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2.  Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study 
The lancet oncology  2012;13(10):1011-1019.
Summary
Background
ALK fusion genes occur in a subset of non-small-cell lung cancers (NSCLCs). We assessed the tolerability and activity of crizotinib in patients with NSCLC who were prospectively identified to have an ALK fusion within the first-in-man phase 1 crizotinib study.
Methods
In this phase 1 study, patients with ALK-positive stage III or IV NSCLC received oral crizotinib 250 mg twice daily in 28-day cycles. Endpoints included tumour responses, duration of response, time to tumour response, progression-free survival (PFS), overall survival at 6 and 12 months, and determination of the safety and tolerability and characterisation of the plasma pharmacokinetic profile of crizotinib after oral administration. Responses were analysed in evaluable patients and PFS and safety were analysed in all patients. This study is registered with ClinicalTrials.gov, number NCT00585195.
Findings
Between Aug 27, 2008, and June 1, 2011, 149 ALK-positive patients were enrolled, 143 of whom were included in the response-evaluable population. 87 of 143 patients had an objective response (60·8%, 95% CI 52·3–68·9), including three complete responses and 84 partial responses. Median time to first documented objective response was 7·9 weeks (range 2·1–39·6) and median duration of response was 49·1 weeks (95% CI 39·3–75·4). The response rate seemed to be largely independent of age, sex, performance status, or line of treatment. Median PFS was 9·7 months (95% CI 7·7–12·8). Median overall survival data are not yet mature, but estimated overall survival at 6 and 12 months was 87·9% (95% CI 81·3–92·3) and 74·8% (66·4–81·5), respectively. 39 patients continued to receive crizotinib for more than 2 weeks after progression because of perceived ongoing clinical benefit from the drug (12 for at least 6 months from the time of their initial investigator-defined disease progression). Overall, 144 (97%) of 149 patients experienced treatment-related adverse events, which were mostly grade 1 or 2. The most common adverse events were visual effects, nausea, diarrhoea, constipation, vomiting, and peripheral oedema. The most common treatment-related grade 3 or 4 adverse events were neutropenia (n=9), raised alanine aminotransferase (n=6), hypophosphataemia (n=6), and lymphopenia (n=6).
Interpretation
Crizotinib is well tolerated with rapid, durable responses in patients with ALK-positive NSCLC. There seems to be potential for ongoing benefit after initial disease progression in this population, but a more formal definition of ongoing benefit in this context is needed.
Funding
Pfizer.
doi:10.1016/S1470-2045(12)70344-3
PMCID: PMC3936578  PMID: 22954507
3.  Diagnostic Assays for Identification of Anaplastic Lymphoma Kinase–Positive Non–Small Cell Lung Cancer 
Cancer  2012;119(8):1467-1477.
In series dominated by adenocarcinoma histology, approximately 5% of non–small cell lung cancers (NSCLCs) harbor an anaplastic lymphoma kinase (ALK) gene rearrangement. Crizotinib, a tyrosine kinase inhibitor with significant activity against ALK, has demonstrated high response rates and prolonged progression-free survival in ALK-positive patients enrolled in phase 1/2 clinical trials. In 2011, crizotinib received accelerated approval from the US Food and Drug Administration (FDA) for the treatment of proven ALK-positive NSCLC using an FDA-approved diagnostic test. Currently, only break-apart fluorescence in situ hybridization testing is FDA approved as a companion diagnostic for crizotinib; however, many other assays are available or in development. In the current review, the authors summarize the diagnostic tests available, or likely to become available, that could be used to identify patients with ALK-positive NSCLC, highlighting the pros and cons of each.
doi:10.1002/cncr.27913
PMCID: PMC3935240  PMID: 23280244
crizotinib; non-small cell lung cancer; anaplastic lymphoma kinase (ALK) gene rearrangements; fluorescence in situ hybridization
5.  Diphtheria Toxin-Epidermal Growth Factor Fusion Protein DAB389EGF for the treatment of bladder cancer 
Purpose
The novel fusion protein, DAB389EGF, is comprised of both the catalytic and translocation domains of diphtheria toxin that are fused to the human epidermal growth factor, providing a targeting and a toxicity component. We tested DAB389EGF for anti-tumor activity in both in vitro and in vivo urinary bladder cancer models.
Experimental Design
Human bladder cancer lines were treated with DAB389EGF and assessed for growth inhibition and clonogenic suppression. Using 6–8 week old female athymic nude mice implanted orthotopically with HTB9 cells, DAB389EGF was administered intravesically twice weekly for two weeks. The response of the luciferase expressing HTB9 cells was monitored via bioluminescence as the primary endpoint..
Results
Treatment response with DAB389EGF was specific and robust, with an IC50 ranging from 0.5 to 15ng/ml in 8 tested bladder cancer cell lines, but greater than 50ng/ml in the EGFR-negative H520 control cell line. Simulating short duration intravesical therapy used clinically, a 2 hour treatment exposure of DAB389EGF (10ng/ml) produced clonogenic suppression in three selected bladder cancer cell lines. In vivo, luciferase activity was suppressed in 5 of 6 mice treated with DAB389EGF (70 μl (1ng/μL) per mouse), as compared to only 1 of 6 mice treated with a control DT fusion protein. Histologic assessment of tumor clearance correlated with the bioluminescent changes observed with DAB389EGF treatment. Immunocompetent mice treated with intravesical DAB389EGF did not demonstrate any non-specific systemic toxicity.
Conclusions
The intravesical delivery of targeted-toxin fusion proteins is a novel treatment approach for non-muscle-invasive urinary bladder cancer. With appropriate targeting, the treatments are effective and well tolerated in vivo.
doi:10.1158/1078-0432.CCR-12-1258
PMCID: PMC3537889  PMID: 23172881
6.  Predictive Biomarkers of Sensitivity to the Aurora and Angiogenic Kinase Inhibitor ENMD-2076 in Preclinical Breast Cancer Models 
Purpose
The Aurora kinases are a family of conserved serine-threonine kinases with key roles in mitotic cell division. As with other promising anti-cancer targets, patient selection strategies to identify a responsive subtype will likely be required for successful clinical development of Aurora kinase inhibitors. The purpose of this study was to evaluate the antitumor activity of the Aurora and angiogenic kinase inhibitor ENMD-2076 against preclinical models of breast cancer with identification of candidate predictive biomarkers.
Experimental Design
Twenty-nine breast cancer cell lines were exposed to ENMD-2076 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. In vitro activity was confirmed in in MDA-MB-468 and MDA-MB-231 triple-negative breast cancer xenografts. Systematic gene expression analysis was used to identify up- and down-regulated pathways in the sensitive and resistant cell lines, including within the triple-negative breast cancer subset.
Results
ENMD-2076 demonstrated anti-proliferative activity against breast cancer cell lines, with more robust activity against cell lines lacking estrogen receptor expression and those without increased HER2 expression. Within the triple-negative breast cancer subset, cell lines with a p53 mutation and increased p53 expression were more sensitive to the cytotoxic and pro-apoptotic effects of ENMD-2076 exposure than cell lines with decreased p53 expression.
Conclusions
ENMD-2076 exhibited robust anticancer activity against models of triple-negative breast cancer and the candidate predictive biomarkers identified in this study may be useful in selecting patients for Aurora kinase inhibitors in the future.
doi:10.1158/1078-0432.CCR-12-1611
PMCID: PMC3537923  PMID: 23136197
triple-negative breast cancer; aurora kinase; ENMD-2076; angiogenesis; vascular endothelial growth factor
7.  Resistance to ROS1 Inhibition Mediated by EGFR Pathway Activation in Non-Small Cell Lung Cancer 
PLoS ONE  2013;8(12):e82236.
The targeting of oncogenic ‘driver’ kinases with small molecule inhibitors has proven to be a highly effective therapeutic strategy in selected non-small cell lung cancer (NSCLC) patients. However, acquired resistance to targeted therapies invariably arises and is a major limitation to patient care. ROS1 fusion proteins are a recently described class of oncogenic driver, and NSCLC patients that express these fusions generally respond well to ROS1-targeted therapy. In this study, we sought to determine mechanisms of acquired resistance to ROS1 inhibition. To accomplish this, we analyzed tumor samples from a patient who initially responded to the ROS1 inhibitor crizotinib but eventually developed acquired resistance. In addition, we generated a ROS1 inhibition-resistant derivative of the initially sensitive NSCLC cell line HCC78. Previously described mechanisms of acquired resistance to tyrosine kinase inhibitors including target kinase-domain mutation, target copy number gain, epithelial-mesenchymal transition, and conversion to small cell lung cancer histology were found to not underlie resistance in the patient sample or resistant cell line. However, we did observe a switch in the control of growth and survival signaling pathways from ROS1 to EGFR in the resistant cell line. As a result of this switch, ROS1 inhibition-resistant HCC78 cells became sensitive to EGFR inhibition, an effect that was enhanced by co-treatment with a ROS1 inhibitor. Our results suggest that co-inhibition of ROS1 and EGFR may be an effective strategy to combat resistance to targeted therapy in some ROS1 fusion-positive NSCLC patients.
doi:10.1371/journal.pone.0082236
PMCID: PMC3862576  PMID: 24349229
8.  Common PIK3CA Mutants and a Novel 3′ UTR Mutation Are Associated with Increased Sensitivity to Saracatinib 
Purpose
Dysregulation of the phosphoinositide 3-kinase (PI3K) and Src signaling pathways commonly occur in colorectal cancer. Mutations in the PIK3CA gene are associated with an increase in severity of disease and worse clinical outcomes. Elevated levels of Src have been identified in premalignant lesions and are suggested to play a central role in tumor progression. Because these pathways appear to enhance tumor growth and metastasis, molecularly targeted agents for both pathways are currently being evaluated in early-phase clinical trials.
Experimental Design
We used colorectal cancer cell lines and a patient-derived explant model to investigate the efficacy of saracatinib. Mutations in the PIK3CA were evaluated to examine the association between mutations in the PIK3CA gene and sensitivity to saracatinib.
Results
We have identified a subset of patients with a PIK3CA (exon 9 and 20) mutation with increased sensitivity to saracatinib. A novel 3′ untranslated region (UTR) mutation was also shown to be associated with increased sensitivity to saracatinib and have a reduced affinity for miR-520a and miR-525a. Importantly, we show that Src inhibition reduces the interaction between Src and p85, subsequently decreasing Akt-dependent signaling.
Conclusion
These results indicate that a personalized approach in targeting Src in PIK3CA-mutant patients with colorectal cancers may prove effective in a subset of patients with this genetic alteration.
doi:10.1158/1078-0432.CCR-11-3167
PMCID: PMC3836589  PMID: 22553375
9.  Gene Array and Fluorescence In situ Hybridization Biomarkers of Activity of Saracatinib (AZD0530), a Src Inhibitor, in a Preclinical Model of Colorectal Cancer 
Purpose
To evaluate the efficacy of saracatinib (AZD0530), an oral Src inhibitor, in colorectal cancer (CRC) and to identify biomarkers that predict antitumor activity.
Experimental Design
Twenty-three CRC cell lines were exposed to saracatinib, and baseline gene expression profiles of three sensitive and eight resistant cell lines in vitro and in vivo were used to predict saracatinib sensitivity in an independent group of 10 human CRC explant tumors using the gene array K-Top Scoring Pairs (K-TSP) method. In addition, fluorescence in situ hybridization (FISH) and immunoblotting determined both Src gene copy number and activation of Src, respectively.
Results
Two of 10 explant tumors were determined to be sensitive to saracatinib. The K-TSP classifier (TOX>GLIS2, TSPAN7>BCAS4, and PARD6G>NXN) achieved 70% (7 of 10) accuracy on the test set. Evaluation of Src gene copy number by FISH showed a trend toward significance (P = 0.066) with respect to an increase in Src gene copy and resistance to saracatinib. Tumors sensitive to saracatinib showed an increase in the activation of Src and FAK when compared with resistant tumors.
Conclusions
Saracatinib significantly decreased tumor growth in a subset of CRC cell lines and explants. A K-TSP classifier (TOX>GLIS2, TSPAN7>BCAS4, and PARD6G>NXN) was predictive for sensitivity to saracatinib. In addition, increased activation of the Src pathway was associated with sensitivity to saracatinib. These results suggest that FISH, a K-TSP classifier, and activation of the Src pathway have potential in identifying CRC patients that would potentially benefit from treatment with saracatinib.
doi:10.1158/1078-0432.CCR-10-0066
PMCID: PMC3805460  PMID: 20682712
10.  Correlations between the percentage of tumor cells showing an ALK gene rearrangement, ALK signal copy number and response to crizotinib therapy in ALK FISH positive non-small cell lung cancer 
Cancer  2012;118(18):4486-4494.
Background
FISH, using break-apart red (3’) and green (5’) ALK probes, consistently shows rearrangements in < 100% of tumor cells in ALK+ NSCLC. Increased copy numbers of fused and rearranged signals also occur. Here we explore correlations between the percentage of ALK positive cells and signal copy number and their association with response to ALK inhibition.
Methods
Ninety ALK + NSCLC cases were evaluated. The percentage of positive cells, pattern of positivity (split, single red, or both) and copy number of fused, isolated red and green signals were recorded. Thirty patients had received crizotinib.
Results
Increased isolated red signal copy number (contributing to both single red and split patterns of positivity) correlated with a higher percentage of ALK+ cells (r = 0.743, p = <0.0001). Mean fused copy number was negatively associated with isolated red signal copy number (r = −0.409, p = <0.0001). Neither percentage cells positive (r = 0.192, p = 0.3), nor copy number of isolated red (r = 0.274, p = 0.195) correlated with maximal tumor shrinkage with crizotinib.
Conclusions
The strong association between increased copy number of of key ALK signals and percentage positive cells suggests that the <100% rate of cellular positivity in ALK+ tumors is due to technical factors not biology. In ALK+ tumors, neither percentage cells positive, nor signal copy number appear to be informative variables for predicting benefit from ALK inhibition. The inverse relationship between fused and isolated red copy number suggests ALK+ may be a distinct ‘near diploid’ subtype of NSCLC developing before significant chromosomal aneusomy occurs.
doi:10.1002/cncr.27411
PMCID: PMC3342464  PMID: 22282074
ALK; FISH; copy number; crizotinib
11.  Oncogene Status Predicts Patterns of Metastatic Spread in Treatment-Naïve Non-Small Cell Lung Cancer 
Cancer  2012;118(18):4502-4511.
Background
The discovery of distinct subsets of non-small cell lung cancer (NSCLC) characterized by activation of driver oncogenes has greatly impacted personalized therapy. We hypothesized that the dominant oncogene in NSCLC would be associated with distinct patterns of metastatic spread in NSCLC at the time of diagnosis.
Methods
209 consecutive patients with stage IV non-squamous NSCLC with an EGFR mutation (N=39), KRAS mutation (N=49), ALK gene rearrangement (N=41), or wild-type for all three (triple negative, N=80) were included. The percentage of patients with metastatic disease at a given site was compared between each molecular cohort (EGFR, KRAS, or ALK) and the triple negative cohort.
Results
ALK gene rearrangement was significantly associated with pericardial disease (OR=4.61, 95% CI 1.30, 16.37, p=0.02) and pleural disease (OR=4.80, 95% CI 2.10, 10.97, p<0.001). Patients with ALK gene rearrangements (OR=5.50, 95% CI 1.76, 17.18, p= 0.003) and patients with EGFR mutations (OR=5.17, 95% CI 1.63, 16.43, p= 0.006) were predisposed to liver metastasis compared to the triple negative cohort. No molecular cohort had a predisposition to pulmonary nodules, adrenal, bone, or brain metastasis compared to the triple negative cohort. The mean number of metastatic disease sites in patients within the ALK rearranged cohort was significantly greater than the triple negative cohort (mean = 3.6 sites vs. 2.5 sites, p<0.0001).
Conclusion
The results support the hypothesis that the dominant molecular oncogenes in NSCLC are associated with different biological behaviors manifesting as distinct patterns of metastatic spread at the time of diagnosis.
doi:10.1002/cncr.27409
PMCID: PMC3370097  PMID: 22282022
metastasis; Non-Small Cell Lung Cancer; EGFR; ALK tyrosine kinase receptor; KRAS
13.  Identifying and Targeting ROS1 Gene Fusions in Non-Small Cell Lung Cancer 
Purpose
Oncogenic gene fusions involving the 3’ region of ROS1 kinase have been identified in various human cancers. In this study, we sought to characterize ROS1 fusion genes in non-small cell lung cancer (NSCLC) and establish the fusion proteins as drug targets.
Experimental Design
A NSCLC tissue microarray (TMA) panel containing 447 samples was screened for ROS1 rearrangement by fluorescence in-situ hybridization (FISH). This assay was also used to screen NSCLC patients. In positive samples, the identity of the fusion partner was determined through inverse-PCR and RT-PCR. In addition, the clinical utility of ROS1 inhibition was assessed by treating a ROS1-positive patient with crizotinib. The HCC78 cell line, which expresses the SLC34A2-ROS1 fusion, was treated with kinase inhibitors that have activity against ROS1. The effects of ROS1 inhibition on proliferation, cell-cycle progression, and cell signaling pathways were analyzed by MTS assay, flow cytometry, and western blotting.
Results
In the TMA panel, 5/428 (1.2%) evaluable samples were found to be positive for ROS1 rearrangement. Additionally, 1/48 patients tested positive for rearrangement, and this patient demonstrated tumor shrinkage upon treatment with crizotinib. The patient and one TMA sample displayed expression of the recently identified SDC4-ROS1 fusion, while two TMA samples expressed the CD74-ROS1 fusion and two others expressed the SLC34A2-ROS1 fusion. In HCC78 cells, treatment with ROS1 inhibitors was anti-proliferative and down-regulated signaling pathways that are critical for growth and survival.
Conclusions
ROS1 inhibition may be an effective treatment strategy for the subset of NSCLC patients whose tumors express ROS1 fusion genes.
doi:10.1158/1078-0432.CCR-12-0550
PMCID: PMC3703205  PMID: 22919003
14.  Anaplastic Lymphoma Kinase Gene Rearrangements in Non-small Cell Lung Cancer are Associated with Prolonged Progression-Free Survival on Pemetrexed 
Hypothesis
To explore whether the progression-free survival (PFS) with pemetrexed differs between anaplastic lymphoma kinase (ALK)-positive and other major molecular subtypes of non-small cell lung cancer.
Methods
In an ALK-enriched population, patients with metastatic non-small cell lung cancer were screened by ALK fluorescence in situ hybridization and for epidermal growth factor receptor (EGFR) and KRAS mutations. Triple-tested, pemetrexed-treated patients (monotherapy or combination therapy) were identified and PFS with pemetrexed captured retrospectively.
Results
Eighty-nine eligible cases were identified (19 ALK fluorescence in situ hybridization positive, 12 EGFR mutant, 21 KRAS mutant, and 37 triple negatives). Eighty-three cases (93%) were adenocarcinomas, two were adenosquamous, one squamous, and three had large cell histology. None of the ALK-positive patients had received crizotinib before pemetrexed. Pemetrexed was first-line therapy in 48% (72% as platinum-based combinations). Median PFS (95% confidence interval) data were EGFR mutant (5.5 months; 1–9), KRAS mutant (7 months; 1.5–10), ALK positive (9 months; 3–12), and triple negative (4 months; 3–5). In a multivariate analysis adjusting for line of therapy, mono- versus platinum and nonplatinum combination therapy, age, sex, histology, and smoking status, the only variable associated with prolonged PFS on pemetrexed was ALK+ (hazard ratio = 0.36 [95% confidence interval: 0.17– 0.73], p = 0.0051).
Conclusions
In this exploratory analysis, ALK-positive patients have a significantly longer PFS on pemetrexed compared with triple-negative patients, whereas EGFR or KRAS mutant patients do not. This information should be considered when sizing randomized studies in ALK-positive patients that involve pemetrexed. Pemetrexed should also be prioritized as a cytotoxic to explore further in patients with known ALK-positive disease.
doi:10.1097/JTO.0b013e31820cf053
PMCID: PMC3626562  PMID: 21336183
Anaplastic lymphoma kinase; Non-small cell lung cancer; Epidermal growth factor receptor; KRAS; Pemetrexed
15.  Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models 
The p21-activated kinase (PAK) family of serine/threonine kinases, which are overexpressed in several cancer types, are critical mediators of cell survival, motility, mitosis, transcription, and translation. In the study presented here, we utilized a panel of colorectal cancer (CRC) cell lines to identify potential biomarkers of sensitivity or resistance that may be used to individualize therapy to the PAK inhibitor PF-03758309. We observed a wide range of proliferative responses in the CRC cell lines exposed to PF-03758309, this response was recapitulated in other phenotypic assays such as anchorage-independent growth, three-dimensional (3D) tumor spheroid formation, and migration. Interestingly, we observed that cells most sensitive to PF-03758309 exhibited up-regulation of genes associated with a mesenchymal phenotype (CALD1, VIM, ZEB1) and cells more resistant had an up-regulation of genes associated with an epithelial phenotype (CLDN2, CDH1, CLDN3, CDH17) allowing us to derive an epithelial-to-mesenchymal transition (EMT) gene signature for this agent. We assessed the functional role of EMT-associated genes in mediating responsiveness to PF-3758309, by targeting known genes and transcriptional regulators of EMT. We observed that suppression of genes associated with the mesenchymal phenotype conferred resistance to PF-3758309, in vitro and in vivo. These results indicate that PAK inhibition is associated with a unique response phenotype in CRC and that further studies should be conducted to facilitate both patient selection and rational combination strategies with these agents.
doi:10.3389/fphar.2013.00035
PMCID: PMC3610060  PMID: 23543898
PAK; PF-3758309; colorectal cancer; intrinsic resistance; EMT
16.  Mechanisms of Resistance to Crizotinib in Patients with ALK Gene Rearranged Non-Small Cell Lung Cancer 
Purpose
Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those that do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in ALK+ non-small cell lung cancer (NSCLC) patients.
Experimental Design
We analyzed tissue obtained from 14 ALK+ NSCLC patients demonstrating evidence of radiologic progression while on crizotinib in order to define mechanisms of intrinsic and acquired resistance to crizotinib.
Results
Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK kinase domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient demonstrated outgrowth of EGFR mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient demonstrated the emergence of an ALK gene fusion negative tumor compared to the baseline sample, but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism.
Conclusions
Crizotinib resistance in ALK+ NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers.
doi:10.1158/1078-0432.CCR-11-2906
PMCID: PMC3311875  PMID: 22235099
oncogene fusion; anaplastic lymphoma kinase; protein kinase inhibitors; drug resistance; non-small cell lung cancer
17.  Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer 
Nature genetics  2012;44(10):1111-1116.
Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein–coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ~27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.
doi:10.1038/ng.2405
PMCID: PMC3557461  PMID: 22941189
18.  HIV-1 TAT INCREASES OXIDANT BURDEN IN THE LUNGS OF TRANSGENIC MICE 
Free radical biology & medicine  2011;51(9):1697-1707.
Chronic human immunodeficiency virus (HIV) infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic alveolitis, and interstitial pneumonitis not attributed to either opportunistic infections or presence of the virus. The Tat (TransActivator of Transcription) protein, a required transactivator for expression of full-length viral genes, is pleiotropic and influences expression of cellular inflammatory genes. Tat-dependent transactivation of cellular genes requires specific mediators, including NF-κB, widely recognized as sensitive to changes in cellular oxidant burden. We hypothesized that overproduction of Tat leads to increased oxidant burden and to alterations of basal inflammatory status as measured by NF-κB activation. We engineered transgenic mouse lines that express Tat (86-amino acid isoform) in the lung under the control of the surfactant protein C promoter (SP-C). Tat-transgenic mice exhibit increased pulmonary cellular infiltration, increased nitrotyrosine and carbonyl protein modifications, increased levels of NF-κB, MnSOD and thioredoxin interacting protein (TxNIP). These data indicate that Tat increases oxidant burden and resets the threshold for inflammation, which may increase susceptibility to secondary injuries.
doi:10.1016/j.freeradbiomed.2011.07.023
PMCID: PMC3188365  PMID: 21855628
HIV; Tat; oxidative stress; NF-κB; inflammation; TxNIP
19.  The detection of circulating tumor cells in metastatic and clinically localized urothelial carcinoma 
Urology  2011;78(4):863-867.
Introduction
The detection of circulating tumor cells (CTC) is prognostic in several cancer types. This trial examines the incidence and prognostic value of CTCs in urothelial cancer (UC).
Materials and Methods
44 subjects with UC were assessed for CTCs using CellSearch® Technology (Veridex, LLC, Raritan, NJ), using 7.5 ml of peripheral blood, sorted by magnetic separation (EpCaM positive, and immunofluorescent staining, (Cytokeratin 8, 18, or 19 positive, CD 45 negative, DAPI positive cells) to identify CTCs.
Results
Five of 30 (17%) subjects with clinically localized and 7 of 14 (50%) patients with metastatic UC had at least 1 detectable CTC, with a range of 1–177 CTCs. Six subjects had 5 or more CTCs. FISH analysis was performed in 20 samples from 18 unique subjects, using the UroVysion probe set. Copy number gains consistent with neoplasm were observed in cases with measurable CTCs, but not in any of the CTC-negative samples tested. With a median follow-up of 337 days, all 7 metastatic subjects with detectable CTCs had died, versus 3 of 7 (43%) metastatic subjects without detectable CTCs.
Conclusions
CTCs were observed in 50% of the metastatic UC patients tested. FISH analysis confirmed the aneusomic chromosomal content in the CTCs. The findings suggest measurable CTCs may be prognostic for shortened survival in metastatic UC patients, although the optimal threshold for a “positive” finding is unknown. CTCs were also detected in a subset of patients with clinically-localized disease, identifying a potential high-risk, pre-operative group for future study.
doi:10.1016/j.urology.2011.05.045
PMCID: PMC3190033  PMID: 21813167
Urinary Bladder Neoplasms Neoplastic Cells; Circulating Tumor Markers; Biological In Situ Hybridization; Fluorescence
20.  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
21.  Increased VEGFR-2 Gene Copy Is Associated With Chemoresistance and Shorter Survival in Patients with Non-small Cell Lung Carcinoma Who Receive Adjuvant Chemotherapy 
Cancer research  2011;71(16):5512-5521.
Vascular endothelial growth factor-2 (VEGFR-2 or KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected NSCLC (n=248). KDR copy number gains (CNGs), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR=5.16; P=0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear HIF-1α in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11 and 21 by PCR-based sequencing; however, two variant SNP genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.
doi:10.1158/0008-5472.CAN-10-2614
PMCID: PMC3159530  PMID: 21724587
22.  Optimizing the Detection of Lung Cancer Patients Harboring Anaplastic Lymphoma Kinase (ALK) Gene Rearrangements Potentially Suitable for ALK Inhibitor Treatment 
Clinical Cancer Research  2010;16(22):5581-5590.
Purpose
Anaplastic lymphoma kinase (ALK) rearrangements, associated with sensitivity to an experimental ALK/MET inhibitor, occur in 3% to 5% of non-small cell lung cancers. Intratumoral fluorescence in situ hybridization (FISH) heterogeneity has been reported. We explored the heterogeneity basis, the requirements for accurately determining ALK FISH positivity, and the effect of enriching the tested population using clinical and molecular factors.
Experimental Design
Lung cancer patients were screened by ALK and MET FISH and for EGFR and KRAS mutations.
Results
Thirteen ALK-positive cases were identified from 73 screened patients. Gene copy number increases occurred together with classic rearrangements. All positive cases were adenocarcinomas, 12 were EGFR/KRAS wild-type, and 1 had a coexistent EGFR exon 20 mutation. No association with MET amplification occurred. ALK positivity was associated with <10-pack-year smoking status (P = 0.0004). Among adenocarcinomas, without KRAS or EGFR mutations, with <10-pack-year history, 44.8% of cases were ALK positive. ALK FISH positivity was heterogeneous, but mean values in tumor areas from ALK-positive patients (54% of cells; range, 22-87%) were significantly higher than in adjacent normal tissue or tumor/ normal areas from ALK-negative patients (mean, 5-7%). Contiguous sliding field analyses showed diffuse heterogeneity without evidence of focal ALK rearrangements. One hundred percent sensitivity and specificity occurred when four or more fields (~60 cells) were counted.
Conclusions
Intratumoral ALK FISH heterogeneity reflects technique, not biology. The clinical activity of ALK/MET inhibitors in ALK-positive patients probably reflects ALK, but not MET, activity. Prescreening by histology, EGFR/KRAS mutations, and smoking status dramatically increases the ALK-positive hit rate compared with unselected series.
doi:10.1158/1078-0432.CCR-10-0851
PMCID: PMC3395226  PMID: 21062932
23.  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
24.  HER Family Receptor Abnormalities in Lung Cancer Brain Metastases and Corresponding Primary Tumors 
Clinical Cancer Research  2009;15(15):4829-4837.
Purpose
To compare the characteristics of HER receptors and their ligands deregulation between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC).
Experimental design
Fifty five NSCLC primary tumors (PT) and corresponding brain metastases (BM) specimens were examined for the immunohistochemical expression of EGFR, phosphorylated (p)–EGFR, Her2, Her3, and p-Her3, and their ligands EGF, TGF-α, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, and neuregulins-1 and -2. Analysis of EGFR copy number using fluorescent in situ hybridization and mutation by PCR-based sequencing was also performed.
Results
Metastases showed significantly higher immunohistochemical expression of EGF (membrane, BM 66.0 vs. PT 48.5; P=0.027; and nucleus, BM 92.2 vs. 67.4; P=0.008), amphiregulin (nucleus, BM 53.7 vs. PT 33.7; P=0.019), p-EGFR (membrane, BM 161.5 vs. PT 76.0; P<0.0001; and cytoplasm, BM 101.5 vs. PT 55.9; P=0.014), and p-Her3 (membrane, BM 25.0 vs. PT 3.7; P=0.001) than primary tumors (PT) did. Primary tumors showed significantly higher expression of cytoplasmic TGF–α (PT 149.8 vs. BM 111.3; P=0.008) and neuregulin-1 (PT 158.5 vs. BM 122.8; P=0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time points.
Conclusions
Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptors-related abnormalities from primary lung tumors.
doi:10.1158/1078-0432.CCR-08-2921
PMCID: PMC3372920  PMID: 19622585
25.  EGFR Abnormalities in the Pathogenesis and Progression of Lung Adenocarcinomas 
To identify the characteristics and sequence of epidermal growth factor receptor (EGFR) abnormalities relevant to the pathogenesis and progression of lung adenocarcinoma, we performed a precise mapping analysis of EGFR mutation, gene copy number and total and phosphorylated EGFR (pEGFR) protein expression for the same tissue sites. We examined normal bronchial and bronchiolar epithelium (NBE) and tumor tissues obtained from 50 formalin-fixed lung adenocarcinomas, including 24 EGFR-mutant primary tumors with nine corresponding lymph node metastases and 26 wild-type primary tumors. NBE in 12/24 (50%) mutant and 3/26 (12%) wild-type tumors harbored EGFR mutation; these NBE also showed lack of EGFR copy number increase and frequent EGFR (69%) and pEGFR (33%) overexpression. EGFR mutation and protein overexpression were more frequent in NBE sites within tumors than in NBE sites adjacent to and distant from tumors, suggesting a localized field effect. Sites with high and low EGFR copy numbers were heterogeneously distributed in six of nine primary tumors and one of eight metastases. EGFR protein overexpression was significantly higher in metastasis sites than in primary tumors. We conclude from our findings that EGFR mutations and protein overexpression are early phenomena in the pathogenesis of lung adenocarcinoma and that EGFR mutation precedes an increase in gene copy number. In EGFR-mutant adenocarcinoma metastases, the higher levels of EGFR overexpression and more homogeneously distributed high gene copy numbers suggest tumor progression. Our findings have important implications for the development of new strategies for targeted chemoprevention and therapy in lung adenocarcinoma using EGFR inhibitors.
doi:10.1158/1940-6207.CAPR-08-0032
PMCID: PMC3369271  PMID: 19138956

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