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1.  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
2.  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
3.  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
4.  Contributions of KRAS and RAL in Non-Small Cell Lung Cancer growth and progression 
Introduction
KRAS mutations are poor prognostic markers for patients with non-small cell lung cancer (NSCLC). RALA and RALB GTPases lie downstream of RAS and are implicated in RAS mediated tumorigenesis. However, their biological or prognostic role in the context of KRAS mutation in NSCLC is unclear.
Methods
Using expression analysis of human tumors and a panel of cell lines coupled with functional in vivo and in vitro experiments, we evaluated the prognostic and functional importance of RAL in NSCLC and their relationship to KRAS expression and mutation.
Results
Immunohistochemical (N=189) and transcriptomic (N=337) analyses of NSCLC patients revealed high RALA and RALB expression was associated with poor survival. In a panel of 14 human NSCLC cell lines, RALA and RALB had higher expression in KRAS mutant cell lines while RALA but not RALB activity was higher in KRAS mutant cell lines. Depletion of RAL paralogs identified cell lines that are dependent on RAL expression for proliferation and anchorage independent growth. Overall, growth of NSCLC cell lines which carry a glycine to cystine (G12C) KRAS mutation were more sensitive to RAL depletion than those with wild-type KRAS. Using gene expression and outcome data from 337 human tumors, RAL-KRAS interaction analysis revealed that KRAS and RAL paralog expression jointly impact patient prognosis.
Conclusion
RAL GTPase expression carries important additional prognostic information to KRAS status in NSCLC patients. Simultaneously targeting RAL may provide a novel therapeutic approach in NSCLC patients harboring G12C KRAS mutations.
doi:10.1097/JTO.0000000000000007
PMCID: PMC3934792  PMID: 24389431
RAL; RAS; non-small cell lung cancer; GTPase; prognosis
5.  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
6.  Fibroblast Growth Factor Receptor 1 as a Putative Therapy Target in Colorectal Cancer 
Digestion  2013;88(3):172-181.
Background/Aims
Resembling a potential therapeutic drug target, fibroblast growth factor receptor 1 (FGFR1) amplification and expression was assessed in 515 human colorectal cancer (CRC) tissue samples, lymph node metastases and CRC cell lines.
Methods
FGFR1 amplification status was determined using fluorescence in situ hybridization. Additionally, we assessed protein levels employing Western blots and immunohistochemistry. The FGFR1 mRNA localization was analyzed using mRNA in situ hybridization. Functional studies employed the FGFR inhibitor NVP-BGJ398.
Results
Of 454 primary CRCs, 24 displayed FGFR1 amplification. 92/94 lymph node metastases presented the same amplification status as the primary tumor. Of 99 investigated tumors, 18 revealed membranous activated pFGFR1 protein. FGFR1 mRNA levels were independent of the amplification status or pFGFR1 protein occurrence. In vitro, a strong antiproliferative effect of NVP-BGJ398 could be detected in cell lines exhibiting high FGFR1 protein.
Conclusion
FGFR1 is a potential therapeutic target in a subset of CRC. FGFR1 protein is likely to represent a central factor limiting the efficacy of FGFR inhibitors. The lack of correlation between its evaluation at genetic/mRNA level and its protein occurrence indicates that the assessment of the receptor at an immunohistochemical level most likely represents a suitable way to assess FGFR1 as a predictive biomarker for patient selection in future clinical trials.
doi:10.1159/000355018
PMCID: PMC4186657  PMID: 24135816
Fibroblast growth factor receptor 1; Colorectal cancer; NVP-BGJ398
7.  Insulin-like growth factor-1 receptor (IGF-1R) as a biomarker for resistance to the tyrosine kinase inhibitor gefitinib in non-small cell lung cancer 
Cellular oncology (Dordrecht)  2013;36(4):277-288.
Background
The insulin-like growth factor-1 receptor (IGF-1R) pathway is known to play a role in the acquisition of resistance to epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). However, its exact role in TKI resistance has so far remained unclear. Here, we interrogated the hypothesis that the IGF-1R may serve as a biomarker for, and may play a role in, intrinsic resistance to the EGFR-specific TKl gefitinib in NSCLC.
Methods
Total-IGF-1R and phosphorylated (p)-IGF-1R expression levels were related to gefitinib sensitivity in 23 NSCLC cell lines. This sensitivity was re-evaluated after knocking down IGF-1R expression and after IGF-1R up-regulation through exogenous IGF-1 expression. The utility of IGF-1R expression as a predictive biomarker was also evaluated by immunohistochemistry (IHC) in 98 primary NSCLC samples from patients treated with gefitinib.
Results
Seventeen of the cell lines tested were resistant to gefitinib, whereas 3 cell lines were sensitive. The three remaining cell lines showed intermediate values. Thirteen resistant cell lines were found to be positive for total-IGF-lR expression, while all the sensitive cell lines were negative, resulting in a positive predictive value (PPV) of 81 % for total-IGF-lR to predict resistance. Seven resistant cell lines exhibited high p-IGF-1R levels, whereas all 3 sensitive cell lines were negative for p-IGF-1R, resulting in a PPV of 100 % for p-IGF-1R to predict resistance. Neither a knock-down of IGF-lR expression nor an activation of the IGF1-R pathway through exogenous IGF-1 expression affected gefitinib sensitivity. In primary NSCLC tissues, IGF-1R expression was found to be significantly higher in patients with progressive disease, i.e., showing gefitinib resistance, as compared to those with a complete or partial response.
Conclusions
IGF-1R acts as a predictor for resistance to gefitinib in NSCLC cell lines and NSCLC patients, but does not seem to play a role in the intrinsic resistance to this drug. High total-IGF-1R and p-IGR-1R levels may predict such a resistance. Since the underlying mechanism does not appear to be related to proliferation induction, alternative pathways should be explored.
doi:10.1007/s13402-013-0133-9
PMCID: PMC4186686  PMID: 23619944
NSCLC; EGFR; IGF-1R; Therapy resistance; Biomarkers
8.  Endobronchial miRNAs as biomarkers in lung cancer chemoprevention 
Lung cancers express lower levels of prostacyclin than normal lung tissues. Prostacyclin prevents lung cancer in a variety of mouse models. A randomized phase II trial comparing oral iloprost (a prostacyclin analogue) to placebo in high-risk subjects demonstrated improvement in bronchial histology in former, but not current, smokers. This placebo-controlled study offered the opportunity for investigation of other potential intermediate endpoint and predictive biomarkers to incorporate into chemoprevention trials.
Matched bronchial biopsies were obtained at baseline (BL) and at 6 months follow-up (FU) from 125 high-risk individuals who completed the trial: 31/29 and 37/28 current/former smokers in the iloprost and placebo arm, respectively. We analyzed the expression of 14 selected miRNAs by qRT-PCR in 496 biopsies.
The expression of seven miRNAs was significantly correlated with histology at BL. The expression of miR-34c was inversely correlated with histology at BL (p<0.0001) and with change in histology at FU (p=0.0003), independent of treatment or smoking status. Several miRNAs were also found to be differentially expressed in current smokers as compared with former smokers. In current smokers, miR-375 was up-regulated at BL (p<0.0001) and down-regulated after treatment with iloprost (p=0.0023). No miRNA at baseline reliably predicted a response to iloprost.
No biomarker predictive of response to iloprost was found. MiR-34c was inversely correlated with BL histology and with histology changes. Mir-34c changes at FU could be used as a quantitative biomarker which parallels histologic response in formalin-fixed bronchial biopsies in future lung cancer chemoprevention studies.
doi:10.1158/1940-6207.CAPR-12-0382
PMCID: PMC4159305  PMID: 23268837
miRNAs; chemoprevention; lung cancer; iloprost
10.  MiR-200c overexpression is associated with better efficacy of EGFR-TKIs in non-small cell lung cancer patients with EGFR wild-type 
Oncotarget  2014;5(17):7902-7916.
Several randomized trials have demonstrated non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations can achieve favorable clinical outcomes on treatment with EGFR tyrosine kinase inhibitors (TKIs). EGFR mutation is considered as a predictive marker for efficacy of EGFR-TKIs in NSCLC. Here we show miR-200c overexpression was correlated with the epithelial phenotype and sensitivity to gefitinib in EGFR wild-type NSCLC cell lines. Up-regulated miR-200c could regain the sensitivity to gefitinib in the EGFR wild-type cell lines and miR-200c could regulate epithelial to mesenchymal transition through PI3K/AKT and MEK/ERK pathways. NSCLC patients at advanced stage (N=150) who received EGFR-TKIs (gefitinib or erlotinib) as second- or third-line therapy from September 2008 to December 2012 were included in the study. In 66 NSCLC patients with wild-type EGFR, high levels of miR-200c expression was associated with higher disease control rate (DCR), longer progression-free survival (PFS) and longer overall survival (OS) compared with low miR-200c expression subgroup. In the subgroup with EGFR mutation, the trend remained the same but not statistically significant. Overall, these findings indicated that miR-200c might be a predictive biomarker for sensitivity to EGFR-TKIs in advanced NSCLC patients with wild-type EGFR.
PMCID: PMC4202169  PMID: 25277203
Non-small cell lung cancer; MiR-200c; Epidermal growth factor receptor; Wild type; Tyrosine-kinase inhibitor
11.  Chemotherapy Outcomes by Histologic Subtypes of Non-Small cell Lung Cancer: Analysis of the Southwest Oncology Group Database for Antimicrotubule-Platinum Therapy 
Clinical lung cancer  2013;14(6):627-635.
Histological subtyping has been advocated to select chemotherapy for patients with advanced stage non-small-cell lung cancer (NSCLC). Data from four randomized trials (S9308, S9509, S9806 and S0003) administering an antimicrotubular agent (a taxane or vinorelbine) plus platinum in patients receiving first line treatment for advanced stage NSCLC were analyzed. Of 1146 patients included in this analysis there was no difference in OS or PFS by histological subtype. Since the great majority of advanced NSCLC patients continue to receive chemotherapy, defining molecular-based predictive markers of responsiveness is warranted.
Objectives
Histologic subtyping has been advocated to select chemotherapy for patients with advanced-stage non-small-cell lung cancer (NSCLC). To determine if histologic subtype was associated with efficacy for the commonly used antimicrotubular (AMT) agents, paclitaxel, docetaxel and vinorelbine plus a platinum compound, we examined the Southwest Oncology Group (SWOG) lung cancer database.
Methods
Data from 4 randomized trials (S9308, S9509, S9806 and S0003) administering an AMT agent plus platinum in patients receiving first-line treatment for advanced stage NSCLC were analyzed. Overall survival (OS) and progression-free survival (PFS) comparisons were performed using Cox proportional hazard regression, adjusting for sex. Median survival times were estimated by Kaplan-Meier.
Results
Of 1146 patients included in this analysis, 640 had adenocarcinoma (56%), 220 had squamous cell carcinoma (19%), 121 had large cell carcinoma (11%) and 165 had NSCLC not otherwise specified (NOS)(14%). Median OS times by histologic subtypes were 8.5, 8.4, 8.2, and 9.6 months, respectively, and median PFS times were 4.2, 4.3, 4.3, and 4.6 months, respectively. No difference in OS or PFS was observed by histologic subtype and, specifically, between nonsquamous and squamous histologies.
Conclusions
This pooled analysis from 4 SWOG trials employing an AMT-platinum regimen did not show a difference in survival outcomes by histologic subtype. Because the majority of patients with advanced NSCLC continue to receive chemotherapy, defining molecular-based predictive markers of responsiveness is warranted.
doi:10.1016/j.cllc.2013.06.010
PMCID: PMC4122504  PMID: 23910067
Chemotherapy outcomes; Histology; Lung cancer
12.  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
13.  A patient tumor transplant model of squamous cell cancer identifies PI3K inhibitors as candidate therapeutics in defined molecular bins 
Molecular oncology  2013;7(4):776-790.
Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV−, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.
doi:10.1016/j.molonc.2013.03.004
PMCID: PMC3760013  PMID: 23607916
Head and neck cancer; Xenografts; Human papillomavirus; EGFR; PI3K; NOTCH1
14.  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
15.  Phase II Trial of Carboplatin, Paclitaxel, Cetuximab and Bevacizumab Followed by Cetuximab and Bevacizumab in Advanced Non-Squamous Non-Small Cell Lung Cancer: SWOG S0536 
Introduction
Cetuximab and bevacizumab have each been demonstrated to prolong survival when added to chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). However, the potential benefit of combining cetuximab and bevacizumab together with a platinum-based doublet had not been explored. We designed this phase II trial to evaluate the safety, tolerability and efficacy of the combination of carboplatin, paclitaxel, cetuximab and bevacizumab in chemotherapy-naïve patients with advanced, non-squamous NSCLC.
Methods
Patients received with up to six cycles of carboplatin (area under curve 6), paclitaxel (200 mg/m2), cetuximab (400 mg/m2 day 1 then 250 mg/m2 weekly) and bevacizumab (15 mg/kg) every 21 days. Patients with an objective response or stable disease received maintenance cetuximab (250 mg/m2 weekly) and bevacizumab (15 mg/kg every 21 days) until disease progression. The primary endpoint was safety as defined by the frequency and severity of hemorrhagic toxicities. Secondary endpoints included response rate (RR), progression-free survival (PFS), overall survival (OS), and toxicity. Molecular biomarkers were assessed in an exploratory manner.
Results
The primary endpoint of grade 4 or higher hemorrhage of 2% (95% confidence interval: 0-7%) met prespecified criteria for safety. Oone hundred ten patients were enrolled. There were 4 treatment-related deaths including lung hemorrhage (2), infection (1), and unknown (1). Median progression-free survival was 7 months and median overall survival was 15 months. The RR was 56% with an overall disease control rate of 77%.
Conclusion
This regimen was safe, feasible and effective as frontline treatment of advanced NSCLC, providing the basis for the ongoing Phase III trial S0819.
doi:10.1097/JTO.0000000000000009
PMCID: PMC4072123  PMID: 24189513
Non-small-cell lung cancer; Paclitaxel; Carboplatin; Cetuximab; Bevacizumab; Frontline
16.  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
17.  Hedgehog signaling alters reliance on EGF receptor signaling and mediates anti-EGFR therapeutic resistance in head and neck cancer 
Cancer research  2013;73(11):3381-3392.
The epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of head and neck squamous cell carcinoma (HNSCC), but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in Vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacological or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four out of six HNSCC patient-derived xenografts; however they eventually re-grew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed re-growth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state delaying or completely blocking tumor recurrence.
doi:10.1158/0008-5472.CAN-12-4047
PMCID: PMC3674118  PMID: 23576557
Head and neck squamous cell cancer; hedgehog pathway; epidermal growth factor receptor pathway; epithelial to mesenchymal transition
18.  EGFR and HER2 Genomic Gain in Recurrent Non-small Cell Lung Cancer After Surgery 
Journal of Thoracic Oncology  2009;4(3):318-325.
Background
Sensitivity to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and frequency of activation mutations in EGFR is lower in Caucasian than Asian non small-cell lung cancer (NSCLC) patients. Increased EGFR gene copy numbers evaluated by fluorescence in situ hybridization (FISH) has been reported as predictor of clinical benefit from EGFR-TKIs in Caucasian NSCLC patients. This study was carried out to verify whether EGFR FISH had similar performance in Japanese patients.
Methods
A cohort of 44 Japanese patients with recurrent NSCLC after surgery was treated with gefitinib 250 mg daily. The cohort included 48% females and 52% never-smokers; 73% had prior chemotherapy and 57% had stage III-IV at the time of surgery. Adenocarcinoma was the most common histology (86%). FISH was performed using the EGFR/Chromosome Enumeration Probe 7 and PathVysion DNA probes (Abbott Molecular). Specimens were classified as FISH positive when showing gene amplification or high polysomy (≥4 copies of the gene in ≥40% of tumor cells). Tumor response to gefitinib was assessed by RECIST for 33 patients with measurable diseases.
Results
Twenty-nine tumors (66%) were EGFR FISH+ and 23 (53%) were HER2 FISH+. Overall response rate was 52%, representing 65% of EGFR FISH+ patients and 29% of EGFR FISH+ patients (p = 0.0777). Survival was not impacted by the EGFR FISH (p = 0.9395) or the HER2 FISH (p = 0.0671) status. EGFR FISH= was significantly associated with HER2 FISH+ (p = 0.015) and presence of EGFR mutation (p = 0.0060). EGFR mutation significantly correlated with response (p < 0.0001) and survival after gefitinib (p = 0.0204). EGFR and HER2 FISH status were not associated with KRAS mutation.
Conclusion
Frequency of EGFR FISH+ status was higher and its predictive power for TKI sensitivity was lower in this Japanese cohort than in Western NSCLC cohorts. These findings support differences in the mechanisms of EGFR pathway activation in NSCLC between Asian and Caucasian populations. Confirmation of these results in larger cohorts is warranted.
doi:10.1097/JTO.0b013e31819667a3
PMCID: PMC3379811  PMID: 19247083
FISH; EGFR; HER2; KRAS; Biomarkers; NSCLC; Tyrosine inhibitors
19.  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
20.  Fluorescence In situ Hybridization Subgroup Analysis of TRIBUTE, a Phase III Trial of Erlotinib Plus Carboplatin and Paclitaxel in Non – Small Cell Lung Cancer 
Clinical Cancer Research  2008;14(19):6317-6323.
Purpose
TRIBUTE was a phase III trial evaluating the addition of erlotinib to carboplatin and paclitaxel as a first-line treatment for advanced non – small cell lung cancer that did not meet its primary end point of improving overall survival. Here, we assess the value of using epidermal growth factor receptor (EGFR) gene copy number in tumor biopsy samples, as determined by fluorescence in situ hybridization (FISH), as a predictor of treatment outcome.
Methods
EGFR FISH analysis was done using LSI EGFR SpectrumOrange/CEP7 Spectrum-Green probe.
Results
Of 275 samples, 245 (89.1%) were successfully analyzed by FISH. One hundred (40.8%) of patients were EGFR FISH(+). Median overall survival was not different between FISH(+) and FISH(−) patients in either the chemotherapy+erlotinib arm or the chemotherapy+placebo arm. In FISH(+) patients, median time to progression (TTP) was 6.3 months in the erlotinib arm versus 5.8 months in the placebo arm (hazard ratio, 0.59; 95% confidence interval, 0.35–0.99; P = 0.0430); in FISH(−) patients, median TTP was 4.6 months versus 6.0 months (hazard ratio,1.42; 95%confidence interval, 0.95–2.14; P = 0.0895; treatment interaction test, P = 0.007). After 6 months of treatment, a notable separation of the TTP curves in favor of erlotinib emerged. Objective response rates were11.6% versus 29.8% in FISH(+) patients (chemotherapy+erlotinib arm versus chemotherapy+placebo arm; P = 0.0495) and 21.8% versus 25.4%, respectively, for FISH(−) patients (P = 0.6954).
Conclusions
EGFR gene copy number by FISH did not predict survival benefit. However, among EGFR FISH(+) patients, TTP was longer in patients who received erlotinib and continued to receive it after completing first-line therapy.
doi:10.1158/1078-0432.CCR-08-0539
PMCID: PMC3368373  PMID: 18829515
22.  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
23.  Increased insulin-like growth factor 1 receptor (IGF1R) protein expression and gene copy number in small cell lung cancer 
Journal of Thoracic Oncology  2010;5(12):1905-1911.
Purpose
Identification of new therapies in small cell lung cancer (SCLC) is urgently needed. IGF1R is a tyrosine kinase receptor implicated in the pathogenesis of several malignancies and is potentially an attractive target for anti-cancer treatment. Knowledge about IGF1R protein expression, gene copy number and the prognostic relevance of these features in SCLC is limited.
Methods
We analyzed IGF1R protein expression and gene copy number in primary tumors from 90 SCLC patients (67 males and 23 females) who underwent pulmonary resection. IGF1R expression assessed by immunohistochemistry (IHC) with H-scores from 0 to 400 was evaluable in 84 patients, and IGF1R gene copy number assessed by silver in-situ hybridization (SISH) technique in 81 patients.
Results
Median H-score for IGF1R protein expression was 88 (range 0-400) and the proportion of positive immunostaining using cut-off H-score of 10 was 74%. Increased IGF1R gene copy number (an average of 4 or more copies per cell) was found in 15 cases (18.5%), 5 of whom (6.2%) showed gene amplification. There was a significant correlation between protein expression and gene copy number (r=0.49, p<0.005). IGF1R expression and gene copy number did not associate with clinicopathological factors such as patient age, tumor size, lymph node involvement, stage and survival.
Conclusions
SCLC is characterized by frequent high IGF1R protein expression, increased gene copy number and occasional occurrence of true gene amplification. These features may have important implications for future anti-IGF1R therapeutic approaches.
doi:10.1097/JTO.0b013e3181f38f57
PMCID: PMC3356161  PMID: 21124078
lung cancer; IGF1R; protein expression; gene copy number; prognosis
24.  Novel EGFR mutation specific antibodies for NSCLC: Immunohistochemistry as a possible screening method for EGFR mutations 
Background
Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) predict better outcome to EGFR tyrosine kinase inhibitors (TKIs). The most common mutations are exon 19 deletions (most frequently E746-A750) and L858R point mutation in exon 21. Here, we evaluated the accuracy of novel EGFR mutation specific antibodies in a Japanese cohort with NSCLC and compared to direct DNA sequencing and clinical outcome.
Materials and methods
Immunohistochemistry (IHC) using antibodies specific for the E746-A750 and L858R mutations in EGFR was performed on tissue microarrays of tumors from 70 gefitinib treated NSCLC patients. Extracted DNA was sequenced for mutational analysis of EGFR exons 18 to 21.
Results
DNA sequencing showed EGFR mutations in 41 patients (58.6%), and exon 19 deletions in 18 patients (25.7%), 61% (11/18) had a deletion in the range of E746-A750) and 12 (17.1%) had exon 21 mutations (L858R). IHC showed, for the E746-A750 and L858R mutations, sensitivity (81.8% and 75%), specificity (100%, 96.6%), PPV (100%, 81.8%) and NPV (96.7%, 94.9%). Analysis for objective response rates (ORR) and survival were not correlated to IHC staining, although the combined staining showed non-significant trends towards better overall survival for patients with EGFR mutations.
Conclusions
The mutation specific IHC antibodies have high sensitivity and specificity for pre-defined EFGR mutations and may be suitable for screening for these pre-defined mutations. However, negative IHC results require further mutation analyses prior to excluding EGFR-targeted therapy.
doi:10.1097/JTO.0b013e3181e9da60
PMCID: PMC2946481  PMID: 20697298
EGFR; Biomarkers; Lung Cancer; NSCLC; Mutation
25.  Insulin-like Growth Factor Receptor 1 (IGF1R) Gene Copy Number Is Associated With Survival in Operable Non–Small-Cell Lung Cancer: A Comparison Between IGF1R Fluorescent In Situ Hybridization, Protein Expression, and mRNA Expression 
Journal of Clinical Oncology  2010;28(13):2174-2180.
Purpose
The purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non–small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis.
Patients and Methods
One hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181).
Results
IGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis.
Conclusion
IGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.
doi:10.1200/JCO.2009.24.6611
PMCID: PMC2860435  PMID: 20351332

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