Frequency and significance of epidermal growth factor receptor mutations detected by PCR methods in patients with non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) is the most important driver gene of non-small cell lung cancer (NSCLC) as EGFR mutations determine the efficacy of EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. In the present study, the comprehensive ability of widely used polymerase chain reaction (PCR) methods to detect EGFR mutations was determined. Among the 35 EGFR mutations detected via the direct sequencing of 73 patients with NSCLC, 11 types were identified in exons 18, 19 and 21. Among the 11 mutation types, all exon 18 and 21 mutations were identified by 2 widely used PCR methods, namely, Scorpion-Amplification Refractory Mutation System and cobas v2. However, among the 9 different exon 19 deletions, 3 types were not identified by the 2 methods. In addition, 25 samples with EGFR mutations were analyzed by the 2 methods, including a sample from a patient with an unidentified exon 19 deletion, the T751_I759 deletion and insertion S; this patient had long-term disease control as a result of EGFR-TKI therapy. The 2 methods could not detect this unidentified deletion, whereas sizing capillary electrophoresis for the comprehensive detection of exon 19 deletions detected this deletion. It is generally thought that patients with exon 19 mutations have higher response rates to EGFR-TKI therapy than patients with exon 21 mutations. The present study confirmed the EGFR mutation status by comparing the mutations with the Catalog Of Somatic Mutations In Cancer, which is the world's largest and most comprehensive resource for analyzing the effects of somatic mutations in human cancers. The predicted frequency of EGFR mutations identified by the 2 methods was 85%. The frequency of mutations detectable by the 2 methods was less for exon 19 than exon 21. Therefore, the results of the present study suggest that decreasing false-negative detection of exon 19 deletions is crucial for the clinical testing of EGFR mutations.

Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines.

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.

Altered Cell-Cycle Control, Inflammation, and Adhesion in High-Risk Persistent Bronchial Dysplasia.

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G2-M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention.Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971-83. ©2018 AACR.

Baseline gene expression predicts sensitivity to gefitinib in non-small cell lung cancer cell lines.

Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.

Combined use of oligonucleotide and tissue microarrays identifies cancer/testis antigens as biomarkers in lung carcinoma.

High density oligonucleotide microarrays (OMAs) have been used recently to profile gene expression in lung carcinoma tissue homogenates. The length of the lists of potentially interesting genes generated by these studies is daunting, and biological and clinical relevance of these lists remains to be validated. Moreover, specific identification of individual biomarkers that might be used for early detection and surveillance has not been the objective of these early studies. We have developed a schema for combining the data derived from the OMA analysis of a few lung cancer cell lines with immunohistochemical testing of tissue microarrays to rapidly identify biomarkers of potential clinical relevance. Initially, we profiled gene expression in lung tumor cell lines using the Affymetrix HG-U95Av2 OMA. RNA from 2 non-small cell lung cancer (NSCLC) cell lines (A549 and H647) and 2 small cell lung cancer (SCLC) cell lines (SHP-77 and UMC-19) were tested. Cells from 1 histologically and cytogenetically normal bronchial epithelial primary culture from a volunteer who had never smoked and 10 samples of histologically unremarkable lung tissue from resection specimens served as normalization controls. Array results were analyzed with Gene Spring software. Results were confirmed by reverse transcription-PCR in an expanded number of cell lines. We then validated the cell line data by immunohistochemical testing for protein using a tissue microarray containing 187 NSCLC clinical samples. Of the 20 most highly expressed genes in the tumor lines, 6 were members of the cancer/testis antigen (CTAG) gene group including 5 MAGE-A subfamily members and NY-ESO-1. SCLC lines strongly expressed all of the MAGE-A genes as well as NY-ESO-1, whereas NSCLC lines expressed a subset of MAGE-A genes at a lower level of intensity and failed to express NY-ESO-1. Reverse transcription-PCR of an extended series of 25 lung cancer cell lines including 13 SCLC, 9 NSCLC, and 3 mesothelioma lines indicated that MAGE-A10 and NY-ESO-1 were expressed only by SCLC, and that MAGE-A1, 3, 6, 12, and 4b were expressed by both SCLC and NSCLC. By immunohistochemistry using the monoclonal antibody 6C1 that recognizes several MAGE-A gene subfamily members, 44% of NSCLC clearly expressed MAGE-A proteins in cytoplasm and/or nucleus. Expression of MAGE-A genes did not correlate with survival but did correlate with histological classification with squamous carcinomas more frequently MAGE-A positive than other NSCLC types (P < 0.00002). We conclude that expression of CTAG gene products, whereas apparently not of prognostic importance, may be useful for early detection and surveillance because of a high level of specificity for central airway squamous and small cell carcinomas.

Overexpression of vascular endothelial growth factor and its receptors in bronchial dypslasia demonstrated by quantitative RT-PCR analysis.

Neoangiogenesis is required for the growth of invasive lung carcinoma, however, the role of angiogenesis in the progression of premalignant changes to carcinoma of the lung is less clear. We have evaluated vascular endothelial growth factor (VEGF) expression and microvessel densities (MVDs) in 62 bronchoscopic biopsies from normal, reactive (basal cell hyperplasia (BCH)) and dysplastic bronchial epithelium and in tissue from twenty-seven invasive lung carcinomas in an effort to demonstrate angiogenic activity in these preneoplastic lesions and determine whether it is associated with increased bronchial epithelial VEGF expression. MVDs and VEGF RNA expression measured by quantitative RT-PCR were found to be elevated in comparison to normal bronchial tissue in bronchial dysplasias and invasive lung carcinomas but not in basal cell hyperplasias. Immunohistochemical (IHC) analyses revealed that expression of VEGF arose predominantly from bronchial epithelium. ELISA analysis of lung tumor tissue showed that elevated VEGF protein expression correlated with VEGF RNA levels (r=0.59, p=0.004). Increased expression of VEGF RNA was also found in histologically normal bronchial mucosa from patients with either dysplasia at other sites or a history of heavy tobacco use suggesting a possible field effect in regard to the elaboration of VEGF. Furthermore, analysis of VEGF isoforms and VEGF receptors by semi-quantitative RT-PCR in dysplastic and invasive lesions revealed characteristic altered patterns of expression in dysplasia and early cancer as compared to normal tissue. These results indicate that angiogenesis develops early in lung carcinogenesis and is associated with overexpression of VEGF.

Prognostic effect of epidermal growth factor receptor gene mutations and the aberrant phosphorylation of Akt and ERK in ovarian cancer.

OBJECTIVES: We herein assessed the influence of Epidermal Growth Factor Receptor (EGFR) gene mutations on EGFR expression levels, downstream mediators such as Akt or ERK, and overall survival in patients with ovarian cancer. STUDY DESIGN: EGFR mutation status was analyzed by direct sequencing in 102 Japanese ovarian cancer patients. The EGFR expression, phosphorylated Akt (pAkt), and phosphorylated ERK (pERK) were determined by immunohistochemistry. RESULTS: Twenty-nine EGFR gene mutations were detected in 24 of 102 patinets (23.5%). EGFR mutations were observed in 27.9% (19/68) in serous adenocarcinomas, 15.0% (3/20) in clear cell adenocarcinomas, and 66.7% (2/3) in mucinous adenocarcinomas, while no mutations were observed in endometrioid adenocarcinomas (0/11). Protein expression of EGFR, pAkt, and pERK were detected in 47 (46.1%), 49 (48%), and 17 (16.7%) of patients, respectively. EGFR gene mutations, EGFR and pERK expression were not associated with a poor prognosis. In a multivariate analysis, a High pAkt expression was found to be a significant predictor for both the progression free survival (p=0.017) and overall survival (P=0.025). CONCLUSION: EGFR gene mutations were frequently observed in not only non-small-cell lung cancer (NSCLC), but also in ovarian cancer in Japanese patients. the selective EGFR inhibitor Gefitinib might therefore offer some benefit in patients with EGFR mutations in ovarian cancer. Our results indicate that the Akt, but not necessarily EGFR, is one of the most important target in the response of the platinum-based chemotherapy and prognosis for ovarian cancer patients.

KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancer.

Treatment of colon carcinoma with the anti-epidermal growth factor receptor antibody Cetuximab is reported to be ineffective in KRAS-mutant tumors. Mutation testing techniques have therefore become an urgent concern. We have compared three methods for detecting KRAS mutations in 59 cases of colon carcinoma: 1) high resolution melting, 2) the amplification refractory mutation system using a bifunctional self-probing primer (ARMS/Scorpion, ARMS/S), and 3) direct sequencing. We also evaluated the effects of the methods of sectioning and coring of paraffin blocks to obtain tumor DNA on assay sensitivity and specificity. The most sensitive and specific combination of block sampling and mutational analysis was ARMS/S performed on DNA derived from 1-mm paraffin cores. This combination of tissue sampling and testing method detected KRAS mutations in 46% of colon tumors. Four samples were positive by ARMS/S, but initially negative by direct sequencing. Cloned DNA samples were retested by direct sequencing, and in all four cases KRAS mutations were identified in the DNA. In six cases, high resolution melting abnormalities could not be confirmed as specific mutations either by ARMS/S or direct sequencing. We conclude that coring of the paraffin blocks and testing by ARMS/S is a sensitive, specific, and efficient method for KRAS testing.