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1.  ROS1 and ALK Fusions in Colorectal Cancer, with Evidence of Intra-tumoral Heterogeneity for Molecular Drivers 
Molecular cancer research : MCR  2013;12(1):111-118.
Activated ALK and ROS1 tyrosine kinases, through gene fusions, has been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma (CRC) specimens using a 4-target, 4-color break-apart fluorescence in situ hybridization (FISH) assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical CRC specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intra-tumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in CRC.
Implications
ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection.
doi:10.1158/1541-7786.MCR-13-0479-T
PMCID: PMC4140177  PMID: 24296758
2.  Using Multiplexed Assays of Oncogenic Drivers in Lung Cancers to Select Targeted Drugs 
IMPORTANCE
Targeting oncogenic drivers (genomic alterations critical to cancer development and maintenance) has transformed the care of patients with lung adenocarcinomas. The Lung Cancer Mutation Consortium was formed to perform multiplexed assays testing adenocarcinomas of the lung for drivers in 10 genes to enable clinicians to select targeted treatments and enroll patients into clinical trials.
OBJECTIVES
To determine the frequency of oncogenic drivers in patients with lung adenocarcinomas and to use the data to select treatments targeting the identified driver(s) and measure survival.
DESIGN, SETTING, AND PARTICIPANTS
From 2009 through 2012, 14 sites in the United States enrolled patients with metastatic lung adenocarcinomas and a performance status of 0 through 2 and tested their tumors for 10 drivers. Information was collected on patients, therapies, and survival.
INTERVENTIONS
Tumors were tested for 10 oncogenic drivers, and results were used to select matched targeted therapies.
MAIN OUTCOMES AND MEASURES
Determination of the frequency of oncogenic drivers, the proportion of patients treated with genotype-directed therapy, and survival.
RESULTS
From 2009 through 2012, tumors from 1007 patients were tested for at least 1 gene and 733 for 10 genes (patients with full genotyping). An oncogenic driver was found in 466 of 733 patients (64%). Among these 733 tumors, 182 tumors (25%) had the KRAS driver; sensitizing EGFR, 122 (17%); ALK rearrangements, 57 (8%); other EGFR, 29 (4%); 2 or more genes, 24 (3%); ERBB2 (formerly HER2), 19 (3%); BRAF, 16 (2%); PIK3CA, 6 (<1%); MET amplification, 5 (<1%); NRAS, 5 (<1%); MEK1, 1 (<1%); AKT1, 0. Results were used to select a targeted therapy or trial in 275 of 1007 patients (28%). The median survival was 3.5 years (interquartile range [IQR], 1.96-7.70) for the 260 patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years (IQR, 0.88-6.20) for the 318 patients with any oncogenic driver(s) who did not receive genotype-directed therapy (propensity score–adjusted hazard ratio, 0.69 [95% CI, 0.53-0.9], P = .006).
CONCLUSIONS AND RELEVANCE
Actionable drivers were detected in 64% of lung adenocarcinomas. Multiplexed testing aided physicians in selecting therapies. Although individuals with drivers receiving a matched targeted agent lived longer, randomized trials are required to determine if targeting therapy based on oncogenic drivers improves survival.
doi:10.1001/jama.2014.3741
PMCID: PMC4163053  PMID: 24846037
3.  Native and rearranged ALK copy number and rearranged cell count in NSCLC: Implications for ALK inhibitor therapy 
Cancer  2013;119(22):3968-3975.
Background
Anaplastic Lymphoma Kinase positive (ALK+) non-small cell lung cancer (NSCLC) responds to ALK inhibitors. Clinically, ≥ 15% cells showing rearrangements by break-apart FISH classify tumors as positive. Increases in native and rearranged ALK copy number also occur.
Methods
1426 NSCLC clinical specimens (174 ALK+ and 1252 ALK negative), and 24 ALK negative NSCLC cell lines were investigated. ALK copy number and genomic status were assessed by FISH.
Results
Clinical specimens with 0–9%, 10–15%, 16–30%, 31–50% and >50% of ALK+ cells were found in 79.3%, 8.5%, 1.4%, 2.7% and 8.1% of cases, respectively. Increased native ALK copy number (≥3 copies/cell in ≥40% cells) was detected in 19% of ALK+ and 62% of ALK negative tumors. In ALK negative tumors, abundant focal amplification of native ALK was rare (0.8%). Other atypical patterns occurred in ~6% of tumors. Mean native ALK copy number ranged from 2.1–6.9 in cell lines and was not correlated with crizotinib sensitivity (IC50s 0.34–2.8 uM) (r=0.279, p=0.1764). Neither native, nor rearranged ALK copy number, nor percentage cells positive correlated with extra-central nervous system progression free survivalin ALK+ patients on crizotinib.
Conclusions
8.5% of cases are below the established positivity threshold by ≤5%. Further investigation of ALK by other diagnostic techniques in such cases may be warranted. Native ALK copy number increases alone are not associated with sensitivity to ALK inhibition in vitro. However, rare complex patterns of increased native ALK in patients should be studied further as atypical rearrangements contained within these may otherwise be missed.
doi:10.1002/cncr.28311
PMCID: PMC3947483  PMID: 24022839
ALK; FISH; copy number; borderline; crizotinib
4.  The dual pathway inhibitor rigosertib is effective in direct-patient tumor xenografts of head and neck squamous cell carcinomas 
Molecular cancer therapeutics  2013;12(10):1994-2005.
The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in head and neck squamous cell cancer (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from 8 HNSCC patients (4 HPV16-positive). HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, western blotting, and IHC. Rigosertib had potent antiproliferative effects on 11 of the 16 HPV− HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in 3/8 HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA activating event (amplification or mutation) and a p53 inactivating event (either HPV16-mediated or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV+ and HPV− HNSCCs focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient for rigosertib-sensitivity.
doi:10.1158/1535-7163.MCT-13-0206
PMCID: PMC3796006  PMID: 23873848
Head and neck squamous cell cancer; Human papilloma virus; Rigosertib; PI3K; TP53
5.  Phase I Study of Oral Rigosertib (ON 01910.Na), a Dual Inhibitor of the PI3K and Plk1 Pathways, in Adult Patients with Advanced Solid Malignancies 
Purpose
To determine the pharmacokinetics (PK), maximum tolerated dose (MTD), safety, and antitumor activity of an oral formulation of rigosertib, a dual phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathway inhibitor, in patients with advanced solid malignancies.
Experimental Design
Patients with advanced solid malignancies received rigosertib twice daily continuously in 21-day cycles. Doses were escalated until intolerable grade ≥ 2 toxicities, at which point the previous dose level was expanded to define the MTD. All patients were assessed for safety, PK, and response. Urinary PK were performed at the MTD. Archival tumors were assessed for potential molecular biomarkers with multiplex mutation testing. A subset of squamous cell carcinomas (SCC) underwent exome sequencing.
Results
Forty-eight patients received a median of 2 cycles of therapy at 5 dose levels. Rigosertib exposure increased with escalating doses. Dose-limiting toxicities were hematuria and dysuria. The most common grade ≥2 drug-related toxicities involved urothelial irritation. The MTD is 560 mg twice daily. Activity was seen in head and neck SCCs (1 complete response, 1 partial response) and stable disease for ≥ 12 weeks was observed in 8 additional patients. Tumors experiencing ≥partial response had PI3K pathway activation, inactivated p53, and unique variants in ROBO3 and FAT1, two genes interacting with the Wnt/β-catenin pathway.
Conclusions
The recommended phase II dose of oral rigosertib is 560 mg twice daily given continuously. Urinary toxicity is the dose-limiting and most common toxicity. Alterations in PI3K, p53, and Wnt/β-catenin pathway signaling should be investigated as potential biomarkers of response in future trials.
doi:10.1158/1078-0432.CCR-13-2506
PMCID: PMC4160109  PMID: 24493827
7.  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
9.  Genetic stability of bone marrow-derived human mesenchymal stromal cells in the Quantum System 
Cytotherapy  2013;15(11):1323-1339.
Background aims
The Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality.
Methods
A rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum.
Results
BM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation.
Conclusions
Quantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates.
doi:10.1016/j.jcyt.2013.05.024
PMCID: PMC4116682  PMID: 23992670
cell proliferation; chromosome; mesenchymal stromal cell; micronucleus; spectral karyotyping; xenograft
11.  ALDH+ tumor-initiating cells exhibiting gain in NOTCH1 gene copy number have enhanced regrowth sensitivity to a γ-secretase inhibitor and irinotecan in colorectal cancer 
Molecular oncology  2012;6(3):370-381.
The Notch signaling pathway has been shown to be upregulated in colorectal cancer (CRC) and important for the self-renewal of cancer stem cells. In this study, we evaluated the efficacy of PF-03084014, a γ-secretase inhibitor, in combination with irinotecan to identify the effects of treatment on tumor recurrence and the tumor-initiating population in our CRC preclinical explant model. The combination of PF-03084014 and irinotecan had the greatest effect at reducing tumor growth on four CRC tumors when compared with treatment with PF-03084014 or irinotecan alone. The combination significantly reduced tumor recurrence in two CRC explants (CRC001 and CRC036) after treatment was discontinued. Both of these tumors exhibited elevated baseline levels of Notch pathway activation as well as an increase in NOTCH1 gene copy number when compared with the two CRC explants (CRC026 and CRC027) where tumors reappeared quickly after termination of treatment. Isolation and injection of aldehyde dehydrogenase (ALDH+ and ALDH−) cells in an in vivo explant model demonstrated that the ALDH+ cell population were tumorigenic. Evaluation of the ALDH+ cells after 28 days of treatment showed that the combination reduced the ALDH+ population in the tumors that did not regrow. Furthermore, ALDH+ cells from CRC001 and CRC027 were injected in vivo and treated immediately for 28 days. Two months after treatment, tumors were evident in the combination treatment group for CRC027 but not for CRC036. These results indicate the combination of PF-03084014 and irinotecan may be effective in reducing tumor recurrence in CRC patients whose tumors exhibit elevated levels of the Notch pathway.
doi:10.1016/j.molonc.2012.03.004
PMCID: PMC4033713  PMID: 22521243
Notch; Cancer stem cell; Gamma-secretase inhibitor; Colorectal cancer
12.  Increased SOX2 Gene Copy Number Is Associated with FGFR1 and PIK3CA Gene Gain in Non-Small Cell Lung Cancer and Predicts Improved Survival in Early Stage Disease 
PLoS ONE  2014;9(4):e95303.
Background
We aimed to investigate prevalence and prognostic role of SOX2, PIK3CA, FGFR1 and BRF2 gene gain in patients with surgically resected non-small cell lung cancer (NSCLC).
Methods
SOX2, PIK3CA, FGFR1 and BRF2 gene copy number was assessed by fluorescence in situ hybridization (FISH) in arrayed tissue cores from 447 resected NSCLCs.
Results
Increased gene copy number (FISH+) for SOX2, PIK3CA, FGFR1 and BRF2 was observed in 23.6%, 29.2%, 16.6% and 14.9% of cases, respectively. FISH+ status for each gene was significantly associated with smoking history, squamous cell carcinoma (SCC) histology, and increased copy number of the other studied genes. Multivariate analysis of overall survival indicated increased SOX2 gene copy number (P = 0.008), stage I-II (P<0.001), and adenocarcinoma or SCC histology (P = 0.016) as independent, favorable prognostic factors. A statistically significant interaction was observed between stage and SOX2 gene status (P = 0.021), indicating that the prognostic impact of SOX2 gene gain differs across stages and is limited to patients with stage I-II disease (HR 0.44, 95% CI: 0.25–0.77; P = 0.004, adjusted for histology).
Conclusions
Increased SOX2 gene copy number is an independent and favorable prognostic factor in surgically resected, early stage NSCLC, regardless of histology. SOX2, PIK3CA, FGFR1 and BRF2 gene gains are likely to occur concurrently, with potentially relevant implications for the development of new therapeutic strategies.
doi:10.1371/journal.pone.0095303
PMCID: PMC3988173  PMID: 24736592
14.  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
15.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
25.  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

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