This study was designed to determine the relationship of cigarette smoking to the frequency and qualitative differences among KRAS mutations in lung adenocarcinomas from Korean patients.
Materials and Methods
Detailed smoking histories were obtained from 200 consecutively enrolled patients with lung adenocarcinoma according to a standard protocol. EGFR (exons 18 to 21) and KRAS (codons 12/13) mutations were determined via direct-sequencing.
The incidence of KRAS mutations was 8% (16 of 200) in patients with lung adenocarcinoma. KRAS mutations were found in 5.8% (7 of 120) of tumors from never-smokers, 15% (6 of 40) from former-smokers, and 7.5% (3 of 40) from current-smokers. The frequency of KRAS mutations did not differ significantly according to smoking history (p=0.435). Never-smokers were significantly more likely than former or current smokers to have a transition mutation (G→A or C→T) rather than a transversion mutation (G→T or G→C) that is known to be smoking-related (p=0.011). In a Cox regression model, the adjusted hazard ratios for the risk of progression with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) were 0.24 (95% CI, 0.14-0.42; p<0.001) for the EGFR mutation and 1.27 (95% CI, 0.58-2.79; p=0.537) for the KRAS mutation.
Cigarette smoking did not influence the frequency of KRAS mutations in lung adenocarcinomas in Korean patients, but influenced qualitative differences in the KRAS mutations.
EGFR; KRAS; pulmonary adenocarcinoma; cigarette smoking; EGFR-tyrosine kinase inhibitors
EGFR mutations underlie the sensitivity of lung cancers to erlotinib and gefitinib and can occur in any patient with this illness. Here we examine the frequency of EGFR mutations in smokers and men.
We determined the frequency of EGFR mutations and characterized their association with cigarette smoking status and male sex.
We tested 2,142 lung adenocarcinoma specimens for the presence of EGFR exon 19 deletions and L858R. EGFR mutations were found in 15% of tumors from former smokers (181 of 1,218; 95% CI, 13% to 17%), 6% from current smokers (20 of 344; 95% CI, 4% to 9%), and 52% from never smokers (302 of 580; 95% CI, 48% to 56%; P < .001 for ever v never smokers). EGFR mutations in former or current smokers represented 40% of all those detected (201 of 503; 95% CI, 36% to 44%). EGFR mutations were found in 19% (157 of 827; 95% CI, 16% to 22%) of tumors from men and 26% (346 of 1,315; 95% CI, 24% to 29%) of tumors from women (P < .001). EGFR mutations in men represented 31% (157 of 503; 95% CI, 27% to 35%) of all those detected.
A large number of EGFR mutations are found in adenocarcinoma tumor specimens from men and people who smoked cigarettes. If only women who were never smokers were tested, 57% of all EGFR mutations would be missed. Testing for EGFR mutations should be considered for all patients with adenocarcinoma of the lung at diagnosis, regardless of clinical characteristics. This strategy can extend the use of EGFR tyrosine kinase inhibitors to the greatest number individuals with the potential for substantial benefit.
Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in EGFR and KRAS appear to be mutually exclusive.
Methods and Findings
We sought to determine whether mutations in KRAS could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in EGFR and KRAS. We show that mutations in KRAS are associated with a lack of sensitivity to either drug.
Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both EGFR and KRAS.
Mutational analysis of the KRAS gene in lung cancer patients treated with two different kinase inhibitors suggests that tumors with KRAS mutations do not respond to these drugs
To determine the proportion of lung adenocarcinomas from East Asian never-smokers who harbor known oncogenic driver mutations.
Patients and Methods
In this surgical series, 52 resected lung adenocarcinomas from never-smokers (< 100 cigarettes in a lifetime) at a single institution (Fudan University, Shanghai, China) were analyzed concurrently for mutations in EGFR, KRAS, NRAS, HRAS, HER2, BRAF, ALK, PIK3CA, TP53 and LKB1.
Forty-one tumors harbored EGFR mutations, three harbored EML4-ALK fusions, two harbored HER2 insertions, and one harbored a KRAS mutation. All mutations were mutually exclusive. Thus, 90% (47 of 52; 95% CI, 0.7896 to 0.9625) of lung adenocarcinomas from never-smokers were found to harbor well-known oncogenic mutations in just four genes. No BRAF, NRAS, HRAS, or LKB1 mutations were detected, while 15 had TP53 mutations. Four tumors contained PIK3CA mutations, always together with EGFR mutations.
To our knowledge, this study represents the first comprehensive and concurrent analysis of major recurrent oncogenic mutations found in a large cohort of lung adenocarcinomas from East Asian never-smokers. Since drugs are now available that target mutant EGFR, HER2, and ALK, respectively, this result indicates that prospective mutation testing in these patients should successfully assign a targeted therapy in the majority of cases.
Our previous study revealed that 90% (47 of 52; 95% CI: 0.79–0.96) of Chinese never-smokers with lung adenocarcinoma harbor known oncogenic driver mutations in just four genes: EGFR, ALK, HER2, and KRAS. Here, we examined the status of known driver mutations specifically in female never-smokers with lung adenocarcinoma.
Tumors were genotyped for mutations in EGFR, KRAS, ALK, HER2, and BRAF. Data on age, stage, tumor differentiation, histological subtypes, and molecular alterations were recorded from 349 resected lung adenocarcinomas from female never-smokers. We further compared the clinicopathological parameters according to mutational status of these genes.
Two hundred and sixty-six (76.2%) tumors harbored EGFR mutations, 16 (4.6%) HER2 mutations, 15 (4.3%) EML4-ALK fusions, seven (2.0%) KRAS mutations, and two (0.6%) BRAF mutations. In univariate analysis, patients harboring EGFR mutations were significantly older (p<0.001), whereas patients harboring HER2 mutations were significantly younger (p=0.036). Higher prevalence of KRAS (p=0.028) and HER2 (p=0.021) mutations was found in invasive mucinous adenocarcinoma (IMA). The frequency of EGFR mutations was positively correlated with acinar predominant tumors (p=0.002). Multivariate analysis revealed that older age at diagnosis (p=0.013) and acinar predominant subtype (p=0.005) were independent predictors of EGFR mutations. Independent predictors of HER2 mutations included younger age (p=0.030) and IMA (p=0.017). IMA (p=0.006) and poor differentiation (p=0.028) were independently associated with KRAS mutations.
The frequency of driver mutations in never-smoking female lung adenocarcinoma varies with histological subtypes and age at diagnosis. These data have implications for both clinical trial design and therapeutic strategies.
Lung adenocarcinoma; Female; Never smoker; EGFR mutation; HER2 mutation; Acinar; Mucinous; Age
In highly susceptible A/J mice, lung adenomas develop spontaneously and can also be induced by the tobacco carcinogen, 3-methylcholanthrene (MCA). If MCA administration is followed by butylated hydroxytoluene (BHT)-elicited chronic inflammation, tumor multiplicity increases. The distribution of Kras codon 12 mutations in these MCA/BHT-induced tumors is strikingly similar to those found in adenocarcinomas from human smokers. Like A/J, Strain BALB/cBy (hereafter cBy) mice also develop few lung tumors unless MCA treatment is followed by BHT. MCA/BHT induced tumors in cBy mice contain predominantly G
D Kras codon 12 mutations, which is the most common mutation in never-smokers. Thus, a single lung carcinogen induces different Kras lung tumor initiating mutations in different strains of mice. These strain effects may be useful for investigating the role of specific Kras mutations in adenocarcinoma pathogenesis in smokers vs. never-smokers, identifying mechanisms that select for certain Kras mutations, and developing new drugs that specifically target cells with different Kras mutations.
The aim of the current study was to determine the clinical significance according to the subtypes of epidermal growth factor receptor (EGFR) mutations and presence of KRAS mutations in operable non-small-cell lung cancer (NSCLC). We sequenced exons 18-21 of the EGFR tyrosine kinase domain and examined mutations in codons 12 and 13 of KRAS in tissues of patients with NSCLC who had undergone surgical resection. EGFR mutations were more frequent in never-smokers than smokers (33% vs. 14%, respectively; p=0.009) and in females than in males (31% vs. 16%, respectively; p=0.036). Mutations in exon 18-19 and 20-21 were found in 10 and 22 patients, respectively. Never-smokers and broncho-alveolar cell carcinoma features were positively associated with a mutation in exon 18-19 (p=0.027 and 0.016, respectively). The five-year survival rate in patients with a mutation in exons 18-19 (100%) was higher than that in patients without such mutation (47%; p=0.021). KRAS mutations were found in 16 patients (12%) and were not related to the overall survival (p=0.742). Patients with an EGFR mutation in exons 18-19 had better survival than patients without such mutation. Subtypes of EGFR mutations may be prognostic factors in patients undergoing curative resection.
Carcinoma, Non-small-cell Lung; Receptor, Epidermal Growth Factor; Genes, Ras; Mutation
We previously showed that 90% (47 of 52; 95% CI, 0.79 to 0.96) of lung adenocarcinomas from East Asian never-smokers harbored well-known oncogenic mutations in just four genes: EGFR, HER2, ALK, and KRAS. Here, we sought to extend these findings to more samples and identify driver alterations in tumors negative for these mutations.
We have collected and analyzed 202 resected lung adenocarcinomas from never smokers seen at Fudan University Shanghai Cancer Center. Since mutations were mutually exclusive in the first 52 examined, we determined the status of EGFR, KRAS, HER2, ALK, and BRAF in stepwise fashion as previously described. Samples negative for mutations in these 5 genes were subsequently examined for known ROS1 fusions by RT-PCR and direct sequencing.
152 tumors (75.3%) harbored EGFR mutations, 12 (6%) had HER2 mutations, 10 (5%) had ALK fusions all involving EML4 as the 5′ partner, 4 (2%) had KRAS mutations, and 2 (1%) harbored ROS1 fusions. No BRAF mutation were detected.
The vast majority (176 of 202; 87.1%, 95% CI: 0.82 to 0.91) of lung adenocarcinomas from never smokers harbor mutant kinases sensitive to available TKIs. Interestingly, patients with EGFR mutant patients tend to be older than those without EGFR mutations (58.3 Vs 54.3, P = 0.016) and patient without any known oncogenic driver tend to be diagnosed at a younger age (52.3 Vs 57.9, P = 0.013). Collectively, these data indicate that the majority of never smokers with lung adenocarcinoma could benefit from treatment with a specific tyrosine kinase inhibitor.
Mutations inducing resistance to anti-epidermal growth factor receptor (EGFR) therapy may have a clinical impact even if present in minor cell clones which could expand during treatment. We tested this hypothesis in lung cancer patients treated with tyrosine kinase inhibitors (TKIs). Eighty-three patients with lung adenocarcinoma treated with erlotinib or gefitinib were included in this study. The mutational status of KRAS and EGFR was investigated by direct sequencing (DS). KRAS mutations were also assessed by mutant-enriched sequencing (ME-sequencing). DS detected KRAS mutations in 16 (19%) of 83 tumors; ME-sequencing identified all the mutations detected by DS but also mutations in minor clones of 14 additional tumors, for a total of 30 (36%) of 83. KRAS mutations assessed by DS and ME-sequencing significantly correlated with resistance to TKIs (P = .04 and P = .004, respectively) and significantly affected progression-free survival (PFS) and overall survival (OS). However, the predictive power of mutations assessed by ME-sequencing was higher than that obtained by DS (hazard ratio [HR] = 2.82, P = .0001 vs HR = 1.98, P = .04, respectively, for OS; HR = 2.52, P = .0005 vs HR = 2.21, P = .007, respectively, for PFS). Survival outcome of patients harboring KRAS mutations in minor clones, detected only by ME-sequencing, did not differ from that of patients with KRAS mutations detected by DS. Only KRAS mutations assessed by ME-sequencing remained an independent predictive factor at multivariate analysis. KRAS mutations in minor clones have an important impact on response and survival of patients with lung adenocarcinoma treated with EGFR-TKI. The use of sensitive detection methods could allow to more effectively identify treatment-resistant patients.
Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices.
We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform.
We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations.
HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.
Mutations in the KRAS gene are associated with poor response to epidermal growth factor receptor inhibitors used in the treatment of metastatic colorectal cancer. Factors influencing KRAS test results in tumor specimens include: tumor heterogeneity, sample handling, slide preparation, techniques for tumor enrichment, DNA preparation, assay design and sensitivity. We evaluated comparability and consistency of KRAS test results among five laboratories currently being used to determine KRAS mutation status of metastatic colorectal cancer specimens in a large, multi-center observational study.
Twenty formalin-fixed paraffin-embedded human colorectal cancer samples from colon resections previously tested for KRAS mutations were selected based on mutation status (6 wild type, 8 codon 12 mutations, and 6 codon 13 mutations). We found good agreement across laboratories despite differences in mutation detection methods. Eighteen of twenty samples (90%) were concordant across all five labs. Discordant results are likely not due to laboratory error, but instead to tumor heterogeneity, contamination of the tumor sample with normal tissue, or analytic factors affecting assay sensitivity.
Our results indicate commercial and academic laboratories provide reliable results for the common KRAS gene mutations at codons 12 and 13 when an adequate percentage of tumor cells is present in the sample.
KRAS; Colorectal cancer; EGFR; Laboratory error
To assess prognostic roles of various KRAS oncogene mutations in colorectal cancer, BRAF mutation status must be controlled for because BRAF mutation is associated with poor prognosis, and almost all BRAF mutants are present among KRAS-wild-type tumors. Taking into account experimental data supporting a greater oncogenic effect of codon 12 mutations compared to codon 13 mutations, we hypothesized that KRAS codon 12 mutated colorectal cancers might behave more aggressively than KRAS-wild-type tumors and codon 13 mutants.
Utilizing molecular pathological epidemiology database of 1261 rectal and colon cancers, we examined clinical outcome and tumor biomarkers of KRAS codon 12 and 13 mutations in 1075 BRAF-wild-type cancers (i.e., controlling for BRAF status). Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for potential confounders, including stage, PIK3CA mutations, microsatellite instability, CpG island methylator phenotype, and LINE-1 methylation.
Compared to patients with KRAS-wild-type/BRAF-wild-type cancers (N=635), those with KRAS codon 12 mutations (N=332) experienced significantly higher colorectal cancer-specific mortality [log-rank P=0.0001; multivariate HR=1.30; 95% confidence interval (CI), 1.02–1.67; P=0.037], whereas KRAS codon 13 mutated cases (N=108) were not significantly associated with prognosis. Among the seven most common KRAS mutations, c.35G>T (p.G12V; N=93) was associated with significantly higher colorectal cancer-specific mortality (log-rank P=0.0007; multivariate HR=2.00, 95% CI, 1.38–2.90, P=0.0003) compared to KRAS-wild-type/BRAF-wild-type cases.
KRAS codon 12 mutations (in particular, c.35G>T), but not codon 13 mutations, are associated with inferior survival in BRAF-wild-type colorectal cancer. Our data highlight the importance of accurate molecular characterization in colorectal cancer.
colon cancer; genetics; oncogenic; molecular diagnostics; personalized medicine; RAF; RAS
Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in ~50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in ~40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.
wild-type Kras2; mutations: lung cancer; loss of heterozygosity; tumor suppressor
Recent evidence suggests that the observed clinical distinctions between lung tumors in smokers and never smokers (NS) extend beyond specific gene mutations, such as EGFR, EML4-ALK, and KRAS, some of which have been translated into targeted therapies. However, the molecular alterations identified thus far cannot explain all of the clinical and biological disparities observed in lung tumors of NS and smokers. To this end, we performed an unbiased genome-wide, comparative study to identify novel genomic aberrations that differ between smokers and NS.
High resolution whole genome DNA copy number profiling of 69 lung adenocarcinomas from smokers (n = 39) and NS (n = 30) revealed both global and regional disparities in the tumor genomes of these two groups. We found that NS lung tumors had a greater proportion of their genomes altered than those of smokers. Moreover, copy number gains on chromosomes 5q, 7p, and 16p occurred more frequently in NS. We validated our findings in two independently generated public datasets. Our findings provide a novel line of evidence distinguishing genetic differences between smoker and NS lung tumors, namely, that the extent of segmental genomic alterations is greater in NS tumors. Collectively, our findings provide evidence that these lung tumors are globally and genetically different, which implies they are likely driven by distinct molecular mechanisms.
Lung cancer patients with mutations in EGFR tyrosine kinase have improved prognosis when treated with EGFR inhibitors. We hypothesized that EGFR mutations may be related to residential radon or passive tobacco smoke.
This hypothesis was investigated by analyzing EGFR mutations in seventy lung tumors from a population of never and long-term former female smokers from Missouri with detailed exposure assessments. The relationship with passive-smoking was also examined in never-smoking female lung cancer cases from the Mayo clinic.
Overall, the frequency of EGFR mutation was 41% [95% Confidence Interval (CI): 32-49%]. Neither radon nor passive-smoking exposure was consistently associated with EGFR mutations in lung tumors.
The results suggest that EGFR mutations are common in female, never-smoking, lung cancer cases from the U.S, and EGFR mutations are unlikely due to exposure to radon or passive-smoking.
EGFR mutations; never-smokers; lung cancer; radon; passive-smoking; second hand smoke; tobacco smoke
The let-7 family of microRNAs are important regulatory molecules in lung cancer. One downstream target of let-7 is the RAS gene family, including KRAS, an important oncogene in the etiology and clinical outcome of lung adenocarcinoma. Recently, a SNP in the let-7 binding region of the KRAS 3’ UTR was identified (termed LCS6). This functional polymorphism alters let-7 binding, resulting in both increased KRAS expression and decreased let-7 exposure. Further, this SNP has been reported as a risk trait for lung cancer risk among low-moderate smokers. Given the functionality of LCS6, we tested the hypothesis that this SNP is associated with the occurrence of KRAS mutation as well as patient survival. Here, we report there is no association between the LCS6 KRAS polymorphism and KRAS mutation. Further, we find no association between the LCS6 polymorphism and lung cancer survival. These unexpected findings imply that this newly reported KRAS-LCS6 polymorphism will have limited clinical utility for NSCLC.
LCS6; let-7; kras; lung cancer
To determine if tumor regression following treatment with gefitinib correlates with the presence of sensitizing mutations in EGFR.
Patients and Methods
Patients with resectable stage I and II non-small cell lung cancer (NSCLC) enriched for the likelihood of EGFR mutation (≤ 15 pack year cigarette smoking history and/or a component of bronchioloalveolar carcinoma) received preoperative gefitinib for 21 days. Tumor specimens were analyzed for EGFR and KRAS mutations and EGFR protein expression and amplification. Patients with ≥ 25% reduction in tumor size measured bidimensionally at 3 weeks and/or patients with an EGFR mutation received adjuvant gefitinib for 2 years post-operatively.
50 patients with stage I/II NSCLC were treated. After 21 days of preoperative gefitinib a response of ≥ 25% was observed in 21/50 (42%) patients. 17/21 patients with a response had an EGFR mutation and 4/21 patients with a response did not (p=0.0001). 25/50 patients were eligible to receive adjuvant gefitinib. With a median follow-up of 44.1 months, 2-year disease free survival for EGFR mutant patients and for those who received adjuvant gefitinib was not statistically different than those who were EGFR wild-type and those who did not receive adjuvant gefitinib. The median disease free and overall survivals have not been reached.
The presence of sensitizing EGFR mutations correlates with radiographic response. A short course of preoperative treatment serves a platform for evaluating activity of new agents and assures sufficient tumor availability for correlative analyses.
Erlotinib is clinically effective in patients with non–small-cell lung cancer (NSCLC) who have adenocarcinoma, are never or limited former smokers, or have EGFR mutant tumors. We investigated the efficacy of erlotinib alone or in combination with chemotherapy in patients with these characteristics.
Patients and Methods
Patients with advanced NSCLC (adenocarcinoma) who were epidermal growth factor receptor tyrosine kinase inhibitor and chemotherapy naive never or light former smokers (smokers of > 100 cigarettes and ≤ 10 pack years and quit ≥ 1 year ago) were randomly assigned to continuous erlotinib or in combination with carboplatin and paclitaxel (ECP) for six cycles followed by erlotinib alone. The primary end point was progression-free survival (PFS). Tissue collection was mandatory.
PFS was similar (5.0 v 6.6 months; P = .1988) in patients randomly assigned to erlotinib alone (arm A; n = 81) or to ECP (arm B; n = 100). EGFR mutation analysis was possible in 91% (164 of 181) of patients, and EGFR mutations were detected in 40% (51 of 128) of never smokers and in 42% (15 of 36) of light former smokers. In arm A, response rate (70% v 9%), PFS (14.1 v 2.6 months), and overall survival (OS; 31.3 v 18.1 month) favored EGFR-mutant patients. In arm B, response rate (73% v 30%), PFS (17.2 v 4.8 months), and OS (38.1 v 14.4 months) favored EGFR-mutant patients. Incidence of grades 3 to 4 hematologic (2% v 49%; P < .001) and nonhematologic (24% v 52%; P < .001) toxicity was greater in patients treated with ECP.
Erlotinib and erlotinib plus chemotherapy have similar efficacy in clinically selected populations of patients with advanced NSCLC. EGFR mutations identify patients most likely to benefit.
Standard cytotoxic chemotherapy is effective for some cancers, but for many others, available treatments offer only a limited survival benefit. Lung adenocarcinoma is one such cancer, responsible for approximately half of lung cancer deaths each year. Development of targeted therapies is thought to hold the most promise for successfully treating this disease, but a targeted approach is dependent on understanding the genomic state of the tumor cells. Exon-directed sequencing of large numbers of lung adenocarcinoma tumor samples has provided an initial low-resolution image of the somatic mutation profile of these tumors. Such cancer sequencing studies have confirmed the high frequency of TP53 and KRAS mutations in lung adenocarcinoma, have found inactivating mutations in known tumor suppressor genes not previously associated with lung adenocarcinoma, and have identified oncogenic mutations of EGFR upon which the first targeted therapy for lung adenocarcinoma patients was based. Additional candidate oncogenes await functional validation. It is anticipated that upcoming whole-exome and whole-genome lung adenocarcinoma sequencing experiments will reveal a more detailed landscape of somatic mutations that can be exploited for therapeutic purposes.
lung adenocarcinoma; EGFR; cancer sequencing; targeted therapy
Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5–8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDA’s indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.
pancreatic cancer; Src; Ras; oncogenic cooperativity
The EML4-ALK fusion gene has been recently identified in a small subset of non-small cell lung cancer (NSCLC) patients who respond positively to ALK inhibitors. The characteristics of the EML4-ALK fusion gene in Chinese patients with NSCLC are poorly understood. Here, we report on the prevalence of EML4-ALK, EGFR status and KRAS mutations in 208 Chinese patients with NSCLC. EGFR mutations were found in 24.5% (51/208) of patients. In concordance with previous reports, these mutations were identified at high frequencies in females (47.5% vs 15.0% in males; P<0.05); never-smokers (42.3% vs 13.9% in smokers; P<0.05), and adenocarcinoma patients (44.2% vs 8.0% in non-adenocarcinoma patients; P<0.05). There were only 2.88% (6/208) patients with KRAS mutations in our study group. We identified 7 patients who harbored the EML4-ALK fusion gene (3.37%, 7/208), including 4 cases with variant 3 (57.1%), 2 with variant 1, and 1 with variant 2. All positive cases corresponded to female patients (11.5%, 7/61). Six of the positive cases were non-smokers (7.69%, 6/78). The incidence of EML4-ALK translocation in female, non-smoking adenocarcinoma patients was as high as 15.2% (5/33). No EGFR/KRAS mutations were detected among the EML4-ALK positive patients. Pathological analysis showed no difference between solid signet-ring cell pattern (4/7) and mucinous cribriform pattern (3/7) in ALK-positive patients. Immunostaining showed intratumor heterogeneity of ALK rearrangement in primary carcinomas and 50% (3/6) of metastatic tumors with ALK-negative staining. Meta-analysis demonstrated that EML4-ALK translocation occurred in 4.84% (125/2580) of unselected patients with NSCLC, and was also predominant in non-smoking patients with adenocarcinoma. Taken together, EML4-ALK translocations were infrequent in the entire NSCLC patient population, but were frequent in the NSCLC subgroup of female, non-smoker, adenocarcinoma patients. There was intratumor heterogeneity of ALK rearrangement in primary carcinomas and at metastatic sites.
The exact influence of statins on gefitinib resistance in human non-small cell lung cancer (NSCLC) cells with KRAS mutation alone or KRAS/PIK3CA and KRAS/PTEN comutations remains unclear. This work found that transfection of mutant KRAS plasmids significantly suppressed the gefitinib cytotoxicity in Calu3 cells (wild-type KRAS). Gefitinib disrupted the Kras/PI3K and Kras/Raf complexes in Calu3 cells, whereas not in Calu3 KRAS mutant cells. These trends were corresponding to the expression of pAKT and pERK in gefitinib treatment. Atorvastatin (1 μM) plus gefitinib treatment inhibited proliferation, promoted cell apoptosis, and reduced the AKT activity in KRAS mutant NSCLC cells compared with gefitinib alone. Atorvastatin (5 μM) further enhanced the gefitinib cytotoxicity through concomitant inhibition of AKT and ERK activity. Atorvastatin could interrupt Kras/PI3K and Kras/Raf complexes, leading to suppression of AKT and ERK activity. Similar results were also obtained in comutant KRAS/PTEN or KRAS/PIK3CA NSCLC cells. Furthermore, mevalonate administration reversed the effects of atorvastatin on the Kras/Raf and Kras/PI3K complexes, as well as AKT and ERK activity in both A549 and Calu1 cells. The in vivo results were similar to those obtained in vitro. Therefore, mutant KRAS-mediated gefitinib insensitivity is mainly derived from failure to disrupt the Kras/Raf and Kras/PI3K complexes in KRAS mutant NSCLC cells. Atorvastatin overcomes gefitinib resistance in KRAS mutant NSCLC cells irrespective of PIK3CA and PTEN statuses through inhibition of HMG-CoA reductase-dependent disruption of the Kras/Raf and Kras/PI3K complexes.
gefitinib; atorvastatin; mutant KRAS; NSCLC
KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.
Mutations in the KRAS gene are one of the most frequent genetic abnormalities in ovarian carcinoma. They are of renewed interest as new epidermal growth factor receptor (EGFR)-targeted therapies are being investigated for use in ovarian carcinoma. As KRAS mutations are associated with poor response and resistance to EGFR-targeting drugs, this study was conducted to obtain more information on the spectrum of KRAS mutations in ovarian carcinoma.
The presence of KRAS mutations in codon 12 and 13 was analyzed in frozen and formalin-fixed paraffin-embedded (FFPE) tissue with a low density biochip platform. 381 malignant (29 borderline malignancy, 270 primary carcinomas, and 82 recurrent carcinomas) and 22 benign tissue samples from a total of 394 patients were examined. KRAS mutational status of each sample was correlated with dignity, FIGO stage, grade, histology, and survival.
KRAS mutations were found in 60 (15%) samples with 58 samples deriving from malignant tissue and 2 samples deriving from benign tissue. In 55 (92%) samples codon 12 was found to be mutated. Frozen and FFPE samples concurred with respect to KRAS mutation status.
KRAS mutation is a common event in ovarian cancer primarily in carcinomas of lower grade, lower FIGO stage, and mucinous histotype. The KRAS mutational status is no prognostic factor for patients treated with standard therapy. However, in line with experience from colorectal cancer and non-small-cell-lung cancer (NSCLC), it may be important for prediction of response to EGFR-targeted therapies.
DNAs from human pancreatic adenocarcinomas were analyzed for the presence of mutations in codons 12, 13 and 61 of the NRAS, KRAS and HRAS gene. Formalin-fixed and paraffin-embedded tissue was used directly in an in vitro amplification reaction to expand the relevant RAS sequences. The mutations were detected by selective hybridization using mutation-specific synthetic oligonucleotides. In 28 of the 30 patients we found a mutation in codon 12 of the KRAS gene. This result confirms the findings of Almoguera et al. [Cell 53 (1988) 549-554] that KRAS mutations occur frequently in adenocarcinomas of the exocrine pancreas. The mutations are predominantly G-T transversions, in contrast to the KRAS mutations in colon tumors which are mainly G-A transitions. Furthermore, in a portion of the tumors the mutation appears to be homozygous.