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

PURPOSE: KRAS mutations are found in approximately 25% of lung adenocarcinomas in Western countries and, as a group, have been 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. EXPERIMENTAL DESIGN: 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. RESULTS: KRAS mutational analysis was done on 482 lung adenocarcinomas, 81 (17%) of which were obtained from patients who had never smoked cigarettes. KRAS mutations were found in 15% (12 of 81; 95% confidence intervals, 8-24%) of tumors from never smokers. Similarly, 22% (69 of 316; 95% confidence intervals, 17-27%) of tumors from former smokers, and 25% (21 of 85; 95% confidence intervals, 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. 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 on 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.     

KRAS gene amplification in colorectal cancer and impact on response to EGFR‐targeted therapy

KRAS mutations are the most common oncogenic event in colorectal cancer (CRC) progression and their occurrence is associated with lack of response to anti epidermal growth factor receptor (EGFR) targeted therapies. Using preclinical models and patients' samples we recently reported that the emergence of KRAS mutations but also KRAS amplification is associated with acquired resistance to the EGFR inhibitors cetuximab or panitumumab. We reasoned that KRAS amplification may also be responsible for primary resistance to these agents. Furthermore, while the prevalence of KRAS mutations has been well established in CRC, little is known about the frequency of KRAS amplification in large CRC series. We performed a screening of 1,039 CRC samples to assess the prevalence of KRAS amplification in this tumor type and further evaluated the role of this genetic alteration on the sensitivity to anti EGFR therapies. We detected KRAS amplification in 7/1,039 (0.67%) and 1/102 evaluable CRC specimens and cell lines, respectively. KRAS amplification was mutually exclusive with KRAS mutations. Tumors or cell lines harboring this genetic lesion are not responsive to anti‐EGFR inhibitors. Although KRAS amplification is an infrequent event in CRC, it might be responsible for precluding response to anti‐EGFR treatment in a small proportion of patients.     

Distribution of KRAS and BRAF Mutations in Metastatic Colorectal Cancers in Turkish Patients

The results of this study demonstrate the potential prognostic and predictive values of KRAS and BRAF gene mutations in patients with colorectal cancer (CRC). It has been proven that KRAS and BRAF mutations are predictive biomarkers for resistance to anti-EGFR monoclonal antibody treatment in patients with metastatic CRC (mCRC). We demonstrated the distribution of KRAS (codons 12, 13 and 61) and BRAF (codon 600) gene mutations in 50 mCRCs using direct sequencing and compared the results with clinicopathological data. KRAS and BRAF mutations were identified in 15 (30%) and 1 (2%) patients, respectively. We identified KRAS mutations in codon 12, 13 and 61 in 73.3% (11/15), 20% (3/15) and 6.67% (1/15) of the positive patients, respectively. The KRAS mutation frequency was significantly higher in tumors located in the ascending colon (p=0.043). Thus, we found that approximately 1/3 of the patients with mCRC had KRAS mutations and the only clinicopathological factor related to this mutation was tumor location. Future studies with larger patient groups should yield more accurate data regarding the molecular mechanism of CRC and the association between KRAS and BRAF mutations and clinicopathological features.     

Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib.

PURPOSE: Epidermal growth factor receptor (EGFR) mutations have been associated with tumor response to treatment with single-agent EGFR inhibitors in patients with relapsed non-small-cell lung cancer (NSCLC). The implications of EGFR mutations in patients treated with EGFR inhibitors plus first-line chemotherapy are unknown. KRAS is frequently activated in NSCLC. The relationship of KRAS mutations to outcome after EGFR inhibitor treatment has not been described. PATIENTS AND METHODS: Previously untreated patients with advanced NSCLC in the phase III TRIBUTE study who were randomly assigned to carboplatin and paclitaxel with erlotinib or placebo were assessed for survival, response, and time to progression (TTP). EGFR exons 18 through 21 and KRAS exon 2 were sequenced in tumors from 274 patients. Outcomes were correlated with EGFR and KRAS mutations in retrospective subset analyses. RESULTS: EGFR mutations were detected in 13% of tumors and were associated with longer survival, irrespective of treatment (P < .001). Among erlotinib-treated patients, EGFR mutations were associated with improved response rate (P < .05) and there was a trend toward an erlotinib benefit on TTP (P = .092), but not improved survival (P = .96). KRAS mutations (21% of tumors) were associated with significantly decreased TTP and survival in erlotinib plus chemotherapy-treated patients. CONCLUSION: EGFR mutations may be a positive prognostic factor for survival in advanced NSCLC patients treated with chemotherapy with or without erlotinib, and may predict greater likelihood of response. Patients with KRAS-mutant NSCLC showed poorer clinical outcomes when treated with erlotinib and chemotherapy. Further studies are needed to confirm the findings of this retrospective subset analysis.     

KRAS mutation detection in Tunisian sporadic coloractal cancer patients with direct sequencing, high resolution melting and denaturating high performance liquid chromatography

The Kirsten Rat Sarcoma (KRAS) oncogene has been introduced recently as a genetic biomarker for metastatic sporadic colorectal cancer prior to anti-EGFR treatment. Identifying patients with KRAS mutations that not respond to EGFR targeted therapies require sensitive, rapid and efficacious routine technique. We have attempted to evaluate the efficiency of three conventional methods: direct sequencing, HRM and DHPLC, to detect mutations in codon 12 and 13 of the KRAS exon2 gene. For this first Tunisian study on KRAS, we detected 45.83% of altered KRAS gene among 48 formalin-fixed paraffin-embedded sporadic colorectal adenocarcinoma patients. The use of HRM-sequencing allowed as enlarging the detected KRAS exon 2 mutations (22/48) in comparison with direct sequencing (17/48). DHPLC was used to confirm results when consensus was not observed between HRM and direct sequencing. This study brings an interesting data concerning an inter-method validation between sequencing and HRM in the investigation of sporadic colorectal cancer biomarker. It also shows that KRAS mutations occur at similar frequencies in Tunisian patients as in other populations; and suggests that the same genes are at play in sporadic CRC cancer, despite ethnic, geographical and environmental differences between countries.     

Frequency of K-RAS and N-RAS Gene Mutations in Colorectal Cancers in Southeastern Iran

Background: K-RAS and N-RAS gene mutations cause resistance to treatment in patients with colorectal cancer. Based on this, awareness of mutation of these genes is considered a clinically important step towards better diagnosis and appropriate treatment. Materials and Methods: Fifty paraffin-embedded blocks of colorectal cancer were obtained from Imam Reza Hospital of Birjand, Iran. Following DNA extraction, the samples were analyzed for common mutations of exons 2, 3 and 4 of KRAS and NRAS genes using real time PCR and pyrosequencing. Results: According to this study, the prevalence of mutations was respectively 28% (14 out of 50) and 2% (1 out of 50) in KRAS and NRAS genes. All the mutations were observed in patients >50 years old. Conclusions: Mutations were found in both KRAS and NRAS genes in colorectal cancers in Iranian patients. Determining the frequency of these mutations in each geographical region may be necessary to benefit from targeted cancer therapy.     

Molecular spectrum of KRAS,BRAF, and PIK3CA gene mutation: determination of frequency,distribution pattern in Indian colorectal carcinoma

Molecular evaluation of KRAS, BRAF, and PIK3CA mutation has become an important part in colorectal carcinoma evaluation, and their alterations may determine the therapeutic response to anti-EGFR therapy. The current study demonstrates the evaluation of KRAS, BRAF, and PIK3CA mutation using direct sequencing in 204 samples. The frequency of KRAS, BRAF, and PIK3CA mutations was 23.5, 9.8, and 5.9 %, respectively. Five different substitution mutations at KRAS codon 12 (G12S, G12D, G12A, G12V, and G12C) and one substitution type at codon 13 (G13D) were observed. KRAS mutations were significantly higher in patients who were >50 years, and were associated with moderate/poorly differentiated tumors and adenocarcinomas. All mutations in BRAF gene were of V600E type, which were frequent in patients who were ≤50 years. Unlike KRAS mutations, BRAF mutations were more frequent in well-differentiated tumors and right-sided tumors. PIK3CA–E545K was the most recurrent mutation while other mutations detected were T544I, Q546R, H1047R, G1049S, and D1056N. No significant association of PIK3CA mutation with age, tumor differentiation, location, and other parameters was noted. No concomitant mutation of KRAS and BRAF mutations was observed, while, interestingly, five cases showed concurrent mutation of KRAS and PIK3CA mutations. In conclusion, to our knowledge, this is the first study to evaluate the PIK3CA mutation in Indian CRC patients. The frequency of KRAS, BRAF, and PIK3CA was similar to worldwide reports. Furthermore, identification of molecular markers has unique strengths, and can provide insights into the pathogenic process and help optimize personalized prevention and therapy.     

慢性粒细胞白血病患者血液肿瘤相关基因突变研究进展

慢性粒细胞白血病（CML）是以Ph染色体异位为特征的骨髓增殖性疾病,酪氨酸激酶抑制剂（TKI）的应用显著改善了CML患者的预后。细胞遗传学和分子学监测用于评估TKI疗效并指导疾病管理已成为CML治疗的重要组成部分。然而,在疾病诊断、转化和耐药性方面扩大基因组分析是CML研究中尚未完全探索的领域。文章旨在探讨CML最初诊断和治疗失败转化时的基因突变频率和类别,分析诊治中基因突变与CML患者预后的关系。     

Both BRAF and KRAS mutations are rare in colorectal carcinomas from patients with hereditary nonpolyposis colorectal cancer

The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal carcinomas. To clarify the extent of BRAF mutations in HNPCC colorectal carcinomas, which are typical mismatch repair deficient carcinomas, we compared the frequency of BRAF mutations between HNPCC, familial adenomatous polyposis (FAP) and sporadic cases. The frequency of KRAS mutations was also compared between these three syndromes. No BRAF mutations were detected in 33 HNPCC colorectal carcinomas, while they were detected in 3 of 26 (12%) FAP carcinomas and 2 of 53 (4%) microsatellite stable sporadic carcinomas. KRAS mutations were detected in 2 of 33 (6%) HNPCC, 9 of 26 (35%) FAP and 18 of 53 (34%) sporadic carcinomas. Such extremely low frequencies of BRAF and KRAS mutations in HNPCC colorectal carcinomas suggest that the participation of RAS-RAF signaling is minor in HNPCC, and that the previously suggested high frequency of BRAF mutations in mismatch repair deficient colorectal carcinomas is not due to mutations of mismatch repair genes.     

Anti-DLL4 inhibits growth and reduces tumor-initiating cell frequency in colorectal tumors with oncogenic KRAS mutations

KRAS mutations are frequent in colorectal cancer (CRC) and are associated with clinical resistance to treatment with the epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies. Delta-like 4 ligand (DLL4) is an important component of the Notch signaling pathway and mediates stem cell self-renewal and vascular development. DLL4 inhibition in colon tumor cells reduces tumor growth and stem cell frequency. Considering the need for new drugs to treat colon cancers with oncogenic KRAS mutations, we examined in this study the efficacy of anti-DLL4 antibodies in KRAS mutant tumors in a panel of early passage colon tumor xenograft models derived from patients. Consistent with clinical findings, mutant KRAS colorectal xenograft tumors were insensitive to the EGFR therapeutic antibody cetuximab, whereas KRAS wild-type tumors responded to cetuximab. In contrast, anti-DLL4 was efficacious against both wild-type and mutant KRAS colon tumors as a single agent and in combination with irinotecan. Further analysis of mutant KRAS tumors indicated that the anti-DLL4/irinotecan combination produced a significant decrease in colon cancer stem cell frequency while promoting apoptosis in tumor cells. Our findings provide a rationale for targeting DLL4-Notch signaling for improved treatment of CRC patients with activating KRAS mutations.     

KRAS as a druggable target in NSCLC: Rising like a phoenix after decades of development failures

Cancers of nearly all lineages harbor alterations that deregulate mitogen-activated protein kinase signaling, a crucial signaling pathway for tumor formation and maintenance. Of these, KRAS mutations are the most frequent gain-of-function alterations found in patients with cancer. In particular they represents the most common molecular alteration detected in non-small cell lung cancer (NSCLC) accounting for up to 25% of all oncogenic mutations. They were identified decades ago and prior efforts to target these proteins have been unsuccessful. KRAS mutation profiles (i.e. frequency of specific codon substitutions) in smokers and never-smokers are distinct and not all KRAS alterations are driver mutations. KRAS has evolved from a mutation with possible predictive value to a therapeutic target with great promise. Here, we will discuss the biology of KRAS in lung cancer and its clinical implications in oncology today and in the foreseeable future.     

High Frequency of KRAS Codon 146 and FBXW7 Mutations in Thai Patients with Stage II-III Colon Cancer

Background: KRAS, NRAS, and BRAF gene mutations are the most clinically relevant and frequently reported incolorectal cancer (CRC). Although data on these genes are frequently reported in several counties, data specific to thesegenes among Thai population are scarce. The aim of this study was to investigate and identify molecular alterationsassociated with colon cancer in Thai population, and to determine the impact of these genetic aberrations on clinicaloutcome. Methods: DNA from 108 archived formalin-fixed, paraffin-embedded (FFPE) tissue samples that histologicallyconfirmed adenocarcinoma of stage II-III colon cancer between 2010 and 2012 at Siriraj Hospital (Bangkok, Thailand)were extracted. Gene mutational analysis was performed by next-generation sequencing (NGS) using an OncomineSolid Tumor DNA kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Results: A total of 22 somatic genemutations were detected. The mutation frequency observed in KRAS, NRAS, BRAF, PIK3CA, and FBXW7 mutationswas 47.2%, 1.9%, 1.9%, 12%, and 14.8%, respectively. KRAS mutation codon 12, 13, 59, 61, 117, and 146 mutationswere identified in 29.6%, 8.3%, 1.8%, 0.9%, 0.0%, and 8.3%, respectively. KRAS Exon 4 had better DFS comparedwith Exon 2 and 3. Conclusions: This study is the first to comprehensively report hotspot mutations using NGS in Thaicolon cancer patients. The most commonly identified gene mutation frequencies among Thai patients (KRAS, NRAS,BRAF, TP53, and PIK3CA) were similar to the gene mutation frequencies reported in Western population, except forsubgroup of KRAS codon 146 and FBXW7 mutations that had a slightly higher frequency.     

Association of p16 homozygous deletions with clinicopathologic characteristics and EGFR/KRAS/p53 mutations in lung adenocarcinoma

PURPOSE: The p16 gene is frequently inactivated in lung adenocarcinoma. In particular, homozygous deletions (HD) have been frequently detected in cell lines; however, their frequency and specificity is not well-established in primary tumors. The purpose of this study was to elucidate the prevalence and the timing for the occurrence of p16 HDs in lung adenocarcinoma progression in vivo. EXPERIMENTAL DESIGN: Multiple ligation-dependent probe amplification was used for the detection of p16 HDs in 28 primary small-sized lung adenocarcinomas and 22 metastatic lung adenocarcinomas to the brain. Cancer cells were isolated from primary adenocarcinoma specimens by laser capture microdissection. HDs were confirmed by quantitative real-time genomic PCR analysis. RESULTS: HDs were detected in 8 of 28 (29%) primary tumors, including 2 of 8 (25%) noninvasive bronchioloalveolar carcinomas, and 5 of 22 (26%) brain metastases, respectively. No significant associations were observed between p16 HDs and gender, age, smoking history, stage, and prognosis. HDs were detected with similar frequencies (17-29%) among adenocarcinomas with epidermal growth factor receptor (EGFR) mutations, with KRAS mutations, and without EGFR/KRAS mutations, and with similar frequencies (22-28%) between adenocarcinomas with and without p53 mutations. CONCLUSIONS: p16 HDs occur early in the development of lung adenocarcinomas and with similar frequencies among EGFR type, KRAS type, and non-EGFR/KRAS type lung adenocarcinomas. Tobacco carcinogens would not be a major factor inducing p16 HDs in lung adenocarcinoma progression.     

The molecular mechanism of chronic myelogenous leukemia and its therapeutic implications: studies in a murine model

Chronic myelogenous leukemia (CML) is a malignant disease resulting from the neoplastic transformation of a hematopoietic stem cell. Generation of the BCR-ABL fusion gene plays an essential role in causing the vast majority of CML. Clinical and laboratory studies have indicated that development of CML involves both the effects of BCR-ABL within its correct target cells and interactions of BCR-ABL target cells with the rest of the in vivo environment, and that the progression of the disease to blast crisis involves multiple genetic alterations. An efficient mouse bone marrow transduction and transplantation model for CML has recently been developed. This review summarizes the analysis of the roles of functional domains and downstream signaling pathways of BCR-ABL, of altered cytokine production, of interferon signaling pathways and of oncogene cooperation in the pathogenesis of CML using this murine model. The in vivo studies of leukemogenesis will help to advance mechanism-based therapies for CML, as well as to understand fundamental rules of leukemogenesis and hematopoiesis.     

Heterogeneity of KRAS,NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer and potential effects on therapy in the CAPRI GOIM trial

BackgroundEvidence suggests that metastatic colorectal carcinoma (mCRC) has a high level of intratumor heterogeneity. We carried out a quantitative assessment of tumor heterogeneity for KRAS, NRAS, BRAF and PIK3CA mutations, in order to assess potential clinical implications.Patients and methodsTumor samples (n = 182) from the CAPRI-GOIM trial of first-line cetuximab + FOLFIRI in KRAS exon-2 wild-type mCRC patients were assessed by next-generation sequencing that allows quantitative assessment of mutant genes. Mutant allelic frequency was normalized for the neoplastic cell content and, assuming that somatic mutations usually affect one allele, the Heterogeneity Score (HS) was calculated by multiplying by 2 the frequency of mutant alleles in neoplastic cells. Therefore, HS virtually corresponds to the fraction of neoplastic cells carrying a specific mutation.ResultsThe KRAS HS ranged between 12 and 260 with mean value of 87.1 and median value of 84.4, suggesting that in most CRC, the majority of neoplastic cells carry mutant KRAS. Similar findings were observed for NRAS (HS range 35.5–146.7; mean 102.8; median 117.1). In contrast, in BRAF (HS range 17.1–120; mean 54.8; median 54.3) and PIK3CA (HS range 14.3–120; mean 59.5; median 47.3) mutant cases, only a fraction of neoplastic cells seem to carry the mutant allele. The response rate was 70% in KRAS mutant patients with an HS <33 (low KRAS; n = 10) and 45.7% in KRAS HS >33 patients (high KRAS; n = 35); median progression-free survival were 7.97 and 8.37 months, respectively. Low-KRAS tumors had a higher frequency of additional mutations in PIK3CA when compared with high-KRAS (6/10 versus 8/35).ConclusionsKRAS and NRAS mutations are usually present in the majority of neoplastic cells, whereas BRAF and PIK3CA mutations often affect a limited fraction of transformed cells. Resistance to cetuximab in low-KRAS patients might be driven by the complex mutational profile rather than KRAS mutation load.     

Colorectal cancer with mutation in BRAF, KRAS, and wild-type with respect to both oncogenes showing different patterns of DNA methylation.

PURPOSE: BRAF mutations are common in sporadic colorectal cancers (CRCs) with a DNA mismatch repair (MMR) deficiency that results from promoter methylation of hMLH1, whereas KRAS mutations are common in MMR proficient CRCs associated with promoter methylation of MGMT. The aim of this study was to further investigate the link between genetic alterations in the RAS/RAF/ERK pathway and an underlying epigenetic disorder. PATIENTS AND METHODS: Activating mutations of BRAF and KRAS were identified and correlated with promoter methylation of 11 loci, including MINT1, MINT2, MINT31, CACNA1G, p16(INK4a), p14(ARF), COX2, DAPK, MGMT, and the two regions in hMLH1 in 468 CRCs and matched normal mucosa. RESULTS: BRAF V599E mutations were identified in 21 (9%) of 234 CRCs, and KRAS mutations were identified in 72 (31%) of 234 CRCs. Mutations in BRAF and KRAS were never found in the same tumor. CRCs with BRAF mutations showed high-level promoter methylation in multiple loci, with a mean number of methylated loci of 7.2 (95% CI, 6.6 to 7.9) among 11 loci examined (P < .0001). Tumors with KRAS mutations showed low-level promoter methylation, and CRCs with neither mutation showed a weak association with promoter methylation, with an average number of methylated loci of 1.8 (95% CI, 1.5 to 2.1) and 1.0 (95% CI, 0.79 to 1.3), respectively. CONCLUSION: In CRC, the methylation status of multiple promoters can be predicted through knowledge of BRAF and, to a lesser extent, KRAS activating mutations, indicating that these mutations are closely associated with different patterns of DNA hypermethylation. These changes may be important events in colorectal tumorigenesis.     

Polymorphic variation in glutathione-S-transferase genes and risk of chronic myeloid leukaemia in the Kashmiri population

Cancer is a complex disease and the genetic susceptibility to it could be an outcome of the inherited difference in the capacity of xenobiotic metabolizing enzymes. Glutathione-S-transferases (GSTs) are phase II metabolizing enzymes whose various genotypes have been associated with increased risk of different types of cancer. Null mutations caused by the deletion of the entire gene result in the absence of the enzymatic activity and increase in the risk of developing cancer including chronic myeloid leukaemia (CML). In the present case-control study we evaluated the effect of null mutations in GSTM1 and GSTT1 genes on the risk of developing CML. The study included 75 CML patients (43 males and 32 females; age (mean ± S.D) 42.3 ± 13.4 years) and unrelated non-malignant controls (76 male and 48 females; age (mean ± S.D) 41.5 ± 12.9). The distribution of GSTM1 and GSTT1 genotypes in CML patients and controls was assessed by multiplex-PCR method. Logistic regression was used to assess the relationship between GSTM1 and GSTT1 genotypes and risk of CML. Chi-square test was used to evaluate the trend in modulating the risk to CML by one or more potential high risk genotype. Although GSTM1 null genotype frequency was higher in CML patients (41%) than in the controls (35%), it did not reached a statistical significance (OD = 1.32, 95% CI: 0.73-2.40; P value = 0.4295). The frequency of GSTT1 null genotypes was higher in the CML patients (36%) than in the controls (21%) and the difference was found to be statistically significant (OD = 2.12, 95% CI: 1.12-4.02; P value = 0.0308). This suggests that the presence of GSTT1 genotype may have protective role against the CML. We found a statistically significant (OD = 3.09, 95% CI: 1.122-8.528; P value = 0.0472) interaction between the GSTM1 and GSTT1 null genotypes and thus individuals carrying null genotypes of both GSTM1 and GSTT1 genes are at elevated risk of CML.     

EGFR mutations, gene amplification, and protein expression and KRAS mutations in primary and metastatic tumors of nonsmall cell lung cancers and their clinical implications: a meta-analysis

A meta-analysis was performed to determine EGFR mutations, gene amplification, and protein expression and KRAS mutations in primary and metastatic nonsmall cell lung cancer (NSCLC). We found that KRAS gene mutation frequencies were higher in primary than in metastatic tumors. There was no significant difference in EGFR mutation frequency between the primary and metastatic tumors. These results suggest that KRAS mutations in primary NSCLC foci may be a more accurate biomarker than in metastases to reflect KRAS mutation status. Combined detection of EGFR and KRAS mutations in primary NSCLC foci appears to be an optimal approach for first-line EGFR-TKI therapy.