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author:("Soh, munich")
1.  A Case of Carcinoma Showing Thymus-Like Differentiation with a Rapidly Lethal Course 
Case Reports in Oncology  2014;7(3):840-844.
A 55-year-old woman underwent a total thyroidectomy for carcinoma showing thymus-like differentiation (CASTLE). The patient was referred to our hospital after the tumor was found to have directly invaded the cervical esophagus and the entire circumference of the trachea. A total thyroidectomy was performed, followed by end-to-end anastomosis of the trachea, suprahyoid release and dissection of bilateral pulmonary ligaments. No major complications, including anastomotic dehiscence or stenosis, were observed. The patient experienced some swallowing disturbances and hoarseness during the perioperative period but fully recovered. Radiotherapy to the neck was performed as an adjuvant therapy. Eleven months after surgery, lower back pain and right leg numbness developed and led to gait inability. Multiple lung and bone recurrences were observed, but no local recurrence. Palliative radiotherapy to the bone metastasis was performed. The patient died of pleural metastasis 14 months after the initial diagnosis of CASTLE.
doi:10.1159/000370306
PMCID: PMC4307009
Carcinoma showing thymus-like differentiation; Infiltrating trachea; Reconstruction
2.  CDKN2A/p16 inactivation mechanisms and their relationship to smoke exposure and molecular features in non-small cell lung cancer 
Introduction
CDKN2A(p16) inactivation is common in lung cancer and occurs via homozygous deletions (HD), methylation of promoter region, or point mutations. While p16 promoter methylation has been linked to KRAS mutation and smoking, the associations between p16 inactivation mechanisms and other common genetic mutations and smoking status are still controversial or unknown.
Methods
We determined all three p16 inactivation mechanisms using multiple methodologies for genomic status, methylation, RNA and protein expression, and correlated them with EGFR, KRAS, STK11 mutations and smoking status in 40 cell lines and 45 tumor samples of primary NSCLC. We also performed meta-analyses to investigate the impact of smoke exposure on p16 inactivation.
Results
p16 inactivation was the major mechanism of RB pathway perturbation in NSCLC, with HD being the most frequent method, followed by methylation and the rarer point mutations. Inactivating mechanisms were tightly correlated with loss of mRNA and protein expression. p16 inactivation occurred at comparable frequencies regardless of mutational status of EGFR, KRAS and STK11, however, the major inactivation mechanism of p16 varied. p16 methylation was linked to KRAS mutation but was mutually exclusive with EGFR mutation. Cell lines and tumor samples demonstrated similar results. Our meta-analyses confirmed a modest positive association between p16 promoter methylation and smoking.
Conclusions
Our results confirm that all of the inactivation mechanisms are truly associated with loss of gene product and identify specific associations between p16 inactivation mechanisms and other genetic changes and smoking status.
doi:10.1097/JTO.0b013e3182a46c0c
PMCID: PMC3951422  PMID: 24077454
p16; CDKN2A; inactivation; homozygous deletion; methylation; lung cancer; adenocarcinoma; meta-analysis
3.  Anti-Cancer Effects of REIC/Dkk-3-encoding Adenoviral Vector for the Treatment of Non-small Cell Lung Cancer 
PLoS ONE  2014;9(2):e87900.
Objectives
REIC/Dkk-3 is down-regulated in a broad range of human cancer cells and is considered to function as a tumor suppressor. We previously reported that REIC/Dkk-3-expressing adenovirus vector (Ad-REIC) induced endoplasmic reticulum (ER) stress and cancer-specific apoptosis in human prostate cancer. In this study, we examined the therapeutic impact of Ad-REIC on non-small cell lung cancer (NSCLC).
Materials and Methods
We examined the anti-tumor effect of Ad-REIC on 25 NSCLC cell lines in vitro and A549 cells in vivo. Two of these cell lines were artificially established as EGFR-tyrosine kinase inhibitor (TKI) resistant sublines.
Results
Ad-REIC-treatment inhibited the cell viability by 40% or more in 13 (52%) of the 25 cell lines at multiplicity of infection (MOI) of 20 (20 MOI). These cell lines were regarded as being highly sensitive cells. The cell viability of a non-malignant immortalized cell line, OUMS-24, was not inhibited at 200 MOI of Ad-REIC. The effects of Ad-REIC on EGFR-TKI resistant sublines were equivalent to those in the parental cell lines. Here, we demonstrated that Ad-REIC treatment activated c-Jun N-terminal kinase (JNK) in NSCLC cell lines, indicating the induction of ER stress with GRP78/BiP (GRP78) up-regulation and resulting in apoptosis. A single intratumoral injection of Ad-REIC significantly inhibited the tumorigenic growth of A549 cells in vivo. As predictive factors of sensitivity for Ad-REIC treatment in NSCLC, we examined the expression status of GRP78 and coxsackievirus and adenovirus receptor (CAR). We found that the combination of the GRP78 and CAR expressional statuses may be used as a predictive factor for Ad-REIC sensitivity in NSCLC cells.
Conclusion
Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. Our study indicated that Ad-REIC has therapeutic potential against NSCLC and that the expression statuses of GRP78 and CAR may predict a potential therapeutic benefit of Ad-REIC.
doi:10.1371/journal.pone.0087900
PMCID: PMC3912155  PMID: 24498395
4.  Novel Germline Mutation in the Transmembrane Domain of HER2 in Familial Lung Adenocarcinomas 
We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing, we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways, we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition, they activated p38, which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic, causing hereditary and sporadic lung adenocarcinomas.
doi:10.1093/jnci/djt338
PMCID: PMC3906987  PMID: 24317180
5.  Intrathoracic irrigation with arbekacin for methicillin-resistant Staphylococcus aureus empyema following lung resection 
OBJECTIVES
Empyema is a well-known complication following lung resection. In particular, empyema caused by methicillin-resistant Staphylococcus aureus (MRSA) is difficult to treat. Here, we present our experience of MRSA empyema treated with local irrigation using arbekacin.
METHODS
Six patients consisted of 4 males and 2 females with an average age of 65.7 years. They developed MRSA empyema following lung resection and were treated at our institution between 2007 and 2011. Cases comprised four primary and one metastatic lung cancer, and 1 patient was a living lung transplantation donor. The surgical procedure consisted of four lobectomies, one segmentectomy and one wedge resection. After diagnosis of MRSA empyema, anti-MRSA drugs were administered intravenously in all cases. In addition, arbekacin irrigation at a dose of 100 mg dissolved in saline was performed after irrigation with saline only.
RESULTS
The average number of postoperative days for the diagnosis of MRSA empyema was 13 (range 4–19). The period of irrigation ranged from 6 to 46 days. Arbekacin irrigation did not induce nephrotoxicity or other complications, and no bacteria resistant to arbekacin was detected in the thoracic cavity. We re-operated on 1 case because he had pulmonary fistula and severe wound infection. At the time of removing the thoracic catheter, MRSA in the pleural effusion disappeared completely in 3 patients. The period until MRSA concentration in the pleural effusion became negative after starting arbekacin irrigation ranged from 4 to 9 days. In the remaining cases, in which MRSA did not disappear, the catheter was removed because of no inflammatory reaction after stopping irrigation and clamping the catheters. All patients were discharged from our institution without thoracic catheterization and no patients had relapsed during the follow-up period ranging from 6 to 44 months.
CONCLUSIONS
Irrigation of the thoracic cavity with arbekacin proved to be an effective, safe and readily available method for treating MRSA empyema following lung resection.
doi:10.1093/icvts/ivs285
PMCID: PMC3422970  PMID: 22718464
Methicillin-resistant Staphylococcus aureus; Empyema; Irrigation; Arbekacin; Lung resection
6.  Impact of aberrant methylation of microRNA-9 family members on non-small cell lung cancers 
Molecular and Clinical Oncology  2012;1(1):185-189.
MicroRNAs (miRs) contribute to cancer development and progression by acting as oncogenes and tumor suppressor genes. miR-9 family members (miR-9s), including miR-9-1, 9-2 and 9-3, have been shown to be oncogenically involved through the downregulation of E-cadherin expression, which promotes the epithelial-mesenchymal transition. Tumor suppressive roles of miR-9s have also been reported to silence miR-9 through methylation, which is associated with an shortened overall survival (OS) period in several types of cancer. In this study, the impact of miR-9s methylation on non-small cell lung cancers (NSCLC) was investigated. In total, 293 resected NSCLC samples were examined and the miR-9s methylation status was determined using a combined bisulfite restriction analysis. miR-9 expression was analyzed by in situ hybridization. Methylation of miR-9-1, 9-2 and 9-3 was present in 20 (7%), 33 (11%) and 34 (12%) of the cases, respectively. Methylation of any miR-9s (miR-9s methylation) was observed in 76 of the cases (26%), and miR-9 expression was silenced in cases with miR-9s methylation. Logistic regression analysis demonstrated that male gender [odds ratio (OR), 2.0; 95% confidence interval (95% CI), 1.1–3.6; P=0.01] and pathologically negative lymph node metastasis (OR, 4.8; 95% CI, 1.4–17.2; P=0.002) were independent relative factors for miR-9s methylation. Additionally, miR-9s methylation [hazard ratio (HR), 4.2; 95% CI, 1.2–27.0; P=0.026] and early pathological stage (HR, 8.3; 95% CI, 2.1–28.6; P=0.004) were found to be independent predictive factors for prolonged OS time by the Cox proportional hazard test. miR-9s methylation which induces expression silencing is common in NSCLC cases without lymph nodal metastasis, suggesting that miR-9s are oncogenically involved in NSCLC carcinogenesis through the promotion of tumor metastasis.
doi:10.3892/mco.2012.18
PMCID: PMC3956236  PMID: 24649145
lung cancer; non-small cell lung cancer; microRNA; miR-9; methylation; in situ hybridization
7.  Induction Chemoradiotherapy Followed by Surgical Resection for Clinical T3 or T4 Locally Advanced Non–Small Cell Lung Cancer 
Annals of Surgical Oncology  2012;19(8):2685-2692.
ABSTRACT
Purpose
To examine the usefulness of trimodality therapy in patients with clinical T3 or T4 (cT3–4) locally advanced non–small cell lung cancer (LA-NSCLC).
Methods
Between 1997 and 2009, a total of 76 LA-NSCLC patients with cT3–4 underwent surgery. Among them, 36 patients underwent induction chemoradiotherapy with docetaxel and cisplatin plus concurrent radiation followed by surgery (IC group). The other 40 patients initially underwent surgery (IS group). The outcomes of the IC and IS groups were then investigated. To minimize possible biases caused by confounding treatment indications, we performed a retrospective cohort analysis by applying a propensity score (PS). Patients were divided into three groups according to PS tertiles, and comparisons between the IC and IS groups were made by PS tertile-stratified Cox proportional hazard models.
Results
For the entire cohort, which had a median follow-up duration of 48 months, the 3- and 5-year overall survival rates were 83.8 and 78.9%, respectively, in the IC group, versus 66.8 and 56.5%, respectively, in the IS group (P = 0.0092). After adjustments for potentially confounding variables, the IC group continued to have a significantly longer overall survival than the IS group (P = 0.0045). In addition, when the analysis was limited to 52 patients with cT3–4N0 or N1 disease, the IC group had a significantly longer overall survival than the IS group after adjustments for confounding variables (P = 0.019).
Conclusions
Our study indicates that trimodality therapy is highly effective in patients with cT3–4 LA-NSCLC.
Electronic supplementary material
The online version of this article (doi:10.1245/s10434-012-2302-x) contains supplementary material, which is available to authorized users.
doi:10.1245/s10434-012-2302-x
PMCID: PMC3404289  PMID: 22396006
8.  Knockdown of Oncogenic KRAS in Non-Small Cell Lung Cancers Suppresses Tumor Growth and Sensitizes Tumor Cells to Targeted Therapy 
Molecular cancer therapeutics  2011;10(2):336-346.
Oncogenic KRAS is found in >25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we performed microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines (HBECs) with and without oncogenic KRAS. We found that while the MAPK pathway is significantly down-regulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-EGFR, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment but may offer possibilities of combining anti-KRAS strategies with other targeted drugs.
doi:10.1158/1535-7163.MCT-10-0750
PMCID: PMC3061393  PMID: 21306997
9.  DNA methylation status of REIC/Dkk-3 gene in human malignancies 
Purpose
The REIC (reduced expression in immortalized cells)/Dkk-3 is down-regulated in various cancers and considered to be a tumor suppressor gene. REIC/Dkk-3 mRNA has two isoforms (type-a,b). REIC type-a mRNA has shown to be a major transcript in various cancer cells, and its promoter activity was much stronger than that of type-b. In this study, we examined the methylation status of REIC/Dkk-3 type-a in a broad range of human malignancies.
Methods
We examined REIC/Dkk-3 type-a methylation in breast cancers, non-small-cell lung cancers, gastric cancers, colorectal cancers, and malignant pleural mesotheliomas using a quantitative combined bisulfite restriction analysis assay and bisulfate sequencing. REIC/Dkk-3 type-a and type-b expression was examined using reverse transcriptional PCR. The relationships between the methylation and clinicopathological factors were analyzed.
Results
The rate of REIC/Dkk-3 type-a methylation ranged from 26.2 to 50.0% in the various primary tumors that were examined. REIC/Dkk-3 type-a methylation in breast cancer cells was significantly heavier than that in the other cell lines that we tested. REIC/Dkk-3 type-a methylation was inversely correlated with REIC/Dkk-3 type-a expression. There was a correlation between REIC/Dkk-3 type-a and type-b mRNA expression. REIC/Dkk-3 type-a expression was restored in MDA-MB-231 cells using 5-aza-2′-deoxycytidine treatment. We found that estrogen receptor–positive breast cancers were significantly more common among the methylated group than among the non-methylated group.
Conclusions
REIC/Dkk-3 type-a methylation was frequently detected in a broad range of cancers and appeared to play a key role in silencing REIC/Dkk-3 type-a expression in these malignancies.
doi:10.1007/s00432-012-1158-6
PMCID: PMC3325424  PMID: 22274868
DNA methylation; REIC/Dkk-3; Breast cancer; Lung cancer; Mesothelioma
10.  Presence of EGFR mutation in pathologically non-malignant specimens from computed tomography-guided lung needle biopsies 
Oncology Letters  2011;3(2):401-404.
Activating mutations of the epidermal growth factor receptor (EGFR) gene are characteristic of non-small cell lung cancer (NSCLC). EGFR mutations were previously detected in histologically normal lung tissue around NSCLC tumors. Computed tomography-guided lung needle biopsy (CTNB) is an accurate and useful technique for the diagnosis of lung tumors. However, pathologically non-malignant cases occasionally become apparent following lung tumor resection. In this study, we determined the EGFR mutational status of lung tumors diagnosed as non-malignant in CTNB specimens, but diagnosed as NSCLC following surgical resection. Between 2000 and 2008, 1,109 CTNBs were performed at Okayama University Hospital. Among them, 15 cases were initially diagnosed as non-malignant by CTNB, but diagnosed as NSCLC following surgical resection as a result of a high likelihood of malignancy by clinical findings. Twelve paired DNAs of CTNB and corresponding resected specimens were available to examine the EGFR mutational status using a mutant-enriched PCR assay. EGFR mutations were detected in one out of 12 CTNB specimens and three of the corresponding resected tumors. This case harbored the same EGFR mutation in the CTNB specimen and resected tumor, but not in the distant corresponding non-malignant lung tissue. Our results indicated that the detection of EGFR mutations may therefore aid the diagnosis of NSCLC in pathologically non-malignant CTNB specimens.
doi:10.3892/ol.2011.471
PMCID: PMC3362587  PMID: 22740920
non-small cell lung cancer; epidermal growth factor receptor; computed tomography-guided lung needle biopsy
11.  The estrogen receptor influences microtubule-associated protein tau (MAPT) expression and the selective estrogen receptor inhibitor fulvestrant downregulates MAPT and increases the sensitivity to taxane in breast cancer cells 
Introduction
Microtubule-associated protein tau (MAPT) inhibits the function of taxanes and high expression of MAPT decreases the sensitivity to taxanes. The relationship between estrogen receptor (ER) and MAPT in breast cancer is unclear. In this study, we examined the correlation of MAPT expression with the sensitivity of human breast cancer cells to taxanes, and the relationship between ER and MAPT.
Methods
The correlation between MAPT expression and sensitivity to taxanes was investigated in 12 human breast cancer cell lines. Alterations in cellular sensitivity to taxanes were evaluated after knockdown of MAPT expression. ER expression was knocked down or stimulated in MAPT- and ER-positive cell lines to examine the relationship between ER and MAPT. The cells were also treated with hormone drugs (tamoxifen and fulvestrant) and taxanes.
Results
mRNA expression of MAPT did not correlate with sensitivity to taxanes. However, expression of MAPT protein isoforms of less than 70 kDa was correlated with a low sensitivity to taxanes. Downregulation of MAPT increased cellular sensitivity to taxanes. MAPT protein expression was increased by stimulation with 17-β-estradiol or tamoxifen, but decreased by ER downregulation and by fulvestrant, an ER inhibitor. The combination of fulvestrant with taxanes had a synergistic effect, whereas tamoxifen and taxanes had an antagonistic effect.
Conclusions
Expression of MAPT protein isoforms of less than 70 kDa is correlated with a low sensitivity to taxanes in breast cancer cells. ER influences MAPT expression and fulvestrant increases the sensitivity to taxanes in MAPT- and ER-positive breast cancer cells.
doi:10.1186/bcr2598
PMCID: PMC2917038  PMID: 20579400
12.  PIK3CA Mutations and Copy Number Gains in Human Lung Cancers 
Cancer research  2008;68(17):6913-6921.
We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non–small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference–mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.
doi:10.1158/0008-5472.CAN-07-5084
PMCID: PMC2874836  PMID: 18757405
13.  Genetic predictors of MEK-dependence in non-small cell lung cancer 
Cancer research  2008;68(22):9375-9383.
Hyperactivated ERK signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS and upstream receptor tyrosine kinases. In order to characterize the MEK/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n= 87) and tumors (n=916) for BRAF mutations. We found that NSCLC cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition, compared to those harboring mutations in EGFR, KRAS, or ALK and ROS kinase fusions. Supporting its classification as a “driver” mutation in the cells in which it is expressed, MEK inhibition in V600EBRAF NSCLC cells led to substantial induction of apoptosis, comparable to that seen with EGFR kinase inhibition in EGFR-mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR-mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF-mutant cell lines were resistant to EGFR inhibition. These data, together with the non-overlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hot-spot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors, and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared to those whose tumors harbor kinase domain mutations in EGFR.
doi:10.1158/0008-5472.CAN-08-2223
PMCID: PMC2649746  PMID: 19010912
BRAF; non-small cell lung cancer; MEK inhibitor; PD0325901; EGFR mutation; gefitinib; erlotinib
14.  MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors 
We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC.
doi:10.1002/ijc.24150
PMCID: PMC2767331  PMID: 19117057
MET; amplification; EGFR; gefitinib; lung cancer
15.  Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells 
PLoS ONE  2009;4(10):e7464.
Background
Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.
Methodology/Principal Findings
We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.
Conclusions
MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.
doi:10.1371/journal.pone.0007464
PMCID: PMC2757721  PMID: 19826477
16.  Sequential Molecular Changes during Multistage Pathogenesis of Small Peripheral Adenocarcinomas of the Lung 
Introduction
We investigated EGFR and KRAS alterations among atypical adenomatous hyperplasia and small lung adenocarcinomas with bronchioloalveolar features to understand their role during multistage pathogenesis.
Methods
Sixty lesions measuring 2 cm or less were studied, including 38 noninvasive lesions (4 atypical adenomatous hyperplasias, 19 Noguchi type A and 15 type B) and 22 invasive lesions (type C) based on the World Health Organization classification and Noguchi’s criteria. EGFR and KRAS mutations were examined using PCR-based assays. EGFR copy number was evaluated using fluorescence in situ hybridization.
Results
EGFR and KRAS mutations were found in 26 (43.3%) and 5 (8.3%) lesions, respectively. Increased EGFR copy number status was identified in 10 lesions (16.7%), both mutant and wild type. EGFR or KRAS mutations were present in 39.5% and 7.9% (respectively) of noninvasive lesions and 50% or 9.1% (respectively) of invasive lesions. EGFR copy number was increased in 7.9% and 31.8% of noninvasive and invasive lesions (P = 0.029). Multivariate analysis revealed that increased EGFR copy number was the only significant factor to associate with invasive lesions (P = 0.035).
Conclusions
EGFR and KRAS mutations occur early during the multistage pathogenesis of peripheral lung adenocarcinomas. By contrast, increased EGFR copy number is a late event during tumor development and plays a role in the progression of lung adenocarcinoma independent of the initiating molecular events.
doi:10.1097/JTO.0b013e318168d20a
PMCID: PMC2758162  PMID: 18379350
Multistage pathogenesis; EGFR; KRAS; Mutation; Amplification
17.  Histone Deacetylase Inhibitor Romidepsin Enhances Anti-Tumor Effect of Erlotinib in Non-small Cell Lung Cancer (NSCLC) Cell Lines 
Introduction
Most epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers (NSCLCs) are sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib or gefitinib, but many EGFR wild type NSCLCs are resistant to TKIs. In this study, we examined the effects of the histone deacetylase inhibitor, romidepsin, in combination with erlotinib, in NSCLC cell lines and xenografts.
Methods
For in vitro studies, nine NSCLC cell lines with varying mutation status and histology were treated with erlotinib and romidepsin alone or in combination. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays were performed to determine the concentration that inhibits 50% (IC50) value of each drug or the combination. For in vivo studies, NCI-H1299 xenografts were inoculated subcutaneously into athymic nude mice. Romidepsin and/or erlotinib were injected intraperitoneally after tumors developed and tumor sizes were measured.
Results
We found that romidepsin increased the sensitivity of erlotinib synergistically in all nine NSCLC cell lines including EGFR and KRAS wild type cell lines, KRAS mutant cell lines, and TKI resistant EGFR mutant cell lines. This effect was partially due to enhanced apoptosis. Furthermore, cotreatment of erlotinib and romidepsin inhibited NCI-H1299 xenograft growth in athymic nude mice.
Conclusions
These observations support a role for the combination of a histone deacetylase inhibitor and a TKI in the treatment of NSCLCs.
doi:10.1097/JTO.0b013e318194fae7
PMCID: PMC2758160  PMID: 19179890
Erlotinib; Romidepsin; Non-small cell lung cancer; Epidermal growth factor receptor; KRAS
18.  Alterations in Genes of the EGFR Signaling Pathway and Their Relationship to EGFR Tyrosine Kinase Inhibitor Sensitivity in Lung Cancer Cell Lines 
PLoS ONE  2009;4(2):e4576.
Background
Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs.
Methodology/Principal Findings
We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3 HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results:
 1. mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance.
 2. mKRAS was associated with increased in vitro resistance to gefitinib.
Conclusions/Significance
Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.
doi:10.1371/journal.pone.0004576
PMCID: PMC2642732  PMID: 19238210

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