpretation of the mutation distribution, we generated interactive 3D heatmaps giving an easily interpretable understanding of the spatial distribution of the analyzed mutations. The method described herein provides an accessible way of describing intra-tumor heterogeneity for a limited number of mutations.
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Affiliation(s)
- Anna Połeć
- Department of Cancer Genetics, Institute for Cancer Research, Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Per Olaf Ekstrøm
- Department of Tumor Biology, Institute for Cancer Research, Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Christian Fougner
- Department of Cancer Genetics, Institute for Cancer Research, Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Therese Sørlie
- Department of Cancer Genetics, Institute for Cancer Research, Radium Hospital, Oslo University Hospital, Oslo, Norway
- Institute for Clinical Medicine, University of Oslo, Oslo, Norway
| | - Jens Henrik Norum
- Department of Cancer Genetics, Institute for Cancer Research, Radium Hospital, Oslo University Hospital, Oslo, Norway.
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KRAS and BRAF somatic mutations in colonic polyps and the risk of metachronous neoplasia. PLoS One 2017; 12:e0184937. [PMID: 28953955 PMCID: PMC5617162 DOI: 10.1371/journal.pone.0184937] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2017] [Accepted: 09/01/2017] [Indexed: 01/09/2023] Open
Abstract
Background & aims High-risk features of colonic polyps are based on size, number, and pathologic characteristics. Surveillance colonoscopy is often recommended according to these findings. This study aimed to determine whether the molecular characteristics of polyps might provide information about the risk of metachronous advanced neoplasia. Methodology We retrospectively included 308 patients with colonic polyps. A total of 995 polyps were collected and tested for somatic BRAF and KRAS mutations. Patients were classified into 3 subgroups, based on the polyp mutational profile at baseline, as follows: non-mutated polyps (Wild-type), at least one BRAF-mutated polyp, or at least one KRAS-mutated polyp. At surveillance, advanced adenomas were defined as adenomas ≥ 10 mm and/or with high grade dysplasia or a villous component. In contrast, advanced serrated polyps were defined as serrated polyps ≥ 10 mm in any location, located proximal to the splenic flexure with any size or with dysplasia. Results At baseline, 289 patients could be classified as wild-type (62.3%), BRAF mutated (14.9%), or KRAS mutated (22.8%). In the univariate analysis, KRAS mutations were associated with the development of metachronous advanced polyps (OR: 2.36, 95% CI: 1.22–4.58; P = 0.011), and specifically, advanced adenomas (OR: 2.42, 95% CI: 1.13–5.21; P = 0.023). The multivariate analysis, adjusted for age and sex, also showed associations with the development of metachronous advanced polyps (OR: 2.27, 95% CI: 1.15–4.46) and advanced adenomas (OR: 2.23, 95% CI: 1.02–4.85). Conclusions Our results suggested that somatic KRAS mutations in polyps represent a potential molecular marker for the risk of developing advanced neoplasia.
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Palomba G, Cossu A, Paliogiannis P, Pazzola A, Baldino G, Scartozzi M, Ionta MT, Ortu S, Capelli F, Lanzillo A, Sedda T, Sanna G, Barca M, Virdis L, Budroni M, Palmieri G. Prognostic role of KRAS mutations in Sardinian patients with colorectal carcinoma. Oncol Lett 2016; 12:1415-1421. [PMID: 27446446 PMCID: PMC4950545 DOI: 10.3892/ol.2016.4798] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2015] [Accepted: 05/16/2016] [Indexed: 12/13/2022] Open
Abstract
The presence of mutations in the KRAS gene is a predictor of a poor clinical response to EGFR-targeted agents in patients affected by colorectal cancer (CRC), but its significance as a global prognostic factor remains unclear. The aim of the present study was to evaluate the impact of the KRAS mutational status on time to first metastasis (TTM) and overall survival (OS) in a cohort of Sardinian CRC patients. A total of 551 patients with metastatic CRC at the time of enrolment were included. Clinical and pathological features of the disease, including follow-up information, were obtained from medical records and cancer registry data. For mutational analysis formalin-fixed paraffin-embedded tissue samples were processed using a standard protocol. The coding sequence and splice junctions of exons 2 and 3 of the KRAS gene were screened for mutations by direct automated sequencing. Overall, 186 KRAS mutations were detected in 183/551 (33%) patients: 125 (67%) were located in codon 12, 36 (19%) in codon 13, and 18 (10%) in codon 61. The remaining mutations (7; 4%) were detected in uncommonly-affected codons. No significant correlation between KRAS mutations and gender, age, anatomical location and stage of the disease at the time of diagnosis was identified. Furthermore, no prognostic value of KRAS mutations was found considering either TTM or OS. When patients were stratified by KRAS mutational status and gender, males were significantly associated with a longer TTM. The results of the present study indicate that KRAS mutation correlated with a slower metastatic progression in males with CRC from Sardinia, irrespective of the age at diagnosis and the codon of the mutation.
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Affiliation(s)
- Grazia Palomba
- Institute of Biomolecular Chemistry, CNR, 07100 Sassari, Italy
| | - Antonio Cossu
- Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, 07100 Sassari, Italy
| | - Panagiotis Paliogiannis
- Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, 07100 Sassari, Italy
| | - Antonio Pazzola
- Oncology Unit, Local Health Unit (ASL1), 07100 Sassari, Italy
| | | | - Mario Scartozzi
- Department of Medical Oncology, University of Cagliari, 09042 Cagliari, Italy
| | - Maria Teresa Ionta
- Department of Medical Oncology, University of Cagliari, 09042 Cagliari, Italy
| | - Salvatore Ortu
- Oncology Unit, Local Health Unit (ASL2), 07026 Olbia, Italy
| | | | | | - Tito Sedda
- Oncology Unit, Local Health Unit (ASL), 09170 Oristano, Italy
| | - Giovanni Sanna
- Department of Medical Oncology, Hospital University (AOU), 07100 Sassari, Italy
| | - Michela Barca
- Oncology Unit, Local Health Unit (ASL4), 08045 Lanusei, Italy
| | - Luciano Virdis
- Oncology Unit, Local Health Unit (ASL7), 09013 Carbonia-Iglesias, Italy
| | - Mario Budroni
- Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, 07100 Sassari, Italy
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Mutation spectra of RAS gene family in colorectal cancer. Am J Surg 2016; 212:537-544.e3. [PMID: 27394063 DOI: 10.1016/j.amjsurg.2016.02.013] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Revised: 01/26/2016] [Accepted: 02/09/2016] [Indexed: 12/14/2022]
Abstract
BACKGROUND The clinicopathologic features and frequency of KRAS mutations in colorectal cancer (CRC) patients have been reported; however, the characteristics and impact of NRAS and HRAS mutations on the survival of CRC patients have seldom been addressed. METHODS Under institutional review board approval, 1,519 CRC patients who underwent surgery were enrolled. Mutation status of RAS was determined by polymerase chain reaction and mass spectrophotometry. RESULTS The frequency of KRAS, NRAS, and HRAS mutations was 39.6%, 4.3%, and 1.7%, respectively. The KRAS mutation was associated with fewer left-sided tumors, fewer poor differentiated tumors, more mucin component, and less lymphovascular invasion. The NRAS or HRAS mutations were not associated with any of the clinicopathologic features examined. After univariate analysis, only NRAS mutation was associated with patients' overall and disease-free survival. However, the association of NRAS with patients' overall and disease-free survival disappeared after stepwise elimination. CONCLUSIONS This study demonstrates the clinicopathologic characteristics of CRC patients with RAS mutations. Patients with NRAS mutation tended to have worse outcomes.
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Rosa B, de Jesus JP, de Mello EL, Cesar D, Correia MM. Effectiveness and safety of monoclonal antibodies for metastatic colorectal cancer treatment: systematic review and meta-analysis. Ecancermedicalscience 2015; 9:582. [PMID: 26557880 PMCID: PMC4631576 DOI: 10.3332/ecancer.2015.582] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2015] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND The effectiveness of chemotherapy (CT) for select cases of metastatic colorectal cancer (MCRC) has been well established in the literature, however, it provides limited benefits and in many cases constitutes a treatment with high toxicity. The use of specific molecular biological treatments with monoclonal antibodies (MA) has been shown to be relevant, particularly for its potential for increasing the response rate of the host to the tumour, as these have molecular targets present in the cancerous cells and their microenvironment thereby blocking their development. The combination of MA and CT can bring a significant increase in the rate of resectability of metastases, the progression-free survival (PFS), and the global survival (GS) in MCRC patients. OBJECTIVE To assess the effectiveness and safety of MA in the treatment of MCRC. METHODS A systematic review was carried out with a meta-analysis of randomised clinical trials comparing the use of cetuximab, bevacizumab, and panitumumab in the treatment of MCRC. RESULTS Sixteen randomised clinical trials were selected. The quality of the evidence on the question was considered moderate and data from eight randomised clinical trials were included in this meta-analysis. The GS and PFS were greater in the groups which received the MA associated with CT, however, the differences were not statistically significant between the groups (mean of 17.7 months versus 17.1 months; mean difference of 1.09 (CI: 0.10-2.07); p = 0.84; and 7.4 versus 6.9 months. mean difference of 0.76 (CI: 0.08-1.44); p = 0.14 respectively). The meta-analysis was not done for any of the secondary outcomes. CONCLUSION The addition of MA to CT for patients with metastatic colorectal cancer does not prolong GS and PFS.
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Affiliation(s)
- Bruno Rosa
- Instituto Nacional de Câncer, Rio de Janeiro 20230-130, Brazil
| | | | | | - Daniel Cesar
- Instituto Nacional de Câncer, Rio de Janeiro 20230-130, Brazil
| | - Mauro M Correia
- Instituto Nacional de Câncer, Rio de Janeiro 20230-130, Brazil
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Martinetti D, Costanzo R, Kadare S, Alimehmeti M, Colarossi C, Canzonieri V, Berretta M, Memeo L. KRAS and BRAF mutational status in colon cancer from Albanian patients. Diagn Pathol 2014; 9:187. [PMID: 25267307 PMCID: PMC4198623 DOI: 10.1186/s13000-014-0187-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2014] [Accepted: 09/07/2014] [Indexed: 11/15/2022] Open
Abstract
Background Numerous clinical studies have shown that anti-EGFR therapies are effective only in a subset of patients with colorectal cancer. Mutations in the KRAS and BRAF genes have been confirmed as negative predictors of the response to EGFR-targeted therapies. In this study we evaluated KRAS and BRAF status in 159 colorectal cancer samples obtained from the University of Tirana. Methods We evaluated KRAS mutations in codons 12, 13, 61, 146 and in codon 600 of BRAF by direct sequencing. 90 patients were male (57%) and 69 female (43%); the patients’ ages ranged from 17 to 85 (median 61.7). 24 patient were stage I, 36 stage II, 84 stage III and 15 stage IV. Results Out of the 159 cases, 28 (17,6%) showed KRAS mutation (13 G12D, 4 G12C, 4 G12V, 3 G12A, 2 G13 D, 1 G12S and 1 A146T), and 10 (6,3%) showed BRAF mutation (all V600E). No significant correlations between KRAS and BRAF mutations and various clinicopathological parameters was found. This is the first report of KRAS and BRAF status in Albanian patients with colorectal carcinoma (CRC) and though the relatively small sample size might not provide enough statistics power. Conclusions The results of KRAS and BRAF mutation analysis could be used in the selection of patients for anti-EGFR therapy. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_187
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[Clinical relevance of the K-ras oncogene in colorectal cancer: experience in a Mexican population]. REVISTA DE GASTROENTEROLOGÍA DE MÉXICO 2014; 79:166-70. [PMID: 25216999 DOI: 10.1016/j.rgmx.2014.07.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/06/2013] [Revised: 06/17/2014] [Accepted: 07/04/2014] [Indexed: 11/20/2022]
Abstract
BACKGROUND Colorectal cancer is frequent in the developed countries, with a cancer-specific mortality rate of 33%. Different biomarkers are associated with overall survival and the prediction of monoclonal treatment effectiveness. The presence of mutations in the K-ras oncogene alters the response to target therapy with cetuximab and could be an independent prognostic factor. AIMS To analyze the difference in survival between patients with mutated K-ras and those with K-ras wild-type status. METHODS Thirty-one clinical records were retrospectively analyzed of patients presenting with colorectal cancer that underwent K-ras sequencing through real-time polymerase chain reaction within the time frame of 2009 to 2012 at the Hospital de Alta Especialidad de Veracruz of the Instituto para la Salud y Seguridad Social de los Trabajadores del Estado (HAEV-ISSSTE). Survival analysis for patients with and without K-ras mutation was performed using the Kaplan Meier method. Contrast of covariates was performed using logarithmic transformations. RESULTS No statistically significant difference was found in relation to survival in the patients with mutated K-ras vs. those with K-ras wild-type (P=.416), nor were significant differences found when analyzing the covariants and survival in the patients with mutated K-ras: ECOG scale (P=.221); age (less than, equal to or greater than 65years, P=.441); clinical stage according to the AJCC (P=.057), and primary lesion site (P=.614). CONCLUSIONS No relation was found between the K-ras oncogene mutation and reduced survival, in contrast to what has been established in the international medical literature. Further studies that include both a larger number of patients and those receiving monoclonal treatment, need to be conducted. There were only 5 patients in the present study that received cetuximab, resulting in a misleading analysis.
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Cabrera-Mendoza F, Gainza-Lagunes S, Castañeda-Andrade I, Castro-Zárate A. Clinical relevance of the K-ras oncogene in colorectal cancer: Experience in a Mexican population. REVISTA DE GASTROENTEROLOGÍA DE MÉXICO (ENGLISH EDITION) 2014. [DOI: 10.1016/j.rgmxen.2014.10.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
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Abstract
The clinical benefits of BRAF inhibition in patients with advanced-stage BRAF-mutant melanoma are now well established. Although the emergence of cutaneous squamous-cell carcinomas (SCCs) and secondary melanomas in patients on BRAF-inhibitor therapy have been well described, reports are emerging of additional secondary premalignant and malignant events, including RAS-mutant leukaemia, the metastatic recurrence of RAS-mutant colorectal cancer and the development of gastric and colonic polyps. In most cases, paradoxical MAPK activation--resulting from the BRAF-inhibitor-mediated homodimerization and heterodimerization of nonmutant RAF isoforms--seems to underlie the development of these secondary tumours. Although evidence supports that therapy with the simultaneous administration of BRAF and MEK inhibitors abrogates the onset of treatment-induced SCCs, whether combination treatment will limit the emergence of all BRAF-inhibitor-driven pathologies is unclear. In this Review, we describe the clinical and mechanistic manifestations of secondary cancers that have thus far been observed to arise as a consequence of BRAF inhibition. We discuss the concept of pre-existing populations of partly transformed cells with malignant potential that might be present in various organ systems, and the rationale for novel therapeutic strategies for the management of BRAF-inhibitor-induced neoplasia.
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Manceau G, Laurent-Puig P. Potential Role of KRAS and Other Mutations in the Adjuvant Therapy of Colorectal Cancer. CURRENT COLORECTAL CANCER REPORTS 2012. [DOI: 10.1007/s11888-012-0133-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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Is K-ras gene mutation a prognostic factor for colorectal cancer: a systematic review and meta-analysis. Dis Colon Rectum 2012; 55:913-23. [PMID: 22810479 DOI: 10.1097/dcr.0b013e318251d8d9] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND : The K-ras gene is one of the commonly mutated oncogenes associated with colorectal cancer. However, its prognostic significance for patients with colorectal cancer remains inconclusive. OBJECTIVE : To derive a more precise estimation of the prognostic significance of K-ras gene mutations, a systematic review and meta-analysis were performed. DATA SOURCES : We searched PubMed, Embase, and the Cochrane databases from January 1992 to November 2011. STUDY SELECTION : The prognostic value of K-ras gene mutations was examined in patients with colorectal cancer who did not receive preoperative chemotherapy or radiation. MAIN OUTCOME MEASURES : The effect of K-ras gene mutations on the overall survival was measured by the HR and 95% CIs. RESULTS : The pooled HR for the association between K-ras gene mutations and overall survival in patients with colorectal cancer was 1.04 (95% CI: 0.99-1.10, p = 0.11). Subgroup analysis showed significant reductions in the overall survival associated with mutations at K-ras codon 12, the articles that reported HR directly, and the studies published before and after 2005, although publication bias was present. All the associations disappeared after adjustment with the trim-and-fill method. The pooled HR of 3 studies examining mutations at K-ras codon 13 was 1.47 (95% CI: 1.09-1.97, p = 0.02), and no publication bias was observed. No significant association was observed in different study regions. LIMITATIONS : The heterogeneity in the study populations is a potential problem, the use of different staging systems or small groups of different stages may contribute to heterogeneity, and residual confounding may have influenced the results in those studies that did not completely adjust for other factors. CONCLUSIONS : Overall K-ras gene mutations seem not to correlate with the prognosis of patients with colorectal cancer. The association remains to be confirmed with a more precise analysis of a large sample.
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Association of folate intake, dietary habits, smoking and COX-2 promotor -765G>C polymorphism with K-ras mutation in patients with colorectal cancer. J Egypt Natl Canc Inst 2012; 24:115-22. [PMID: 22929917 DOI: 10.1016/j.jnci.2012.05.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2011] [Accepted: 05/15/2012] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Understanding the role of environmental and molecular influences on the nature and rate of K-ras mutations in colorectal neoplasms is crucial. COX-2 polymorphisms -765G>C may play a role in carcinogenic processes in combination with specific life-style conditions or dependent on the racial composition of a particular population. If mutational events play an important role in colorectal carcinogenesis sequence, one can hypothesize that modification of these events by life-style or other factors would be a useful prevention strategy. AIM OF WORK To explore the association between K-ras mutation and potential variables known or suspected to be related to the risk of colorectal cancer (CRC) as well as determining the possible modulating effect of the COX-2 polymorphism, -765G>C. SUBJECTS AND METHODS The study was conducted on 80 patients with colorectal cancer from Tropical Medicine and Gastrointestinal Tract endoscopy Departments and those attending clinic of the National Cancer Institute, Cairo University during the period extending from April 2009 to March 2010. Full history taking with emphasis on the risk factors of interest, namely age, sex, family history, smoking and dietary history. Serum CEA and CA19-9, RBCs folic acid and occult blood in stool were done to all samples. K-ras protooncogene mutation at codon 12 (exon 1) and cyclooxygenase 2 (COX-2) -765G>C polymorphism were determined by PCR-RFLP. RESULTS The K-ras mutation was positive in 23 (28.7%) patients. COX-2 polymorphism revealed GG in 62.5%, GC in 26.2 % and CC genotype was found in 11.3 % of cases. The mean red blood cell folic acid level was lower in the K-ras positive group (100.96±51.3 ng/ml) than the negative group (216.6±166.4 ng/ml), (P<0.01). Higher folate levels were found in males than females (median=173 ng/ml and 85 ng/ml; respectively, P=0.002) with adjusted odds ratio (OR) of 0.984. Only, the RBCs folate (P=0.0018) followed by gender (P=0.036) contributed significantly in the discrimination between patients prone to develop K-ras mutation and those who are not. CONCLUSION RBC folic acid was significantly deficient in CRC (colorectal cancer) patients with K-ras mutations in comparison with CRC patients free of the mutations, suggesting that folic acid may be a risk factor for K-ras mutation development.
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Abstract
AIMS Epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) mutations have been identified as predictors of response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer. We aimed to screen the mutations of both genes in gastric carcinoma to detect the suitability of EGFR TKIs for patients with gastric carcinoma. METHODS We screened EGFR mutation in exons 19-21 and KRAS mutation in exon 2 in 58 gastric adenocarcinomas from China using high resolution melting analysis (HRMA). Positive samples were confirmed by DNA sequencing. RESULTS Three EGFR missense mutations (5.2%) and 22 single nucleotide polymorphisms (SNP, Q787Q, 37.9%) were identified. To our knowledge, we report for the first time three mutation patterns of EGFR, Y801C, L858R and G863D, in gastric carcinoma. Two samples with EGFR mutation were mucinous adenocarcinoma. These three samples were collected from male patients aged over 75 years old. The frequency of KRAS mutation was 10.3% (6/58). The exclusiveness of EGFR and KRAS mutations was proven for the first time in gastric cancer. CONCLUSIONS Gastric carcinoma of the mucinous adenocarcinoma type collected from older male patients may harbour EGFR mutations. The small subset of gastric adenocarcinoma patients may respond to EGFR TKIs.
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Stefanius K, Ylitalo L, Tuomisto A, Kuivila R, Kantola T, Sirniö P, Karttunen TJ, Mäkinen MJ. Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma. Histopathology 2011; 58:679-92. [PMID: 21457162 PMCID: PMC3107946 DOI: 10.1111/j.1365-2559.2011.03821.x] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2010] [Accepted: 07/05/2010] [Indexed: 12/12/2022]
Abstract
AIMS To define the occurrence of KRAS and BRAF mutations, microsatellite instability (MSI), and MGMT and hMLH1 methylation and expression in colorectal serrated adenocarcinoma. METHODS AND RESULTS KRAS codon 12/13 and 59/61 and BRAF V600E mutations, MSI, and MGMT and hMLH1 methylation and expression in 42 serrated adenocarcinomas and 17 serrated adenomas were compared with those in 59 non-serrated colorectal carcinomas (CRCs) and nine adenomas. KRAS and BRAF mutations were observed in 45% and 33% of serrated adenocarcinomas and in 27% and 0% of non-serrated CRCs (P < 0.001). The KRAS c12G→A transition was the predominant type of mutation in serrated adenocarcinomas. Forty-two per cent of BRAF-mutated serrated adenocarcinomas showed high-level MSI (MSI-H) (P = 0.075), 100% showed hMLH1 methylation (P = 0.001) and 90.9% showed MGMT methylation (P = 0.019). Fifty-six per cent of serrated adenocarcinomas with microsatellite stability/low-level microsatellite instability harboured KRAS mutations. In non-serrated cancers, KRAS mutations were not associated with MSI status. CONCLUSIONS A high combined mutation rate (79-82%) of KRAS and BRAF in serrated adenomas and adenocarcinomas indicates that mitogen-activated protein kinase activation is a crucial part of the serrated pathway. BRAF mutations are specific for serrated adenocarcinoma and identify a subset of serrated adenocarcinomas with gene methylation and a tendency for MSI-H. A high frequency of KRAS mutations in serrated adenocarcinomas suggests that a significant proportion of KRAS-mutated CRCs originate from serrated precursors, thus challenging the traditional model of Vogelstein.
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Affiliation(s)
- Karoliina Stefanius
- Department of Pathology, Institute of Diagnostics, University of Oulu, Oulu, Finland
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Shen H, Yuan Y, Hu HG, Zhong X, Ye XX, Li MD, Fang WJ, Zheng S. Clinical significance of K-ras and BRAF mutations in Chinese colorectal cancer patients. World J Gastroenterol 2011; 17:809-16. [PMID: 21390154 PMCID: PMC3042662 DOI: 10.3748/wjg.v17.i6.809] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2010] [Revised: 01/04/2011] [Accepted: 01/11/2011] [Indexed: 02/06/2023] Open
Abstract
AIM: To identify and assess mutations in the K-ras and BRAF genes in a cohort of Chinese patients with colorectal cancer (CRC) for their association with various clinicopathological parameters and prognosis.
METHODS: Genomic DNA was isolated from frozen tissues. Pyrosequencing analysis was conducted to detect mutations in the K-ras (codons 12, 13, and 61) and BRAF genes (codon 600). Statistical analysis was carried out using SPSS-15.0 software.
RESULTS: Among the 118 colorectal cancer patients, we detected 41 (34.7%) mutations in the K-ras gene. Mutation frequencies at codon 12 and codon 13 were 23.7% (28/118) and 10.2% (12/118), respectively. Only one patient harbored a point mutation at codon 61 (0.8%, 1/118). Gender was the only factor that showed an obvious relationship with K-ras gene mutation (female 44.7% vs male 28.2%, P = 0.037). Other clinicopathological features, such as age, location of the tumor, tumor differentiation, Tumor, Node and Metastases classification, and the Union for International Cancer Control staging, showed no positive relationship with K-ras gene mutations. No significant correlation was observed between the presence of K-ras mutations (codons 12, 13, and 61) and the survival of the patients. BRAF mutations were rare, and only two patients (1.7%) harbored a detectable mutation at codon 600.
CONCLUSION: K-ras gene mutation is a common event in our 118 Chinese CRC patients, with an obvious relationship with gender. However, it seems not to be an independent prognostic factor in CRC patients. The BRAF gene is rarely mutated in Chinese CRC patients.
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Wu WKK, Law PTY, Lee CW, Cho CH, Fan D, Wu K, Yu J, Sung JJY. MicroRNA in colorectal cancer: from benchtop to bedside. Carcinogenesis 2010; 32:247-53. [PMID: 21081475 DOI: 10.1093/carcin/bgq243] [Citation(s) in RCA: 118] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Colon carcinogenesis represents a stepwise progression from benign polyps to invasive adenocarcinomas and distant metastasis. It is believed that these pathologic changes are contributed by aberrant activation or inactivation of protein-coding proto-oncogenes and tumor suppressor genes. However, recent discoveries in microRNA (miRNA) research have reshaped our understanding of the role of non-protein-coding genes in carcinogenesis. In this regard, a remarkable number of miRNAs exhibit differential expression in colon cancer tissues. These miRNAs alter cell proliferation, apoptosis and metastasis through their interactions with intracellular signaling networks. From a clinical perspective, polymorphisms within miRNA-binding sites are associated with the risk for colon cancer, whereas miRNAs isolated from feces or blood may serve as biomarkers for early diagnosis. Altered expression of miRNA or polymorphisms in miRNA-related genes have also been shown to correlate with patient survival or treatment outcome. With further insights into miRNA dysregulation in colon cancer and the advancement of RNA delivery technology, it is anticipated that novel miRNA-based therapeutics will emerge.
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Affiliation(s)
- William K K Wu
- Institute of Digestive Diseases, The Chinese University of Hong Kong, Hong Kong, China
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17
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Kamisawa T, Horiguchi SI, Hayashi Y, Yun X, Yamaguchi T, Tsuruta K, Sasaki T. K-ras mutation in the major duodenal papilla and gastric and colonic mucosa in patients with autoimmune pancreatitis. J Gastroenterol 2010; 45:771-8. [PMID: 20157749 DOI: 10.1007/s00535-010-0211-y] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/12/2009] [Accepted: 01/15/2010] [Indexed: 02/04/2023]
Abstract
BACKGROUND Pancreatic cancer occurs in some patients with autoimmune pancreatitis (AIP). Significant K-ras mutations are frequently detected in the pancreas of AIP patients. AIP may be a pancreatic lesion of IgG4-related systemic disease. Gastric and colonic cancer can occur during the follow up of AIP patients. We examined K-ras mutations in the major duodenal papilla and gastric and colonic mucosa of AIP patients. METHODS K-ras analysis and/or immunohistochemical study was performed on the tissues of the major duodenal papilla (n = 8), gastric mucosa (n = 5), colonic mucosa (n = 3), pancreas (n = 5), common bile duct (n = 5), and gallbladder (n = 4) of 12 AIP patients. RESULTS Significant K-ras mutations were detected in the major duodenal papilla of 4 of 8 cases [GAT (n = 4)], in the gastric mucosa of 2 of 4 cases [AGT (n = 2)], and in the colonic mucosa of 2 of 3 cases [GAT (n = 2)]. Significant K-ras mutations were detected in the pancreas of all 5 cases [GAT (n = 5), in the common bile duct of 4 cases (GAT (n = 2), TGT (n = 1), and GCT/TGT (n = 1)], and in the gallbladder epithelium of 3 cases [GAT (n = 1), GCT (n = 1), and GTT (n = 1)]. K-ras mutations were detected in the organs associated with IgG4-related fibroinflammation with abundant infiltration of T lymphocytes and forkhead box P3-positive cells. CONCLUSIONS Significant K-ras mutations were frequently detected in the major duodenal papilla and gastric and colonic mucosa of AIP patients. AIP patients may have risk factors for gastric and colonic cancer, but the mechanisms of K-ras mutation and its clinical implications are not clear.
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Affiliation(s)
- Terumi Kamisawa
- Department of Internal Medicine, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8677, Japan.
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18
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Deschoolmeester V, Baay M, Specenier P, Lardon F, Vermorken JB. A review of the most promising biomarkers in colorectal cancer: one step closer to targeted therapy. Oncologist 2010; 15:699-731. [PMID: 20584808 PMCID: PMC3228001 DOI: 10.1634/theoncologist.2010-0025] [Citation(s) in RCA: 116] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2010] [Accepted: 05/01/2010] [Indexed: 02/06/2023] Open
Abstract
Rapidly growing insights into the molecular biology of colorectal cancer (CRC) and recent developments in gene sequencing and molecular diagnostics have led to high expectations for the identification of molecular markers to be used in optimized and tailored treatment regimens. However, many of the published data on molecular biomarkers are contradictory in their findings and the current reality is that no molecular marker, other than the KRAS gene in the case of epidermal growth factor receptor (EGFR)- targeted therapy for metastatic disease, has made it into clinical practice. Many markers investigated suffer from technical shortcomings, resulting from lack of quantitative techniques to capture the impact of the molecular alteration. This understanding has recently led to the more comprehensive approaches of global gene expression profiling or genome-wide analysis to determine prognostic and predictive signatures in tumors. In this review, an update of the most recent data on promising biological prognostic and/or predictive markers, including microsatellite instability, epidermal growth factor receptor, KRAS, BRAF, CpG island methylator phenotype, cytotoxic T lymphocytes, forkhead box P3-positive T cells, receptor for hyaluronic acid-mediated motility, phosphatase and tensin homolog, and T-cell originated protein kinase, in patients with CRC is provided.
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Affiliation(s)
- Vanessa Deschoolmeester
- Laboratory of Cancer Research and Clinical Oncology, Department of Medical Oncology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.
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19
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Wu CH, Shih YW, Chang CH, Ou TT, Huang CC, Hsu JD, Wang CJ. EP4 upregulation of Ras signaling and feedback regulation of Ras in human colon tissues and cancer cells. Arch Toxicol 2010; 84:731-40. [PMID: 20571779 DOI: 10.1007/s00204-010-0562-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2009] [Accepted: 05/07/2010] [Indexed: 01/24/2023]
Abstract
Previous studies indicate that COX-2 and prostaglandin E(2) (PGE(2)) receptor subtypes are involved in intestinal carcinogenesis and activation of downstream pathways. In this report, we try to understand the association of PGE(2) receptor and K-ras cellular mechanism during the development of colorectal cancer. We collected 21 colorectal cancer patients and compared the protein expression of tumor tissues and normal mucosa tissues by using immunoblot. Furthermore, we transferred empty vector and pcDNA-K-ras into Ras-HT29 colon cancer cells. Result showed that phosphorylation of Akt and EP(1)/EP(4) were over-expressed in the colorectal tumor tissue. K-ras induces HT29 cells proliferation through the expressions of COX-2, EP1/EP4, pAkt, GSK3beta and increases Tcf transcriptional factor activation. Additionally, Ras protein was suppressed when treated with EP(4) inhibitor in Ras-HT29 cell. In cell cycle assay, K-ras mutation causing cell cycle S phase was prolonged with an increase in the G2/M phase ratio. In conclusion, we suggested that Ras overexpression leads to cell proliferation through activating Ras/PI3K/GSK3beta/EP(4) PGE(2) receptor signals and caused a feedback regulation of Ras by EP4 in colorectal tumor progression.
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Affiliation(s)
- Cheng-Hsun Wu
- Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan, Republic of China
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20
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Abubaker J, Bavi P, Al-Haqawi W, Sultana M, Al-Harbi S, Al-Sanea N, Abduljabbar A, Ashari LH, Alhomoud S, Al-Dayel F, Uddin S, Al-Kuraya KS. Prognostic significance of alterations in KRAS isoforms KRAS-4A/4B and KRAS mutations in colorectal carcinoma. J Pathol 2009; 219:435-445. [PMID: 19824059 DOI: 10.1002/path.2625] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2009] [Accepted: 09/03/2009] [Indexed: 12/12/2022]
Abstract
Somatic KRAS mutation is an early well-known event in colorectal carcinogenesis but a complete understanding of RAS function and dysfunction in colorectal cancer is still to come. Our aim was to study the incidence of KRAS mutation; KRAS splice variants: KRAS4A and KRAS4B; and their relationships with various clinico-pathological characteristics in colorectal cancer (CRC).In this study, 285 CRC cases were analysed for KRAS mutation by direct DNA sequencing followed by immunohistochemical analysis after validation with real-time PCR assay, to study the protein expression of KRAS4A and -4B isoforms. KRAS gene mutations were seen in 80/285 CRCs (28.1%) and of the mutated cases, the majority of the mutations were seen in codon 12 (81.2%) as opposed to codon 13 (18.8%). CRCs with KRAS mutations were associated with a poor overall survival (p = 0.0009). Furthermore, KRAS mutations at codon 12 were associated with a poor overall survival of 64.4% at 5 years compared with a 5-year overall survival of 75.8% and 78.2% with codon 13 mutation and absence of KRAS mutations, respectively (p = 0.0025). KRAS4A protein expression was predominantly seen in the cytoplasm, while KRAS4B protein was nuclear. KRAS4A overexpression was significantly associated with left colon, histology subtype of adenocarcinoma, p27kip1, and cleaved caspase3 expression. Interestingly, KRAS4A overexpression was associated with a better overall survival (p = 0.0053). On the other hand, KRAS4B overexpression (33.2%) was significantly associated with larger tumour size (p = 0.0234) and inversely correlated with p27kip1 protein (p = 0.0159). Both KRAS mutation and KRAS4A were independent prognostic markers in a multivariate analysis with age, gender, stage, differentiation, and MSI status. Our results highlight the differential role of KRAS isoforms in CRC, their utility as a prognostic biomarker, and underline the importance of KRAS alterations as a potential therapeutic target for CRC.
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Affiliation(s)
- Jehad Abubaker
- Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Saudi Arabia
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21
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Neumann J, Zeindl-Eberhart E, Kirchner T, Jung A. Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathol Res Pract 2009; 205:858-62. [PMID: 19679400 DOI: 10.1016/j.prp.2009.07.010] [Citation(s) in RCA: 266] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2009] [Accepted: 07/09/2009] [Indexed: 02/07/2023]
Abstract
Mutation analysis of the KRAS oncogene is now established as a predictive biomarker in colorectal cancer (CRC). Large prospective clinical trials have shown that only CRCs with wild-type KRAS respond to anti-epidermal growth factor receptor (EGFR) treatment. Therefore, mutation analysis is mandatory before treatment, and reliable benchmarks for the frequency and types of KRAS mutations have to be established for routinely testing large numbers of metastatic CRCs. A thousand and eighteen cases (879 primary tumors and 139 metastases) of metastatic colorectal cancer were analyzed for the KRAS mutational status of codons 12 and 13 of the KRAS gene by genomic sequencing in a routine setting. Results were analyzed separately for specimens derived from primary tumors and metastases. KRAS mutations in codons 12 and 13 were present in 39.3% of all analyzed CRCs. The most frequent types of mutations were glycine to aspartate on codon 12 (p.G12D, 36.0%), glycine to valine on codon 12 (pG12V, 21.8%), and glycine to aspartate on codon 13 (p.G13D, 18.8%). They account for 76.6% of all mutations and prevail in primary tumors and distant metastases, indicating a robustness of the KRAS mutational status during neoplastic dissemination. The frequency of KRAS mutations and the preponderance of three types of mutations in codons 12 and 13 in a large, unselected cohort of metastatic CRC confirm the previous data of small and selected CRC samples. Thus, a mutation frequency of 40% and a cluster of three mutation types (p.G12D, pG12V, and p.G13D) in primaries and metastases can be defined as benchmarks for routine KRAS analyses.
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Affiliation(s)
- Jens Neumann
- Department of Pathology, Ludwig-Maximilians Universität München, Thalkirchner Strasse 36, 80337 Munich, Germany.
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22
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Effect of COX2 -765G>C and c.3618A>G polymorphisms on the risk and survival of sporadic colorectal cancer. Cancer Causes Control 2009; 20:1421-9. [DOI: 10.1007/s10552-009-9368-1] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2009] [Accepted: 05/11/2009] [Indexed: 12/01/2022]
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23
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van Krieken JHJM, Jung A, Kirchner T, Carneiro F, Seruca R, Bosman FT, Quirke P, Fléjou JF, Plato Hansen T, de Hertogh G, Jares P, Langner C, Hoefler G, Ligtenberg M, Tiniakos D, Tejpar S, Bevilacqua G, Ensari A. KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program. Virchows Arch 2008; 453:417-31. [PMID: 18802721 DOI: 10.1007/s00428-008-0665-y] [Citation(s) in RCA: 220] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2008] [Revised: 08/21/2008] [Accepted: 08/21/2008] [Indexed: 12/21/2022]
Abstract
Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30-40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.
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Affiliation(s)
- J H J M van Krieken
- Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, 6500, HB, The Netherlands.
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24
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Abstract
Even though liver metastasis accounts for the vast majority of cancer deaths in patients with colorectal cancer (CRC), fundamental questions about the molecular and cellular mechanisms of liver metastasis still remain unanswered. Determination of gene expression profiles by microarray technology has improved our knowledge of CRC molecular pathways. However, defined gene signatures are highly variable among studies. Expression profiles and molecular markers have been specifically linked to liver metastases mechanistic paths in CRC. However, to date, none of the identified signatures or molecular markers has been successfully validated as a diagnostic or prognostic tool applicable to routine clinical practice. To obtain a genetic signature for liver metastasis in CRC, measures to improve reproducibility, to increase consistency, and to validate results need to be implemented. Alternatives to expression profiling with microarray technology are continuing to be used. In the recent past, many genes codifying for proteins that are directly or indirectly involved in adhesion, invasion, angiogenesis, survival and cell growth have been linked to mechanisms of liver metastases in CRC.
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25
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Poehlmann A, Kuester D, Meyer F, Lippert H, Roessner A, Schneider-Stock R. K-ras mutation detection in colorectal cancer using the Pyrosequencing technique. Pathol Res Pract 2007; 203:489-97. [PMID: 17629419 DOI: 10.1016/j.prp.2007.06.001] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/11/2007] [Accepted: 06/05/2007] [Indexed: 10/23/2022]
Abstract
The identification of gene mutations is a critical goal for the assessment of diagnosis and prognosis in cancer disease, particularly by direct sequencing. Pyrosequencing is a straightforward, non-electrophoretic DNA sequencing method using the luciferase-luciferin light release as a signal for nucleotide incorporation into a PCR template DNA. In this study, we aimed to investigate mutations in the K-ras gene using Pyrosequencing technology, because its reliable chemistry and robust detection mechanism allow for rapid, real-time detection of sequencing events. For the simultaneous detection of the predominant K-ras codons 12 and 13 mutations, we established a sequencing protocol based on the design of a single PCR primer pair and a single sequencing primer. The assay has been validated with DNA from 65 colorectal carcinomas. Furthermore, analysis of the rare K-ras codon 61 mutation was included. In 29% (19/65) of the patients, the K-ras gene was found to be mutated, whereas codons 12 and 13 were most frequently affected (18/65, 27.7%). Mutations with the highest frequency were G-->A transitions (12/19, 63%), followed by G-->T transversions (5/19, 26%). Overall survival was significantly shorter in patients with a tumor containing K-ras codon 12 mutations than in those without K-ras codon 12 mutations (p=0.024). In conclusion, we found Pyrosequencing to be a suitable technology for fast detection of hot-spot mutations in the K-ras oncogene. We demonstrated an important relationship between K-ras codon 12 mutations and overall survival in colorectal cancer patients.
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Affiliation(s)
- Angela Poehlmann
- Department of Pathology, Otto-von-Guericke University, Magdeburg, Germany
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26
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Tassi E, Wellstein A. The angiogenic switch molecule, secreted FGF-binding protein, an indicator of early stages of pancreatic and colorectal adenocarcinoma. Semin Oncol 2007; 33:S50-6. [PMID: 17178288 PMCID: PMC1781498 DOI: 10.1053/j.seminoncol.2006.10.014] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Tumor angiogenesis has been related to the initiation as well as progression toward more aggressive behavior of human tumors. We will discuss genetic events underlying the initiation and progression of colorectal and pancreatic adenocarcinoma with a particular focus on the modulation of angiogenesis. A secreted fibroblast growth factor (FGF) binding protein (FGF-BP), which is an extracellular chaperone molecule for FGFs, has been shown to enhance FGF-mediated biochemical and biologic events and to be a crucial rate-limiting factor for tumor-dependent angiogenesis. Histochemical and in situ hybridization studies with archival samples show that FGF-BP is induced early during the initiation of colorectal and pancreatic adenocarcinoma. We will discuss the potential of this secreted protein as a serum marker to identify at-risk subjects.
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Affiliation(s)
- Elena Tassi
- Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
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27
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Tassi E, Wellstein A. Tumor angiogenesis: initiation and targeting - therapeutic targeting of an FGF-binding protein, an angiogenic switch molecule, and indicator of early stages of gastrointestinal adenocarcinomas -. Cancer Res Treat 2006; 38:189-97. [PMID: 19771241 DOI: 10.4143/crt.2006.38.4.189] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Tumor angiogenesis has been related to the initiation as well as progression toward more aggressive behavior of human tumors. In particular, the activity of angiogenic factors is crucial for tumor progression. We previously characterized a secreted fibroblast growth factor-binding protein (FGF-BP) as a chaperone molecule, which binds to various FGFs, enhances FGF-mediated biochemical and biologic events and importantly is a crucial rate-limiting factor for tumor-dependent angiogenesis. We generated monoclonal antibodies that target FGF-BP protein and used them as a tool to evaluate frequency and pattern of FGF-BP expression during the malignant progression of pancreas and colorectal carcinoma in archival tissue samples. We found that FGF-BP is dramatically upregulated during the initiation of colorectal and pancreatic adenocarcinoma. Crucial genetic events underlying the initiation and progression of colorectal and pancreatic adenocarcinoma with a particular focus on the modulation of angiogenesis and antiangiogenic therapies are discussed. We propose that the upregulation of the secreted FGF-BP protein during early phases of pancreas and colon cancer could make this protein a possible serum marker indicating the presence of high-risk premalignant lesions. Furthermore, the biological activity of FGF-BP is neutralized by monoclonal antibodies suggesting the potential for antibody-based therapeutic targeting.
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Affiliation(s)
- Elena Tassi
- Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
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28
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Einspahr JG, Martinez ME, Jiang R, Hsu CH, Rashid A, Bhattacharrya AK, Ahnen DJ, Jacobs ET, Houlihan PS, Webb CR, Alberts DS, Hamilton SR. Associations of Ki-ras proto-oncogene mutation and p53 gene overexpression in sporadic colorectal adenomas with demographic and clinicopathologic characteristics. Cancer Epidemiol Biomarkers Prev 2006; 15:1443-50. [PMID: 16896030 PMCID: PMC3547362 DOI: 10.1158/1055-9965.epi-06-0144] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
In colorectal tumorigenesis, Ki-ras proto-oncogene mutation often occurs early in the adenoma-adenocarcinoma sequence, whereas mutation of the p53 gene is associated with late progression to carcinoma. We evaluated the relationship of demographic and clinicopathologic characteristics to Ki-ras mutation and p53 gene product overexpression in 1,093 baseline sporadic colorectal adenomas from 926 individuals enrolled in a phase III recurrence prevention trial. Ki-ras mutation was found in 14.7% of individuals and p53 overexpression was found in 7.0% of those tested. Multivariate analysis found older age, rectal location, and villous histology to be independently associated with Ki-ras mutation. Individuals with an advanced adenoma (>or=1 cm or high-grade dysplasia or villous histology) had a 4-fold higher likelihood of Ki-ras mutation [odds ratios (OR), 3.96; 95% confidence intervals (CI), 2.54-6.18]. Ki-ras mutations in codon 12 and of the G-to-A transition type were more frequent in older individuals, whereas G-to-T transversion was more frequent in rectal adenomas than in the colon. Multivariate analysis showed that previous history of a polyp (P = 0.03) was inversely associated with p53 overexpression. Large adenoma size (>or=1 cm), high-grade dysplasia, and villous histology were independently associated with p53 overexpression, with the strongest association for advanced adenomas (OR, 7.20; 95% CI, 3.01-17.22). Individuals with a Ki-ras mutated adenoma were more likely to overexpress p53 (OR, 2.46; 95% CI, 1.36-4.46), and 94.8% of adenomas with both alterations were classified as advanced (P <or= 0.0001). Our large cross-sectional study supports the role of both Ki-ras and p53 in the progression of adenomas and shows that their molecular pathogenesis differs by anatomic location, age, and mucosal predisposition as evidenced by previous history of a polyp.
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Affiliation(s)
- Janine G Einspahr
- Department of Medicine, Arizona Cancer Center, P.O. Box 245024, Tucson, AZ 85724, USA.
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Akagi K, Uchibori R, Yamaguchi K, Kurosawa K, Tanaka Y, Kozu T. Characterization of a novel oncogenic K-ras mutation in colon cancer. Biochem Biophys Res Commun 2006; 352:728-32. [PMID: 17150185 DOI: 10.1016/j.bbrc.2006.11.091] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2006] [Accepted: 11/15/2006] [Indexed: 12/23/2022]
Abstract
Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity.
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Affiliation(s)
- Kiwamu Akagi
- Molecular Diagnosis and Cancer Prevention Division, Saitama Cancer Center, 818 Komuro Ina, Kitaadachigun, Saitama 362-0806, Japan.
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30
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Lin JK, Chang SC, Wang HS, Yang SH, Jiang JK, Chen WC, Lin TC, Li AFY. Distinctive clinicopathological features of Ki-ras mutated colorectal cancers. J Surg Oncol 2006; 94:234-41. [PMID: 16900509 DOI: 10.1002/jso.20438] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
BACKGROUND AND OBJECTIVES We explored the relationship between the mutation pattern of Ki-ras and the clinicopathological features of colorectal cancers (CRCs). METHODS Relationships between clinicopathological parameters and Ki-ras mutation status were analyzed in 255 CRC patients using the chi-square and student t-tests. Kaplan-Meier survival curves were compared using the log-rank test. RESULTS Ki-ras mutation occurred in 43.9% of tumors, and 83% affected codon 12. The most frequent mutations were GGT->GAT (Gly->Asp) (37.5%), followed by GGT->GTT (Gly->Val) (31.3%), both in codon 12. The frequency of Ki-ras mutation was similar for different tumor stages (38.2-47.8%). The mucin component of tumors was significantly associated with Ki-ras mutation. The 4-year overall and disease-free survival was 61% and 54%, respectively, for patients with Ki-ras mutated tumors, and 73% and 60% for patients with nonmutated tumors (not statistically significant). Patients with Ki-ras mutated tumors had lower plasma folate (24 ng/dl) than those bearing nonmutated tumors (37 ng/dl). Patients with G->T Ki-ras mutations had the lowest folate level (22 ng/dl), followed by those with G->A mutations (25 ng/dl). CONCLUSIONS Ki-ras mutated colorectal tumors have a higher mucin production and higher differentiation, and are associated with lower plasma folate levels and a relatively poorer disease outcome.
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Affiliation(s)
- Jen-Kou Lin
- Division of Colon & Rectal Surgery, Department of Surgery, Taipei Veterans General Hospital, National Yang-Ming University, Taipei, Taiwan.
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31
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Alsop K, Mead L, Smith LD, Royce SG, Tesoriero AA, Young JP, Haydon A, Grubb G, Giles GG, Jenkins MA, Hopper JL, Southey MC. Low somatic K-ras mutation frequency in colorectal cancer diagnosed under the age of 45 years. Eur J Cancer 2006; 42:1357-61. [PMID: 16765042 DOI: 10.1016/j.ejca.2006.02.023] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2006] [Revised: 02/16/2006] [Accepted: 02/20/2006] [Indexed: 10/24/2022]
Abstract
Somatic mutation of K-ras is known to be a common event in colorectal cancer tumourigenesis however its association with age at onset has not been widely explored. In this study, we have analyzed tumours from a population-based study of colorectal cancer diagnosed before the age of 45 years, in which cases had been previously screened for germ-line mismatch repair gene mutations and for microsatellite instability. We used a micro-dissection and sequencing approach to search for somatic K-ras mutations in codons 12, 13 and 61 in 101 early-onset colorectal cancers. Six (6%) somatic K-ras mutations were detected; five in codon 12 (4 G>T transitions and 1 G>A) and one in codon 13 (G>A transition). All codon 12 mutations were identified in microsatellite stable tumours and the codon 13 mutation was identified in a MSI-high tumour. Four cases with K-ras mutations had no reported family history of colorectal cancer and two had some family history of colorectal cancer. None were known to carry a germ-line mutation in hMSH2, hMLH1, hMSH6 or hPMS2. The role of somatic K-ras mutations in early-onset colorectal cancer carcinogenesis appears to be minor, in contrast to its significant role in colorectal cancer of later age of onset.
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Affiliation(s)
- Kathryn Alsop
- Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Vic., Australia
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Lüchtenborg M, Weijenberg MP, Wark PA, Saritas AM, Roemen GMJM, van Muijen GNP, de Bruïne AP, van den Brandt PA, de Goeij AFPM. Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study. BMC Cancer 2005; 5:160. [PMID: 16356174 PMCID: PMC1334229 DOI: 10.1186/1471-2407-5-160] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2005] [Accepted: 12/15/2005] [Indexed: 01/15/2023] Open
Abstract
Background The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. Methods In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Results Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. Conclusion CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.
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Affiliation(s)
- Margreet Lüchtenborg
- Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Epidemiology, Maastricht University, Maastricht, The Netherlands
| | - Matty P Weijenberg
- Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Epidemiology, Maastricht University, Maastricht, The Netherlands
| | - Petra A Wark
- Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands
| | - A Merdan Saritas
- Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Pathology, Maastricht University, Maastricht, The Netherlands
| | - Guido MJM Roemen
- Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Pathology, Maastricht University, Maastricht, The Netherlands
| | - Goos NP van Muijen
- Department of Pathology, University Medical Centre St. Radboud, Nijmegen, The Netherlands
| | - Adriaan P de Bruïne
- Research Institute Growth and Development (GROW), Department of Pathology, Maastricht University, Maastricht, The Netherlands
| | - Piet A van den Brandt
- Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Epidemiology, Maastricht University, Maastricht, The Netherlands
| | - Anton FPM de Goeij
- Research Institute Growth and Development (GROW), Department of Pathology, Maastricht University, Maastricht, The Netherlands
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Friday BB, Adjei AA. K-ras as a target for cancer therapy. Biochim Biophys Acta Rev Cancer 2005; 1756:127-44. [PMID: 16139957 DOI: 10.1016/j.bbcan.2005.08.001] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2005] [Revised: 07/29/2005] [Accepted: 08/01/2005] [Indexed: 11/30/2022]
Abstract
The central role K-, H- and N-Ras play in regulating diverse cellular pathways important for cell growth, differentiation and survival is well established. Dysregulation of Ras proteins by activating mutations, overexpression or upstream activation is common in human tumors. Of the Ras proteins, K-ras is the most frequently mutated and is therefore an attractive target for cancer therapy. The complexity of K-ras signaling presents many opportunities for therapeutic targeting. A number of different approaches aimed at abrogating K-ras activity have been explored in clinical trials. Several of the therapeutic agents tested have demonstrated clinical activity, supporting ongoing development of K-ras targeted therapies. However, many of the agents currently being evaluated have multiple targets and their antitumor effects may not be due to K-Ras inhibition. To date, no selective, specific inhibitor of K-ras is available for routine clinical use. In this review, we will summarize the structure and function of K-ras with attention to its role in tumorigenesis and discuss the successes and failures of the various strategies designed to therapeutically target this important oncogene.
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Affiliation(s)
- Bret B Friday
- Division of Medical Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
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Smakman N, Borel Rinkes IHM, Voest EE, Kranenburg O. Control of colorectal metastasis formation by K-Ras. Biochim Biophys Acta Rev Cancer 2005; 1756:103-14. [PMID: 16098678 DOI: 10.1016/j.bbcan.2005.07.001] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2005] [Revised: 06/22/2005] [Accepted: 07/14/2005] [Indexed: 12/21/2022]
Abstract
Mutational activation of the K-Ras proto-oncogene is frequently observed during the very early stages of colorectal cancer (CRC) development. The mutant alleles are preserved during the progression from pre-malignant lesions to invasive carcinomas and distant metastases. Activated K-Ras may therefore not only promote tumor initiation, but also tumor progression and metastasis formation. Metastasis formation is a very complex and inefficient process: Tumor cells have to disseminate from the primary tumor, invade the local stroma to gain access to the vasculature (intravasation), survive in the hostile environment of the circulation and the distant microvascular beds, gain access to the distant parenchyma (extravasation) and survive and grow out in this new environment. In this review, we discuss the potential influence of mutant K-Ras on each of these phases. Furthermore, we have evaluated the clinical evidence that suggests a role for K-Ras in the formation of colorectal metastases.
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Affiliation(s)
- Niels Smakman
- Department of Surgery G04-228, University Medical Center Utrecht, Heidelberglaan 100, PO Box 85500, 3508GA Utrecht, The Netherlands
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Wu CM, Tang R, Wang JY, Changchien CR, Hsieh LL. Frequency and spectrum of K-RAS codons 12 and 13 mutations in colorectal adenocarcinomas from Taiwan. ACTA ACUST UNITED AC 2005; 158:55-60. [PMID: 15771905 DOI: 10.1016/j.cancergencyto.2004.08.030] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2004] [Revised: 08/18/2004] [Accepted: 08/19/2004] [Indexed: 11/26/2022]
Abstract
Mutations in codons 12 and 13 of the K-RAS oncogene are detected at a remarkably high frequency in colorectal adenocarcinoma and are believed to be a critical event in oncogenesis. In the present study, we evaluated colorectal tumor specimens from Taiwan for mutations in K-RAS codons 12 and 13 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct DNA sequencing. Mutations were found in 48 of 181 (26.5%) tumors, 30 mutations were G-->A transitions (62.5% of all mutations), 14 were G-->T transversions (29.2%), and only 4 were G-->C transversions (8.3%). Similar relative mutation frequencies and spectra were found regardless of the sex of the patient, the tumor grade, or the tumor stage. The high frequency of transitions among K-RAS mutation suggests that G/T mismatches play an important role in the oncogenesis of colorectal adenocarcinoma, implying that alkylating carcinogens may be involved in the colorectal carcinogenesis. Although the frequency of mutation (26.5%) appears to be lower than those reported in the United States (40%), France (49%), and the Netherlands (34%), the spectrum of point mutations in codons 12 and 13 of the K-RAS gene in the Taiwan Chinese population appears to be similar. The reason for these results may be that diet and ethnicity are not rate limit factors in controlling the spectra of mutations but influence on the frequency of K-RAS mutations in human colorectal adenocarcinomas.
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Affiliation(s)
- Chi-Ming Wu
- Graduate Institute of Basic Medical Science, Chang Gung University, Taoyuan 333, Taiwan.
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Dieterle CP, Conzelmann M, Linnemann U, Berger MR. Detection of isolated tumor cells by polymerase chain reaction-restriction fragment length polymorphism for K-ras mutations in tissue samples of 199 colorectal cancer patients. Clin Cancer Res 2004; 10:641-50. [PMID: 14760087 DOI: 10.1158/1078-0432.ccr-1355-02] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The aim of this study was to identify K-ras mutations as marker for isolated tumor cells in liver, lymph node, and bone marrow specimens of colorectal cancer patients. To detect these, a PCR-RFLP assay was used with a sensitivity exceeding that of routine histopathology by at least 1 order of magnitude. In addition, the ratio of mutated versus wild-type alleles was determined by an internal standard. Of 199 patients, 74 (37.5%) were found to bear a K-ras-positive tumor. Of these, 60 (81%) were mutated in codon 12 and 14 (19%) in codon 13 (P < 0.001). In addition, 14 organs were found K-ras positive, 13 of which were from 12 patients with a K-ras-positive tumor (16%) and 1 from a patient with a K-ras-negative tumor (0.8%). Eight patients exhibited liver involvement and 6 showed lymph node involvement. Remarkably, no bone marrow specimen was found K-ras positive (P < 0.017 versus liver involvement). Sequence analysis of tumor DNA revealed that GGT (Gly) was replaced by GAT (Asp; 35%), GTT (Val; 32%), AGT (Ser; 13%), GCT (Ala; 10%), TGT (Cys; 8%), and CGT (Arg; 2%) for codon 12, and by GAC (Asp) as the only type of mutation for codon 13. In colorectal carcinomas the ratio of K-ras mutated versus wild-type alleles ranged over 4 orders of magnitude (10(0)-10(-4), median: 10(-2)) and was correlated with both, residual tumor load (R1/2; P = 0.028) and distant metastasis (M1; P = 0.057). These results show that detection of K-ras mutated alleles by PCR-RFLP in patients with colorectal carcinoma may aid in the identification of isolated tumor cells. High ratios of K-ras alleles were correlated with certain negative prognostic parameters (R,M). In accord with its function as a primary filter for colorectal carcinoma cells, the liver was more often contaminated with K-ras-positive cells than bone marrow.
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Affiliation(s)
- Christoph P Dieterle
- Unit of Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg, Germany
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González-Aguilera JJ, Oliart S, Azcoita MM, Fernández-Peralta AM. Simultaneous mutations in K-ras and TP53 are indicative of poor prognosis in sporadic colorectal cancer. Am J Clin Oncol 2004; 27:39-45. [PMID: 14758132 DOI: 10.1097/01.coc.0000045920.49210.7a] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
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.
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Klump B, Nehls O, Okech T, Hsieh CJ, Gaco V, Gittinger FS, Sarbia M, Borchard F, Greschniok A, Gruenagel HH, Porschen R, Gregor M. Molecular lesions in colorectal cancer: impact on prognosis? Original data and review of the literature. Int J Colorectal Dis 2004; 19:23-42. [PMID: 12827409 DOI: 10.1007/s00384-003-0499-7] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 03/14/2003] [Indexed: 02/04/2023]
Abstract
BACKGROUND In the Dukes' B and C stages of colorectal carcinoma there are considerable variations in the observed courses of the disease. Since post-operative chemotherapy in patients with Dukes' C (node-positive) colon carcinoma has been demonstrated to be effective in improving overall-survival, a more exact prognosis assessment gains additional significance and therapeutic relevance. DISCUSSION One also hopes to derive improved prognostic factors from the clarification of the molecular pathogenesis. Because of its frequency and the accessibility and recognizability of its developmental stages colorectal carcinoma is among the best investigated of all solid tumors. Despite a multitude of suggested molecular candidate markers none of these changes has yet been able enter the everyday life of the clinic. However, it is to be expected that some of the molecular alterations presently discussed will gain importance before long in the clinical treatment of patients with colorectal carcinoma. CONCLUSION Considering also our own findings, this review presents the latest developments in the scientific discussion of the tumor suppressor/oncogenes p53, k-ras, and DCC, biochemical determinants of the 5-fluorouracil metabolism, and defects of the DNA repair system.
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Affiliation(s)
- B Klump
- Department of Internal Medicine I, University Hospital, Otfried-Müller-Strasse 10, 72076, Tübingen, Germany.
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McDermott U, Longley DB, Johnston PG. Molecular and biochemical markers in colorectal cancer. Ann Oncol 2003; 13 Suppl 4:235-45. [PMID: 12401696 DOI: 10.1093/annonc/mdf665] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
MESH Headings
- Antigens, Neoplasm/analysis
- Antigens, Neoplasm/metabolism
- Antigens, Tumor-Associated, Carbohydrate/analysis
- Antigens, Tumor-Associated, Carbohydrate/metabolism
- Biomarkers, Tumor/analysis
- Biomarkers, Tumor/metabolism
- CA-19-9 Antigen/genetics
- Carcinoembryonic Antigen/analysis
- Carcinoembryonic Antigen/metabolism
- Colorectal Neoplasms/diagnosis
- Colorectal Neoplasms/genetics
- Female
- Gene Expression Regulation, Neoplastic
- Genes, p53/genetics
- Genes, ras/genetics
- Genetic Markers/genetics
- Genetic Predisposition to Disease
- Genetic Testing/standards
- Genetic Testing/trends
- Humans
- Male
- Microsatellite Repeats
- Sensitivity and Specificity
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Affiliation(s)
- U McDermott
- Department of Oncology, Cancer Research Centre, Queen's University Belfast, Belfast, Northern Ireland
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40
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Longley DB, McDermott U, Johnston PG. Clinical significance of prognostic and predictive markers in colorectal cancer. THE PHARMACOGENOMICS JOURNAL 2003; 2:209-16. [PMID: 12196910 DOI: 10.1038/sj.tpj.6500124] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- D B Longley
- Department of Oncology, Cancer Research Centre, Queen's University Belfast, Belfast, Northern Ireland
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41
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Bjørheim J, Gaudernack G, Ekstrøm PO. Melting gel techniques in single nucleotide polymorphism and mutation detection: From theory to automation. J Sep Sci 2002. [DOI: 10.1002/1615-9314(20020701)25:10/11<637::aid-jssc637>3.0.co;2-l] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Bjørheim J, Minarik M, Gaudernack G, Ekstrøm PO. Mutation detection in KRAS Exon 1 by constant denaturant capillary electrophoresis in 96 parallel capillaries. Anal Biochem 2002; 304:200-5. [PMID: 12009696 DOI: 10.1006/abio.2002.5629] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Mutations in KRAS exon 1 oncogene are frequently found in colon carcinomas. A correlation between the mutated KRAS and the prognosis and outcome of treatment of colon cancer patients was reported in the literature. The object of our work was to establish a high-throughput method with high sensitivity to enable screening of tumor mutation status of KRAS exon 1 in large groups of colon cancer patients. KRAS exon 1 sequences from DNA isolated from 191 sporadic colon cancers were PCR amplified using one primer labeled with fluorescein and a second primer extended by a GC-clamp. After PCR amplification samples were subjected to automated 96-array constant denaturant capillary electrophoresis using a modified MegaBACE 1000 sequencing instrument. Mutant samples were identified by characteristic peak patterns. The sensitivity of detection of a mutant allele in a background of the wild-type alleles was 0.3%. Using the 96-array instrument a typical screening of 191 samples for KRAS mutation status could be performed within 2 h. A KRAS exon 1 mutation was found in 66 of 191 (34.6%) of the samples. The 96-array constant denaturant capillary electrophoresis provides an opportunity for the high-sensitivity screening of large cancer populations for KRAS exon 1 mutations.
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Affiliation(s)
- Jens Bjørheim
- Section for Immunotherapy, The Norwegian Radium Hospital, 0310 Oslo, Norway.
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Fischer H, Stenling R, Rubio C, Lindblom A. Differential expression of aquaporin 8 in human colonic epithelial cells and colorectal tumors. BMC PHYSIOLOGY 2001; 1:1. [PMID: 11231887 PMCID: PMC32180 DOI: 10.1186/1472-6793-1-1] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/04/2000] [Accepted: 01/23/2001] [Indexed: 11/10/2022]
Abstract
BACKGROUND The gene expression pattern in tumor cells differs from that in corresponding normal cells. In order to identify differentially expressed genes in colorectal tumors and normal colorectal epithelium, a differential display experiment was used to compare RNA expression in normal and tumor tissue samples. RESULTS One gene fragment was expressed only in normal tissue and not, or to a much lesser extent, in the adenomas, carcinomas and cancer cell lines. The isolated gene fragment was identical to Aquaporin 8 (AQP8), a water channel protein. In situ hybridization demonstrated that AQP8 was expressed in the cells facing the lumen in the normal colonic epithelium. CONCLUSION Our result suggests that the expression of AQP8 is a marker of normal proliferating colonic epithelial cells and suggest these cells to be involved in fluid transport in the colon.
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Affiliation(s)
- Heléne Fischer
- Dept. Molecular Medicine, Karolinska Institute, Stockholm, Sweden
| | - Roger Stenling
- Dept. of Pathology, Umeå University Hospital, Umeå, Sweden
| | - Carlos Rubio
- Dept. of Pathology, Karolinska Hospital, Stockholm, Sweden
| | - Annika Lindblom
- Dept. Molecular Medicine, Karolinska Institute, Stockholm, Sweden
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Luna-Pérez P, Segura J, Alvarado I, Labastida S, Santiago-Payán H, Quintero A. Specific c-K-ras gene mutations as a tumor-response marker in locally advanced rectal cancer treated with preoperative chemoradiotherapy. Ann Surg Oncol 2000; 7:727-31. [PMID: 11129419 DOI: 10.1007/s10434-000-0727-0] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Forty percent of patients with colorectal cancer develop mutations in the K-ras gene. OBJECTIVE Our objective was to evaluate whether the presence of c-K-ras gene mutations is a useful tumor-response marker in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy. MATERIAL AND METHODS Thirty seven patients with locally advanced rectal cancer were treated with preoperative chemoradiotherapy. Four to six weeks later, surgery was performed. Specimens were classified according to the UICC-AJC classification. A segment of the tumor was obtained to analyze specific c-K-ras gene mutations. Restriction fragment length polymorphism (RFLP) and single strand confirmation polymorphism (SSCP) techniques were used with a set of probes to detect specific c-K-ras mutations in codons 12, 13, and 61. The 37 patients were divided into Group A (with mutations) and Group B (without mutations). RESULTS All 37 patients completed the scheduled treatment. Group A consisted of 12 patients, whose tumors were classified and specific c-K-ras mutations were located as follows: eight in codon 12, two in codon 13, and one in codon 61. Group B consisted of 25 patients. The tumors were classified and there were more early-stage tumors in Group A, whereas in Group B there were more advanced-stage tumors (P = .05, respectively). The mean follow-up was 36.2+/-18.3 months. All Group A patients survived, whereas 8 of the 25 patients in Group B died due to progressive metastatic disease. Survival in Group A was 100%, whereas in Group B it was 59% (P = .03). CONCLUSIONS The presence of specific c-K-ras mutations is an indicator of tumor response in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy and surgery. Therefore, responding patients may be more amenable to less radical surgical procedures based on c-K-ras mutations.
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Affiliation(s)
- P Luna-Pérez
- Colorectal Service, Surgical Department, Hospital de Oncología, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México DF.
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Inganäs M, Byding S, Eckersten A, Eriksson S, Hultman T, Jorsback A, Löfman E, Sabounchi F, Kressner U, Lindmark G, Tooke N. Enzymatic Mutation Detection in the P53 Gene. Clin Chem 2000. [DOI: 10.1093/clinchem/46.10.1562] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
Abstract
Abstract
Background: The enzymatic mutation detection (EMD) assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves preformed heteroduplex molecules at mismatch sites, forming two shorter fragments that can be resolved by gel electrophoresis. The method can be used to detect single and multiple base changes, as well as insertions and deletions.
Methods: The sensitivity, specificity, and positional accuracy of mutation detection by EMD with the PASSPORTTM Mutation Scanning Kit were assessed in a blind fashion for three analytical platforms (radioactive detection and automated laser sequencers ALFexpress and ABI PRISM 377). PCR products of 703 bp covering codons 188–393 of the P53 gene were prepared from colorectal tumor samples and analyzed by EMD; the results were compared to data from cDNA sequencing. A 1362-bp PCR product prepared from IL4r gene was used to test detection of multiple base changes in long PCR products.
Results: The sensitivity for detection of mutations using EMD exceeded 90%, and the specificity exceeded 80% on all analysis platforms. The method localized 90% of mutations to within two codons and four codons for automated laser sequencers and detection by radioactivity, respectively. The method detected at least five mismatches in heteroduplexes >1 kb.
Conclusions: The EMD system facilitates efficient detection of genetic variation in fragments exceeding 1 kb irrespective of location and type. The technology is particularly well suited to the detection of mutations in genes frequently mutated at unpredictable locations.
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Affiliation(s)
- Mats Inganäs
- Amersham Pharmacia Biotech, SE-751 84 Uppsala, Sweden
| | - Sara Byding
- Amersham Pharmacia Biotech, SE-751 84 Uppsala, Sweden
| | - Ann Eckersten
- Amersham Pharmacia Biotech, SE-751 84 Uppsala, Sweden
| | | | - Tomas Hultman
- Amersham Pharmacia Biotech, SE-751 84 Uppsala, Sweden
| | | | - Esfir Löfman
- Amersham Pharmacia Biotech, SE-751 84 Uppsala, Sweden
| | | | - Ulf Kressner
- Department of Surgery, Uddevalla Hospital, SE-541 80 Uddevalla, Sweden
| | - Gudrun Lindmark
- Department of Surgery, University Hospital of Northern Sweden, SE-901 85 Umeå, Sweden
| | - Nigel Tooke
- Amersham Pharmacia Biotech, SE-751 84 Uppsala, Sweden
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46
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A Novel Ras Antagonist Regulates Both Oncogenic Ras and the Tumor Suppressor p53 in Colon Cancer Cells. Mol Med 2000. [DOI: 10.1007/bf03402049] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022] Open
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Salahshor S, Kressner U, P�hlman L, Glimelius B, Lindmark G, Lindblom A. Colorectal cancer with and without microsatellite instability involves different genes. Genes Chromosomes Cancer 1999. [DOI: 10.1002/(sici)1098-2264(199911)26:3<247::aid-gcc9>3.0.co;2-h] [Citation(s) in RCA: 91] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
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48
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Chen TJ, Boles RG, Wong LJC. Detection of Mitochondrial DNA Mutations by Temporal Temperature Gradient Gel Electrophoresis. Clin Chem 1999. [DOI: 10.1093/clinchem/45.8.1162] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Abstract
Background: A unique requirement for the molecular diagnosis of mitochondrial DNA (mtDNA) disorders is the ability to detect heteroplasmic mtDNA mutations and to distinguish them from homoplasmic sequence variations before further testing (e.g., sequencing) is performed. We evaluated the potential utility of temporal temperature gradient gel electrophoresis (TTGE) for these purposes in patients with suspected mtDNA mutations.
Methods: DNA samples were selected from patients with known mtDNA mutations and patients suspected of mtDNA disorders without detectable mutations by routine analysis. Six regions of mtDNA were PCR amplified and analyzed by TTGE. Electrophoresis was carried out at 145 V with a constant temperature increment of 1.2 °C/h. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR/allele-specific oligonucleotide or PCR/restriction fragment length polymorphism analysis.
Results: In the experiments using patient samples containing various amounts of mutant mtDNA, TTGE detected as little as 4% mutant heteroplasmy and identified heteroplasmy in the presence of a homoplasmic polymorphism. In 109 specimens with 15 different known mutations, TTGE detected the presence of all mutations and distinguished heteroplasmic mutations from homoplasmic polymorphisms. When 11% of the mtDNA genome was analyzed by TTGE in 104 patients with clinically suspected mitochondrial disorders, 7 cases of heteroplasmy (≈7%) were detected.
Conclusions: TTGE distinguishes heteroplasmic mutation from homoplasmic polymorphisms and appears to be a sensitive tool for detection of sequence variations and heteroplasmy in patients suspected of having mtDNA disorders.
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Affiliation(s)
- Tian-Jian Chen
- Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC 20007
- Division of Medical Genetics, Children’s Hospital Los Angeles, and Department of Pediatrics, University of Southern California, School of Medicine, Los Angeles, CA 90027
| | - Richard G Boles
- Division of Medical Genetics, Children’s Hospital Los Angeles, and Department of Pediatrics, University of Southern California, School of Medicine, Los Angeles, CA 90027
| | - Lee-Jun C Wong
- Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC 20007
- Division of Medical Genetics, Children’s Hospital Los Angeles, and Department of Pediatrics, University of Southern California, School of Medicine, Los Angeles, CA 90027
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Beránek M, Bures J, Palicka V, Jandík P, Langr F, Nejedlá E. A relationship between K-ras gene mutations and some clinical and histologic variables in patients with primary colorectal carcinoma. Clin Chem Lab Med 1999; 37:723-7. [PMID: 10510729 DOI: 10.1515/cclm.1999.111] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Mutations in the Kirsten ras 2 (K-ras) gene were described as early events in the process of colorectal carcinogenesis. The aim of this study was to find a possible relationship between the presence of K-ras mutation in samples of primary colorectal carcinomas and the clinico-pathological data of the investigated patients. Mutation in codon 12 of the K-ras gene was determined in 18 of 53 colorectal carcinomas (34%) in our group of patients. The presence of K-ras gene mutations was not related to gender, age of subject at diagnosis, staging or cancer location (p > 0.05). Sixteen of the 42 (38%) moderately differentiated carcinomas, and two of the eight (25%) well differentiated carcinomas contained K-ras mutation in codon 12, but none of the three poorly differentiated carcinomas contained the mutation. Moderately differentiated tumours contained an aspartate code GAT (in eight cases), a valine code GTT (in six cases), an alanine code GCT (in one case) and a serine code AGT (in one case) in codon 12. Well differentiated tumours contained only the valine code GTT (two cases). Our results show that the frequency of mutations in the K-ras gene in carcinomas in Central Europe is not different from the frequencies found in other parts of the world. The homogeneous incidence of K-ras mutation does not seem to be related to ethnic factors, dietary habits, or the composition of the diet.
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Affiliation(s)
- M Beránek
- Institute of Clinical Biochemistry and Diagnostics, University Hospital, Hradec Králové, Czech Republic
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Abstract
Molecular prognostic markers are molecules produced by either the tumor or the host (patient) whose expression is associated with the clinical outcome. Three types of molecular markers exist that characterize different aspects of the tumor : host relationship: (1) tumor biology, (2) tumor burden and (3) host response. The first type of marker is measured within the primary or metastatic tumor mass and defines the aggressiveness of the cancer and its ability to respond to therapy. The other two types of markers are usually measured in the blood and assess concentrations of circulating tumor products or cytokines that may be involved in host resistance to the cancer. In this brief review we will define each type of marker, provide examples of their current utility and then describe how these markers may be useful.
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Affiliation(s)
- J M Jessup
- Department of Surgery, University of Pittsburgh Medical Center, PA 15213, USA.
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