Dhananjay Chitale

MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib

In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (P = 0.007, Fishers Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFRT790M mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFRT790M mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.

Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1.

Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1_G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET-positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors.

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.

Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

An integrated genomic analysis of lung cancer reveals loss of DUSP4 in EGFR-mutant tumors

To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This showed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with underexpression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling, and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of a reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers.

Novel MEK1 Mutation Identified by Mutational Analysis of Epidermal Growth Factor Receptor Signaling Pathway Genes in Lung Adenocarcinoma

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

EGFR Mutations in Lung Adenocarcinomas: Clinical Testing Experience and Relationship to EGFR Gene Copy Number and Immunohistochemical Expression

Lung adenocarcinomas responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors possess EGFR mutations and often increased EGFR copy number. We prospectively studied 334 clinical cases using polymerase chain reaction-based assays to detect deletions within exon 19 and the L858R mutation in exon 21, which together account for approximately 90% of EGFR mutations. Seventy-eight (23%) of these tumors had an EGFR mutation, with 55 (71%) exon 19 deletions and 23 (29%) exon 21 L858R mutations. We were able to compare mutant and normal EGFR alleles and found a preferential amplification of the mutant allele. The association of mutations with EGFR amplification (determined by chromogenic in situ hybridization) and EGFR expression (determined by immunohistochemistry) was further examined in a subset of 60 tumors. EGFR amplification (> or =5 EGFR signals per nucleus) was seen in 15 of 29 (52%) EGFR-mutated tumors but in only five of 31 (6%) non-mutated tumors (P = 0.006). EGFR overexpression was strongly associated with amplification but was statistically independent of EGFR mutation. Most patients with EGFR mutations (17 of 29, 59%) never smoked compared with 13% (four of 31) of patients lacking such mutations (P = 0.0003). The association of amplification with smoking status was marginal and was nonexistent with EGFR expression. Thus, these results indicate that EGFR amplification, preferentially of the mutant allele, often accompanies EGFR mutation, whereas EGFR immunohistochemical staining associates with amplification but cannot predict EGFR mutation status.

Frequency of EGFR and KRAS mutations in lung adenocarcinomas in African Americans.

Introduction: The detection of mutations in the epidermal growth factor receptor (EGFR) gene, which predict sensitivity to treatment with EGFR tyrosine kinase inhibitors, represents a major advance in the treatment of lung adenocarcinoma. KRAS mutations confer resistance to EGFR-tyrosine kinase inhibitors. The prevalence of these mutations in African American patients has not been thoroughly investigated. Methods: We collected formalin-fixed, paraffin-embedded material from resected lung adenocarcinomas from African American patients at three institutions for DNA extraction. The frequencies of EGFR exon 19 deletions, exon 21 L858R substitutions, and KRAS mutations in tumor specimens from African American patients were compared with data in white patients (n = 476). Results: EGFR mutations were detected in 23 of the 121 specimens from African American patients (19%, 95% confidence interval [CI]: 13–27%), whereas KRAS mutations were found in 21 (17%, 95% CI: 12–25%). There was no significant difference between frequencies of EGFR mutations comparing African American and white patients, 19% versus 13% (61/476, 95% CI: 10–16%; p = 0.11). KRAS mutations were more likely among whites, 26% (125/476, 95% CI: 23–30%; p = 0.04). Conclusions: This is the largest study to date examining the frequency of mutations in lung adenocarcinomas in African Americans. Although KRAS mutations were somewhat less likely, there was no difference between the frequencies of EGFR mutations in African American patients, when compared with whites. These results suggest that all patients with advanced lung adenocarcinomas should undergo mutational analysis before initiation of therapy.

Expression of cancer-testis antigens in endometrial carcinomas using a tissue microarray.

Cancer–testis (CT) antigens are expressed in a variety of malignant tumors, but in normal adult tissue, they are only expressed in testicular germ cells. Owing to this tumor-associated expression pattern, these antigens are of major interest as potential targets for immunotherapy and possibly for diagnostic purposes. This study was performed to analyze the expression of four CT antigens, NY-ESO-1, MAGE-A3, MAGE-A4, and CT7/MAGE-C1, in endometrial carcinoma using immunohistochemistry, and to correlate expression with histologic subtypes, grade, and expression of WT1 and p53. Formalin-fixed paraffin-embedded tissues of 130 endometrial carcinomas of the following types and grades were analyzed using a tissue microarray: 85 endometrioid carcinomas (FIGO grade 1, 39; grade 2, 11; and grade 3, 35), 18 papillary serous carcinomas, 12 clear cell carcinomas, 13 malignant mixed mullerian tumors, one mucinous adenocarcinoma, and one undifferentiated carcinoma. The following anti-CT monoclonal antibodies/antigens were studied by immunohistochemistry: monoclonal antibody ES121/NY-ESO-1, monoclonal antibody M3H67/MAGE-A3, monoclonal antibody 57B/MAGE-A4, and monoclonal antibody CT7-33/CT7. The CT expression data were compared to WT1 and p53 protein expression as analyzed in a previous study. Positive staining with anti-CT monoclonal antibodies was graded as follows: focal, <5% positive cells; 1+, 5–25% cells; 2+, 26–50% cells; 3+, 51–75%; and 4+, >75% cells. The 3+ and 4+ staining patterns were considered homogeneous patterns of potential clinical significance and were scored positive for statistical analysis. In low-grade tumors, the most immunoreactivity was seen with mAb M3H67 but little labeling was observed with the other monoclonal antibodies. In high-grade tumors, monoclonal antibodies M3H67 (25%), 57B (23%), and CT7-33 (20%) showed the highest reactivity, while ES121 showed the lowest immunoreactivity (6%). The staining pattern was mostly heterogeneous. Statistical significance was found solely for the correlation of monoclonal antibody 57B staining and p53 expression. No correlation was found for any anti-CT monoclonal antibody staining and clinical stage or for anti-CT staining and WT1 expression. CT antigens CT7, MAGE-A3 and MAGE-A4, but not NY-ESO-1, are expressed in high-grade endometrial carcinomas, and expression of MAGE-A4 is correlated with the presence of overexpressed p53.