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1.  Increased EGFR Gene Copy Number Detected by Fluorescent In Situ Hybridization Predicts Outcome in Non–Small-Cell Lung Cancer Patients Treated With Cetuximab and Chemotherapy 
Journal of Clinical Oncology  2008;26(20):3351-3357.
Purpose
Epidermal growth factor receptor (EGFR) gene copy number detected by fluorescent in situ hybridization (FISH) has proven to be useful for selection of non–small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors. Here, we evaluate EGFR FISH as a predictive marker in NSCLC patients receiving the EGFR monoclonal antibody inhibitor cetuximab plus chemotherapy.
Patients and Methods
Two hundred twenty-nine chemotherapy-naive patients with advanced-stage NSCLC were enrolled onto a phase II selection trial evaluating sequential or concurrent chemotherapy (paclitaxel plus carboplatin) with cetuximab.
Results
EGFR FISH was assessable in 76 patients with available tumor tissue and classified as positive (four or more gene copies per cell in ≥ 40% of the cells or gene amplification) in 59.2%. Response (complete response/partial response) was numerically higher in FISH-positive (45%) versus FISH-negative (26%) patients (P = .14), whereas disease control rate (complete response/partial response plus stable disease) was statistically superior (81% v 55%, respectively; P = .02). Patients with FISH-positive tumors had a median progression-free survival time of 6 months compared with 3 months for FISH-negative patients (P = .0008). Median survival time was 15 months for the FISH-positive group compared with 7 months for patients who were FISH negative. (P = .04). Furthermore, survival favored FISH-positive patients receiving concurrent therapy.
Conclusion
These results are the first to suggest that EGFR FISH is a predictive factor for selection of NSCLC patients for cetuximab plus chemotherapy. Prospective validation of these findings is warranted.
doi:10.1200/JCO.2007.14.0111
PMCID: PMC3368372  PMID: 18612151
2.  Fluorescence In Situ Hybridization, Immunohistochemistry, and Next-Generation Sequencing for Detection of EML4-ALK Rearrangement in Lung Cancer 
The Oncologist  2015;20(3):316-322.
The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement in lung cancer is fluorescence in situ hybridization (FISH); however, FISH may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by immunohistochemistry should be strongly considered, and next-generation sequencing is recommended for borderline cases.
Background.
The U.S. Food and Drug Administration-approved method for detecting EML4-ALK rearrangement is fluorescence in situ hybridization (FISH); however, data supporting the use of immunohistochemistry (IHC) for that purpose are accumulating. Previous studies that compared FISH and IHC considered FISH the gold standard, but none compared data with the results of next-generation sequencing (NGS) analysis.
Materials and Methods.
We studied FISH and IHC (D5F3 antibody) systematically for EML4-ALK rearrangement in 51 lung adenocarcinoma patients, followed by NGS in case of discordance.
Results.
Of 51 patients, 4 were positive with FISH (7.8%), and 8 were positive with IHC (15.7%). Three were positive with both. NGS confirmed that four of the five patients who were positive with IHC and negative with FISH were positive for ALK. Two were treated by crizotinib, with progression-free survival of 18 and 6 months. Considering NGS as the most accurate test, the sensitivity and specificity were 42.9% and 97.7%, respectively, for FISH and 100% and 97.7%, respectively, for IHC.
Conclusion.
The FISH-based method of detecting EML4-ALK rearrangement in lung cancer may miss a significant number of patients who could benefit from targeted ALK therapy. Screening for EML4-ALK rearrangement by IHC should be strongly considered, and NGS is recommended in borderline cases. Two patients who were negative with FISH and positive with IHC were treated with crizotinib and responded to therapy.
doi:10.1634/theoncologist.2014-0389
PMCID: PMC4350802  PMID: 25721120
EML4-ALK; Non-small cell lung cancer; Fluorescence in situ hybridization; Immunohistochemistry; Next-generation sequencing
3.  ROS1 Immunohistochemistry Among Major Genotypes of Non—Small-Cell Lung Cancer 
Clinical lung cancer  2014;16(2):106-111.
Identification of ROS1 rearrangements in patients with lung cancer allows them to benefit from targeted therapy. We compared immunohistochemistry (IHC) with more cumbersome methods such as fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction for identification of ROS1 rearrangements in patients with lung adenocarcinoma (n = 33). Our results showed that IHC is a sensitive (100%) and specific (100%) method to identify ROS1 rearrangements in patients with lung cancer.
Background
ROS1 gene fusions cause several cancers by constitutively activating the ROS1 tyrosine kinase receptor. ROS1-targeted inhibitor therapy improves survival in the approximately 1% to 2% of patients with lung adenocarcinoma with ROS1 gene fusions. Although fluorescence in situ hybridization (FISH) is the standard diagnostic procedure for detecting ROS1 rearrangements, we studied immunohistochemistry (IHC).
Materials and Methods
ROS1 IHC was performed on a selected cohort of 33 lung adenocarcinoma whole tissue specimens with alterations in the EGFR (n = 5), KRAS (n = 5), ERBB2 (HER2) (n = 3), ROS1 (n = 6), ALK (n = 5), and RET (n = 3) genes and pan-negative (n = 6) detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and FISH.
Results
In the cohort of 33 specimens, both ROS1 gene fusion using RT-PCR and high ROS1 protein expression using IHC were detected in 6 specimens. Of these 6 specimens, 5 were also positive by FISH for ROS1 gene rearrangements. All 27 lung cancer specimens that were negative for ROS1 rearrangements by genetic testing had no to low ROS1 protein expression.
Conclusion
We have optimized ROS1 IHC and scoring to provide high sensitivity and specificity for detecting ROS1 gene rearrangements in whole tissue. ROS1 IHC could be a practical and cost-effective method to screen for ROS1 gene rearrangements.
doi:10.1016/j.cllc.2014.10.003
PMCID: PMC4770803  PMID: 25467930
Adenocarcinoma; Biomarker; Gene rearrangement; NSCLC; ROS1
5.  EGFR Expression and Survival in Patients Given Cetuximab and Chemoradiation for Stage III Non-Small Cell Lung Cancer: A Secondary Analysis of RTOG 0324 
Purpose
We investigated whether expression of epidermal growth factor receptor (EGFR) was associated with survival and disease control in this secondary analysis of a phase II trial of cetuximab+chemoradiation for stage III non-small cell lung cancer.
Methods
Patients received cetuximab weekly before and during radiation (63 Gy/35 fractions/7 weeks) with weekly carboplatin+paclitaxel. We analyzed EGFR expression by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in pretreatment biopsy specimens and compared findings with overall and progression-free survival (OS, PFS) and time to progression (TTP).
Results
Specimens for IHC and FISH were collected from 51 and 45 of 87 evaluable patients. Pretreatment characteristics did not differ for patients with (n=51) or without (n=36) EGFR IHC data, or with (n=45) or without (n=42) FISH data. However, patients without IHC data had worse OS (HR=1.63, P=0.05), worse PFS (HR=1.88, P=0.008), and worse TTP [HR=1.99, P=0.01] than those with IHC data. EGFR protein expression was not related to pretreatment characteristics or OS; FISH-positive disease was associated with better performance status but not with OS, PFS, or TTP.
Conclusions
Surprisingly, outcomes differed not by EGFR expression but by the availability of samples for analysis, underscoring the importance of obtaining biopsy samples in such trials.
doi:10.1016/j.radonc.2014.06.012
PMCID: PMC4169722  PMID: 25042878
Clinical trials identifier NCT00081302; RTOG 0324; cetuximab; inoperable lung cancer; Erbitux
6.  FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity Across All Lung Cancer Histologies 
Purpose
FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous-cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically-relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer.
Experimental Design
Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray comprised of resected lung tumors was submitted to FGFR1 GCN and mRNA analyses and the results were validated with TCGA lung cancer data.
Results
14/58 cell lines exhibited ponatinib sensitivity (IC50 values ≤ 50 nM) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations.
Conclusions
FGFR1 dependency is frequent across various lung cancer histologies and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types.
doi:10.1158/1078-0432.CCR-13-3060
PMCID: PMC4062100  PMID: 24771645
7.  Fibroblast Growth Factor Receptor 1 and Related Ligands in Small-Cell Lung Cancer 
Journal of Thoracic Oncology  2015;10(7):1083-1090.
Introduction:
Small-cell lung cancer (SCLC) accounts for 15% of all lung cancers and has been understudied for novel therapies. Signaling through fibroblast growth factors (FGF2, FGF9) and their high-affinity receptor has recently emerged as a contributing factor in the pathogenesis and progression of non–small-cell lung cancer. In this study, we evaluated fibroblast growth factor receptor 1 (FGFR1) and ligand expression in primary SCLC samples.
Methods:
FGFR1 protein expression, messenger RNA (mRNA) levels, and gene copy number were determined by immunohistochemistry (IHC), mRNA in situ hybridization, and silver in situ hybridization, respectively, in primary tumors from 90 patients with SCLC. Protein and mRNA expression of the FGF2 and FGF9 ligands were determined by IHC and mRNA in situ hybridization, respectively. In addition, a second cohort of 24 SCLC biopsy samples with known FGFR1 amplification by fluorescence in situ hybridization was assessed for FGFR1 protein expression by IHC. Spearman correlation analysis was performed to evaluate associations of FGFR1, FGF2 and FGF9 protein levels, respective mRNA levels, and FGFR1 gene copy number.
Results:
FGFR1 protein expression by IHC demonstrated a significant correlation with FGFR1 mRNA levels (p < 0.0001) and FGFR1 gene copy number (p = 0.03). The prevalence of FGFR1 mRNA positivity was 19.7%. FGFR1 mRNA expression correlated with both FGF2 (p = 0.0001) and FGF9 (p = 0.002) mRNA levels, as well as with FGF2 (p = 0.01) and FGF9 (p = 0.001) protein levels. There was no significant association between FGFR1 and ligands with clinical characteristics or prognosis. In the second cohort of specimens with known FGFR1 amplification by fluorescence in situ hybridization, 23 of 24 had adequate tumor by IHC, and 73.9% (17 of 23) were positive for FGFR1 protein expression.
Conclusions:
A subset of SCLCs is characterized by potentially activated FGF/FGFR1 pathways, as evidenced by positive FGF2, FGF9, and FGFR1 protein and/or mRNA expression. FGFR1 protein expression is correlated with FGFR1 mRNA levels and FGFR1 gene copy number. Combined analysis of FGFR1 and ligand expression may allow selection of patients with SCLC to FGFR1 inhibitor therapy.
doi:10.1097/JTO.0000000000000562
PMCID: PMC4467588  PMID: 26020126
Small-cell lung cancer; Fibroblast growth factor receptor 1; Fibroblast growth factor 2; Fibroblast growth factor 9
8.  Lung Master Protocol (Lung-MAP)—A Biomarker-Driven Protocol for Accelerating Development of Therapies for Squamous Cell Lung Cancer: SWOG S1400 
The Lung Master Protocol (Lung-MAP, S1400) is a groundbreaking clinical trial designed to advance the efficient development of targeted therapies for squamous cell cancer (SCCA) of the lung. There are no approved targeted therapies specific to advanced lung SCCA, although The Cancer Genome Atlas (TCGA) project and similar studies have detected a significant number of somatic gene mutations/amplifications in lung SCCA, some of which are targetable by investigational agents. However, the frequency of these changes is low (5–20%), making recruitment and study conduct challenging in the traditional clinical trial setting. Here we describe our approach to development of a biomarker-driven phase 2/3 multi-substudy “Master Protocol,” employing a common platform (Next Generation DNA Sequencing) to identify actionable molecular abnormalities, followed by randomization to the relevant targeted therapy versus standard of care.
doi:10.1158/1078-0432.CCR-13-3473
PMCID: PMC4654466  PMID: 25680375
9.  Is there clinical value to prognostic signatures in early stage NSCLC? 
Summary
pAMPK and pmTOR favorably predicted outcome in early NSCLC. The differences were small. Phosphoprotein lability makes routine clinical use and validation difficult. Protein IHC is unlikely to be clinically useful and numerous efforts to create predictive models to select resected patients for therapy have been unsuccessful.
doi:10.1158/1078-0432.CCR-13-3387
PMCID: PMC3998101  PMID: 24583801
10.  Independent validation test of the vote-counting strategy used to rank biomarkers from published studies 
Biomarkers in medicine  2015;9(8):751-761.
Aim
Vote counting is frequently used in meta-analyses to rank biomarker candidates, but to our knowledge, there have been no independent assessments of its validity. Here, we used predictions from a recent meta-analysis to determine how well number of supporting studies, combined sample size and mean fold change performed as vote-counting strategy criteria.
Materials & methods
Fifty miRNAs previously ranked for their ability to distinguish lung cancer tissue from normal were assayed by RT-qPCR using 45 paired tumor-normal samples.
Results
Number of supporting studies predicted biomarker performance (p = 0.0006; r = 0.44), but sample size and fold change did not (p > 0.2).
Conclusion
Despite limitations, counting the number supporting studies appears to be an effective criterion for ranking biomarkers. Predictions based on sample size and fold change provided little added value. External validation studies should be conducted to establish the performance characteristics of strategies used to rank biomarkers.
doi:10.2217/BMM.15.39
PMCID: PMC4770796  PMID: 26223535
biomarkers; lung cancer; meta-analysis; microRNAs; miRNAs; statistical methods
11.  Epidermal Growth Factor Receptor Immunohistochemistry 
Cancer  2008;112(5):1114-1121.
BACKGROUND
The ISEL (Iressa Survival Evaluation in Lung Cancer) clinical trial evaluated the efficacy of gefitinib versus placebo in pretreated nonsmall-cell lung cancer patients. Two different antibodies, scoring systems, and cutoff points of epidermal growth factor receptor (EGFR) protein expression were compared to predict response and survival of enrolled patients.
METHODS
EGFR expression was assessed in tumor samples by immunohistochemistry using the Dako EGFR pharmDx kit (scoring percent of tumor cells with positive staining) and Zymed monoclonal antibody clone 31G7 (scoring staining index derived from proportion of positive cells times staining intensity).
RESULTS
Data for EGFR expression were available for 379 patients for Dako and 357 patients for Zymed antibody (22% and 21%, respectively, of trial population). Objective response rates in gefitinib-treated EGFR-positive patients defined with various cutpoints with Dako antibody varied between 8% and 12%, and with Zymed antibody between 10% and 13%. Lower cutoff points with Dako antibody provided the best discrimination between EGFR-positive and EGFR-negative patients for survival hazard ratios comparing gefitinib to placebo, with a significant treatment/cutoff point interaction for 10% cutoff point (P = .049). A similar but less apparent trend was noted for Zymed antibody, although the discrimination between hazard ratios was not significant for any cutoff point analyzed.
CONCLUSIONS
Assessment with the Dako PharmDx kit and percentage of cells with positive staining may provide more accurate prediction of differential effect on survival with gefitinib than assessment with Zymed antibody and staining index. Using higher cutpoints to define positivity does not improve test discrimination.
doi:10.1002/cncr.23282
PMCID: PMC3355966  PMID: 18219661
nonsmall-cell lung cancer; epidermal growth factor receptor; immunohistochemistry; phase 3 trial; cutoff point
12.  Classification of lung cancer histology by gold nanoparticle sensors 
We propose a nanomedical device for the classification of lung cancer (LC) histology. The device profiles volatile organic compounds (VOCs) in the headspace of (subtypes of) LC cells, using gold nanoparticle (GNP) sensors that are suitable for detecting LC-specific patterns of VOC profiles, as determined by gas chromatography–mass spectrometry analysis. Analyzing the GNP sensing signals by support vector machine allowed significant discrimination between (i) LC and healthy cells; (ii) small cell LC and non–small cell LC; and between (iii) two subtypes of non–small cell LC: adenocarcinoma and squamous cell carcinoma. The discriminative power of the GNP sensors was then linked with the chemical nature and composition of the headspace VOCs of each LC state. These proof-of-concept findings could totally revolutionize LC screening and diagnosis, and might eventually allow early and differential diagnosis of LC subtypes with detectable or unreachable lung nodules.
doi:10.1016/j.nano.2011.10.001
PMCID: PMC4745892  PMID: 22033081
Sensor; Gold nanoparticle; Lung cancer; Histology; Volatile organic compound
13.  Is the third generation EGFR TKIs the solution for making EGFR mutant NSCLC a curable disease? 
doi:10.3978/j.issn.2218-6751.2014.11.07
PMCID: PMC4367675  PMID: 25806320
14.  Hepatic Metastases is Associated with Poor Efficacy of Erlotinib as 2nd/3rd Line Therapy in Patients with Lung Adenocarcinoma 
Background
Hepatocyte growth factor (HGF)-mediated mesenchymal-to-epithelial transition factor (MET) gene amplification is a common mechanism for acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). MET gene amplification has also been associated with hepatic metastases in patients with lung cancer. The aim of this study was to investigate whether hepatic metastases are associated with decreased efficacy of erlotinib in patients with adenocarcinoma.
Material/Methods
A cohort of 329 patients with stage IV lung adenocarcinoma, known EGFR mutation status, and who received treatment with erlotinib in the 2nd or 3rd line setting were enrolled into this study over a period of 4 years between January 2011 and January 2015. The cohort was stratified based on the presence or absence of hepatic metastases and the efficacy of erlotinib was defined based on disease control rate (DCR) and progression-free survival (PFS).
Results
Hepatic metastases were present in 220 of the 329 enrolled lung adenocarcinoma patients. EGFR-activating mutations (exon 19 deletion or an exon 21 L858R mutation) were identified in 113 (34.3%) patients. The DCR was significantly lower in the hepatic metastases group than in patients without hepatic metastases (39.5% vs. 51.4% P=0.045). In patients with hepatic metastases, median PFS was 2.3 months in the EGFR mutation-positive group versus 1.4 months in the EGFR mutation-negative group (95% CI 1.3–3.3 vs. 1.3–1.5; P=0.055). Of note, erlotinib therapy in patients with hepatic metastases was complicated by elevated alanine transaminase (ALT) levels.
Conclusions
Hepatic metastasis in patients with lung adenocarcinoma predicts poor response to erlotinib as a 2nd/3rd line therapy. Combination therapy, for example with MET-TKI, may be a good choice for patients with liver metastases with poor prognosis.
doi:10.12659/MSM.896607
PMCID: PMC4734673  PMID: 26811313
Adenocarcinoma; Genes, erbB-1; Neoplasm Metastasis
15.  An International Interpretation Study Using the ALK IHC Antibody D5F3 and a Sensitive Detection Kit Demonstrates High Concordance between ALK IHC and ALK FISH and between Evaluators 
Introduction
The goal of personalized medicine is to treat patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or toxic therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK-positive non–small-cell lung cancer (NSCLC) tumors, but to date the only approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate a new automated standardized ALK IHC assay.
Methods
Archival NSCLC tumor specimens (n =103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc., Tucson, AZ). Specimens were scored binarily as positive if strong granular cytoplasmic brown staining was present in tumor cells. IHC results were compared with the FISH results and interevaluator comparisons made.
Results
Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%), and accurate relative (93%) to the ALK FISH results. Similar results were observed using a majority score. IHC negativity was scored by seven of seven and six of seven evaluators on three and two FISH-positive cases, respectively. IHC positivity was scored on two FISH-negative cases by seven of seven readers. There was agreement among seven of seven and six of seven readers on 88% and 96% of the cases before review, respectively, and after review there was agreement among seven of seven and six of seven on 95% and 97% of the cases, respectively.
Conclusions
On the basis of expert evaluation the ALK IHC test is sensitive, specific, and accurate, and a majority score of multiple readers does not improve these results over an individual reader’s score. Excellent inter-reader agreement was observed. These data support the algorithmic use of ALK IHC in the evaluation of NSCLC.
doi:10.1097/JTO.0000000000000115
PMCID: PMC4186652  PMID: 24722153
Non–small-cell lung cancer; Anaplastic lymphoma kinase; Immunohistochemistry; Fluorescence in situ hybridization; Companion diagnostics; Biomarkers; Crizotinib
16.  Volatile fingerprints of cancer specific genetic mutations 
We report on a new concept for profiling genetic mutations of (lung) cancer cells, based on the detection of patterns of volatile organic compounds (VOCs) emitted from cell membranes, using an array of nanomaterial-based sensors. In this in-vitro pilot study we have derived a volatile fingerprint assay for representative genetic mutations in cancer cells that are known to be associated with targeted cancer therapy. Five VOCs were associated with the studied oncogenes, using complementary chemical analysis, and were discussed in terms of possible metabolic pathways. The reported approach could lead to the development of novel methods for guiding treatments, so that patients could benefit from safer, more timely and effective interventions that improve survival and quality of life while avoiding unnecessary invasive procedures. Studying clinical samples (tissue/blood/breath) will be required as next step in order to determine whether this cell-line study can be translated into a clinically useful tool.
doi:10.1016/j.nano.2013.01.008
PMCID: PMC4186653  PMID: 23428987
Lung cancer; Genetic; Mutation; Volatile organic compound; Sensor
17.  Clinical and comparative utility of afatinib in non-small cell lung cancer 
The first targeted agents approved for non-small cell lung cancer (NSCLC) treatment, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, have an impressive activity in the presence of activating mutations of the EGFR gene. However, all patients develop acquired resistance principally through secondary mutations (T790M), HER2 amplification, MET amplification, and other molecular aberrations. An attempt to overcome EGFR TKI resistance has been through the development of irreversible blockers. Afatinib is an irreversible inhibitor of the tyrosine kinase activity of all members of the HER family. The pharmacologic properties of afatinib (formation of covalent bonds, inhibition of other family members, and in vitro and in vivo activity on T790M mutation positive tumors) made this drug particularly appealing to study in clinic. Therefore, an intense program of clinical research (LUX-Lung program) was started and clinical results have shown very encouraging activity profiles in patients harboring EGFR activating mutations and in those with acquired resistance to reversible TKIs.
doi:10.2147/BTT.S40567
PMCID: PMC4003149  PMID: 24790411
NSCLC; EGFR; tyrosine kinase inhibitor; afatinib
18.  Non-Invasive Breath Analysis of Pulmonary Nodules 
INTRODUCTION
The search for non-invasive diagnostic methods of lung cancer has led to new avenues of research, including the exploration of the exhaled breath. Previous studies have shown that lung cancer can in principle be detected through exhaled breath analysis. This study evaluated the potential of exhaled breath analysis for the distinction of benign and malignant pulmonary nodules (PNs).
METHODS
Breath samples were taken from 72 patients with PNs in a prospective trial. Profiles of volatile organic compounds (VOCs) were determined by (i) gas chromatography/mass spectrometry (GC-MS) combined with solid phase microextraction (SPME) and by (ii) a chemical nanoarray.
RESULTS
53 PNs were malignant and 19 were benign with similar smoking histories and co-morbidities. Nodule size (mean +/− SD) was 2.7±1.7 vs. 1.6±1.3 cm (p=0.004) respectively. Within the malignant group, 47 were NSCLC and 6 were SCLC. Thirty had early stage disease and 23 had advanced disease. GC-MS analysis identified a significantly higher concentration of 1-octene in the breath of lung cancer, and the nanoarray distinguished significantly between benign vs. malignant PNs (p<0.0001; accuracy 88±3%), between adeno- and squamous- cell carcinomas (p<0.0001; 88±3%) and between early stage and advanced disease (p<0.0001; 88±2%).
CONCLUSIONS
In this pilot study, breath analysis discriminated benign from malignant PNs in a high-risk cohort based on lung cancer related VOC profiles. Further, it discriminated adeno-and squamous- cell carcinoma and between early vs. advanced disease. Further studies are required to validate this non-invasive approach, using a larger cohort of patients with PNs detected by CT.
doi:10.1097/JTO.0b013e3182637d5f
PMCID: PMC3444531  PMID: 22929969
Lung cancer; pulmonary nodules; diagnosis; breath; nanoarray
19.  Long non-coding RNA UCA1 induces non-T790M acquired resistance to EGFR-TKIs by activating the AKT/mTOR pathway in EGFR-mutant non-small cell lung cancer 
Oncotarget  2015;6(27):23582-23593.
The aim of this study was to explore the role of long non-coding RNA UCA1 (urothelial cancer-associated 1) in acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). In our study, UCA1 expression was significantly increased in lung cancer cells and patients with acquired resistance to EGFR-TKIs. Over-expression of UCA1 was significantly associated with a shorter progression-free survival (PFS) [13.0 vs. 8.5 months, P < 0.01] in tumors with respond to EGFR-TKIs. The significant relationship was not observed in patients with T790M mutation (10.5 vs. 12.0 months, P = 0.778), but in patients with non-T790M (19.0 vs. 9.0 months, P = 0.023). UCA1 knockdown restored gefitinib sensitivity in acquired resistant cells with non-T790M and inhibited the activation of the AKT/mTOR pathway and epithelial-mesenchymal transition (EMT). The mTOR inhibitor was effective in UCA1-expressing cell PC9/R. Inhibiting mTOR could change the expression of UCA1, although there was no significant difference. In conclusion, the influence of over-expression of UCA1 on PFS for patients with acquired resistance to EGFR-TKIs was from the subgroup with non-T790M mutation. UCA1 may induce non-T790M acquired resistance to EGFR-TKIs by activating the AKT/mTOR pathway and EMT.
PMCID: PMC4695138  PMID: 26160838
UCA1; EGFR-TKIs; acquired resistance; non-small cell lung cancer
20.  Southwestern Oncology Group Phase II Trial (S0526) of Pemetrexed in Bronchioloalveolar Carcinoma Subtypes of Advanced Adenocarcinoma 
Clinical lung cancer  2013;14(4):351-355.
Bronchioloalveolar carcinoma (BAC) subtypes of lung adenocarcinoma represent approximately 10% to 15% of new non-small-cell lung cancer cases (NSCLC). Based on preclinical data supporting the relevance of alpha-folate receptors in BAC, this trial was designed to assess pemetrexed in patients with this pathologic subtype of lung adenocarcinoma. This trial demonstrated that single-agent pemetrexed is active against this histologic subset.
Background
Pemetrexed, a multitargeted antifolate drug, is an active agent in non-small-cell lung cancer (NSCLC), especially adenocarcinomas. Based on preclinical data supporting the relevance of alpha-folate receptors in adenocarcinoma of the bronchioloalveolar carcinoma (BAC) subtype, this trial was designed to assess pemetrexed in patients with this pathologic subtype of lung adenocarcinoma.
Patients and Methods
Patients with histologically confirmed stage IIIB (with malignant pleural effusion) or stage IV adenocarcinoma with BAC features or pure BAC were eligible. Treatment consisted of pemetrexed, 500 mg/m2, administered intravenously every 21 days.
Results
Of 27 patients enrolled, 24 were eligible and assessable for adverse events: Toxicity was primarily hematologic, consisting of leukopenia/neutropenia, thrombocytopenia, and anemia. The median follow-up among patients still alive (n =8) was 35 months (range, 26–47 months). Among 17 patients with measurable disease, the response rate was 23% (all partial responses; 95% confidence interval [CI], 10%–56%). The median progression-free survival (PFS) and overall survival (OS) were 6 and 25 months, respectively.
Conclusion
Pemetrexed is active and well tolerated and, in patients with adenocarcinoma BAC subtypes, likely related to its underlying mechanism of action as a multitargeted antifolate drug.
doi:10.1016/j.cllc.2012.12.004
PMCID: PMC4567037  PMID: 23415808
Lung adenocarcinoma; Bronchioloalveolar carcinoma (BAC); Pemetrexed
21.  International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma 
Introduction
Adenocarcinoma is the most common histologic type of lung cancer. To address advances in oncology, molecular biology, pathology, radiology, and surgery of lung adenocarcinoma, an international multidisciplinary classification was sponsored by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society. This new adenocarcinoma classification is needed to provide uniform terminology and diagnostic criteria, especially for bronchioloalveolar carcinoma (BAC), the overall approach to small nonresection cancer specimens, and for multidisciplinary strategic management of tissue for molecular and immunohistochemical studies.
Methods
An international core panel of experts representing all three societies was formed with oncologists/pulmonologists, pathologists, radiologists, molecular biologists, and thoracic surgeons. A systematic review was performed under the guidance of the American Thoracic Society Documents Development and Implementation Committee. The search strategy identified 11,368 citations of which 312 articles met specified eligibility criteria and were retrieved for full text review. A series of meetings were held to discuss the development of the new classification, to develop the recommendations, and to write the current document. Recommendations for key questions were graded by strength and quality of the evidence according to the Grades of Recommendation, Assessment, Development, and Evaluation approach.
Results
The classification addresses both resection specimens, and small biopsies and cytology. The terms BAC and mixed subtype adenocarcinoma are no longer used. For resection specimens, new concepts are introduced such as adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) for small solitary adenocarcinomas with either pure lepidic growth (AIS) or predominant lepidic growth with ≤5 mm invasion (MIA) to define patients who, if they undergo complete resection, will have 100% or near 100% disease-specific survival, respectively. AIS and MIA are usually nonmucinous but rarely may be mucinous. Invasive adenocarcinomas are classified by predominant pattern after using comprehensive histologic subtyping with lepidic (formerly most mixed subtype tumors with nonmucinous BAC), acinar, papillary, and solid patterns; micropapillary is added as a new histologic subtype. Variants include invasive mucinous adenocarcinoma (formerly mucinous BAC), colloid, fetal, and enteric adenocarcinoma. This classification provides guidance for small biopsies and cytology specimens, as approximately 70% of lung cancers are diagnosed in such samples. Non-small cell lung carcinomas (NSCLCs), in patients with advanced-stage disease, are to be classified into more specific types such as adenocarcinoma or squamous cell carcinoma, whenever possible for several reasons: (1) adenocarcinoma or NSCLC not otherwise specified should be tested for epidermal growth factor receptor (EGFR) mutations as the presence of these mutations is predictive of responsiveness to EGFR tyrosine kinase inhibitors, (2) adenocarcinoma histology is a strong predictor for improved outcome with pemetrexed therapy compared with squamous cell carcinoma, and (3) potential life-threatening hemorrhage may occur in patients with squamous cell carcinoma who receive bevacizumab. If the tumor cannot be classified based on light microscopy alone, special studies such as immunohistochemistry and/or mucin stains should be applied to classify the tumor further. Use of the term NSCLC not otherwise specified should be minimized.
Conclusions
This new classification strategy is based on a multidisciplinary approach to diagnosis of lung adenocarcinoma that incorporates clinical, molecular, radiologic, and surgical issues, but it is primarily based on histology. This classification is intended to support clinical practice, and research investigation and clinical trials. As EGFR mutation is a validated predictive marker for response and progression-free survival with EGFR tyrosine kinase inhibitors in advanced lung adenocarcinoma, we recommend that patients with advanced adenocarcinomas be tested for EGFR mutation. This has implications for strategic management of tissue, particularly for small biopsies and cytology samples, to maximize high-quality tissue available for molecular studies. Potential impact for tumor, node, and metastasis staging include adjustment of the size T factor according to only the invasive component (1) pathologically in invasive tumors with lepidic areas or (2) radiologically by measuring the solid component of part-solid nodules.
doi:10.1097/JTO.0b013e318206a221
PMCID: PMC4513953  PMID: 21252716
Lung; Adenocarcinoma; Classification; Histologic; Pathology; Oncology; Pulmonary; Radiology; Computed tomography; Molecular; EGFR; KRAS; EML4-ALK; Gene profiling; Gene amplification; Surgery; Limited resection; Bronchioloalveolar carcinoma; Lepidic; Acinar; Papillary; Micropapillary; Solid; Adenocarcinoma in situ; Minimally invasive adenocarcinoma; Colloid; Mucinous cystadenocarcinoma; Enteric; Fetal; Signet ring; Clear cell; Frozen section; TTF-1; p63
22.  Correlation between MET Gene Copy Number by Silver in Situ Hybridization and Protein Expression by Immunohistochemistry in Non-Small-Cell Lung Cancer 
Journal of Thoracic Oncology  2012;7(2):340-347.
Purpose
The MET receptor is involved in the pathogenesis and progression of non-small-cell lung cancer (NSCLC). Clinical trials with MET inhibitors in NSCLC are planned with patient selection based on immunohistochemistry (IHC) and/or gene copy number assessment. Therefore, a detailed understanding of relationship between these markers and prognosis is essential.
Methods
This study included tumors from 189 NSCLC patients who underwent pulmonary resection (median follow-up 5.3 years). MET expression was evaluated by IHC on tissue microarrays and scored according to hybrid (H) score (range: 0–400) and by scoring system used in the MetMAb trial (≥50% of cells with moderate or strong staining). MET gene copy number was assessed by silver in-situ hybridization (SISH, N=140 patients).
Results
Median MET IHC H-score was 60 (range: 0–400; N=174). There were no associations between clinical and pathological characteristics, disease-free or overall survival according to median value (P=0.36 and P=0.38, respectively) or other cut-points. According to MetMAb scoring criteria, IHC positivity rate was 25%, again with no associations to clinico-pathological features nor survival. In 140 tumors evaluable for MET copy number, three (2.1%) showed gene amplification and 14 (10%) had tumors with average of 5 or more copies/nucleus. There were no associations of MET copy number with clinical characteristics, disease-free or overall survival with any analyzed cut-points. Correlation between MET copy number and protein expression was significant (Pearson’s r=0.42, P<0.0001).
Conclusions
There is a significant correlation between MET protein expression and MET gene copy number in operable NSCLC, but neither is associated with prognosis.
doi:10.1097/JTO.0b013e318240ca0d
PMCID: PMC3358920  PMID: 22237262
MET gene copy number; MET protein expression; prognosis; non-small- cell lung cancer
23.  The TOPICAL study should be more topical! 
doi:10.3978/j.issn.2218-6751.2012.12.01
PMCID: PMC4367651  PMID: 25806207
24.  EGFR Protein Expression in Non-Small Cell Lung Cancer Predicts Response to an EGFR Tyrosine Kinase Inhibitor – A Novel Antibody for Immunohistochemistry or AQUA Technology 
Clinical Cancer Research  2011;17(24):7796-7807.
Introduction
Epidermal growth factor receptor (EGFR) protein expression in non-small cell lung cancer (NSCLC) is not recommended for predicting response to EGFR tyrosine kinase inhibitors (TKIs) due to conflicting results, all using antibodies detecting EGFR external domain (ED). We tested the predictive value of EGFR protein expression for response to an EGFR TKI using an antibody that detects the intracellular domain (ID) and compared fluorescence-based Automated QUantitative Analysis (AQUA) technology to immunohistochemistry (IHC).
Methods
Specimens from 98 gefitinib-treated NSCLC Japanese patients were evaluated by IHC (n=98/98) and AQUA technology (n=70/98). EGFR ID- (5B7) and ED-specific antibodies (3C6 and 31G7) were compared.
Results
EGFR expression evaluated with 5B7 was significantly higher in responders versus non-responders to gefitinib both with IHC and with AQUA. ED-specific antibodies did not significantly predicted response. Using AQUA and ID-specific antibody resulted in the best prediction performance with a positive and negative predictive value (PPV/NPV) for responders of 50% and 87%, respectively. EGFR expression with ID-specific antibody and AQUA also predicted responders in EGFR mutated patients. Increased EGFR expression with the ID antibody associated with increased median PFS (11.7 months vs 5.0, Log-rank p=0.034) and OS (38.6 vs 14.9, p=0.040), from gefitinib therapy.
Conclusions
EGFR protein expression using an ID-specific antibody specifically predicts response to gefitinib in NSCLC patients, including in EGFR mutated patients, and increased PFS/OS from gefitinib. These data suggest that the choice of diagnostic antibody and methodology matters to predict response and outcome to specific therapies. The potential clinical application needs further validation.
doi:10.1158/1078-0432.CCR-11-0209
PMCID: PMC3266947  PMID: 21994417
EGFR; Biomarker; Lung Cancer; NSCLC; AQUA technology; IHC; EGFR TKI
25.  A Randomized, Phase II, Biomarker-Selected Study Comparing Erlotinib to Erlotinib Intercalated With Chemotherapy in First-Line Therapy for Advanced Non–Small-Cell Lung Cancer 
Journal of Clinical Oncology  2011;29(26):3567-3573.
Purpose
Erlotinib prolongs survival in patients with advanced non–small-cell lung cancer (NSCLC). We report the results of a randomized, phase II study of erlotinib alone or intercalated with chemotherapy (CT + erlotinib) in chemotherapy-naïve patients with advanced NSCLC who were positive for epidermal growth factor receptor (EGFR) protein expression and/or with high EGFR gene copy number.
Patients and Methods
A total of 143 patients were randomly assigned to either erlotinib 150 mg daily orally until disease progression (PD) occurred or to chemotherapy with paclitaxel 200 mg/m2 intravenously (IV) and carboplatin dosed by creatinine clearance (AUC 6) IV on day 1 intercalated with erlotinib 150 mg orally on days 2 through 15 every 3 weeks for four cycles followed by erlotinib 150 mg orally until PD occurred (CT + erlotinib). The primary end point was 6-month progression-free survival (PFS); secondary end points included response rate, PFS, and survival. EGFR, KRAS mutation, EGFR fluorescent in situ hybridization and immunohistochemistry, and E-cadherin and vimentin protein levels were also assessed.
Results
Six-month PFS rates were 26% and 31% for the two arms (CT + erlotinib and erlotinib alone, respectively). Both were less than the historical control of 45% (P = .001 and P = .011, respectively). Median PFS times were 4.57 and 2.69 months, respectively. Patients with tumors harboring EGFR activating mutations fared better on erlotinib alone (median PFS, 18.2 months v 4.9 months for CT + erlotinib).
Conclusion
The feasibility of a multicenter biomarker-driven study was demonstrated, but neither treatment arms exceeded historical controls. This study does not support combined chemotherapy and erlotinib in first-line treatment of EGFR-selected advanced NSCLC, and the patients with tumors harboring EGFR mutations had a better outcome on erlotinib alone.
doi:10.1200/JCO.2010.34.4929
PMCID: PMC3179254  PMID: 21825259

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