Types Of KRAS Mutations Research Articles

A novel and simple KRAS mutation detection method, FRET-Based Preferential Homoduplex Formation Assay (f-PHFA): Results from a multicenter, clinical evaluation trial.

476 Background: KRAS gene mutation in colorectal tumors is predictive of nonresponse to anti-epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (CRC). The F-PHFA has been developed as a simple and accurate mutation detection assay especially for somatic mutations. In this study, we evaluated KRAS mutation detection by F-PHFA which detects 7 common mutations in codon 12 and 13. This study was a multicenter, clinical evaluation trial designed to evaluate the efficacy of the F-PHFA method forKRAS testing in patients with CRC. Primary endpoint was the concordance rate of KRAS mutation between F-PHFA and direct sequencing with manual micro-dissection (MMD) to enrich the tumor concentration. Secondary endpoints included the concordance rate between F-PHFA and direct sequencing without MMD. Methods: The key eligibility criteria included histologically confirmed colorectal adenocarcinoma with adequate tumor samples. The materials were formalin-fixed paraffin-embedded (FFPE) 8 unstained slides at 10 um and 2 matching hematoxylin-eosin (HE)-stained slide, which were sent to the central pathology review to confirm the tumor content and to mark the tumor area. Genomic DNA was extracted using QIAamp DNA FFPE Tissue from both whole tissue of slide and tissue with MMD. F-PHFA and direct sequencing were conducted independently in two laboratories. Results: FFPE specimens from 165 patients with colorectal adenocaricinoma were registered. Success rates on KRAS tests of F-PHFA both of with or without MMD were 100% (165/165). However, those of direct sequencing were 99.4% (164/165). The concordance rate between F-PHFA and direct sequencing were 99.4% (163/164) for both of with MMD and without MMD. Only one discordant sample had a rare mutation other than the 7 common mutations. Total assay time was about 3.5 hrs. Also 20 ng of DNA was enough to examine 7 types of KRAS mutations. Conclusions: We confirmed that F-PHFA and direct sequencing shows high concordance rate regardless of MMD. F-PHFA for KRAS mutation detection method is very suitable for clinical use in terms of easiness and accuracy.

9093 POSTER The Role of Specific KRAS Mutation Types in Response to Treatment by EGFR Inhibitors

9093 POSTER The Role of Specific KRAS Mutation Types in Response to Treatment by EGFR Inhibitors

Clinical outcome of KRAS-mutated advanced non-small cell lung cancer (NSCLC) patients (pts): A mono-institutional analysis.

7580 Background: In the present analysis we evaluated the clinical outcome of KRAS mutated advanced non-small cell lung cancer (NSCLC) patients (pts) treated at a single Institution from April 2007 to December 2010. Methods: KRAS mutational status was assessed on tumor tissue collected at the time of first diagnosis (either primary or metastatic). KRAS (exons 2 and 3) gene was amplified by nested PCR and sequenced in both sense and antisense direction using 3500 Genetic Analyzer. Retrospective chart review was approved by the local Ethics Committee. Results: 64 pts with an identified KRAS mutation were evaluable for the analysis. The majority of them (87.5%) had a good performance status (ECOG 0), while 82.5% were former or current smokers. All pts except for one who was treated with single-agent gemcitabine, received a platinum-based doublet mainly including either gemcitabine (56,5%) or pemetrexed (25%). Overall, 82% of pts received at least two lines of chemotherapy. Three types of KRAS mutations were identified, the most common being codon 12 (78.1%) mutation, followed by codon 13 (12.5%) and codon 61 (9.4%) mutations, respectively. Of the 52 (81,2%) who were evaluable for response 2 complete responses (3,8%), 21 partial responses (40,4%), 13 disease stabilizations (25%) and 16 progressive diseases (30,8%) were observed. Median overall survival (OS) from the beginning of treatment was 39 months, with no statistically significant differences according to the type of K-RAS mutation (p< 0,2). Median OS and first-line PFS were significantly higher in responders versus non-responders (55.4 months versus 24.8 months for OS and 11.2 months versus 4.8 months for first-line PFS, p<0.009). Conclusions: KRAS mutated NSCLC pts do not represent a population with homogeneous clinical characteristics. Response to treatment seems to predict benefit from first-line chemotherapy in terms of OS and first-line PFS. The excellent OS rate observed in the whole group might be the result of a population highly selected for good clinical prognostic factors. Prospective studies of KRAS mutations and sensitivity to chemotherapy are warranted.

The role of specific KRAS mutation types in response to treatment by EGFR inhibitors.

e18006 Background: KRAS mutations can be found in 15 to 25% of lung cancers, frequently in adenocarcinomas and their presence is associated with smoking. The role of KRAS mutations in biological targeted therapy by EGFR inhibitors is ambiguous. While some reports clearly assigned mutated KRAS as negative prognostic factor and negative predictor, others did not confirm such notion. In our work we aimed to elucidate the role of specific type of KRAS mutation in outcome of patients with advanced NSCLC threated with EGFR-TKI therapy. Methods: 443 patients with NSCLC were examined. 38 patients, threated with erlotinib or gefitinib in maintenance setting, were further evaluated. 30 patients were suffering from adenocarcinoma, 7 patients had squamous cell carcinoma including 1 patient with poorly diferentiated carcinoma. 37 patients were smokers and only 1 patient was never smoker. Statistical significance was scrutinized by a Log rank test at a 95% confidence level. Results: KRAS mutation was detected in 66 patients. Glycine to cystein substitution at codon 12 [G12C] was the most frequent type of KRAS mutation, detected in 24 patients. Outcome of patients with G12C [n=24] and patients with other specific type of mutation [nonG12C] [n=14] was compared. Median of TTP in the G12C group was 4,3 weeks in comparison with 9,0 weeks in the nonG12C group [p 0.009]. Median of TTP among patients with adenocarcinoma in the G12C group was 4,1 weeks in comparison with 9,0 weeks in the nonG12C group [p 0.007]. Median of OS in the G12C group was 9,3 weeks in comparison with 12,1 weeks in the nonG12C group [p 0.068], median of OS among patients with adenocarcinoma in the G12C group was 9,3 weeks in comparison with 10,6 weeks in the nonG12C group [p 0.095]. Analysis of OS was influenced by different oncological threatment after EGFR-TKI, different quality of basic supportive care and higher incidence of brain matestasis in the nonG12C group. Conclusions: G12C KRAS mutation is a strong negative predictor for EGFR-TKI threatment. Other specific types of KRAS mutation didn’t prove such a negative predictive value and TTP was comparable to patients bearing wild type KRAS gene.

Abstract 2264: K-ras mutations in colorectal cancer may predict a pattern of organ-specific metastasis

Abstract Background: Activating mutations of the K-ras oncogene in colorectal cancer (CRC) are associated with resistance to EGFR antibodies and poor prognosis. We systematically analyzed clinical and pathological characteristics associated with K-ras mutations. Methods: Patients with CRC referred to the phase I program for targeted therapy were analyzed for K-ras mutations using PCR-based DNA sequencing. Patients with mutant K-ras were matched with wild-type (wt) K-ras controls. Multiple clinical and pathological characteristics were analyzed to determine, whether there is a phenotype associated with K-ras mutations. Results: A total of 151 patients with CRC were identified; 61 with K-ras mutations; 80 with wt K-ras and 10 with unknown K-ras status. Patients with K-ras mutations compared with wt K-ras had more frequently lung metastases (80% vs. 63%, p = 0.03). Patients whose tumors had K-ras mutations had higher number of metastatic sites of disease (3.18 vs. 2.65; p = 0.013). There were no associations between K-ras mutations and gender, age at diagnosis, liver metastases, peritoneal metastases, lymphatic involvement or Duke stage at diagnose. Patients with K-ras wt had a trend for longer median survival (283 vs. 222 weeks, p=0.09). There was no difference in the median survival from diagnosis in patients with different types of K-ras mutations (codon 12, codon 13, and codon 61). Dukes staging was the only statistically significant prognostic factor on multivariate analysis (p &amp;lt; 0.05). Conclusions: K-ras mutations in CRC in comparison with wt K-ras were associated with more frequent lung metastases and shorter survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2264. doi:10.1158/1538-7445.AM2011-2264

Relationship between K-ras oncogene mutations in portal vein blood of patients with colorectal cancer and liver metastases

Objective To detect mutations of K-ras oncogene in portal vein blood of patients with colorectal cancer, and to find out the relationship between mutated K-ras oncogene and liver metastases in colorectal cancer. Methods Forty-eight patients with colorectal cancer were screened for the mutations of K-ras oncogene in tissue samples from their tumors, portal vein blood, proximally adjacent mucosa and 8metastatic liver biopsies by real-time fluorescence quantitative polymerase chain reaction (PCR) and DNA sequencing. The results were analyzed with their clinical data. Results Sixteen of the 48 patients with colorectal cancer had K-ras point mutations at codon 12 in their portal vein blood, and 17 of 48 patients had K-ras mutations in their primary tumors, but only 4 of 48 patients had K-ras mutations in proximally adjacent mucosa. There was no significant difference in rate of K-ras mutation between tumor tissues and portal vein blood (P ＞ 0. 05 ), but significant difference was found between the tumor tissue and the proximally adjacent mucosa ( P ＜0. 05 ). The rate of K-ras mutations in portal vein blood of colorectal cancer with liver metastases (70. 0% ) was higher than that of without liver metastases (23.7%). Sixteen cases of mutated K-ras in portal vein blood showed mutations in tumor tissues. Patients without mutated K-ras in tumor tissue had no mutations in their portal vein blood and proximally adjacent mucosa. In 5 of 8 patients with simultaneous liver metastasis, mutated K-ras oncogenes were detected in portal vein blood, and the type of K-ras mutation detected in the tumor tissue was accord with that in metastatic liver biopsies. Two patients with mutated K-ras detected in their portal vein blood had no liver metastases during perioperation, but liver metastases were diagnosed by CT at the postoperative month 6 and 9 respectively. The main types of K-ras mutations at codon 12 included GGT to GAT and GGT to GTT. No one had point mutation at codon 13. Conclusion Mutated K-ras detected in both cancer tissue and portal vein blood may indicate livermetastases from colorectal cancer. Key words: Colorectal neoplasms; Ras gene; Mutation; Hepatic metastasis

Retrospective study to elucidate a K-ras phenotype in patients with colorectal cancer.

462 Background: Activating mutations of the K-ras oncogene in colorectal cancer (CRC) are associated with resistance to EGFR antibodies and poor prognosis. Clinical and pathological characteristics associated with K-ras mutations have not been systematically analyzed. Methods: Patients with CRC referred to the phase I program for targeted therapy were analyzed for K-ras mutations using PCR-based DNA sequencing. Patients with mutant K-ras were matched with wild-type (wt) K-ras controls. Multiple clinical and pathological characteristics were analyzed to determine, whether there is a phenotype associated with K-ras mutations. Results: A total of 107 patients with CRC were identified; 58 with K-ras mutations; and 49 with wt K-ras. Patients with K-ras mutations compared with wt K-Ras had more frequently lung metastases (80% vs. 57%, p = 0.013). There was a trend towards higher number of metastatic sites in K-ras mutant patients compared with wt K-ras (3.16 vs. 2.69; p = 0.07). There were no associations between K-ras mutations and gender, age at diagnosis, liver metastases, peritoneal metastases, and lymphatic involvement. The median time from diagnosis to development of metastatic disease was similar in K-ras mutant and K-ras wt patients (194 vs. 196 weeks, p = 0.43). The median survival from diagnosis was also similar in K-ras mutant and K-ras wt patients (254 vs. 223 weeks, p = 0.32). There was no difference in the median survival from diagnosis in patients with different types of K-ras mutations (codon 12, codon 13, and codon 61). Dukes staging was the only statistically significant prognostic factor on multivariate analysis (p &lt; 0.05). Conclusions: K-ras mutations in CRC in comparison with wt K-ras were associated with more frequent lung metastases. K-ras mutations did not impact survival. No significant financial relationships to disclose.

Mutation of k-ras mutation and evaluation of anti-EGFR monoclonal antibody combined with chemotherapy in Chinese colorectal cancer patients

Objective To observe the k-ras mutation rate of colorectal cancer in China, and assess the effect and toxicity in advanced colorectal cancer (CRC) patients (pts) receiving chemotherapy combined with monoclonal antibody against Epidermal Growth Factor Receptor (EGFR). Methods The k-ras mutation of 139 samples collected from our hospital were tested by pyrophosphoric acid sequencing. Twenty-three advanced colorectal cancer patients were treated with chemotherapy combined with anti-EGFR monoclonal antibody, including 3 initial treated and 20 retreated. In the total 23 patients, 18 were treated with cetuximab and chemotherapy, and S were treated with nimotuzumab and chemotherapy. Cetuximab was taken with 400 mg/m2 first time, and then 250 mg/m2 every week. Nimotuzumab was taken with 400 mg first time, and then 200 mg every week. Eight patients of the total 23 received anti-EGFR monoclonal antibody combined with irinotecan, 12 with FOLFIRI, 3 with FOLFOX4. Results k-ras mutation type (mt) was detected in 39.6 % (55/139) of pts, k-ras wild type (wt) was 60.4 % (94/139). In 22 effect evaluable patients, 5 received PR and 9 SD, and RR and DCR were 22.7 % and 63.6 %, respectively. TTP was 124 days. Thirteen of the 22 patients tested k-ras mutation, of the 11 k-ras mt pts, 4 received PR, 4 SD and 3 PD. Two patients of k-ras mt received PD after 2 cycles treatment. Acneform eruptions were observed in 15 patients of 18 pts who received cetuximab and paronychia in 3 pts. Eruption or paronychia was not observed in all patients who received nimotuzumab. Grade 3 hypersensitivity were occured in 2 patients with cetuximab, and one of them alternated nimotuzumab in next cycle didn't get hypersensitivity any more. Conclusion The mutation rate of k-ras in Chinese colorectal cancer patients was similar with westerners. The effect of cetuximab combined with chemotherapy on advanced colorectal cancer was reliable. Nimotuzumab combined with chemotherapy worth to be studied further because of the promising effect and mild toxicity. Key words: Colorectal neoplasms; k-ras mutation; Cetuximab; Nimotuzumab

Effect of tumor-specific KRAS mutational status on impact of anti-EGFR therapy in non-small cell lung cancer (NSCLC).

7564 Background: The RAS/RAF/MEK/ERK pathway is a therapeutic target in both colorectal (CRC) and NSCLC cancer, but while mutated KRAS is a negative prognostic marker and a negative predictor for anti-EGFR therapy in CRC, its role in lung cancer is quite ambiguous. KRAS is a pool of mutations mostly occurring in codons 12 and 13 but differing for the replaced basis and for the aminoacid substitution. In CRC the most expressed KRAS mutation is G12D. We hypothesized a different KRAS mutational status in NSCLC patients, bearing a potential different profile in the tumour response to treatment which we tested in vitro. Methods: TAILOR (NCT00637910) is a multicenter Italian phase III trial powered to evaluate a differential effect of all biological markers in selecting patients for erlotinib and docetaxel as second line. Consecutive patients are registered and molecularly profiled centrally. KRAS and EGFR mutations were detected by direct sequencing. Therefore, we identified all KRAS mutations types. The EGFR-wt NCI-H1299 lung cell line was transfected with expression plasmids encoding all different identified KRAS mutations and tested in vitro for the activity of cisplatin, taxanes, erlotinib and pemetrexed. Results: To date, KRAS and EGFR mutational status is available on 161 of 256 patients registered into the trial. We found six types of KRAS mutation in 35 patients (20%): G12C (45.7%) G12D (20%) G12V (20%) G12A (5.7%) G13C (5.7%) G13D (2.8%). Preliminary data, in the NCI- H1299 transfected cell lines, indicate that specific KRAS mutations are associated to different sensitivity or resistance drug profiles. Conclusions: Final results will be presented at the congress and clearly suggest that the specific mutational status of KRAS gene in NSCLC seems to differ from that reported for CRC. This clinical observation, with in vitro results suggesting different patterns of sensitivity/resistance to EGFR inhibition in transfected cell lines, might explain the ambiguous role of KRAS as a predictor to anti-EGFR therapy in NSCLC. Final results of the randomized TAILOR study will definetely allow to verify this suggestive hypothesis. No significant financial relationships to disclose.

KRAS mutations (MTs) in non-small cell lung cancer (NSCLC) versus colorectal cancer (CRC): Implications for cetuximab therapy.

10529 Background: KRAS MTs have been associated with a diversity of biologic functions, and have both prognostic and predictive consequences in NSCLC and CRC. Little data exist comparing the specifics of KRAS MTs in the two cancers. In CRC, benefit from the cetuximab is mainly limited to patients whose tumors are KRAS wild-type (WT). However, recent studies show no such predictive value in NSCLC. We hypothesized that the unique molecular biology and tobacco carcinogen-related etiology of NSCLC contribute to a divergence in KRAS MT type and EGFR-related dependency. Here, we compare KRAS and EGFR status in NSCLC versus (vs) CRC in the large Response Genetics (RGI) database. Methods: KRAS MTs were assessed in 2,693 specimens (838 CRC and 1165 NSCLC) using DNA from microdissected formalin fixed paraffin embedded tumor specimens. EGFR MTs were determined in 649 NSCLCs. A PCR approach with direct sequencing was used to detect 7 different base substitutions in KRAS and 28 EGFR MTs. Statistics: Fisher's Exact Test. Results: KRAS MTs were found in 39% of CRC and 23% of NSCLC specimens (p < 0.001). EGFR MTs were identified in 16% of NSCLCs. In 447 specimens with both assays, EGFR and KRAS MTs were largely exclusive; 4 specimens harbored both (p < 0.001). The ratio of base transversions to transitions was 3.27 in NSCLC versus 0.77 in CRC (p < 0.001). With regard to histology, in NSCLC KRAS MTs were observed in 28.7% (186/649) of adenocarcinomas vs 3.5% (7/198) of squamous cell carcinomas (p < 0.001). Tobacco carcinogenesis-associated G>T transversions (codon 12 GGT>TGT plus GGT>GTT) comprised 61% of KRAS MTs in NSCLC vs 39% in CRC (p < 0.001). The GGT>GAT transition was most prevalent in CRC at 33.3%. Conclusions: This analysis of the RGI database demonstrates differences between NSCLC vs CRC in both incidence and type of KRAS MTs. Increased frequencies of DNA KRAS transversions were seen in NSCLC, likely linked to tobacco exposure. Distinct MT patterns and biologic function may contribute to differences in predictive value for cetuximab therapy between NSCLC and CRC. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics Amgen, AstraZeneca, Biodesix, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genoptix, GlaxoSmithKline, Lilly, Merck, Novartis, Pfizer, Response Genetics, sanofi-aventis Response Genetics DxS, Response Genetics Abbott Laboratories, Bristol-Myers Squibb, Genentech, Lilly, Merck, Novartis, OSI, Pfizer

Radiosensitization Effect of STI-571 on Pancreatic Cancer Cells In Vitro

To examine STI-571-induced radiosensitivity in human pancreatic cancer cells in vitro. Three human pancreatic cancer cell lines (Bxpc-3, Capan-1, and MiaPaCa-2) exhibiting different expression levels of c-Kit and platelet-derived growth factor receptor beta (PDGFRbeta) and showing different K-ras mutation types were used. For evaluation of the antitumor activity of STI-571 in combination with radiation, clonogenic survival assays, Western blot analysis, and the annexin V/propidium iodide assay with microscopic evaluation by 4',6-diamidino-2-phenylindole were conducted. Dramatic phosphorylated (p)-c-Kit and p-PDGFRbeta attenuation, a modest dose- and time-dependent growth inhibition, and significant radiosensitization were observed after STI-571 treatment in view of apoptosis, although the levels of growth inhibition and increased radiosensitization were different according to cell lines. The grades of radiosensitivity corresponded to the attenuation levels of p-c-Kit and p-PDGFRbeta by STI-571, particularly to those of p-c-Kit, and the radiosensitivity was partially affected by K-ras mutation in pancreatic cancer cells. Among downstream pathways associated with c-Kit or PDGFRbeta, p-PLCgamma was more closely related to radiosensitivity compared with p-Akt1 or p-extracellular signal-regulated kinase 1. STI-571 enhances radiation response in pancreatic cancer cells. This effect is affected by the attenuation levels of p-c-Kit or p-PDGFRbeta, and K-ras mutation status. Among them, p-c-Kit plays more important roles in the radiosensitivity in pancreatic cancer compared with p-PDGFRbeta or K-ras mutation status.

Frequency and Type of KRAS Mutations in Routine Diagnostic Analysis of Metastatic Colorectal Cancer

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. & 2009 Elsevier GmbH. All rights reserved.

Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer

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.

Different types of K-Ras mutations are conversely associated with overall survival in patients with colorectal cancer

A glycine to valine substitution at codon 12 (G12V) in Kirsten-Ras (K-Ras) gene has been associated with reduced overall survival in colorectal cancer patients; however, the effect of other K-Ras mutations than G12V still remains unclear. Therefore, we investigated the role of different K-Ras mutations on overall survival in a homogeneous, large patient cohort with standardized therapy and uniform analysis of K-Ras mutation status. The study included 342 patients with histopathologically proven colorectal cancer. Survival data were provided by the federal agency for statistics in Austria. Occurrence of K-Ras mutations at codons 12, 13 and 61 were determined by capillary sequencing. The overall K-Ras mutation frequency in carcinoma tissue was 28%. Carriers of the G12V mutation at the K-Ras gene showed a significantly decreased overall survival compared to carriers of the wild-type [HR=2.56 (1.15-5.69)]. Other mutations than G12V were associated with better overall survival compared to wild-type [HR=0.44 (0.2-0.99)]. In conclusion, for the first time, our study showed clearly that different types of K-Ras mutations are conversely associated with overall survival in patients with colorectal cancer.

Correlation of history of tobacco consumption and type of k-ras mutation in patients (pts) with pancreatic adenocarcinoma (PC)

15557 Background: Approximately 90% of PC harbor mutations in k-ras codon 12. Smoking (Sm) is the most important risk factor for development of PC. Ras genes are critical targets for chemical carcinogens and a relationship between tobacco carcinogens and codon 12 k- ras mutations has been suggested. In lung adenocarcinoma, k-ras mutations have been identified preferentially in patients with a positive Sm history. In PC, a correlation of Sm history with specific k-ras mutations remains to be defined. Our aim was to evaluate whether a correlation between Sm history and k-ras mutation in pts with PC exists. We specifically looked at the correlation between Sm history and aspartic acid (D), valine (V), arginine (R), and serine (S) k-ras mutations. Methods: We retrospectively obtained the Sm history in a cohort of pts enrolled on a prospective pilot trial of k-ras vaccination in resected PC. Genomic DNA samples were analyzed for codon 12 k-ras mutations using multiplex competitive oligonucleotide probe ligatio...

Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma

8006 Background: KRAS mutations are found in ∼ 25% of lung adenocarcinomas in Western countries and, as a group, are strongly associated with cigarette smoking. These mutations are predictive of poor prognosis in resected disease as well as resistance to treatment with erlotinib or gefitinib. In KRAS, transversions (substituting the purine G with a pyrimidine) are more common than transitions (substituting the purine G with A) and identify a molecular signature for the carcinogenic effects of cigarette smoke. Methods: We determined the frequency and type of KRAS codon 12 and 13 mutations and characterized their association with cigarette smoking history in patients with lung adenocarcinomas. Comparisons were made using Fisher’s exact test. Results: KRAS mutational analysis was performed on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12/81; 95% CI 8%-24%) of tumors from never smokers. Twenty-two% (69/316; 95% CI 17%-27%) of tumors from former smokers, and 25% (21/85; 95% CI 16%-35%) of tumors from current smokers had KRAS mutations. The frequency of KRAS mutation was not associated with age, gender, or smoking history. The number of pack years of cigarette smoking did not predict an increased likelihood of KRAS mutations. When the type of KRAS mutation was examined, never smokers were significantly more likely than former or current smokers to have a transition mutation (G–>A) rather than the transversion mutations known to be smoking related (G–>T or G–>C; p<0.0001). Conclusions: Based upon our data, KRAS mutations are not rare among never smokers with lung adenocarcinoma and such patients have a distinct KRAS mutation profile. The etiologic and biological heterogeneity of KRAS mutant lung adenocarcinomas is worthy of further study. Mutation Nucleotide Former/current Never Total G12A GGT–>GCT 13 0 13 G12C GGT–>TGT 38 0 38 G12V GGT–>GTT 20 1 21 G13C GGC–>TGC 2 0 2 G13D GGC–>GAC 1 0 1 G12D GGT–>GAT 15 10 25 G12S GGT–>AGT 1 1 2 Total 90 12 No significant financial relationships to disclose.

P53 mutations in colorectal cancer from northern Iran: Relationships with site of tumor origin, microsatellite instability and K‐ras mutations

CRC-associated P53 mutations have not been studied extensively in non-Western countries at relatively low CRC risk. We examined, for the first time, 196 paraffin-embedded CRC cases from Northern Iran for mutations in P53 exons 5-8 using PCR-direct sequencing. P53 status and mutation site/type were correlated with nuclear protein accumulation, clinicopathologic variables and data on K-ras mutations and high-level microsatellite instability (MSI-H). We detected 96 P53 mutations in 87 (44.4%) cases and protein accumulation in 84 cases (42.8%). P53 mutations correlated directly with stage and inversely with MSI-H. Distal CRCs were more frequently mutated at major CpG hotspot codons [248 (8/66, 12.1%), 175 (7/66, 10.6%), and 245 (7/66, 10.6%)], while in proximal tumors codon 213, emerged as most frequently mutated (5/28, 17.9% vs. 3/66, 4.5%, P = 0.048). Transitions at CpGs, the most common mutation type, were more frequent in non-mucinous (25% vs. 10.4% in mucinous, P = 0.032), and distal CRC (27% vs. 12.5% in proximal, P = 0.02), and correlated with K-ras transversions. Transitions at non-CpGs, second most common P53 mutation, were more frequent in proximal tumors (15.6% vs. 4.7% in distal, P = 0.01), and correlated with K-ras transitions and MSI-H. Overall frequency and types of mutations and correlations with P53 accumulation, stage and MSI-H were as reported for non-Iranian patients. However P53 mutation site/type and correlations between P53 and K-ras mutation types differed between proximal and distal CRC. The codon 213 P53 mutation that recurred in proximal CRC was previously reported as frequent in esophageal cancer from Northern Iran.

Differences in K-ras and p53 gene mutations among pancreatic adenocarcinomas associated with regional environmental pollution

Variations in genetic mutations in pancreatic carcinoma between different geographical regions have not been studied extensively, especially in developing countries where pancreatic cancer is relatively rare. We studied the molecular pathology of 54 pancreatic adenocarcinomas from Egyptian patients residing in a heavily polluted region of the eastern Nile River delta and compared the findings with 45 tumors from patients residing in low-pollution regions. Rates of K-ras mutation in codon 12 and of p53 mutation in exons 5-8 were higher in tumors of patients from the high-pollution region as compared with the low-pollution regions (61.5 versus 34.2%, respectively, for K-ras, P = 0.01; 25.9 versus 11.6%, respectively, for p53, P = 0.08). There were also distinct differences in the specific types of K-ras and p53 mutations between the two regions. The ratio of G-to-T k-ras transversion mutation (codon 12) relative to wild-type was significantly higher in tumors from the high-pollution region (0.90) than tumors from the non-pollution site (0.28) (P = 0.03). Relative to tumors with wild-type, the ratio of p53 mutations in exons 5, 7 or 8 to wild-type in tumors from the high-pollution region was significantly higher than the ratio from the non-pollution site (0.28 versus 0.03, P = 0.01). Logistic regression showed that G-to-T transversion mutation in K-ras was predicted by the region of residence of the patients. Our study reveals that there are differences in the frequencies and types of K-ras and p53 mutations found in pancreatic adenocarcinomas of patients in high-pollution and low-pollution regions in Egypt and suggests that environmental factors may explain these differences. We speculate that gene-environment interactions in pancreatic carcinogenesis also occur in other populations.

Differing molecular pathology of pancreatic adenocarcinoma in Egyptian and United States patients

Variations in genetic mutations in pancreatic carcinoma between different populations have not been studied extensively, especially in developing countries where pancreatic cancer is rare. We studied the molecular pathology of 44 pancreatic carcinomas from patients residing in a heavily polluted region in the Nile River delta and compared the findings with tumors from 44 United States (US) patients. We evaluated K-ras mutations in codon 12, p53 mutations in exons 5-8, and Gadd45a mutations in exons 1 and 4. Overall, rates of K-ras, p53 and Gadd45 mutations were not statistically different in tumors of patients from Egypt and the US (67.4 vs. 63.4%; 27.3 vs. 36.4% and 9.1 vs. 4.5%, respectively). However, there were distinct differences in the specific types of K-ras and p53 mutations between the 2 groups. In K-ras, G --> T transversion mutation was more frequent in the tumors from Egypt than from the US (58.6 vs. 26.9%), whereas G --> C transversion was detected in 26.9% of US tumors but none from Egypt (p = 0.003). We also found a trend toward differences in the p53 exons in which mutations occurred, with higher frequency of exon 5 mutation and lower frequency of exon 6 mutation in Egyptian tumors. Logistic regression showed that K-ras G --> T transversion mutations and p53 exon 6 mutations were predicted by the country of residence of the patients. Our study identifies that there are differences in the types of mutations found in tumors from pancreatic carcinoma patients in Egypt and the US, and suggests that environmental factors may explain these differences.

K- ras mutation influences macroscopic features of gastric carcinoma

Gastric carcinoma is classified morphologically as type 1 to 4. Type 1 is defined as a polypoid tumor; types 2 and 3 are defined as ulcerated tumors with polypoid growth or gastric wall infiltration, respectively, and type 4 tumors are defined as flat. This morphological classification is important because biological characteristics differ between the four morphological types, but little is known about genetic differences between them. One hundred eight gastric tumors were classified macroscopically as type 1 to 4. Tumoral DNA was microdissected from paraffin-embedded tissue sections. PCR amplification of exon 1 of a K-ras containing codons 12 and 13 was performed. K-ras amplicons were dot-blotted onto nylon filters and hybridized with radiolabeled oligomer primers. A K-ras mutation was found in 20 of 108 gastric cancers. A significant relationship of K-ras mutation with polypoid cancer was found. The frequency of K-ras mutation was 6/14 (43%), 8/29 (28%), 2/11 (18%), and 4/54 (7%) in type 1 to 4 tumors, respectively. K-ras mutation was correlated with well-differentiated tumors. Of various types of K-ras mutations, 12 Asp often was seen in type 1 and 2 gastric cancers (well-demarcated, elevated tumors), while 12 Val and 12 Ser were often seen in type 3 and 4 cases (infiltrating carcinomas). K-ras mutations occur prominently in type 1 and type 2 gastric cancers.