Jenifer L. Marks

Frequency and Distinctive Spectrum of KRAS Mutations in Never Smokers with Lung Adenocarcinoma

Purpose:KRAS mutations are found in ∼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.

Prognostic and therapeutic implications of EGFR and KRAS mutations in resected lung adenocarcinoma

Background: Somatic mutations in EGFR (exons 19 and 21) and KRAS (exon 2) are found in lung adenocarcinomas and have potential prognostic value in patients with advanced disease. These mutations also have therapeutic significance, as they predict for sensitivity and resistance, respectively, to EGFR tyrosine kinase inhibitor therapy. Whether EGFR and KRAS mutations also have an impact on survival in patients who undergo lung resection for curative intent in the absence of targeted therapy has not been established. Methods: We analyzed the clinical characteristics and outcomes data for 296 patients who underwent resection at our institution for stage I–III lung adenocarcinoma. Tumors were assessed for both EGFR and KRAS mutations by established methods. Results: EGFR and KRAS mutations were found in tumors from 40 (14%) and 50 (17%) patients, respectively. Patients with EGFR mutant tumors were more likely to be never smokers (48%), present with stage I disease (88%), and had a 90% (95% confidence interval [CI] 70–97%) 3-year overall survival, whereas patients with KRAS mutant tumors were more likely to be former/current smokers (92%), present with locally advanced disease (40%), and had a 66% (95% CI 48–79%) 3-year overall survival. Conclusions: EGFR and KRAS mutations define distinct molecular subsets of resected lung adenocarcinoma. Because EGFR and KRAS mutations also predict whether tumors are sensitive or resistant, respectively, to EGFR tyrosine kinase inhibitors, they can readily be used in clinical trials to help guide the administration of specific types of adjuvant therapy.

Acquired Resistance to Epidermal Growth Factor Receptor Kinase Inhibitors Associated with a Novel T854A Mutation in a Patient with EGFR-Mutant Lung Adenocarcinoma

Purpose: Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are associated with sensitivity of lung adenocarcinomas to the EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. Acquired drug resistance is frequently associated with a secondary somatic mutation that leads to the substitution of methionine for threonine at position 790 (T790M). We aimed to identify additional second-site alterations associated with acquired resistance. Experimental Design: Tumor samples were obtained from 48 patients with acquired resistance. Tumor cell DNA was analyzed for EGFR kinase domain mutations. Molecular analyses were then done to characterize the biological properties of a novel mutant EGFR allele. Results: A previously unreported mutation in exon 21 of EGFR, which leads to substitution of alanine for threonine at position 854 (T854A), was identified in one patient with a drug-sensitive EGFR L858R–mutant lung adenocarcinoma after long-term treatment with tyrosine kinase inhibitors. The T854A mutation was not detected in a pretreatment tumor sample. The crystal structure analyses of EGFR suggest that the T854 side chain is within contact distance of gefitinib and erlotinib. Surrogate kinase assays show that the EGFR T854A mutation abrogates the inhibition of tyrosine phosphorylation by erlotinib. Such resistance seems to be overcome by a new irreversible dual EGFR/HER2 inhibitor, BIBW 2992. Conclusions: The T854A mutation is the second reported second-site acquired resistance mutation that is within contact distance of gefitinib and erlotinib. These data suggest that acquired resistance to ATP-mimetic EGFR kinase inhibitors may often be associated with amino acid substitutions that alter drug contact residues in the EGFR ATP-binding pocket.

Mutational Analysis of EGFR and Related Signaling Pathway Genes in Lung Adenocarcinomas Identifies a Novel Somatic Kinase Domain Mutation in FGFR4

Background Fifty percent of lung adenocarcinomas harbor somatic mutations in six genes that encode proteins in the EGFR signaling pathway, i.e., EGFR, HER2/ERBB2, HER4/ERBB4, PIK3CA, BRAF, and KRAS. We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this signaling pathway that could contribute to lung tumorigenesis. Methodology/Principal Findings We analyzed genomic DNA from a total of 261 resected, clinically annotated non-small cell lung cancer (NSCLC) specimens. The coding sequences of 39 genes were screened for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Mutations were considered to be somatic only if they were found in an independent tumor-derived PCR product but not in matched normal tissue. Sequencing of 9MB of tumor sequence identified 239 putative genetic variants. We further examined 22 variants found in RAS family genes and 135 variants localized to exons encoding the kinase domain of respective proteins. We identified a total of 37 non-synonymous somatic mutations; 36 were found collectively in EGFR, KRAS, BRAF, and PIK3CA. One somatic mutation was a previously unreported mutation in the kinase domain (exon 16) of FGFR4 (Glu681Lys), identified in 1 of 158 tumors. The FGFR4 mutation is analogous to a reported tumor-specific somatic mutation in ERBB2 and is located in the same exon as a previously reported kinase domain mutation in FGFR4 (Pro712Thr) in a lung adenocarcinoma cell line. Conclusions/Significance This study is one of the first comprehensive mutational analyses of major genes in a specific signaling pathway in a sizeable cohort of lung adenocarcinomas. Our results suggest the majority of gain-of-function mutations within kinase genes in the EGFR signaling pathway have already been identified. Our findings also implicate FGFR4 in the pathogenesis of a subset of lung adenocarcinomas.

EGFR Mutant Lung Adenocarcinomas in Patients with Germline BRCA Mutations

To the Editor: We have been treating a 47-year-old man of European descent who was diagnosed with metastatic lung adenocarcinoma at the age of 43. The patient is a “never smoker” (i.e., a person who smoked less than 100 cigarettes in a lifetime) whose tumor was found to harbor a somatic mutation in epidermal growth factor receptor (EGFR) gene (H773V774HVdup in exon 20). His family history is significant for breast cancer in maternal relatives and lung cancer in his maternal grandfather who also never smoked. The pedigree for this patient is depicted in Figure 1. The patient was also found to have a germline mutation in breast cancer 2 (BRCA2) in exon 25 (c.9641insT). This frameshift mutation leads to premature truncation of the protein at amino acid 3149. We subsequently identified a man of European descent with a minimal smoking history who was diagnosed with an EGFR mutant (exon 19 deletion) stage IIIB lung adenocarcinoma at the age of 74. The patient’s daughter, mother, and maternal aunt all had breast or ovarian cancers. DNA from the patient and daughter harbored the same BRCA2 8867del5 mutation, which leads to premature truncation of the BRCA2 protein at amino acid 2904. Mutations in the kinase domain of EGFR are more commonly found in lung adenocarcinomas from never smokers, East Asian populations, and in tumors of adenocarcinoma histology.1 Why certain patients are susceptible to the development of EGFR mutant tumors is currently unknown. Given the nature of EGFR kinase domain mutations (especially inframe multinucleotide deletions and duplications), we hypothesized that genetic susceptibility to DNA repair defects could predispose certain individuals to the development of EGFR mutant tumors. Consistent with this notion, mice carrying in the Brca1 locus a hypomorphic modification (Brca1tr) that eliminates the C-terminal half of the protein product develop bronchioloalveolar lung tumors.2 On Institutional Review Boardapproved protocols, we screened 110 Jewish patients with lung cancer and found that three harbored germline BRCA mutations (2.7%). This rate is similar to the 2.5% expected in the general Ashkenazi population. None of their lung tumors harbored EGFR mutations. Conversely, we screened 13 patients of Ashkenazi Jewish descent whose lung tumors harbored EGFR mutations and found that none had germline BRCA1/2 mutations. Thus, we did not find a clear epidemiologic association between EGFR mutant lung cancers and BRCA mutation carriers. In the future, larger association studies and further molecular analysis of individual cases like these could provide a clue to the etiology of EGFR mutations in lung cancer.